ION-CAPTURE REAGENTS AND METHODS FOR PERFORMING BINDING ASSAYS

RELATED APPLICATIONS

The present application is a continuation in part application of U.S. Application Serial No. 08/014,048 filed February 5, 1993.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of binding assay devices and methods. In particular, the present invention relates to novel methods and products useful in the performance of immunoassays.

Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of substances of interest or clinical significance which may be present in biological liquids or other materials . Such substances are commonly referred to as "analytes" and can include antibodies, antigens, drugs, and hormones.

Immunoassay techniques take advantage of the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of antigens which are pathogenic or foreign to the organisms. These antibodies and antigens, i.e.. immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism which can be used in vitro to determine the presence or concentration of that particular antigen in a biological sample.

There are several known immunoassay methods using immunoreactants, wherein at least one of the immunoreactants

is labeled with a detectable component so as to be analytically identifiable. For example, the "sandwich" or "two-site" technique may involve the formation of a ternary complex between an an ^f ._ gen and two antibodies. A convenient method of detecting complex formation in such a technique is to provide one labeled antibody and an unlabeled antibody bound to a solid phase support such that the complex can readily be isolated. In this example, the amount of labeled antibody associated with the solid phase is directly proportional to the amount of analyte in the test sample.

An alternative technique is the "competitive" assay. In one example of a competitive assay, the capture mechanism again may use an antibody attached to an insoluble solid phase, but a labeled analyte (rather than a labeled antibody) competes with the analyte present in the test sample for binding to the immobilized antibody. Similarly, an immobilized analyte can compete with the analyte of interest for a labeled antibody. In these competitive assays, the quantity of captured labeled reagent is inversely proportional to the amount of analyte present in the sample.

Despite their great utility, there are disadvantages with such assay methods. First, the heterogeneous reaction mixture of soluble and insoluble reagents, and liquid test sample, can retard the kinetics of the reaction. In comparison to a liquid phase reaction wherein all reagents are soluble, i.e.. a homogeneous reaction mixture, the heterogeneous reaction mixture can require longer incubation periods for equilibrium to be reached in the reaction mixture between the insoluble solid phase system, the free analyte in the test sample, the soluble labeled reagent, and the newly formed insoluble complex. Second, conventional methods of attaching binding members to the solid phase material, such as adsorption of antibody to the solid phase, can produce a solid phase which will readily bind substances other than the analyte. This is referred to as nonspecific binding and can

interfere with the detection of a positive result. Third, with conventional immobilization methods, separate batches of manufactured solid phase reagents can contain variable amounts of immobilized binding member.

SUMMARY OF THE INVENTION

The present invention provides novel capture reagents to facilitate the observation of a detectable signal by separating the analyte and/or indicator reagent from the other assay reagents or test sample. The capture reagents can comprise one or more anionic molecules attached to a specific binding member. The present invention also involves activated polymeric anionic molecules; a method for modifying terminal amino groups on polymeric anionic molecules; and a method for detecting an analyte in a test sample. A readily adapted anionic molecule is an activated polymeric anionic molecule having the formula:

O II

X-(NH-CH-C) _n -NH-CH-COO ^"

(CH ₂ ) _Z (CH ₂ ) _Z

COO ^" COO ^" W,n»2)

wherein n is about 50 to about 1000; z is about 1 to about 6; W is chosen from H+, Na+, K+, Li+, amine salts such as H4+, and derivatives thereof; and X is an amine-reactive moiety, a thiol-reactive moiety or a thiol moiety with which the specific binding member will react. Alternatively, X can represent a specific binding member which has been activated to bind the polymeric anionic molecule. Activation methods are also described by which one or more reactive groups are formed upon the specific binding member or the polymeric anion.

The specific binding member component of the capture reagent can be either a hapten or a acromolecule. The charged capture reagent enables homogeneous assay and separation reactions wherein reaction complexes, as well as unreacted capture reagent, can be removed from the reaction mixture by contacting the mixture with an oppositely charged solid phase. Virtually any binding assay (sandwich assays, competitive assays, indirect assays using ancillary specific binding members, inhibition assays, etc.) can be adapted to use the novel capture reagents.

The present invention brings two advantages to binding assays: (a) the use of liquid phase kinetics in the binding reaction facilitates the formation of a complex from the homogeneous mixture of analyte and assay reagents, and (b) it increases the potential number of complexes that can be immobilized on the solid support.

The invention can also be used in a separation procedure wherein the capture reagent is conjugated to a charged substance. A liquid sample suspected of containing the analyte to be separated is mixed with the capture reagent in solution to form a charged complex. Following the binding reaction, the solution is contacted to an oppositely charged solid phase to attract, attach, and separate the newly formed complex and unreacted capture reagent from the liquid sample.

If liquid phase kinetics are not sought, the present invention also provides an efficient method of immobilizing binding members on a solid phase through ionic interaction between oppositely charged molecules.

DETAILED. DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The assay methods and reagents of the present invention can be used in a variety of assay formats. The present invention is not limited to immunoreactive assays. Any assays using specific binding reactions between the analyte and assay reagents can be performed. Definitions

The following definitions are applicable to the present invention.

The term "specific binding member, " as used herein, refers to a member of a specific binding pair, i.e.. two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to antigen and antibody-specific binding pairs, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and capture nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence) , complementary peptide sequences including those formed by recombinant methods, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member. For example, a derivative or fragment of the analyte, i.e.. an analyte-analog, can be used so long as it has at least one epitope in common with the analyte. Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof including those formed by recombinant DNA methods or peptide synthesis. An antibody can be a monoclonal or polyclonal antibody, a recombinant protein or a mixture (s) or fragment (s) thereof, as well as a mixture of an antibody and other specific binding members. The details of the preparation of such antibodies and their suitability for use

as specific binding members are well known to those skilled in the art.

The term "hapten, " as used herein, refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.

The term "test sample," as used herein, refers to virtually any liquid sample. The test sample can be derived from any desired source, such as a physiological fluid, for example, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid or the like. The liquid test sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous liquids, or the like; methods of treatment can also involve separation, filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. Besides physiological fluids, other liquid samples such as water, food products and the like can be used. In addition, a solid can be used once it is modified to form a liquid medium.

"Analyte, " as used herein, is the substance to be detected in or separated from the test sample using the present invention. The analyte can be any substance for which there exists a naturally occurring specific binding member or for which a specific binding member can be prepared. In addition, the analyte may bind to more than one specific binding member. "Analyte" also includes any antigenic substances, haptens, antibodies, and combinations thereof. The analyte can include a protein, a peptide, an amino acid, a hormone, a steroid', a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.

The term "analyte-analog, " as used herein, refers to a substance which cross-reacts with an analyte-specific binding member, although it may do so to a greater or lesser extent than does the analyte itself. The analyte-analog can include a modified analyte as well as a fragmented or synthetic portion of the analyte molecule so long as the analyte-analog has at least one epitopic site in common with the analyte of interest.

The term "label," as used herein, refers to any substance which is attached to a specific binding member and which is capable of producing a signal that is detectable by visual or instrumental means. Various suitable labels for use in the present invention can include chromogens; catalysts; fluorescent compounds; chemiluminescent compounds; radioactive labels; direct visual labels including colloidal metallic and non-metallic particles, dye particles, enzymes or substrates, or organic polymer latex particles; liposomes or other vesicles containing signal producing substances; and the like.

A large number of enzymes suitable for use as labels are disclosed in U.S. Patent No. 4,275,149, columns 19-23, herein incorporated by reference. An example of an enzyme/substrate signal producing system useful in the present invention is the enzyme alkaline phosphatase and the substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate, or derivative or analog thereof, or the substrate 4- methylumbelliferyl-phosphate.

In an alternative signal producing system, the label can be a fluorescent compound where no enzymatic manipulation of the label is required to produce a detectable signal. Fluorescent molecules such as fluorescein, phycobiliprotein, rhodamine and their derivatives and analogs are suitable for use as labels in this reaction.

In an especially preferred embodiment, a visually detectable, colored particle can be used as the label component of the indicator reagent, thereby providing for a

direct colored readout of the presence or concentration of the analyte in the sample without the need for further signal producing reagents. Materials for use as the colored particles are colloidal metals, such as gold, and dye particles as disclosed in U.S. Patent Nos. 4,313,734 and 4,373,932. The preparation and use of non-metallic colloids, such as colloidal selenium particles, are disclosed in U.S. Patent No. 4,954,452. The use of colloidal particle labels in immunochromatography is disclosed in U.S. Patent No. 5,120,643. Organic polymer latex particles for use as labels are disclosed in co-owned and copending U.S. Patent Application Serial No. 07/248,858 filed September 23, 1988.

A "signal producing component," as used herein, refers to any substance capable of reacting with another assay reagent or the analyte to produce a reaction product or signal that indicates the presence of the analyte and that is detectable by visual or instrumental means. "Signal production system, " as used herein, refers to the group of assay reagents that are needed to produce the desired reaction product or signal. For example, one or more signal producing components can be used to react with a label and generate the detectable signal, i.e.. when the label is an enzyme, amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes to produce a detectable reaction product.

An "indicator reagent," as used herein, refers to a label attached to a specific binding member. The indicator reagent produces a detectable signal at a level relative to the amount of an analyte in the test sample. Generally, the indicator reagent is detected or measured after it is captured on the solid phase material, but the unbound indicator reagent can also be measured to determine the result of an assay.

The specific binding member of the indicator reagent is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as in a competitive assay, or to

an ancillary specific binding member as in an indirect assay. The label, as described above, enables the indicator reagent to produce a detectable signal that is related to the amount of analyte in the test sample. The specific binding member component of the indicator reagent enables the indirect binding of the label to the analyte, to an ancillary specific binding member or to the capture reagent. The selection of a particular label is not critical, but the label will be capable of generating a detectable signal either by itself, such as a visually detectable signal generated by colored organic polymer latex particles, or in conjunction with one or more additional signal producing components, such as an enzyme/substrate signal producing system. A variety of different indicator reagents can be formed by varying either the label or the specific binding member; it will be appreciated by one skilled in the art that the choice involves consideration of the analyte to be detected and the desired means of detection.

A "capture reagent," as used herein, refers to an unlabeled specific binding member attached to a charged substance. The attachment of the components is essentially irreversible and can include covalent mechanisms. The capture reagent is used to facilitate the observation of the detectable signal by substantially separating the analyte and/or the indicator reagent from other assay reagents and the remaining test sample.

The specific binding member can be a small molecule, such as a hapten or small peptide, so long as the attachment to the charged substance does not interfere with the binding member's binding site. The specific binding member of the capture reagent is -specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay. The charged substance can include anionic and

cationic substances. The charged substances may be monomeric or polymeric. In a preferred embodiment, the charged substance is a linear anionic polymer. Examples of anionic polymers include polyglutamic acid (PGA) , anionic protein or derivatized protein such as albumin, anionic polysaccharides such as heparin or alginic acid, polyaspartic acid, polyacrylic acid, and polyamino acids having a net negative charge at an appropriate pH (such as a pH in the range of 4 to 10) . Anionic polymers having a molecular weight of about 5000 to about 500,000 daltons are preferred. The specific binding member can be joined to more than one charged monomer or polymer to increase the net charge associated with the capture reagent.

The attachment of the charged substance to the specific binding member typically does not result in either intermolecular or intramolecular crosslinking which causes an obstacle in conducting the claimed assay methods. Coupling reagents which mediate the attachment of the charged substance and specific binding member by condensation of specific functional groups can be used. For instance, commercially available (from SIGMA Chemical Co.) carbodiimide compounds such as N,N' -dicyclohexylcarbodiimide, and l-ethyl-3- (3- dimethylamino-propyl) carbodiimide can be employed. As described in Examples 2, 15, 16, and 17, these carbodiimide compounds conjugate antibody and polyglutamic acid (PGA) by condensing the amino-groups on antibody and carboxylic acid residues on PGA to form amide linkages. Those skilled in the art will appreciate that intermolecular cross-linking between a binding member and charged substance, such as antibody and PGA, can be controlled by empirically varying the charge ratio of reactants in the conjugation reaction mixture.

The binding affinity of the charged substance to the oppositely charged solid phase should be higher than that of the binding member, analyte, indicator reagent and other materials present in the assay reaction mixture. Assay

protocols (such as one step and two step) , sample pretreatment, and assay sensitivity and range requirements may vary from analyte to analyte, and it will be appreciated by those skilled in the art that the selection of charged substance for the capture reagent in a given assay will be determined empirically.

Another technique that can be employed to avoid or reduce crosslinking is selecting a charged substance and a method of attaching it to the binding member so that there is a single covalent link between the charged substance and the specific binding member. This selection is within the skill in the art and can be made without undue experimentation. If the charged substance is a polyanion, crosslinking may also be avoided or reduced by selecting a polyanion which does not contain multiple activated groups. The examples of conjugating polymeric anions to binding members are described in detail immediately below.

In one embodiment of the present invention, a negatively charged capture reagent can be prepared by conjugating a specific binding member to one or more activated polymeric anionic molecules and conjugate bases thereof represented by the general formula:

O

II

X-(NH-CH-C) _n -NH-CH-COO ^'

(CH ₂ ) _Z (CH ₂ ) _Z COO ^' COO ^" W ₍ ( _n n ₊ +2)

wherein n is about 50 to about 1000; z is about 1 to about 6; is chosen from H+, Na+, K+, Li+, a ine salts such as NH4+, and derivatives thereof; and X is virtually any reactive group or moiety having a reactive group that enables the chemical binding of the specific binding member and the polymer. X can be an amine-reactive group or moiety, a thiol-reactive group

or moiety, or a thiol group or moiety represented by -A-SH wherein A is a spacer arm. For example, a specific binding member having an amino group can be conjugated to an activated PGA anionic molecule having an amine-reactive moiety. The amine-reactive moieties enable the binding of the activated polymer to an amino group on a specific binding member and include, but are not limited to, those represented by the following formulas:

NH o 0 NH

II π n II

-A-C-O- R" A-O-C-CI . A-CH and the addition salts of A-C-O- R"

wherein m is two or three, R' is a sulfur stabilizer and R" is an aliphatic or aryl group. Sulfur stabilizers include, but are- not limited to, 2-pyridyl, 4-pyridyl and 5-nitro-2-pyridyl groups. "A" represents a spacer of about one to about thirty atoms including, but not limited to, carbon, nitrogen, sulfur and oxygen atom chains and combinations thereof such as polyether, polymethylene and polyamide, as well as aromatic spacers such as phenylene.

