IMPROVED ENHANCEMENT OF CHEMILUMINEBCENT ASSAYS

This application is a continuation-in-part of U.S. Patent Application Serial No. 07/806,928, itself a divisional application of U.S. Patent Application Serial No. 07/574,786, issued as U.S. Patent 5,112,960. The entire disclosure of U.S. Patent 5,112,960 is incorporated herein by reference. This application is also a continuation-in-part application of U.S. Patent Application Serial No. 07/959,531, currently pending, the entire disclosure of which is incorporated hereby reference.

Technical Field

This invention pertains to the enhancement of chemiluminescent assays for analytes in a sample, generally a biological sample, based on the use of 1,2-dioxetanes as chemiluminescent substrates for enzyme-labeled targets or probes. The chemiluminescence of the dioxetane reporter molecule can be enhanced by the addition of a water-soluble quaternary polymer. Further enhancement can be achieved by additionally including in the sample an additive which improves the ability of the water-soluble quaternary polymer to sequester the dioxetane within hydrophobic regions formed by the water-soluble polymer thereby suppressing or avoiding water-induced light quenching reactions.

Background Art

Chemiluminescent assays for the detection of the presence or concentration of an analyte in a sample, generally a biological sample, have received increasing attention in recent years as a fast, sensitive and easily read method of conducting bioassays. In such assays, a

che iluminescent compound is used as a reporter molecule, the reporter molecule chemiluminescing in response to the presence or the absence of the suspected analyte.

A wide variety of chemiluminescent compounds have been identified for use as reporter molecules. One class of compounds receiving particular attention is the 1,2- dioxetanes. 1,2-dioxetanes can be stabilized by the addition of a stabilizing group to at least one of the carbon molecules of the dioxetane ring. An exemplary stabilizing group is spiro-bound adamantane. Such dioxetanes can be further substituted at the other carbon position with an aryl moiety, preferably phenyl or naphthyl, the aryl moiety being substituted by an oxygen which is in turn bound to an enzyme-labile group. When contacted by an enzyme capable of cleaving the labile group, the oxyanion of the dioxetane is formed, leading to decomposition of the dioxetane and spontaneous chemiluminescence. A wide variety of such dioxetanes are disclosed in U.S. Patent 5,112,960. That patent focuses on dioxetanes which bear a substituent on the adamantyl- stabilizing group, such as halo substituents, alkyl groups, alkoxy groups and the like. Such dioxetanes represent an advance over earlier-recognized dioxetanes, such as 3-(4- methoxyspiro[1,2-dioxetane-3,2 '-tricyclo]-3.3.1.1 ^3,7 ]decan]- 4-yl)phenyl phosphate, and in particular, the disodium salt thereof, generally identified as AMPPD. The chlorine- substituted counterpart, which converts the stabilizing adamantyl group from a passive group which allows the decomposition reaction to go forward to an active group which gives rise to enhanced chemiluminescence signal due to faster decomposition of the dioxetane anion, greater signal-to-noise values and better sensitivity, is referred to as CSPD. Other dioxetanes, such as the phenyloxy-β-D- galactopyranoside (AMPGD) are also well known, and can be used as reporter molecules. These dioxetanes, and their preparation, do not constitute an aspect of the invention herein, per se.

Assays employing these dioxetanes can include conventional assays, such as Southern, Northern and Western blot assays, DNA sequencing, ELISA, as well as other liquid phase and mixed phase assays. In general, the assay consists of binding the target, if present in the sample, with a complex bearing an enzyme capable of cleaving the enzyme labile group of the dioxetane. In DNA assays, the target is bound by a DNA probe with an enzyme covalently linked thereto, the probe being admixed with the sample immobilized on a membrane, to permit hybridization. Thereafter, excess enzyme complex is removed, and dioxetane added to the hybridized sample. If hybridization has occurred, the dioxetane will be triggered by the bound enzyme, leading to decomposition of the dioxetane, and chemiluminescence. In liquid phase assays, the enzyme is frequently conjugated or complexed with an antibody responsive to the target analyte, unbound complex being removed, and the dioxetane added, chemiluminescence being produced by the decomposition of the dioxetane triggered by the amount of enzyme present. In cases where the enzyme itself is the target analyte, the dioxetane need only be added to the sample. Again, a wide variety of assay modalities has been developed, as disclosed in U.S. Patent 5,112,960, as well as U.S. Patent 4,978,614.