Alternatively, a specific binding member having a thiol group can be conjugated to an activated polymer having a thiol-reactive moiety. The thiol-reactive moieties include, but are not limited to, those represented by the following formulas:

wherein A is a spacer of about one to about thirty atoms as described above. In yet another alternative, a specific binding member having a thiol-reactive group can be linked to an activated polymer having a thiol moiety such as -A-SH.

The negatively charged capture reagents of some of the following Examples were formed by reacting the desired specific binding member with an activated PGA molecule having modified terminal amino groups. Briefly, the modification method involved: (1) dissolving the PGA in a solvent (e.σ.. a water miscible aprotic solvent such as dioxane, dimethylformamide, l-methyl-2-pyrrolidinone and dimethyl sulfoxide) ; (2) adding a proton absorbing reagent (e,σ.. 4- methyl morpholine) in the amount of about one equivalent per titratable carboxylic acid; (3) adding about a 2 to about a 100 molar excess of an amine-reactive modification reagent (e.σ.. 1, 4-phenylene diisothiocyanate dissolved in dimethylformamide); (4) reacting the mixture; and (5) removing the unreacted amine-reactive modification reagent. Suitable proton absorbing reagents include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide or lithium hydroxide, and tertiary amines such as 4-methyl morpholine and triethylamine.

The polymeric anionic molecule or the specific binding member will include one or more amino, carboxyl or thiol groups or can be activated by the incorporation of an

amino, carboxyl or thiol group thereby enabling the chemical linking of the specific binding member with the polymeric anionic molecule. "Activated species" refer to specific binding members and polymeric anionic molecules which contain a reactive group through the incorporation of a linking or other activating agent. The amine-reactive modification reagents are a subclass of those reagents used to "activate" a specific binding member or polymeric anionic molecule, i.e.. to prepare the specific binding member or the polymeric anionic molecule for chemical linking. Activating agents also include thiol introducing agents such as the thiolanes (such as 2-iminothiolane) , succinimidyl mercaptoacetates (such as N- succinimydl-S-acetylmercaptoacetate) , and disulfide compounds which are subsequently reduced to a thiol. The thiol introducing agents can be used to activate specific binding members for subsequent reaction with a thiol-reactive group. Amine-reactive modification reagents include, but are not limited to, bifunctional linking or coupling agents, such as succinic anhydride analogs, iminothiolane analogs, homobifunctional reagents and heterobifunctional reagents, which enable the chemical linking of the specific binding member and the polymeric anionic molecule. Examples of homobifunctional reagents can be represented by the formula X- A-X wherein X is an amine-reactive group and A is a spacer of about one to about thirty atoms. Examples of heterobifunctional reagents can be represented by the formula X-A-Y wherein X is an amine-reactive group, Y is a thiol- reactive moiety, a thiol moiety or a thiol precursor and A is a spacer of about one to about thirty atoms as described above. Proteinaceous specific binding members with cysteine residues at the protein's active site can have their activity decreased by the addition of a coupling agent, therefore the cysteine residues in the active site must be protected, by means known in the art, prior to reacting the protein with the coupling agent.

The term "coupling agent, " as used herein, includes bifunctional linking or coupling agents, i.e.. molecules containing two reactive groups or "ends," which may be tethered by a spacer. The reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens. The heterobifunctional linking reagents have two different reactive ends, e.g.. an amino-reactive end and a thiol-reactive end, while homobifunctional reagents have two similar reactive ends, e.g.. -bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds, and NHS homobifunctional crosslinkers such as disuccinimidyl suberate (DSS) as well as the water soluble analogs, sulfo-NHS esters (Pierce 1989 Handbook and General Catalog; Pierce Chemicals, Rockford, IL, 61105-9976) .

Other commercially available homobifunctional linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA) ; dimethyl pimelimidate dihydrochloride (DMP) ; and dimethyl suberimidate dihydrochloride (DMS) . The iminothiolane analogs can be represented by the general formula:

NH

A S

wherein A is a spacer of about 1 to about 5 atoms, e.g.. 2- iminothiolane (Traut's Reagent) .

Commercially available heterobifunctional reagents suitable for use in the present invention include, but are not

limited to, maleimido-NHS active esters coupling agents such as m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS); succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC); succinimidyl 4- (p-maleimidophenyl)butyrate (SMPB) and derivatives thereof, including sulfosuccinimidyl derivatives such as sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane- 1-carboxylate (sulfo-SMCC) ; m-maleimidobenzoyl- sulfosuccinimide ester (sulfo-MBS) and sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate (sulfo-SMPB) .

Other suitable heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N- succinimiαyl (4-iodacetyl) -aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl (4- iodoacetyl) -aminobenzoate (sulfo-SIAB) . Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3- (2-pyridyldithio) -propionate (SPDP) .

Yet another group of coupling agents includes the extended length heterobifunctional coupling agents described in U.S. Patent Nos. 5,002,883 and 4,994,385 which are incorporated by reference herein. The extended length heterobifunctional coupling agents include maleimido-NHS active ester reagents wherein the spacer is represented by the formula:

O

II

- (amino acid) _n - C - R ^•

wherein the amir .. acid is a substituted or unsubstituted amino acid, having from three to ten carbon atoms in a straight chain; n is from one to ten; and R is an alkyl, cycloalkyl, alkyl-cycloalkyl or an aromatic carboxylic ring. The term alkyl-cycloalkyl includes alkyl groups linked to cycloalkyl ring structures where the alkyl group links the cycloalkyl to a maleimide or carbonyl group. The term alkyl includes

straight or branched alkyl groups, preferably lower alkyl groups having from one to six carbon atoms.

If a spacer is present, the spacer can be any molecular chain that is non-reactive, stable and non-binding to the analyte or other specific binding members with which it will be used. The length of the spacer can be varied and can range from the size of a single atom to the sizes disclosed in U.S. Patent Nos. 5,002,883 and 4,994,385 or larger.

The choice of the amine-reactive modification reagent, thiol introducing agent or other activating agent is not critical, but one skilled in the art can determine without undue experimentation suitable or preferred agents for use with the particular polymeric anionic molecule and specific binding member to be used in the diagnostic assay. Therefore, it will be appreciated by those skilled in the art that the coupling agent or activating agent used in a given assay will generally be determined empirically.

Suitable thiol-reactive moieties of the heterobifunctional reagents include, but are not limited to, those represented by the following formulas:

Suitable thiol precursor moieties include, but are not limited to, those represented by the following formulas:

Suitable amine-reactive moieties include, but are not limited to, those represented by the following formulas:

NH O O NH

II 11 11 II

-C-O- R" . O-C-CI . -CH • and the addition salts of -C-O-R" .

wherein m is 2 or 3, R' is a sulfur stabilizer, as described above, and R" is an aliphatic or aryl group.

In yet another embodiment of the present invention, a specific binding member having an amine-reactive group (e.g.. an activated specific binding member) can be conjugated to a terminal amino group of the polymeric anionic molecule. Briefly, an example of a conjugation procedure involves: (1) dissolving PGA in a solvent (e.g. , a water miscible aprotic solvent such as dioxane, dimethylformamide, l-methyl-2- pyrrolidinone and dimethyl sulfoxide) ; (2) adding a proton absorbing reagent (e.g.. an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, or lithium hydroxide, or a tertiary amine such as -methyl morpholine or triethylamine) in the amount of about one equivalent per titratable carboxylic acid; (3) adding about a 2 to about a 100 molar excess of amine-reactive specific binding member (e.g.. phosgene-activated phenylcyclidine or phenylcyclidine-

4-chloroformate) ; (4) reacting the mixture and (5) removing the unreached amine-reactive specific binding member. Suitable examples of amine-reactive groups on specific binding members include, but are not limited to,

NH II O O NH

I I II I I

^■ A-C-O- R" , A-O-C-CI . A-CH and the addition salts of A-C-O-R"

wherein A is a spacer of about one to about thirty atoms as described above, m is two or three, R' is a sulfur stabilizer and R" is an aliphatic or aryl group.

An example of the preparation of a negatively charged capture reagent involves the reaction of a specific binding member (SBM) having an amino group and an activated PGA having an amine-reactive moiety. The resulting reaction and reaction product can be illustrated as follows:

SBM-NH ₂ +

t

An "ancillary specific binding member, " as used herein, refers to any member of a specific binding pair which is used in the assay in addition to the specific binding members of the capture reagent and the indicator reagent. For example, in an indirect assay an ancillary specific binding member may bind the analyte as well as a second specific binding member to which the analyte itself could not attach, or as in an inhibition assay the ancillary specific binding member may be a reference binding me'iber as described below. One or more ancillary specific bindi - g members can be used in an assay.

A "solid phase, " as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its

intrinsic charge and ability to attract the capture reagent, e.g.. methylated wool, nylons, and special glasses having a positive charge.

The solid phase can include any suitable porous material. By "porous" is meant that the material is one through which liquids can flow and can easily pass. In the present invention, the solid phase can include a fiberglass, cellulose, or nylon pad for use in a pour and flow-through assay device having one or more layers containing one or more of the assay reagents; a dipstick for a dip and read assay; a test strip for wicking (e.g. , paper) or thin layer chromatographic (e.g.. nitrocellulose) techniques; or other porous material well known to those skilled in the art. The solid phase, however, is not limited to porous materials. The solid phase can also comprise polymeric or glass beads, microparticles, tubes, sheets, plates, slides, wells, tapes, test tubes, or the like, or any other material which has an intrinsic charge or which can retain a charged substance.

Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used as a solid phase including polysaccharides, e.g., cellulose materials such as paper and cellulose derivatives such as cellulose acetate and nitrocellulose; silica; inorganic materials such as deactivated alumina, diatomaceous earth, MgS04, or other inorganic finely divided material uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring (e.g.. cotton) and synthetic (e.g. , nylon) ; porous gels such as silica gel, agarose, dextran, and gelatin; polymeric films such as polyacrilamide; and the like. The solid phase should have reasonable strength or strength can be provided by means of a support, and it should not interfere with the production of a detectable signal.

Preferred solid phase materials include a porous fiberglass material, such as a "Whatman 934-AH" filter paper, which has a nominal thickness of 0.33 mm, or the disposable IMX™ wedge and TESTPACK™ (fiber matrix) devices of Abbott Laboratories (Abbott Park, IL) . The thickness of such material is not critical, and will be a matter of choice, largely based upon the properties of the sample or analyte being assayed, such as the fluidity of the test sample.

To change or enhance the intrinsic charge of the solid phase, a charged substance can be coated directly to the material or onto microparticles which are then retained by a solid phase support material. Alternatively, microparticles alone can be used as the charged solid phase. Particles can serve as the solid phase, by being retained in a column or being suspended in the mixture of soluble reagents and test sample, or the particles themselves can be retained and immobilized by a solid phase support material. By "retained and immobilized" is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material. The particles can be selected by one skilled in the art from any suitable type of particulate material composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials.

It is preferable that a charged substance is retained on the solid phase so that there is a relatively uniform charge distributed throughout the solid phase. The solid phase should retain a charged substance that is at least equally and oppositely charged with respect to the charged substance of the capture reagent. For example, an anionic substance can be bound to the capture reagent, and a cationic substance can be retained on the solid phase, or vice versa. Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used as the cationic substance.

In a preferred embodiment, a cationic substance is retained on the solid phase. In a more preferred embodiment, the cationic substance is a polymeric substance. Even more preferably, the cationic polymer is a linear polymer. Although not fully understood, it is believed that higher affinity binding is achieved using linear polymers due to the linear pairing of the oppositely charged molecules. The polymeric substance may be a homopolymer or a copolymer, and preferably, has a relatively high molecular weight, for instance, above about 100,000 daltons. It is also preferable that the polymeric cation substance has little, if any, binding affinity for the indicator reagent employed in the assay. Selection of suitable cationic substances is most critical for assays requiring high sensitivity and extensive washings of the solid phase (such as in two step assays) .

In one embodiment of the invention, the polymeric cationic substance is a quaternary ammonium compound. The invention contemplates that a variety of quaternary ammonium compounds may be employed. Quaternary ammonium compounds having about 1% to about 10% nitrogen (exclusive of counter ion) in the form of quaternary ammonium moiety are preferred. Examples of linear quaternary ammonium polymeric compounds that may be employed include but are not limited to, commercially available compounds known as MERQUAT®, CELQUAT®, GAFQUAT®, and MAGNIFLOC®.

MERQUAT® is commercially available from Calgon, Pittsburgh, PA. The MERQUAT® compound is a homopolymer of dimethyldiallylammonium chloride and has a nitrogen content of about 8.75%. MERQUAT® has a molecular weight of about 10 ^s to about lθ6 daltons, and has about one positive charge per 161 dalton. The structure of MERQUAT® is shown below:

n = 620-6200

CELQUAT® is commercially available from National Starch & Chemical Corporation, Bridgewater, NJ. CELQUAT® is a copolymer of hydroxyethylcellulose and dimethyldiallylammonium chloride. it has a molecular weight of about IO ⁵ dalton and has a nitrogen content of about 1% to 2%. The compound has about one positive charge per 700 - 1400 dalton.

GAFQUAT® is commercially available from GAF Corporation, Wayne, NJ. It is the reaction product of diethyl sulfate and a copolymer of vinylpyrrolidone and dimethylaminoethylmethacrylate. The compound has a molecular weight of about IO ⁵ to about 10 ⁶ dalton. The chemical structure of GAFQUAT® is shown below:

MAGNIFLOC® is commercially available from _C YTEC Industries, Indianapolis, IN. The compound is a copolymer of dime hylamine and epichlorohydrin and has a nitrogen content

of about 10.2%. M _A G _N IFLOC® has about one positive charge per 137 dalton. _T he structure of MAGNIFLOC® is shown below:

n= 1500-2200

Other types of polymeric cation substances are commercially available, and include but are not limited to hexadimethrine bromide (POLYBRENE®) and diethylaminoethyl- dextran, both available from Sigma Chemical Company, St.

Louis, MO.

In preferred embodiments of the invention, MER ^Q UAT® or MAGNIFLOC® is retained on the solid phase. Applicants have found that these polymeric cationic substances provide increased capturing efficiency for polyanionic charged capture reagent, particularly PGA charged capture reagent.

The anionic and cationic substances may be selected on the basis of charge density. If quaternary ammonium compounds such as MERQUAT® are utilized for coating the solid phase, selection of the polyanion substance is also made in view of the analyte being assayed. Typically, the total charge of the solid phase should be at least equal to the total charge of the charged capture reagent in the reaction mixture. It is preferred, however, to have greater total positive charge on the solid phase as compared to the total charged polyanion in the capture reagent. Applicants have found that an optimal amount of MERQUAT® employed in an assay is stoichiometrically about 50 to about 150 times in excess the amount of polyanion charged capture reagent. Methods for retaining the cationic charged substance on the solid phase

are known in the art, and are described in detail in the Examples below.

To achieve the highest sensitivity in the assay, it is desirable to keep non-specific binding as low as possible. As demonstrated in Examples 1-13 below, nonspecific binding is typically not a problem using the presently disclosed ion- capture techniques. However, prevention of nonspecific binding may be accomplished in several ways. For instance, the anionic and cationic substances may be selected so that they are the dominant charges in the reaction mixture. It is known in the art that polyanion/polycation binding consists of high binding affinities due to the extended linear conformation of the polyanion and polycation (in contrast to most protein molecules which are in globular form) and the cooperative nature of the binding shown by the multiple pairing of oppositely charged groups on the polymers. Those skilled in the art will appreciate that most biological materials (such as antibodies, antibody-enzyme conjugates, and antibody- antigen complexes) bound to a polycation can be easily replaced by a polyanion material with a higher binding affinity to the polycation material fSee. e.σ.. Peterson et al. , Ion-Exchanσe Displacement Chromatoσraphv of Proteins. Usinσ Narrow Ranσe Carboxymethyldextrans and a New Index of Affinit ^y . 12j2:271-282 (1983)].

Nonspecific binding can also be reduced or avoided by including a nonspecific binding blocker in the indicator reagent. For example, a polyanionic blocker (such as dextran sulfate or carboxymethyl cellulose) can be included in the indicator reagent to inhibit the binding of indicator reagent to the positively charged solid phase. It was unexpectedly discovered that the nonspecific binding blocker could be a free polyanion even when the capture reagent used in the assay involved a polyanionic substance conjugated to a specific binding member. It would have been expected that the presence of a free or unbound polyanion would prevent, or at least

reduce, the immobilization of the capture reagent on the solid phase. It was found, however, that the nonspecific blocker was more effective in inhibiting the direct, nonspecific binding of indicator reagent to the solid phase than it was in reducing the attachment of the polyanionic capture reagent to the polycationic solid phase. Suitable nonspecific binding blockers include, but are not limited to, dextran sulfate, heparin, carboxymethyl dextran, carboxymethyl cellulose, pentosan polysulfate, inositol hexasulfate and β-cyclodextrin sulfate. Nonspecific binding blockers and their use in the disclosed assays is further described in Examples 14 and 15 below.

The amount of polyanionic nonspecific binding blocker added to the indicator reagent can be greater than the amount of polyanionic substance contained in the capture reagent. It was found that free polyanionic nonspecific binding blocker could be added to the indicator reagent in amounts 40,000 times the amount of polyanionic substance used in the capture reagent. Generally, the preferred amount of polyanionic blocker added to the indicator reagent is 50 to 14,000 times the amount of polyanionic substance used in the capture reagent. For two step sandwich assays, the preferred amount of polyanionic blocker added to the indicator reagent is 1000 to 2000 times that contained in the capture reagent.

An appropriate range of use of the nonspecific binding blocker can be determined for each analyte of interest. For example, in an assay to detect thyroid stimulating hormone (TSH) wherein dextran sulfate was added to the indicator reagent as a free polyanionic nonspecific binding blocker, suitable amounts of free polyanion ranged from 233 to 19,000 times that of the capture reagent, or about 0.1-8% dextran sulfate. As illustrated in Examples 15, 16, and 17, the preferred nonspecific binding blocker as well as the preferred amount of nonspecific binding blocker can be optimized for each analyte of interest and each assay format.

Depending upon the analyte of interest and the desired assay configuration, the preferred nonspecific binding blocker, as well as the optimization of its concentration and whether it is included as a component of another assay reagent, is selected by empirical techniques which can be performed without undue experimentation by one of ordinary skill in the art.

Uses For Ion-Caoture Reaσents

In accordance with the method of the present invention, a sandwich assay can be performed wherein a soluble capture reagent can include an analyte-specific binding member which has been bound to a charged substance such as an anionic substance. The ionic species can be a monomer or a polymer. The capture reagent is contacted with a test sample, suspected of containing the analyte, and an indicator reagent comprising a labeled analyte-specific binding member. The reagents can be mixed simultaneously or added sequentially, either singly or in combination. For instance, in a one step assay, the capture reagent and indicator reagent are mixed simultaneously with the test sample. In a two step assay, the capture reagent is mixed with the test sample and the indicator reagent is subsequently contacted with the reaction mixture. A binding reaction results in the formation of a capture reagent/analyte/indicator reagent complex. The assay also comprises the step of separating the resultant complexes from the reaction mixture by using a solid phase that is either oppositely charged with respect to the capture reagent or that retains an oppositely charged substance, for example a cationic substance. In this example, the oppositely charged solid phase attracts and attaches capture reagent/analyte/indicator reagent complexes, as well as unreacted capture reagent, through the ionic interaction of the anionic and cationic substances. The complexes, if any, retained on the solid phase are then detected by examining the

solid phase for the indicator reagent. If analyte is present in the sample, then label will be bound to the solid phase material. The amount of label on the solid phase is proportional to the amount of analyte in the sample. The only major limitation inherent in the sandwich assay is the requirement for the analyte to have a sufficient size and appropriately orientated epitopes to permit binding of at least two specific binding members.

The present invention also can be used to conduct a competitive assay. In a competitive configuration, the soluble capture reagent again includes a specific binding member which has been attached to a charged substance, such as an anionic polymer. The capture reagent is contacted with both test sample and an indicator reagent that includes a second binding member which has been labeled with a signal generating compound. The assay can be performed as a one step assay or two step assay, as described above. Either the capture reagent and analyte can compete in binding to the indicator reagent (e.g.. the capture reagent and analyte are antigens competing for a labeled antibody) , or the indicator reagent and analyte can compete in binding to the capture reagent (e.g.. the indicator reagent is a labeled antigen which competes with the antigen analyte for binding to the antibody capture reagent) . A competitive binding reaction occurs resulting in the formation of soluble complexes of (1) capture reagent/analyte or indicator reagent/analyte and (2) capture reagent/indicator reagent. The soluble capture reagent/analyte complexes, capture reagent/indicator reagent complexes and/or unreacted capture reagent are removed from the reaction mixture by contacting the reaction mixture with the oppositely charged solid phase, for example, a cationic substance on a solid phase. The complexes and unreacted capture reagent are retained on the solid phase through the ionic interaction of the opposite charges. The complexes retained on the solid phase can be detected via the label of

the indicator reagent. In the competitive assay, the amount of label that becomes bound to the solid phase is inversely proportional to the amount of analyte in the sample. Thus, a positive test sample will generate a negative signal. The competitive assay is advantageously used to determine the presence of small molecule analytes, such as small peptides or haptens, which have a single epitope with which to bind a specific binding partner.

For example, in an assay for theophylline, an anti- theophylline antibody (either monoclonal or polyclonal) can be conjugated with an anionic substance to form a soluble capture reagent, and a competition for binding to that antibody can be established between the soluble labeled theophylline (i.e.. indicator reagent) and the unlabeled theophylline of the test sample. After incubation, the homogeneous reaction mixture can be contacted to a cation-coated solid phase. The ionic interaction between the oppositely charged ionic species of the capture reagent and the solid phase separates the formed complexes and unreacted capture reagent from the reaction mixture. The signal from the indicator reagent can then be detected. In this example, increased theophylline levels in the test sample will result in decreased label bound to the solid phase.

The present invention can also be used in indirect immunoassays using one or more ancillary specific binding members. For example, an indirect sandwich immunoassay with the formation of a capture reagent/analyte/anti-analyte antibody/indicator reagent complex can be performed, wherein the indicator reagent is a specific binding partner for the ancillary specific binding member which is specific for the analyte. In a further example, the capture reagent may include a specific binding partner for the ancillary specific binding member which is specific for the analyte.

In addition, the present invention can be used in an inhibition assay, such as the measurement of an antibody by

inhibiting the detection of a reference antigen. For example, the capture reagent can include an antibody/anion conjugate and the indicator reagent can be a labeled antibody. The test sample, suspected of containing an antibody analyte, is mixed with a reference antigen with which the capture reagent and indicator reagent can form a detectable sandwich complex that can be immobilized upon a solid phase. The degree of inhibition of antigen uptake by the capture reagent is proportional to the amount of antibody analyte in the test sample, thus, as the concentration of the antibody analyte increases, the less reference antigen is available to complete the immobilized sandwich complex.

In general, once complex formation occurs between the analyte and capture and indicator reagents, the solid phase is used as a separation mechanism; the homogeneous reaction mixture is contacted with the solid phase, and the newly formed complexes, and unreacted capture reagent, are retained on the solid phase through the ionic interaction of the opposite charges of the solid phase and the capture reagent. If the user is not concerned with liquid phase kinetics, the capture reagent can be pre-immobilized on the solid phase to form a "capture situs," i.e.. that region of the solid phase having one or more capture reagents non- diffusively attached thereto.

The present invention can also be used for separating a substance from a liquid sample. For example, the capture reagent and solid phase can be used without an indicator reagent for the sole purpose of separating an analyte from a test sample. Furthermore, the capture reagent can be contacted with a soluble second charged substance which is oppositely charged with respect to the capture reagent. The second charged substance is not retained on the solid phase prior to contacting the sample to the solid phase material, but it attracts and attaches to the capture reagent

such that the resultant assay complexes are retained on the solid phase.

When the complex of charged capture reagent and analyte (and/or indicator reagent), and any unreacted capture reagent, is contacted to the oppositely charged solid phase, the ionic attraction of the oppositely charged anion and cation species governs the efficiency of the separation of the complexes and unreacted capture reagent from the reaction mixture. Using the disclosed ion capture techniques, analyte may be determined or quantitated without further processing steps such as centrifugation or filtration. The ionic interaction of the anionic and cationic substances can be selected to provide a greater attraction than the immunological attraction of antibody for antigen, particularly when multiple polycationic and polyanionic species are included in the capture reagent and solid phase. A further advantage is that the "ion-capture" technique minimizes the nonspecific adsorption of interfering substances onto the solid phase, thereby offering improved accuracy of analysis. The ion-capture technique thereby enables the performance of an assay having a highly specific separation method, minimal nonspecific binding, and high sensitivity.

EXAMPLES

The following Examples illustrate preferred ways of making the novel materials of the present invention and performing assay procedures using those materials. The Examples, however, are intended only to be illustrative, and are not to be construed as placing limitations upon the scope of the invention, which scope is defined solely by the appended claims.

Example 1

Sandwich Assay For Carcinoembrvonic Antigen (CEA) a. Preparation Of An Anti-CEA Antibgdγ-PGA ca t re Reagent

The following sequence of steps describes the chemistry employed for the preparation of an antibody/polyglutamic acid (PGA) conjugate, i.e.. an antibody/anionic polymer capture reagent.

Preparation of a traceable anionic polymer: The sodium salt of PGA (one gram; 7.14 x 10 ^~ 5 mole; average molecular weight [MW] 14,000; Sigma Chemical Company, St. Louis, MO) was converted to 3- (2-pyridyl-dithio)propionyl-PGA (PDP-PGA) by the method of Tsukada et al. (JNCI; 73; 721-729, 1984) with the following procedural modifications. The PDP- PGA was not reduced to the free sulfhydryl prior to the thiopropyl SEPHAROSE® 6B isolation. Instead, the PDP-PGA was dissolved in 0.1 M Na phosphate and 1 mM EDTA (pH 6.5) and stirred with thiopropyl SEPHAROSE® 6B (60 ml; 30 grams; Pharmacia Chemicals, Uppsala, Sweden) . After dialysis and lyophilization, a 24% yield of the PDP-PGA conjugate was obtained (0.244 grams; 1.72 x IO ^-5 mole) .

To ensure that the disulfide was maintained during the ensuing chemistries, the thiopyridyl group was exchanged for a 5-thio-2-nitrobenzoate (TNB) protecting group. A 100 mole excess of 1, 4-dithiothreitol (MW 154.2) was added to a solution of the PDP-PGA (20 mg; 1.42 x 10 ^~6 mole) dissolved in 0.1 M sodium phosphate (4.0 ml; pH 7), and the reaction was run for one hour at 40°C. The mixture was diluted to ten milliliters with 5.0 mM sodium acetate, 0.14 M NaCl, and 1.0 mM EDTA (pH 5.5) and dialyzed in 2000 molecular weight cut off (MWCO) tubing against the dilution buffer. Dialysis was continued against distilled water, followed by lyophilization.

was obtained. At this point, a UV/VIS scan was taken to determine the number of fluoresceins per PGA molecule (i.e.. loading). A value of 0.81 fluoresceins per PGA was calculated for this preparation.