It has been well known that light-quenching reactions will occur if the dioxetane decomposition goes forward in a protic solvent, such as water. As the samples suspected of containing or lacking the analyte in question are generally biological samples, these assays generally take place in an aqueous environment. The light-quenching reactions therefor may substantially reduce the chemiluminescence actually observed from the decomposition of the dioxetane. In assays involving low-level detections of particular analytes, such as nucleic acids, viral antibodies and other proteins, particularly those prepared in solution or in solution-solid phase systems, the reduced chemiluminescence observed, coupled with unavoidable background signals, may

reduce the sensitivity of the assay such that extremely low levels cannot be detected. One method of addressing this problem is the addition of water-soluble macromolecules, which may include both natural and synthetic molecules, as is disclosed in detail in U.S. Patent 5,145,772. The disclosure of this patent is incorporated herein, by reference. To similar effect, U.S. Patent 4,978,614 addresses the addition of various water-soluble "enhancement" agents to the sample, although the patent speaks to the problem of suppressing non-specific binding in solid state assays. In U.S. Patent 5,112,960, preferred water-soluble polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride) (TMQ) poly(vinyl-benzyltributylammonium chloride) (TBQ) and poly(vinylbenzyl-dimethylbenzylammonium chloride) (BDMQ) are identified as water-soluble polymeric quaternary ammonium salts which enhance chemiluminescence by the suppression of non-specific binding.

Notwithstanding the advances in technology addressed by these assays, it remains a goal of the industry to provide chemiluminescent assays of greater sensitivity, to determine the presence, concentration or both of an analyte in a sample which is generally biological, and therefor, in an assay in an aqueous environment. 1,2-dioxetane compounds have already been developed which show excellent potential as reporter molecules for such chemiluminescent assays. To be used in extremely low-level detection, however, and/or to improve reliability to provide for machine readability, it is necessary to further improve the enhancement of the chemiluminescence of the 1,2-dioxetane molecules produced in aqueous preparations.

Disclosure of the Invention

Applicants' invention addresses the above goals in two embodiments. As noted above, it has been previously recognized that the addition of water-soluble polymeric

ammonium salts to the aqueous sample improves or enhances chemiluminescence of 1,2-dioxetanes. Applicants have discovered that this enhancement is achieved, at least in part, through the formation of hydrophobic regions in which the dioxetane oxyanion is sequestered. Decomposition in these hydrophobic regions enhances chemiluminescence, because water-based light quenching reactions are suppressed. Among the recognized water-soluble quaternary polymer salts employed, TBQ provides unexpectedly superior enhancement, through this hydrophobic region-forming mechanism.

The chemiluminescent enhancement achieved by the addition of water-soluble polymeric quaternary polymer salts can be further improved by the inclusion, in the aqueous sample, of an additive, which improves the ability of the quaternary polymeric salt to sequester the dioxetane oxyanion and the resulting excited state emitter reporting molecule in a hydrophobic region. Thus, the combination of the polymeric quaternary salt and the additive, together, produce an increase in enhancement far beyond that produced separately by the addition of the polymeric quaternary salt, or the additive, which, when a surfactant or water- soluble polymer itself, may enhance chemiluminescence to a limited degree. The synergistic combination of the polymeric quaternary salt and additives gives enhancement effects making low-level, reliable detection possible even in aqueous samples through the use of 1,2-dioxetanes. The polymeric quaternary salts, coupled with the additives, are sufficiently powerful enhancers to show dramatic 4 and 5- fold increases at levels below 0.005 percent down to 0.001 percent. Increased signal, and improved signal/noise ratios are achieved by the addition of further amounts of the polymeric quaternary salt, the additive, or both, in amounts up to as large as 50 percent or more. In general, levels for both polymeric quaternary salt and additive can be preferably within the range of 0.05 - 25 percent, more preferably from 0.1 - 15 percent by weight.