Antibody activation: The monoclonal antibody, an anti-CEA antibody was maleimide activated per the method of Tuskada et al. (JNCI: 73; 721-729, 1984) with the following exceptions. The antibody concentration was one mg/ml, and a 150 mole excess of N-succinimidyl m-(N-maleimido) benzoate (SMBE, MW 314.3; Sigma) was used. It was determined experimentally that a 150 mole excess was necessary to introduce between three and five maleimide groups to the anti- CEA antibody. Clean-up was performed using the Meares et al. centrifuge method (Analytical Biochemistry: 1142; 68-78, 1984) with Sephadex G-50/80 (Sigma) in three milliliter syringe columns. The number of maleimides per antibody was determined using the titration method of Liu et al. (Biochemistry: 18; 690-696, 1979). It was found that 4.6 maleimides were introduced per antibody during this antibody activation.

The thiopropyl-fluorescein-labeled PGA was then reacted with the maleimide derived antibody to yield the antibody/PGA conjugate appropriate for a carcinoembryonic antigen ion-capture immunoassay. The maleimide-activated antibody (1.0 mg; 6.25 x IO" ⁹ mole) in 0.1 M sodium phosphate (1.0 to 2.0 ml; pH 7.0) was pH adjusted to 6.5 with 1.0 N HCl. Then, a 10 mole excess of HS-PGA/EDA-FI (approximately 1.0 mg) in 0.1 M sodium phosphate (100 μl) was added to the activated antibody preparation. The conjugation was run overnight with gentle stirring at room temperature. The mixture was diluted to ten milliliters in 0.1 M sodium phosphate (pH 7.0) and dialyzed in 50,000 MWCO tubing against 0.001 M Na phosphate (pH 7.0) followed by lyophilization. The dry material was redissolved in distilled water (0.25 ml) and high performance liquid chromatography (HPLC) fractionated for the largest peak

at A280. The chromatography was performed using a Bio-Sil TSK250 (Bio-Rad Laboratories, Richmond, California) 300 mm x 7.5 mm column, eluted at one milliliter/minute with 50 mM sodium sulfate, 20 mM sodium phosphate, and 0.3 M NaCl (pH 6.8) .

The largest peak was assayed for protein content using Bio-Rad's Bradford assay with a bovine IgG standard. The peak contained 95.5 μg/ml protein equating to 5.97 x IO" ⁷ molar protein (IgG MW 160,000). By scanning the UV/VIS and taking the absorbance at 494 nm, it was determined that this fraction also contained 2.12 x 10 ^~ 6 molar fluorescein. The equation of the molar fluorescein gave 3.6 fluoresceins per antibody molecule. Knowing that there were 0.81 fluoresceins per PGA molecule, this equated to 4.4 PGA molecules conjugated to each antibody. The peak fraction was frozen and subsequently used in the assay.

An important aspect of the above described chemistries is that there exists but a single site of attachment between each polymeric anion and the antibody. The solitary covalent link between the two circumvents the potential intermolecular and intramolecular crosslinking that could occur if a polymeric anion having multiple activated groups were employed.

As an alternative to the above capture reagent example, a cationic derived antibody could also be formed for use in conjunction with an anionic solid phase material.

b. Prenaration Of The Solid Phase

The solid phase fibrous matrix of a disposable IMX™ (Abbott Laboratories) wedge was coated with a polymeric quaternary compound to give the solid phase a positive charge. CELQUAT® L-200, a water soluble cellulose derivative, was used. A 1% aqueous solution of CELQUAT® L-200 (50 μl) was applied to the solid phase material, followed by a wash of

diluent containing 300 mM NaCl, 50 mM Tris and 0.1% NaN3 (75 μl; pH 7.5) .

c. Preparation Of The Indicator Reaσent

The indicator reagent consisted of a conjugate of alkaline phosphatase and anti-CEA antibody fragment, which binds to a different epitope than the antibody specified in the capture reagent. The alkaline phosphatase-labeled anti-CEA antibody fragment was in a buffer containing: 50 mM Tris, 50 mM NaCl, 1.0 mM MgCl2, 0.1 mM ZnCl2, 5.0 mM sodium tartrate,

0.5% calf skin gelatin, and 3% mouse serum.

d. Immunoassay Protocol - Determination Of CEA The indicator reagent (70 μl) was placed into a reaction well. Then, buffered capture reagent (20 μl of anti- CEA/PGA conjugate in a buffer of 50 mM, a2S04, 20 mM sodium phosphate, and 300 mM NaCl at pH 6.8) was added to the well. A 35 μl specimen containing CEA was added to the well, and the homogeneous reaction mixture was incubated for 20 minutes at 34.5°C. Four different specimens were run in the assay, each of which was a CEA calibrator from the Abbott Laboratories CEA enzyme immunoassay kit. An aliquot of each reaction mixture (100 μl) was then applied to the quat-treated solid phase material, followed by three 75 μl washes of diluent. Finally, an enzyme substrate (70 μl; 1.2 mM 4-methylumbelliferyl- phosphate in a solution of 100 mM AMP, 1.0 mM MgCl2, 0.1% aN3, and 4.0 mM tetramisole at pH 10.3) was added at 34.5°C for reaction with the indicator reagent, and the resulting rate of fluorescence was measured. The dose-response results of the assay are shown in Table 1. The results demonstrate that as the CEA tefet sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 1

CEA ion-Capture Sandwich Assay

Capture Reagent: Anti-CEA Antibody-PGA Conjugate

Indicator Reagent: Alkaline Phosphatase-Labeled

Anti-CEA Antibody Fragment

CEA (ng/ml) Rate (counts/sec/sec)

0 37

4 170

30 931

80 2398

Example 2 Competi ive inhibition Assay Of Mouse Immunoσlobulin a. Preparation Of An IαG-PGA Capture Reaσent

A protein-A affinity purified mouse monoclonal immunoglobulin G was coupled to negatively charged PGA using a water-soluble carbodiimide reagent (l-ethyl-3- (3- dimethylamino-propyl) carbodiimide; EDCI) according to the following procedures .

Fluorescein-labeled PGA (10 mg; FI-PGA) was added to an ice-cold solution of the antibody (4.8 mg/ml) in phosphate- buffered saline (PBS; 75 mM KH2PO and 300 mM NaCl at pH 7.2).

To that solution was added a freshly prepared ice-cold solution of EDCI (100 μl; 10 mg/ml), and the resultant reaction mixture was allowed to warm to room temperature with continuous stirring for 2.5 hours. An additional freshly prepared ice-cold solution of EDCI (50 μl; 100 mg/ml) was then added to the reaction mixture with rapid stirring. The reaction mixture was stirred for another 1.5 hours. The mixture was then fractionated by gel filtration chromatography using a Spherogel TSK-3000SWG column (2.15 cm x 30 cm) fitted

with a Spherogel TSK-G guard column (2.15 cm x 7.5 cm; Beckman Instruments, Inc., Fullerton, CA 92634). The column was eluted with PBS at a flow rate of five milliliters/minute. The PGA/antibody ratio of these pools was determined by quantitating the fluorescence in the FI-PGA conjugates of the antibody. The results are shown in Table 2.

Table 2 Mouse IgG-PGA Conjugates Prepared Using EDCI Pool Peak Molecular PGA/Antibody

Weight

I 420,000 3.8

II 280,000 4.1

III 220,000 5.5

b. Preparation Of The Solid Phase

A porous fibrous matrix material was coated with a polymeric quaternary ammonium compound (GAFQUAT® 755N; GAF Corporation) to form the solid phase. An aqueous solution of 0.5% GAFQUAT® 755N (50 μl) was applied to the surface of the material, followed by a water wash (75 μl) .

c. Binding Of The Indicator Reagent To The Capture Reagent

The indicator reagent, an alkaline phosphatase conjugate of sheep anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA 19390), was diluted in Tris-buffered saline containing 1% fish gelatin [25 mM Tris (hydroxymethyl) aminomethane and 100 mM NaCl, pH 7.5]. The capture reagent of PGA/mouse monoclonal antibody conjugate (Pool I of Table 2) was similarly treated. Two hundred microliters of each reagent was added to a series of test tubes which were then incubated at 37°C for 30 minutes. An

aliquot of the reaction mixture (75 μl) was applied to the quat-treated solid phase material, immediately followed by three 150 μl washes of Tris-buffered saline. Finally, an enzyme substrate (70 μl of 1.2 mM 4- methylumbelliferylphosphate in a solution of 100 mM AMP, 1 mM MgCl2, 0.1% NaN3, and 4 mM tetramisole; pH 10.3) was added to the materials at 32.7°C, and the resulting rate of fluorescence was measured. The results of the experiment are summarized in Tables 3 and 4.

Table 3

Dose Response Of Capture Reagen /Indicator Reagent Binding

PGA/Antibody* fl-lσ/ml) Rat Of Fluorescence (counts/sec/sec)

10 1559

1 816

0.1 179

0.01 70

0 36

* The initial concentrations of PGA-coupled antibody before mixing with a 1000-fold diluted alkaline phosphatase-labeled sheep anti-mouse immunoglobulin.

Table 4

Dose Response Of Indicator Reagent/Indicator Reagent* Binding

Rate Of fluorescence (connts/sec/se .

IO ² 5062

103 796

10* 93

IO ⁵ 10

10 ⁶ 5

The initial concentration of PGA-coupled antibody before mixing with alkaline phosphatase-labeled sheep anti-mouse immunoglobulin was five μg/ml.

The indicator reagent titer is the reciprocal of the dilution of the reagent stock.

d. Enhancement of Antigen/Antibody Complex Binding to Solid Phase

The following assay was conducted to show enhancement of capturing mouse IgG/Alkaline phosphatase-sheep anti-Mouse IgG complexes by attaching polyglutamic acid to Mouse IgG and coating the solid phase with GAFQUAT® 755N. Capture reagent (pool I) was prepared as described in Section (a) above. The amounts of MIgG were serially diluted to 10, 1, 0.1, and 0.01 microgram/ml, respectively. The solid phase was prepared as described in Section (b) above except that porous fibrous matrix material without the coating of GAFQUAT® 755N was used for the NO QUAT controls. The capture reagent and indicator reagent were reacted under the conditions described in Section (c) above. The results are shown in Table 3(a) below.

Table 3 (a) Rate of Fluorescence Intensity (counts/sec/sec)

Col. 1 Col. 2 Col. 3 Col. 4

MIgG MIgG-PGA MIgG-PGA MIgG MIgG (μg/ml) QUAT NO QUAT NO PGA NO PGA QUAT NO QUAT

10 1491 90 35 71 1626 81 31 77

1 820 84 34 75 812 85 35 71

0.1 152 79 32 69 207 76 32 71

0.01 81 72 27 57 58 75 27 61

0 30 67 26 59 42 74 23 59

The low signals in columns 3 and 4 demonstrate that there was virtually no binding of indicator reagent (alkaline

phosphatase-sheep an i-MIgG) on the solid phase. Formation of MIgG/indicator complexes when the MIgG was not conjugated to the polyanion was not sufficient to specifically bind the complexes to the cation coated solid phase, thus, resulting in a very low specific signal. The marginal increase of signals in column 2 compared to columns 3 and 4 shows that even the tremendously increased amount of negative charge (from the PGA) attached to the complexes was still insufficient to facilitate the binding of the complexes to the solid phase when the solid phase was not coated with the polycationic QUAT compound. In column 1, where the solid phase was coated with the polycationic QUAT, the signal was directly proportional to the amount of MIgG-PGA present in the reaction mixture which was brought into contact with the positively charged solid phase. These results demonstrate that the capturing of the complexes (MlgG/sheep anti-MIgG) is most efficient when the capture reagent (MIgG) is covalently attached to PGA and the QUAT compound is coated on the solid phase. The data in Table 3(a) also clearly confirm that the polycation binding affinity of most biological materials such as antibodies, antibody- enzyme conjugates, and antibody-antigen complexes is lower than that of polyanionic molecules and their complexes.

e. Competitive Inhibition Assay For Mouse IgG

The capture reagent and indicator reagent were prepared as described above. All of the reagents were diluted in Tris-buffered saline containing 1% fish gelatin. The indicator reagent was diluted 1000-fold from the stock solution, and the capture reagent was diluted to 10 μg/ml. In a series of test tubes, 150 μl each of appropriately diluted indicator reagent, capture reagent, and mouse monoclonal antibody were mixed. The mixtures were incubated at 37°C for 30 minutes. Aliquots of the mixtures (75 μl) were applied to the quat-treated solid phase materials, immediately followed by three 150 μl washes of Tris-buffered saline. An enzyme

substrate (70 μl of 1.2 mM 4-methylumbelliferylphosphate in a solution of 100 mM AMP, 1 mM MgCl2, 0.1% NaN3 , and 4.0 mM tetramisole; pH 10.3) was then added to the solid phase at

32.7°C, and the resulting rate of fluorescence was measured.

The results of this example illustrating a competitive inhibition assay for mouse IgG are shown in Table 5. The results demonstrate that as the mouse monoclonal antibody concentration increased there was a corresponding decrease in the formation of capture reagent/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 5

Inhibition Of Indicator Reagent Binding Due To Mouse Monoclonal Antibody

Capture Reagent: PGA/Mouse Monoclonal IgG Conjugate

Indicator Reagent: Alkaline Phosphatase- Sheep Anti-Mouse Immunoglobulin Conjugate

Mouse IgG (μg/ml) Rate Of Fluorescence (counts/sec/sec)

0 no

3.3 x 10 ^~3 106

3.3 x IO ^"2 98

3.3 x 10" ¹ 67

3.3 36

33 10

Example 3

Sandwich Assay For Human Cho ionic Gonadotropin (hCG) a. Preparation Of The Capture Reagent

A highly negatively charged albumin derivative was prepared and coupled to anti-hCG antibodies to form the capture reagent according to the following procedures.

Modification of rabbit serum albumin to form a negatively charged protein derivative: Rabbit serum albumin (RSA) was extensively succinylated and coupled with para- azobenzenesulfonate by the procedure of Jou et al. , (Methods in Enzymology: Vol. 92, Part E; 257-276, Academic Press, 1983) . Two percent RSA in phosphate-buffered saline (PBS, 14 ml, pH 8.0) was mixed with 5% succinic anhydride in para- dioxane (2.28 ml) . The pH was maintained at 8 by the addition of 1.0 N NaOH. The reaction mixture was stirred at room temperature for 30 minutes. Hydroxylamine hydrochloride was added (0.6 g) and the pH of the solution was adjusted to 9.5 by adding an appropriate amount of 5 N NaOH. The mixture was then dialyzed against water. The resultant SUC55-RSA was coupled to para-azobenzenesulfonate according to the following reactions.

A suspension of para-azobenzenesulfonate acid (0.15 mole, 26 mg) in 1 N HCl (0.8 ml) was cooled in an ice bath and treated with 1 N NaN02 (0.2 ml) for 30 minutes with rapid stirring. The resultant diazonium salt solution was added by drops to the ice cooled SUCg5~RSA solution with rapid stirring. The pH of the reaction mixture was maintained at 11 by the addition of 1.0 N NaOH. The dark red reaction mixture was stirred and allowed to warm to room temperature for one hour before it was extensively dialyzed against water. The resultant Sp-SUCg5~RSA anionic derivatized protein was kept refrigerated until used.

Preparation of anti-hCG F(ab')2 fragments: Anti-hCG F(ab')2 fragments were prepared according to the method of Nisonoff et al. (Arch. Biochem. Biophy. : 89; 230-244, 1960) from affinity purified goat anti-hCG antibodies. A portion of affinity purified -antibody solution in phosphate buffered saline (pH 7.2) was acidified to pH 4 by adding acetic acid. The preferred concentration of antibodies at this point was one mg/ml. Pepsin was added to reach a final concentration of 20 μg/ml. The mixture was incubated at 37°C overnight. The

reaction was stopped by adding 6.0 N NaOH to bring the reaction mixture to a pH of 7.5. The digested antibody fragments solution was concentrated to 20 mg/ml. The F(ab')2 fragments were purified by gel-filtration high performance liquid chromatography using a Spherogel TΞK-3000SWG column (2.15 cm x 30 cm) fitted with a Spherogel TSK-G guard column (2.15 cm x 7.5 cm) .

Preparation of anti-hCG TNB-Fab' fragments: Anti- hCG Fab' fragments were prepared and derivatized into a thiol- reactive form according to a modification of the methods of Parham et al. (J. Immunol. Method. : 53:133-173, 1982) and Brennan et al. (Science: 229: 81-83, 1985) . With stirring, a solution (158 μl) of 0.1 M NaAsθ2 containing 20 M EDTA was added to 1.28 ml of goat F(ab')2 (goat anti-human chorionic gonadotropin antibody fragment, 16 mg/ml) containing trace ¹²⁵ -F(ab')2 i- ⁿ PBS. The reductive cleavage reaction was started by adding 0.1 M cystein-HCl (158 μl) . The reaction mixture was overlayed with nitrogen and incubated with stirring at 37°C for one hour. The reaction was then quenched by adding 19 mg of 5, 5 ' -dithiobis- (2-nitrobenzoic acid) . After stirring overnight at room temperature, the mixture was chromatographed on a PD-10 column (Pharmacia Inc., Piscataway, NJ) preequilibrated with PBS, and then chromatographed on a size exclusion high performance liquid chromatography column [Spherogel TSK-2000SWG column (2.15 cm x 30 cm) fitted with a Spherogel TSK-G guard column (2.15 cm x 7.5 cm)]. The purified thionitrobenzoate derivative of Fab' (TNB-Fab') was concentrated to 7.9 mg/ml using a CX-10 ultrafiltration unit (Millipore Corp., Bedford, MA).

Coupling of anti-hCG TNB-Fab' fragments to SP-SUC55-

RSA: a solution of 1 M dithiothreitol (DTT; 86 μl) was added to a solution (4.2 ml) containing Sp-SUCg5-RSA (2.2 mg/ml) in 37.5 mM sodium phosphate, 150 mM NaCl, and 2.0 mM EDTA (pH 6.8). The mixture was incubated at 37°C for three hours and then at room temperature overnight. The resulting reaction

mixture was chromatographed on a 2.5 cm x 20 cm column packed with SEPHADEX® G-25 (Pharmacia Inc.) and preequilibrated with 75 mM sodium phosphate, 300 mM NaCl, and 2.0 mM EDTA (pH 6.8) . A 2ml portion of the pooled fractions of reduced SP-SUC55-RSA

(0.48 mg/ml) was mixed with anti-hCG TNB Fab' (0.15 ml; 7.9 mg/ml) . The mixture was stirred at room temperature overnight. The reaction mixture was then treated with 100 mM iodoacetic acid (107 μl) and stirred for one hour at room temperature. The Fab' -SP-SUC55-RSA conjugated was purified by size exclusion high performance liquid chromatography using a Spherogel TSK-3000SWG column (2.15 cm x 30 cm) fitted with a Spherogel TSK-G guard column (2.15 cm x 7.5 cm) .

Coupling of anti-hCG antibodies to Sp-SUCg5~

RSA: a solution (27 μl) of 30 mM succinimidyl 4- (N-maleimido- methyl) -cyclohexane-1-carboxylate in N,N-dimethylformamide was added to 2.25 ml of affinity purified goat arti-hCG antibody (3 mg/ml) in PBS. The resulting reaction mixture was stirred for one hour at room temperature and then chromatographed on a PD-10 column preequilibrated with 75 mM sodium phosphate, 300 mM NaCl, and 2.0 mM EDTA (pH 6.8) . A 1.8 ml portion of the pooled fractions of modified antibodies (1.6 mg/ml) was mixed with 3ml of the DTT-reduced Sp-SUCg5~RSA (0.48 mg/ml) .

After stirring at room temperature overnight, the reaction was quenched by adding 100 mM iodoacetic acid (0.25 ml) and stirring at room temperature for one hour. The antibody Sp- SUCgs-RSA conjugate was purified by size exclusion high performance liquid chromatography in the manner described above.

b. Preparation Of The Indicator Reagent

The indicator reagent consisted of an alkaline phosphatase-goat anti-hCG antibody conjugate (prepared by coupling anti-hCG antibody to periodate activated alkaline phosphatase) in an assay buffer containing 25 mM Tris

(hydroxymethyl) aminomethane, 100 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 0.07% NaN3, and 1% fish gelatin at pH 7.5.

c. Sandwich Immunoassay Protocol For hCG

The ion-capture immunoassay protocol included the use of a solid phase prepared substantially in accordance with the method described in Example 2, the indicator reagent (alkaline phosphatase-goat anti-hCG antibody conjugate), one of two different capture reagents (goat anti-hCG Fab'-Sp- SUCg ₅ -RSA and goat anti-hCG IgG-Sp-SUCg5-RSA) as prepared in

Example 3.a. above, and a purified hCG standard solution. All reagents were appropriately diluted (as determined by a titer curve) in the assay buffer. Equal volumes (750 μl) of the indicator reagent and hCG sample solution were placed in a series of test tubes. After incubation at 37°C for 30 minutes, a 125 μl aliquot of each incubated mixture was mixed in a separate tube with an equal volume of a capture reagent. The resulting mixtures were incubated for 30 minutes. The assay mixture (75 μl) was then added to each solid phase material. The solid phase materials were then washed three times with 150 μl amounts of washing buffer [25 mM Tris (hydroxymethyl) aminomethane, 100 mM NaCl, 1.0 mM MgCl2, 0.1 mM ZnCl2, and 0.07% NaN3 at pH 7.5]. An enzyme substrate (70 μl of 1.2 mM 4-methylumbelliferylphosphate in a solution of 100 mM AMP, 1.0 mM MgCl2, 0.1% NaN3, and 4.0 mM tetramisole at pH 10.3) was then added to the solid phase materials. The resulting rate of fluorescence was measured at 32.7°C. The results of the experiment are summarized in Table 6. The results demonstrate that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 6 hCG Ion-Capture Sandwich Assay Comparing Different Capture Reagents

Indicator Reagent: hCG-Specific Goat igG-Alkaline Phosphatase

Rate Of Fluorescence (counts/sec/sec) hCG-Specific Capture Reagents hCG ( mIU/r nl Goat IgG-Sp-ΞUC ₆₅ -RΞA Goat Fab' -Sp-SUCgς-RSA

0 63 64

12 . . 5 96 110

25 121 134

50 146 166

100 182 212

Example 4 Indirect Sandwich Ion-Caoture Immunoassay For hCG

The indirect ion-capture immunoassay included the use of a solid phase prepared substantially as described in Example 2 above, an indicator reagent of alkaline phosphatase- sheet anti-mouse IgG conjugate (Jackson immunoResearch Laboratories, Inc.), a capture reagent of goat anti-hCG Fab'- Sp-SUCg5~RSA as prepared in Example 3, an ancillary specific binding member of mouse monoclonal anti-hCG antibodies (ImmunoSearch; Thomas River, NJ 08753), and a purified hCG standard solution. The ancillary specific binding member was used to bind with the analyte and the indicator reagent. All reagents were appropriately diluted in the assay buffer. Equal volumes (150 μl) of the indicator reagent, hCG sample solution, and ancillary specific binding member were placed in a series of test tubes. After incubation at 37°C for five minutes, a 150 μl portion of capture reagent was added to each

tube. The resulting mixtures were incubated for five minutes, The assay mixture (200 μl) was then added to each prepared solid phase material. The solid phase materials were then washed with washing buffer and treated with an enzyme substrate solution in the same manner as described in Example 3 above. The resulting rate of fluorescence was measured at 32.7°C. The results of the assay are summarized in Table 7. The results demonstrate that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/ancillary specific binding member/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 7

Ion-Capture Indirect Sandwich Assay For hCG

Capture Reagent: Goat Anti-hCG Fab' -Sp-SUCg5~RSA

Indicator Reagent: Sheep Anti-Mouse IgG-Alkaline Phosphatase

Ancillary Specific Binding Member: Mouse Monoclonal Anti-hCG Antibody hCG (mlU/ml) Rate Of Fluorescence (counts/sec/sec)

0 13

1.5 18

3.3 27

6.3 40

12.6 70

25.0 112

50.0 230

100.0 443

200.0 732

Example 5

Indirect Sandwich Ion-Capture Immunoassay For hCG Using Two Ancillary Specific Binding Members

The ion-capture immunoassay protocol included the case of a solid phase prepared substantially in accordance

with the method described in Example 2, an indicator reagent of alkaline phosphatase-sheep anti-mouse IgG conjugate (Jackson ImmunoResearch Laboratories, Inc.), an ancillary specific binding member of mouse monoclonal anti-hCG antibodies (ImmunoSearch; Thomas River, NJ 08753), and a purified hCG standard solution. Additionally, the protocol used a second ancillary specific binding member of affinity purified goat anti-hCG antibodies and a capture reagent of rabbit anti-goat IgG-Sp-SUCg5~RSA. The capture reagent was prepared by coupling affinity purified rabbit anti-goat IgG (Cappel; Cochranville, PA 19330) to Sp-SUCg5~RSA according to the procedure described in Example 3 above. All reagents were appropriately diluted in the assay buffer. Equal volumes (100 μl) of the indicator reagent, hCG sample solution, and first ancillary specific binding member were placed in a series of test tubes. After incubation (37°C for ten minutes) the second ancillary specific binding member (100 μl) was added and the incubation was continued (at 37°C for an additional five minutes) . Finally, capture reagent (100 μl) was added to each tube. The resulting mixtures were incubated for five minutes. The assay mixture (200 μl) was then added to each prepared solid phase material. The solid phase materials were then washed with washing buffer, treated with enzyme substrate solution, and measured for the rate of fluorescence in the same manner as described in Example 3 above. The results of the assay are summarized in Table 8. The results demonstrate that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/ancillary specific binding member/analyte/ ancillary specific binding member/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 8

Ion-Capture Indirect Sandwich Assay For hCG

Capture Reagent: Rabbit Anti-Goat lgG-Sp-SUCg5~RSA

Indicator Reagent: Sheep Anti-Mouse IgG-Alkaline Phosphatase

Ancillary Specific Binding Member: Mouse Monoclonal Anti-hCG Antibody

Ancillary Specific Binding Member: Goat Anti-hCG Antibodies

Rate Of Fluorescence (counts/sec/sec) Goat anti-hCG (ng/ml) hCG (40 mlU/ml) Negative Control (0 mlU/ml)

250 3499 36

150 3708 34

50 3543 33

25 3155 30

Example 6

Ion-Capture Immunoassay For An i-Progesterone Antibody a. Preparation of PGA-Labeled Goat Anti-Mouse Capture Reagent

The following sequence of steps describes the chemistry employed for the preparation of an antibody/polyglutamic acid conjugate.

Conversion of PGA-sodium salt to the free acid form: The sodium salt of PGA (200 mg; 1.47 x 10 ^~5 mole; average MW 13,600; Sigma Chemical Company, St. Louis, MO) was stirred with a cation exchange resin (AG50W-X8; 13 grams; Bio-Rad, Richmond, CA) in 60 milliliters of water for three hours. The supernatent was decanted, filtered, and evaporated providing an 80% yield of the free acid form of PGA as a white powder (137 mg; average MW 11,620) .

Preparation of isothiocyanate-PGA (ITC-PGA) : To a solution of the free acid form of PGA (65 mg; 5.6 x 10 ^~ 6 mole)

in dimethylformamide (DMF; 2 ml) was added triethylamine (100 μl; 7.2 x 10 ^~4 mole) and 1, 4-phenylenediisothiocyanate (110 mg; 5.7 x IO ^-4 mole; Aldrich Chemical Company, Milwaukee, WI) . After stirring overnight at room temperature, acetic acid (100 μl; 1.7 x 10"3 mole) was added, and the reaction mixture was then evaporated. Methylene chloride (25 ml) was added to the residue, and after stirring for two hours the mixture was filtered to yield the ITC-PGA as a white powder (101 mg) .

The ITC-PGA (295 μg; 2.5 x 10 ^~8 mole; in 40 μl of 20% DMF/0.1 M sodium phosphate at pH 7.0) was added to a buffered solution of goat anti-mouse IgG (200 μg; 1.25 x 10 ^~ 9 mole; Sigma Chemical Company; in 40 μl of 0.1 M sodium phosphate at pH 7) to form the PGA- abeled goat anti-mouse capture reagent. After stirring at room temperature for two days, 0.1 M Tris (20 μl; pH 7.4) was added and the resulting mixture was stored at 2 to 8°C until used.

b. Immunoassay For

Anti-Progesterone Antibody

The anti-progesterone antibody ion-capture immunoassay included the use of solid phase materials coated with a polymeric quaternary compound as described in Example 1. A 60 μl sample was added to a reaction well. The samples consisted of a monoclonal anti-progesterone antibody at concentrations of 0, 5, 50, 100, 250, and 500 ng/ml in phosphate-buffered saline (PBS, 50 mM sodium phosphate, 99 mM NaCl, 0.1% NaN3, at pH 7.4) Next, 20 μl of PBS were added to the reaction well, followed by 20 μl of the buffered indicator reagent, progesterone labeled with alkaline phosphatase (3 μg/ml in a Tris buffer of 50 mM Tris, pH 7.4, 150 mM NaCl, 1% aN3 _# 1 mM MgCl2, 0.1 mM ZnCl2, and 1% BSA) . After incubating the mixture at 34.5°C for ten minutes, the capture reagent was added (20 μl; PGA-labeled goat anti-mouse antibody at a 1/100 dilution in PBS of the stock solution described above) . The mixture was then incubated an additional ten minutes at

34.5°C. A 100 μl aliquot of the mixture was then applied to the solid phase material, followed by three 75 μl washes of diluent. Lastly, the enzyme substrate solution (70 μl; 1.2 mM 4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 1 mM MgCl2, 0.1% NaN3 , and 4.0 mM tetramisole at pH 10.3) was added to the solid phase, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 9. The results demonstrate that as the anti- progesterone antibody test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 9

Ion-Capture Assay For Mouse Monoclonal Anti-Progesterone Antibody

Capture Reagent: PGA-Labeled Goat Anti-Mouse Antibody

Indicator Reagent: Alkaline Phosphatase-Labeled Progesterone

Anti-Progesterone (ng/ml) Rate Of Fluorescence (counts/sec/sec)

0 9

5 31

50 254

100 441

250 1191

500 2721

Example 7

Indirect Competitive Ion-Capture Immunoassay For Progesterone

The solid phase was prepared substantially in accordance with the method described in Example 1. A 60 μl sample of various concentrations of progesterone in PBS was mixed with 20 μl of progesterone-labeled alkaline phosphatase

indicator reagent (0.4 μg/ml in the Tris buffer of Example 4) and 20 μl of mouse anti-progesterone antibody as an ancillary specific binding member (0.3 μg/ml in PBS) . After incubating the mixture at 34.5°C for ten minutes, 20 μl of the PGA- labeled goat anti-mouse antibody capture reagent were added as described in Example 6 above. The resulting mixture was incubated an additional ten minutes at 34.5°C. A 100 μl aliquot of the mixture was then applied to the solid phase material, followed by three washes of diluent. Lastly, the enzyme substrate solution (70 μl; 1.2 mM 4- methylumbelliferylphosphate in a solution of 100 mM AMP, 1 mM MgCl2, 0.1% NaN3, and 4.0 mM tetramisole at pH 10.3) was added to the solid phase, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 10. The results demonstrate that as the progesterone test sample concentration increased there was a corresponding decrease in the formation of capture reagent/ancillary specific binding member/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 10

Ion-Capture Indirect Competitive Assay For Progesterone

Capture Reagent: PGA-Labeled Goat Anti-Mouse Antibody

Indicator Reagent: Alkaline Phosphatase-Labeled Progesterone

Ancillary Specific Binding Member: Mouse Anti-Progesterone Antibody

Progesterone Rate Of Fluorescence (counts/sec/sec) (ng/ml)

0 1203

1.88 277

3.75 145

7.5 67

15 30

30 16

Example 8

Activation Of Poly-L-Glutamic Acid For The Formation Of Anionic Capture Reagents

The following sequence of steps describes the chemistry used for the bulk preparation of protein-PGA conjugates for the formation of negatively charged capture reagents .

a. Conversion Of PGA-Sodium Salt To The Free Acid Form

The sodium salt of PGA (100 mg; 7.35 x 10 ^~6 mole; average MW 13,600; Sigma) was stirred overnight with a hydrogen form cation exchange resin (50 equivalents/glutamate residue; AG50W-X8; Bio-Rad) . The resin previously had been swelled and washed in distilled water, and finally resuspended in distilled water (20 ml/7 gms dry weight of beads) . The supernatent was removed and lyophilized providing a 90% yield of the free acid form of PGA (PGAFA) as a white powder (80 mg; average MW 11,620) . The free acid form was used to obtain solubility in organic solvents.

b. Preparation Of ITC-PGAFA

The PGAFA was dissolved in solvent (DMF at ten milligrams/milliliter) . A proton absorbing reagent (4-methyl morpholine) was added to the solution in the amount of about one equivalent per titratable free carboxylic acid. Next, about a 100 mole excess of an amine-reactive modification reagent (1, 4-phenylene diisothiocyanate [DITC] in sufficient DMF to dissolve it) was added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was rotavaporated to near dryness, and methylene chloride (25 ml) was added dropwise to precipitate

the ITC-PGAFA. The flocculent precipitate was centrifuged, and the methylene chloride and unreacted DITC were removed.

The precipitation/ centrifugation process was repeated until substantially no detectable DITC remained. The DITC was detected using thin layer chromatography on silica slides developed in methylene chloride; ITC-PGAFA remains at the origin, DITC moves with the solvent front. The remaining solid was vacuum dried to yield the ITC-PGAFA as a yellow powder. c. Coupling Of ITC-PGAFA To

Protein To Make Capture Reagents

The ITC-PGAFA (at about a 1 to about a 20 mole excess to the protein) was dissolved in 0.2 M sodium phosphate buffer at pH 8.5 with the volume held as low as possible. The pH was adjusted to 8.5 as necessary. The desired protein was added to this solution and incubated overnight at 37°C. The preparations were then fractionated using HPLC on either an analytical TSK 400 Bio-Rad column (7.5 x 300 mm, at a 1 ml/min flow rate) for 1-2 milligram protein preparations, or a TSK 4000 Beckman column (31.5 x 300 mm, at a 5 ml/min flow rate) for 2-10 milligram protein preparations. The elution buffer contained 0.1 M sodium phosphate and 0.3 M NaCl at pH 6.8. Fractions were tested and appropriately combined. The amino acid content was determined for those fractions containing protein so that the coupling efficiency for the various proteins at various coupling ratios could be determined. The results of the determinations are presented in Table 11.

Table 11

Coupling Efficiencies Of ITC-PGAFA With Various Proteins

PGA Molar PGA Chain

Protein Excess Number

Anti-CEA antibody 1 0.77 monoclonal 1.0 mg 5 1.7

10 3.1 20 8.6

Goat anti-rabbit antibody monoclonal 1.0 mg 5 1.8

Anti-βhCG antibody 10 4.6 monoclonal 1.0 mg 15 5.2 monoclonal 10 mg 15 7.8

Anti-digoxin antibody monoclonal 1.0 mg 15 8.1 monoclonal 5.0 me 15 5.5

Goat anti-mouse antibody polyclonal 1.0 mg 15 4.3

Anti-T4 antibody monoclonal 1.0 mg 15 6.9

Anti-T4 antibody polyclonal 7.0 mg 15 13.8

Rabbit Serum Albumin loaded with Theophylline 15 7.8

Column 1 of Table 11 lists the quantity of protein used in the reactions to form the various capture reagents. Column 2 lists the mole excess of activated ITC-PGAFA that was reacted with the Column 1 protein. Column 3 provides the number of PGA chains attached per antibody by the reaction, calculated by amino acid analysis based upon a 40,000 average MW and 305 repeating glutamate residues.

Example 9

Theophylline Ion-Capture Competitive Assay-Antigen Capture Format a. Preparation Of Theophylline Capture Reagent

The activation of theophylline was accomplished by dissolving theophylline-butylate (10 mg; MW 280.29; 3.57 x 10" 5 moles) in methylene chloride (3.0 ml) . A three mole excess of dicyclohexyl-carbodiimide (22 mg; MW 206.3) and a three mole excess of N-hydroxysuccinimide (12.3 mg; MW 115.09) were added, and the reaction mixture was stirred over night at room temperature. The mixture was filtered to remove dicyclohexylurea and was rotavaporated to dryness to yield ten milligrams of N-succinimidyl-theophylline-butylate ⁽ theophylline-butylate-oSu) .

The free acid of polyglutamic acid (NH2-PGAFA; 1.4 mg; MW 11,798; 1.19 x IO ^"7 moles) was dissolved in DMF (0.5 ml) and NMM (1.1 mg; MW 101.15; 1.07 x 10 ^~5 moles) . The theophylline-butylate-oSu (10 mg; at 1 mg/0.5 ml DMF) was added, and the reaction mixture was stirred overnight at room temperature. Unbound theophylline was removed by dialysis against a 0.1 M Na phosphate buffer at pH 7.0. The theophylline content of the resulting capture reagent was analyzed, a: the results demonstrated that 3.9 theophylline molecules were attached per PGA chain. The theophylline-PGA capture reagent, which was capable of binding with anti- theophylline antibody, was then diluted to 3 μg/ml in an assay buffer containing 25 mM Tris, 100 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 0.1% NaN ₃ , and 1% fish gelatin at pH 7.2.

b. Preparation Of The Solid Phase

A fiber matrix was coated with a polymeric quaternary compound to provide the solid phase with a positive charge. CELQUAT® L-200, a water soluble cellulose derivative,

was used. A 0.5% aqueous solution of CELQUAT® L-200 (50 μl) containing 10 mM NaCl (50 μl) was applied to the solid phase material.

c. Preparation Of The Indicator Reagent

The indicator reagent consisted of a conjugate of alkaline phosphatase and anti-theophylline antibody, made substantially in accordance with the protocol described in Example 3.b. The indicator reagent was appropriately diluted ⁽ as determined by titer curve) in the assay buffer to give 0.17 micrograms of antibody/milliliter.

d. Immunoassay Protocol

The indicator reagent (200 μl) was placed within a series of reaction tubes. A theophylline standard solution ⁽ 200 μl; theophylline-butylate diluted to 0.6, 1.2, 2.3, 4.9, 9.9, 99.2, and 992 μg/ml in 50 mM Tris, 300 mM NaCl and 0.1% N N3 at pH 7.2) was then added to each tube. The mixture was incubated ten minutes at 37°C. Capture reagent (200 μl) was added to each tube, and the reaction mixtures were incubated ten minutes at 37°C. An aliquot of each reaction mixture (200 μl) was applied to the quat-treated solid phase material, followed by one wash with diluent (75 μl) . An enzyme substrate ⁽ 70 μl 1.2 mM 4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 1.0 mM MgCl2, 0.1% NaN ₃ , and 4.0 mM tetramisole at pH 10.3) was added at 32°C for reaction with the indicator reagent, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 12. The results demonstrate that as the theophylline test sample concentration increased there was a corresponding decrease in the formation of capture reagent/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 12

Theophylline Ion-Capture Competitive Assay-Antigen Capture Format

Capture Reagent: Theophylline-PGA

Indicator Reagent: Alkaline Phosphatase- Labeled Anti-Theophylline Antibody

Theophylline (ng/ml) Rate Of Fluorescence (counts/sec/sec)

0 255

0.6 250

1.2 212

2.5 202

4.9 196

9.9 168

99.2 68

992 16

Example 10

Phenylcyclidine Ion-Capture Competitive Assav-Antigen Capture Format a. Preparation Of Phenylcyclidine Capture Reagent

4-Hydroxy-Phenylcyclidine (1.1 mg; MW 259.37; 4.24 x 10 ^~ 6 moles) was dissolved in tetrahydrofuran (THF; 0.5 ml). One-half milliliter of 10% phosgene in benzene was added (130 mole excess) . The reaction was allowed to proceed at room temperature for 2.5 hours. The solvent was evaporated under a stream of nitrogen to yield a residue of phenylcyclidine-4- chloroformate.

The phenylcyclidine-4-chloroformate (1.1 mg) was dissolved in THF (0.5 ml). To this was added NH2-PGAFA (1.7 g; MW 11,798; 1.19 x 10 ^~7 moles) dissolved in l-methyl-2- pyrrolidinone (0.5 ml). The reaction was carried out overnight at room temperature and then rotavaporated to dryness. The product was dissolved in 0.1 M sodium phosphate (1.5 ml, pH 7.0). The precipitate was filtered, and the cloudy aqueous filtrate was extracted with methylene chloride

until clear. The phenylcyclidine-PGA capture reagent, which was capable of binding with anti-phenylcyclidine antibody, was then diluted to 5 μg/ml in an assay buffer as described in Example 9.

b. Preparation Of The Solid Phase

The solid phase was prepared substantially in accordance with the method described in Example 9.

c. Preparation Of The Indicator Reagent

The indicator reagent consisted of a conjugate of alkaline phosphatase and anti-phenylcyclidine antibody. The indicator reagent was diluted 1/250 in the assay buffer as described in Example 9.

d. Immunoassay Protocol

The indicator reagent (140 μl) was mixed with a series of samples (50 μl each) containing known amounts of phenylcyclidine (0.0, 25, 60, 120, 250 and 500 ng/ml prepared in human urine) , and the mixtures were incubated for ten minutes at 32°C. The phenylcyclidine-PGA capture reagent (100 μl) was added, and the reaction mixtures were incubated for ten minutes. An aliquot of each reaction mixture (200 μl) was applied to a solid phase material. The solid phase was then washed, two times. An enzyme substrate (70 μl; as described in Example 9) was added, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 13. The results demonstrate that as the phenylcyclidine test sample concentration increased there was a corresponding decrease in the formation of capture reagent/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 13

Phenylcyclidine Ion-Capture Competitive Assay-Antigen Capture Format

Capture Reagent: Phenylcyclidine-PGA

Indicator Reagent : Alkaline Phosphatase- Labeled Anti-Phenylcyclidine Antibody

Phenylcyclidine (ng/ml) Rate Of Fluorescence (counts/sec/sec)

0 570

25 133

60 60 120 33 250 18 500 9

)le 11

Digoxin Ion-Capture Competitive Assay - Antigen Capture Format a. Preparation Of a Digoxin-IgG-PGA Capture Reagent

The digoxin-IgG-PGA capture reagent was prepared substantially in accordance with the method described in Example 8.c, with the following procedural modifications. The ITC-PGA (5 mg; 1.25 x IO" ⁷ mole; in 1.0 ml of 0.1 M sodium phosphate at pH 8.5) was added to a buffered solution of rabbit IgG-digoxin (1 mg; 6.25 x IO ^-9 mole; in 1.45 ml of 0.1 M sodium phosphate and 0.3 M NaCl at pH 8.5) to form the capture reagent. The solution was stirred and incubated overnight at 37°C. The preparation was then fractionated using HPLC on a BioSil 400 (Bio-Rad 300 mm x 7.5 mm gel filtration column) and eluted at one milliliter/minute with 0.1 M sodium phosphate and 0.3 M NaCl at pH 6.8. The digoxin- IgG-PGA capture reagent, which was capable of binding with anti-digoxin antibody, was then diluted to 3 μg/ml in an assay buffer as described in Example 9.

/US95/03168

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b. Preparation Of The Solid Phase

The solid phase was prepared substantially in accordance with the method described in Example 9.

c. Preparation Of The Indicator Reagent

The indicator reagent consisted of a conjugate of alkaline phosphatase and mouse anti-digoxin antibody (immuno- search; Emeryville, California 94608) . The indicator reagent was diluted to 33.3 ng/ml in the assay buffer as described in Example 9.

d. Immunoassay Protocol

The indicator reagent (200 μl) was mixed with a series of samples (200 μl) containing known amounts of digoxin

(0.5, 1.0, 2.5, 5.0 and 50.0 ng/ml prepared in normal human serum) . The mixtures were incubated for 15 minutes at 37°C. The digoxin-IgG-PGA capture reagent (200 μl) was added, and the reaction mixtures were incubated for 15 minutes. An aliquot of each reaction mixture (200 μl) was applied to the solid phase material, followed by a wash. An enzyme substrate

(70 μl; as described in Example 9) was added, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 14. The results demonstrate that as the digoxin test sample concentration increased there was a corresponding decrease in the formation of capture reagent/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 14

Digoxin Ion-Capture Competitive Assay-Antigen Capture Format

Capture Reagent: Digoxin-IgG-PGA

Indicator Reagent: Alkaline Phosphatase- Labeled Anti-Digoxin Antibody

Digoxin (ng/ml) Rate Of Fluorescence (counts/sec/sec)

0 115

0.5 101

1.0 91

2.5 74

5.0 60

50.0 14

Example 12

Digoxin Ion-Capture Competitive Assay - Antibody Capture Format a. Preparation Of The Indicator Reagent

The indicator reagent consisted of a conjugate of alkaline phosphatase and digoxin (Immuno-search) . The indicator reagent was diluted to 1/100 in the assay buffer as described in Example 9.

b. Immunoassay Protocol

The anti-digoxin-PGA capture reagent (200 μl, prepared substantially in accordance with the protocol described in Example 8.c) was mixed with a series of samples (200 μl each) containing known amounts of digoxin as described in Example 11. The mixtures were incubated for 15 minutes at 37°C. The indicator reagent (200 μl) was added, and the reaction mixtures were incubated for 15 minutes. An aliquot of each reaction mixture (200 μl) was applied to the solid phase (prepared as described in Example 9), followed by a wash. An enzyme substrate (70 μl; as described in Example 9)

was added, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 15. The results demonstrate that as the digoxin test sample concentration increased there was a corresponding decrease in the formation of capture reagent/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase decreased.

Table 15

Digoxin Ion-Capture Competitive Assay-Antigen Capture Format

Capture Reagent: Anti-Digoxin Antibody-PGA

Indicator Reagent: Alkaline Phosphatase-Labeled Digoxin

Digoxin (ng/ml ) Rate Of Fluorescence ( counts /sec/sec )

0 85

0.5 68

1.0 48

2.5 23

5.0 10

50.0 1

Example 13

Alternative Ion-Capture Sandwich Assay For hCG a. Preparation Of The Capture Reagent

An anti-hCG antibody-PGA capture reagent was prepared substantially in accordance with the method described in Example 8.c. above.

b. Preparation Of The Solid Phase

A fiber matrix was wetted with buffer (80 μl; containing 300 mM NaCl, 50 mM Tris and 0.1% NaN3 at pH 7.5).

The matrix was coated with a 0.5% aqueous solution of CELQUAT®

L-200 (50 μl; containing 10 mM NaCl) followed by a second wash with buffer. c. Preparation Of The Indicator Reagent

The indicator reagent consisted of a conjugate of alkaline phosphatase and goat anti-hCG antibody (made substantially in accordance with the protocol described in Example 3.b) . The indicator reagent was appropriately diluted (as determined by titer curve) in assay buffer containing 25 mM Tris, 100 mM NaCl, 1 mM MgCl2 , 0.1 mM ZnCl2, 0.1% Na ₃ , 5% goat serum and 1% fish gelatin at pH 7.2.

d. Immunoassay Protocol

The indicator reagent (140 μl) was mixed with a series of samples (50 μl) containing known amounts of hCG in normal human serum. The mixtures were incubatec for 10 minutes at 31-32°C. The anti-hCG antibody-PGA capture reagent (100 μl) was added, and the reaction mixtures were incubated for 10 minutes. An aliquot of each reaction mixture (200 μl) was applied to the solid phase material, followed by a wash. An enzyme substrate (70 μl; as described in Example 9) was added, and the resulting rate of fluorescence was measured. The results of the assay are shown in Table 16. The results demonstrate that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased.

Table 16 hCG Ion-Capture Sandwich Assay

Capture Reagent: Anti-hCG Antibody-PGA

Indicator Reagent: Alkaline Phosphatase-Labeled Anti-hCG Antibody

Rate Of Fluorescence (counts/sec/sec) hCG-Specific Capture Reagents hCG (mlU/ml) hCG-ITC-PGA

0 22

8 38

40 116

100 236

550 644

200,000 2058

e. Capture efficiency of anti-hCG-PGA

An assay was conducted using the reagents and protocols described in Sections (a-c) above with the exception that a trace amount of ιl25 radio-labeled anti-hCG-PGA was included in the capture reagent. Radio-labeling was performed using the method described by Hunter et al., Nature. 194:495 (1962) . The anti-hCG-PGA (17μg/ml) was labeled at a specific radio-activity of 70,000 cpm/μg.

After the rate of fluorescence intensity increase was measured at the end of the assay protocol, the radioactivity of anti-hCG-PGA on the solid phase material was also measured by means of a gamma counter (Auto-Logic, Abbott Laboratories, North Chicago, IL) . The results are shown in Table 16 (a) .

Table 16 (a)

Capture of Radiolabeled anti-hCG-PGA on the

Cationic Solid Phase Material hCG (mlU/ml) % anti-hCG-PGA Rate of captured fluorescence

(counts/sec/ sec)

0 97.9 +/- 0.7 213 +/- 1

40 100.7 +/- 1.0 1,334 +/- 7

The data shows that the presence or absence of analyte (hCG) has essentially no effect on the efficiency of capture of the polyanion charged capture reagent. In the assay, there was near quantitative recovery of both free and complexed polyanion conjugated capture reagent on the positively charged solid phase matrix. The essentially quantitative recovery of the radio-labeled capture reagent

(anti-hCG-PGA) was independent of its complex formation with analyte (hCG) and indicator reagent (alkaline phosphatase-goat anti-hCG) . The presence of analyte, however, had a marked effect on the capture of indicator reagent used to detect the analyte by reading the rate of fluorescence increase.

Example 14

Ion-capture Flow-Through Device

For a Two-Step hCG Assay a. Preparation Of The Solid Phase

Test sample application pads (glass fiber matrix) were treated with various concentrations of an aqueous solution of MERQUAT®-100 polymeric ammonium compound, 100 mM Tris, 100 mM sodium chloride, 0.1% fish gelatin, 0.1% sucrose and 0.1% sodium azide. The application pads were allowed to dry, and the pads were overlaid upon a layer of absorbent material. Substantially the same procedure was used to prepare a flow-through solid phase device treated with CELQUAT® L-200 polymeric compound. Alternative devices were prepared by

treating the application pad with MERQUAT® -100 polymeric quaternary ammonium compound (a cationic homopolymer of dimethyldiallylammonium chloride, 0.5% in water) immediately before use.

b. Preparation Of The Indicator Reagent

The indicator reagent was a conjugate of goat anti- ^β -hCG antibody and alkaline phosphate, diluted in 1% Brij®-35 polyoxyethylene (23) lauryl ether (Sigma), 100 mM Tris, 500 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 0.1% NaN3 and 0.5% non-fat dry milk at pH 7.2. The indicator reagent was filtered through a 0.22μm filter before use.

In alternative indicator reagent preparations, dextran sulfate (MW 5,000) or heparin was included as a nonspecific binding blocker. The blocker was used to enhance the signal-to-noise ratio by inhibiting the binding of the labeled antibody to non-analyte.

c. Preparation Of The Capture Reagent

A monoclonal anti-β-hCG antibody-PGA capture reagent was prepared substantially in accordance with the method described in Example 8.c. above. Every five milliliters of the coupling reaction mixture was fractionated on a gel filtration chromatography column (2.4 x 54 cm, at a 0.4 ml/minute flow rate) . The elution buffer contained 0.1 M sodium phosphate, 0.3M NaCl and 0.05% NaN3 , at pH 8.5. The polymeric anion/antibody conjugate was diluted with 25 mM Tris, 100 mM NaCl, 1 mM MgCl2> 0.1 mM ZnCl2, 0.1% NaN3 , ^10% normal mouse serum and 1% fish gelatin at pH 7.2. The capture reagent was filtered through a 0.22 μm filter before use. d. Immunoassay Protocol

The capture reagent (80μl) was mixed with an equal volume of test sample containing a known amount of hCG in normal human serum. The mixture was incubated at

approximately 31-32° for approximately twelve minutes. The specific binding reaction resulted in the formation of a capture reagent/analyte complex.

Each reaction mixture (80 μl) was then applied to a flow-through device, followed by a wash with Tris buffered saline (75 μl) . The indicator reagent (50 μl) was then applied to the solid phase device and incubated for twelve minutes. The device was then washed two times.

An enzyme substrate (70 μl; 1.2 mM 4- methylumbelliferyl-phosphate in a solution of 100 mM AMP, 0.01% EDTA, 0.1% NaN3, and 4.0 mM tetramisole at pH 10.3) was added, and the resulting rate of fluorescence was measured. The results of the assay are shown in Tables 17-19. The results demonstrated that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex, and therefore, the amount of detectable label bound to the solid phase increased. The results show that the signal to noise ratio is improved by including a nonspecific binding blocker in the indicator reagent. Furthermore, the results demonstrated that the cationic homopolymer of dimethyldiallylammonium chloride was a preferred polymeric cation for the preparation of the solid phase for use in two- step assays wherein the device is subjected to one or more washings, e.g. the MERQUAT®-100 polymeric ammonium compound has a nitrogen content of about 8% (exclusive of counter ion) , whereas the CELQUAT® H-100 polymeric compound has a nitrogen content of about 1% (exclusive of counter ion) .

Table 17 hCG Ion-capture two-step Sandwich Assay

Capture reagent: anti-β-hCG antibody-PGA (0.5 μg/test)

Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody (with and without nonspecific binding blocker)

Solid phase: coated with a cationic homopolymer of dimethyldiallylammonium chloride immediately before use

Rate of fluorescence (counts/sec/sec) hCG (mlU/m 2% dextran sulfate no blocker 0 68 255

100 1028 1104

Table 18 hCG Ion-capture two-step Sandwich Assay

Capture reagent: anti-β-hCG antibody-PGA (0.5 μg/test)

Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody (with blocker)

Solid phase: with varying cationic polymer concentration

Rate of fluorescence (counts/sec/sec) MEROUAT®-100 polymeric ammonium compound ! (%w/v)

hCG mTTJ/ml 0.02 0.04 0.2 0.6

0 34 31 26 30 39 100 514 578 627 661 647

Table 19 hCG ion-capture two-step Sandwich Assay

Capture reagent: anti-β-hCG antibody-PGA

Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody

Solid phase: with 0.125% CELQUAT® H-100 polymer compound

Rate of fluorescence (counts/sec/sec) Quantity of capture antibody (ll/test)

hCG ( mTTT /τπ 1 1 0.266 0.402 0-652

0 86 100 115 100 186 202 259

Example 15 Ion-capture Flow-Through Device for Thyroid Stimulating

Hormone (TSH) Assay a. Preparation of the solid phase

An application pad (glass fiber matrix) was treated with an aqueous solution of MERQUAT®-100 polymeric ammonium compound substantially in accordance with the procedure described in Example 14.a. The pad was then overlaid upon a layer of absorbent material to complete the flow-through solid phase device.

b. Preparation of the indicator reagent

The indicator reagent was a conjugate of goat anti- ^β -hCG antibody and alkaline phosphatase, diluted in 1% Brij®- 35 polyoxyethylene (23) lauryl ether, 1% fish gelatin, 100 mM Tris, 500 mM NaCl, 1 mM MgCl2, 0.1% NaN ₃ and 0.5% non-fat dry milk at pH 7.2. The indicator reagent was filtered through a 0.22 μm filter before use. Dextran sulfate (0.5%,MW 5,000) was added as a nonspecific binding blocker.

-73 -

c. Preparation of the capture reagent

The capture reagent was prepared by coupling a Protein A purified monoclonal anti-TSH antibody with carboxymethylamylose (CMA; Polysciences, Inc. , Warrington, PA) . Coupling was performed using a water-soluble carbodiimide reagent (l-ethyl-3- (3-dimethylaminopropyl) carbodiimide; EDCI) substantially in accordance with the following procedure.

The coupling mixture contained an antibody solution (2 ml; 1 mg/ml in MES buffer [25 nM,2-(N-

Morpholino)ethanesulfonic acid] pH 5.5) and CMA (1.6 ml; 10 mg/ml in MES buffer) . To the solution was added, with stirring, a freshly prepared EDCI solution (40 μl; 100 mg/ml in MES buffer) . The reaction mixture was stirred at room temperature for 40 minutes. The reaction was quenched by adding a 25% glycine solution (67 μl), and the product was then fractionated by gel filtration chromatography using a TSKgel G4000SW column (2.15 cm x 30 cm) fitted with a TSKguard column SW (2.15 cm x 7.5 cm; Anspec Co., Ann Arbor, Michigan) . The column was eluted with PBS (0.1 M sodium phosphate, 0.3 M NaCl and 0.05% sodium azide, at pH 6.8) . The purified Antibody/CMA capture reagent was diluted in a diluent containing 50 mM Tris, 300 mM NaCl, 1% bovine serum albumin, 2.5% fish gelatin and 0.1% NaN3, at pH 7.5.

d. Immunoassay protocol

The capture reagent (30 μl) and Tris buffered saline (100 μl; 500 mM Tris, 300 mM NaCl and 0.1% NaH3) were mixed with a test sample (50 μl) containing a known amount of hCG in normal human serum. The reaction mixture was incubated at approximately 33-34° for approximately ten minutes. The specific binding reaction resulted in the formation of a capture reagent/analyte complex.

An aliquot of each reaction mixture (140 μl) was applied to a solid phase device, followed by a wash with Tris

buffered saline (150 μl) . The indicator reagent (70 μl) was applied to the device and incubated for approximately ten minutes. The device was then washed two times with buffer (100 μl each). The enzyme substrate (70 μl; 1.2 mM 4- methylumbelliferyl-phosphate in a solution of 100 mM AMP, 0.01% EDTA, 0.1% NaN3, and 4.0 mM tetramisole at pH 10.3) was added, and the resulting rate of fluorescence was measured.

The results of the assay are shown in Table 20. The results demonstrated that as the concentration of TSH in the test sample increased, there was a corresponding increase in the formation of capture reagent/analyte/indicator reagent complex. Therefore, the amount of detectable label bound to the solid phase increased as the concentration of analyte increased. The results also demonstrated that the combination of MERQUAT®-100 polymeric ammonium compound with polyacrylic acid or with carboxy-methylamylose provided a solid phase and capture reagents which were advantageously used in two-step assay where the device is subject to one or more washings or manipulations.

Table 20

TSH ion-capture Two-step Sandwich Assay

(using polyacrylic acid or carboxymethylamylose polyanions)

Rate of fluorescence (counts/sec/sec)

TSH (m- tU/ml ) carboxymethvlamvlose polvacrvlic acid

0 7.1 6.4

0 . 5 13.3 12.1

2 .0 34.7 28.7

10 . . 0 147.5 119.0

40 . , 0 513.9 442.6

100. , 0 1121.6 995.5

e. TSH capturing efficiency

Radioiodinated TSH was used in the assay protocol, as described in Example 15.d, to demonstrate the more efficient TSH capturing of CMA-coupled antibodies than that of polyaspartic- and polyglutamic-coupled antibodies. The coupling of antibodies to the polyanions was performed substantially in accordance with the method described above (Example 15.c.) After the rate of fluorescence was measured at the end of the assay protocol, the radioactivity of TSH captured on the solid phase material was also measured by means of a gamma counter (Auto-Logic, Abbott Laboratories, North Chicago, IL) . The results of this procedure are demonstrated in Table 20 (a) .

Table 20 (a)

Capture of Radiolabeled TSH in the

Cationic Solid Phase Material

Polyanion-coupled Rate of fluorescence anti-TSR antibody % TSH captured (counts/sec/sec)

Carboxymethyl- amylose 7.0 662

Polyaspartic Acid 1.5 37

Polyglutamic Acid 2.0 57

Example 16

Ion-Capture Flow-Through Device For A One-Step hCG Assay a. Preparation Of The Solid Phase

A glass fiber matrix was treated with an aqueous solution of MERQUAT®-100 polymeric ammonium compound substantially in accordance with the procedure described in

Example 14(a) above. The pad was then overlaid upon a layer of absorbent material to complete the device.

b. Preparation Of The Indicator Reagent

The indicator reagent was a goat anti-β-hCG antibody conjugated to alkaline phosphatase and diluted in 3.33% Brij®- 35 polyoxyethylene (23) lauryl ether, 5 mM Tris, 1 mM MgCl2, 0.1 mM ZnCl ₂ , 0.1% NaN3 and 5% fish gelatin at pH 7.2. The indicator reagent was filtered through a 0.2 μrr filter before use. In alternative indicator reagent preparations, carboxymethyl cellulose (MW 250,000) or carboxymethyl dextran was included as a nonspecific binding blocker.

c. Preparation Of The Capture Reagent

A monoclonal anti-hCG antibody-PGA capture reagent was prepared substantially in accordance with the method described in Example 15(c) above. The polymeric anion/antibody conjugate was diluted with 3.33% Brij®-35 polyoxyethylene (23) lauryl ether, 5 mM Tris, 500 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 0.1% Na ₃ , and 5% fish gelatin at pH

7.2. The enzyme substrate was 1.2 mM 4-methylumbelliferyl- phosphate in a solution of 100 mM AMP, 0.01% EDTA, 0.1% NaN3, and 4.0 mM tetramisole at pH 10.3

d. Immunoassay Protocol

The capture reagent (50 μl) , indicator reagent (55 μl) and sample diluent buffer (35 μl; 75% normal calf serum, 25% normal goat serum and 0.2% NaN3, filtered through a 0.22 μm filter before use) were mixed with a test, sample (30 μl) containing a known amount of hCG in normal human serum. The mixture was incubated at approximately 33-34°C for approximately fourteen minutes. The specific binding reaction resulted in the formation of a capture reagent/analyte/indicator reagent complex.

An aliquot of each reaction mixture (110 μl) was then applied to a solid phase device, followed by two washes with Tris buffered saline (75 μl) . The enzyme substrate (65 μl) was added, and the resulting rate of fluorescence was measured.

The results of the assay are shown in Table 21. The results demonstrated that as the hCG test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte/indictor reagent complex, and therefore, the amount of detectable label bound to the solid phase increased. Furthermore, the results show that the signal to noise ratio was improved when a free polyanionic substance was included in the indicator reagent as a nonspecific binding blocker, even though the capture reagent was a polymeric anion/antibody conjugate.

Table 21 hCG Ion-Capture Sandwich Assay

Capture Reagent: Anti-hCG antibody-PGA

Indicator Reagent: Alkaline Phosphatase-Labeled Anti-hCG Antibody

Rate of fluorescence (counts/sec/sec) hCG

(mlU/ml ) 0. 0.01 0.25 -5.

0 37.2 23.8 17.2 13.3

10 76.8 58.4 48.8 42.1

1000 1803.6 1665.4 1692.2 1507.2

Rate of fluorescence (counts/sec/sec) % of carboxymethyl dextran in indicator reaσent hCG (mlV/ml) Q. o.oi 0.25 £

0 35.6 30.0 17.8 14.8

10 75.2 68.4 54.7 49.8

1000 1826.6 1851.2 1739.5 1646.6

Example 17

Ion-Capture Flow-Through Device For A Total T3 (Triiodothyronine) Competitive Assay a. Preparation Of The Solid Phase

Test sample application pads (glass fiber matrix) were treated with various concentrations of an aqueous solution of CELQUAT® L-200 polymeric quaternary ammonium compound or MERQUAT®-100 polymeric ammonium compound, 100 mM Tris, 100 mM sodium chloride, 0.1% fish gelatin, 0.1% sucrose and 0.1% sodium azide. The application pads were allowed to dry, and the pads were overlaid up:n a layer of absorbent material to form the individual assay devices.

b. Preparation Of The Indicator Reagent

The indicator reagent was a conjugate of T3 and alkaline phosphatase, diluted in 50 mM Tris, 100 mM NaCl, 1.0 mM MgCl2, 0.1 mM ZnCl2 and 1.0% bovine serum albumin at pH

7.5. Dextran sulfate (MW 5,000) was included as a nonspecific binding blocker. The blocker was used to enhance the signal- to-noise ratio by inhibiting the binding of the labeled antibody to non-analyte.

c. Preparation Of The Capture Reagent

The capture reagent, an anti-T3 antibody coupled to polyaspartic acid (PAA-anti-T3 antibody) , polyacrylic acid (PAcA-anti-T3 antibody) or carboxymethyl cellulose (CMA-anti- T3 antibody) anionic polymer molecules, was prepared substantially in accordance with the method described in the Example 15(c) EDCI coupling method, with the exception that no chromatographic filtration of the capture reagent was performed. The capture reagent was diluted with 800 mM Tris, 50 mM NaCl, 0.1% aN3, 0.01% furosemide, 0.1% Tween-20, 1.0% bovine serum albumin and 0.08 mg/ml goat IgG at pH 7.4.

d. Immunoassay Protocol

The capture reagent (50 μl) was mixed with an equal volume of test sample, containing a known amount of Total T3, and sample diluent buffer (150 μl) . The reaction mixture was incubated for approximately 15 minutes. The specific binding reaction resulted in the formation of a capture reagent/analyte complex.

Each reaction mixture (150 μl) was then applied to a solid phase. The indicator reagent (60 μl) was then applied to the solid phase and incubated for eight minutes. The device was then washed two times. An enzyme substrate (50 μl) was added, and the resulting rate of fluorescence was measured.

In an alternative assay format, the solid phase was also washed prior to the addition of the indicator reagent. In yet another assay format, the capture reagent and test sample were combined and incubated, followed by the addition of indicator reagent and further incubation prior to placing an aliquot of the reaction mixture on the solid phase.

The polyelectrolyte interaction of the capture reagent and the oppositely charged solid phase resulted in the immobilization of capture reagent and capture reagent complexes on the solid phase devices. An enzyme substrate (70 μl; 1.2 mM 4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 0.01% EDTA, 0.1% NaN3, and 4.0 mM tetramisole at pH

10.3) was added, and the resulting rate of fluorescence was measured.

In each assay, the results demonstrated that as the Total T3 test sample concentration increased there was a corresponding increase in the formation of capture reagent/analyte complex, and therefore, the amount of detectable label bound to the solid phase decreased. Furthermore, the results show that the signal to noise ratio

is improved by including a nonspecific binding blocker, dextran sulfate, in the indicator reagent.

Table 22

Total T3 Competitive Assay

Calibration Data Comparing One- tep And Two-Step Assay Protocols

Protocol: One-Step Two-Step

Precoated Solid Phase: 0.5% CELQUAT® 0.2% MERQUAT®

Capture Antibody: ITC-PGA Anti-T3 EDAC-PAA an i-T3

(per test) Antibody (0.25 μg) Antibody (0.02 μg)

Indicator Reagen No Blocker With 0.1% dextran sulfa

Calibrators Concentration nσ/ml totsl T3 Rare Of Fluorescence (counts/sec/sec)

0 518 616

0.5 386 513

1.0 310 403

2.0 218 260

4.0 113 109

8.0 ^"" 1 48

Table 23

Total T3 Competitive Two-Step Assay

Comparison Of Indicator Reagents With And Without A Non-Soecific Binding Blocker

Precoated Solid Phase: 0.2% MERQUAT® 0.2% MERQUAT®

Capture Antibody: EDAC-PAA anti-T3 EDAC-PAA anti-T3

(per test) Antibody (0.02 μg) Antibody (0.02 μg)

Indicator Reagent: No Blocker With 0.1% dextran sulfate

T3 alkaline phosphatase dilution: 1:400 1:150

Calibrators Concentration nσ/ml total T3 Rate Of Fluorescence (counts/sec/sec)

0 641 536

2.0 361 220

8.0 81 39

-81-

Table 24

Total T3 Competitive Assay

Calibration Data Comparing Different T3 Capture Reagents

Capture Antibody: PAA-anti-T3 PAcA-anti-T3 CMA-anti-T3

(per test) antibody antibody antibody

(0.013 μg) (0.015 μg) (0.013 μg)

Calibrations

Concentration nσ/ml total T3 Rate of fluorescence (counts/sec/sec)

0 509 544 507

0.5 401 443 394

1.0 332 344 322

2.0 203 219 204

4.0 94 107 99

8.0 47 57 51

It will be appreciated by one skilled in the art that the concepts of the present invention are equally applicable to any separation techniques or binding assays (wherein the signal generating ability of the label is not altered during the binding reaction) by using oppositely charged solid phase materials and capture reagents. The embodiments described in detail herein are intended as examples rather than as limitations. Thus, the description of the invention is not intended to limit the invention to the particular embodiments described, but it is intended to encompass all equivalents and subject matter within the spirit and scope of the invention as described above and as set forth in the following claims.