Brief Description of the Invention

The assays and kits of this invention employ water- soluble chemiluminescent 1,2-dioxetanes. As noted above, these dioxetanes are well established in the art, and their identity and preparation does not constitute a novel aspect of this invention, per se. In general, any chemiluminescent dioxetane which exhibits sufficient stability in water to conduct the assay, and which may be caused to decompose and chemiluminesce by interaction with an enzyme, and cleavage, by the enzyme, of an enzyme labile group inducing the decomposition, can be used in connection with this invention. Typically, the 1,2-dioxetanes useful in this invention will have the general formula:

R ¹ is C.,-C ₂₀ alkyl or C,..,-, aryl or aralkyl; is phenyl or naphthyl; R ² is meta-substituted or non-conjugated on Y with respect to the dioxetane, and is OX, wherein;

X is an enzyme cleavable group which, when cleaved, leaves the dioxetane phenoxy or naphthoxy anion; Z = H, Cl, other halogens, or alkoxy groups.

Preferred dioxetanes include AMPPD, and in particular, its disodium salt, as well as CSPD and its disodium salt. Methods of preparing these dioxetanes are disclosed in the aforereferenced commonly-assigned patents, as well as,

e.g., U.S. Patent 4,962,162, assigned to Wayne State University. The preparation, purification and isolation of the dioxetanes does not constitute a novel aspect of the invention disclosed and claimed herein per se.

The dioxetane is added to the sample which has been mixed with an enzyme complex which will bind to or otherwise co-act with the target analyte, if present in the sample. The dioxetane is therefore the substrate for the enzyme, the enzyme-catalyzed cleavage of the labile groups of the substrate from the body of the dioxetane resulting in the unstable oxyanion, and subsequent decomposition of the dioxetane. Where the target analyte is the enzyme itself, the dioxetane is added directly to the sample, either as drawn, or after preliminary purification to reduce turbidity. Where the target analyte is other than the trigger enzyme, the enzyme is complexed with a binding moiety, such as DNA probe or antibody, so as to bind to any target analyte present in the sample. The amount of chemiluminescence detected will be responsive both to the analyte in the sample, and the amount of analyte in the sample.

To enhance the che iluminescent signal, and improve signal/noise ratio to permit discrimination between background signals and positive target-responsive signals at very low levels, a water-soluble enhancement agent is added to the sample prior or concomitant with the introduction of the dioxetane.

The enhancement agents of this invention, are based, in general, on polymeric onium salts, particularly quaternary salts based on phosphonium, sulfonium and, preferably, ammonium moieties. The polymers have the general formula I shown below:

— (-CH ₂

,3 ^

In this formula each of R ¹ , R ² and R ³ can be a straight or branched chain unsubstituted alkyl group having from 1 to 20 carbon atoms, inclusive, e.g., methyl, ethyl, n- butyl, t-butyl, hexyl, or the like; a straight or branched chain alkyl group having from 1 to 20 carbon atoms, inclusive, substituted with one or more hydroxy, alkoxy, e.g., methoxy, ethoxy, benzyloxy or polyoxethylethoxy, aryloxy, e.g., phenoxy, amino or substituted amino, e.g., methylamino, amido, e.g., acetamido or ureido, e.g., phenyl ureido; or fluoroalkane or fluoroaryl, e.g., heptafluorobutyl, groups, an unsubstituted monocycloalkyl group having from 3 to 12 carbon ring carbon atoms, inclusive, e.g., cyclohexyl or cyclooctyl, a substituted monocycloalkyl group having from 3 to 12 ring carbon atoms, inclusive, substituted with one or more alkyl, alkoxy or fused benzo groups, e.g., methoxycyclohexyl or 1,2,3,4- tetrahydronaphthyl, a polycycloalkyl group having 2 or more fused rings, each having from 5 to 12 carbon atoms, inclusive, unsubstituted or substituted with one or more alkyl, alkoxy or aryl groups, e.g., l-adamantyl or 3- phenyl-1-adamantyl, an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms .in toto. unsubstituted or substituted with one or more alkyl, aryl, fluorine or hydroxy groups, e.g., phenyl, naphthyl, pentafluorophenyl, ethylphenyl, benzyl, hydroxybenzyl,

phenylbenzyl or dehydroabietyl; at least two of R,, R _j and R ₃ , together with the quaternary atom to which they are bonded, can form a saturated or unsaturated, unsubstituted or substituted nitrogen-containing, phosphorus-containing or sulfur-containing ring having from 3 to 5 carbon atoms, inclusive, and 1 to 3 heteroatoms, inclusive, and which may be benzoannulated, e.g., l-pyridinium, l-(3-alkyl or aralkyl)imidazolium, morpholino, alkyl morpholinium, alkylpiperidiniu , N-acylpiperidinium, piperidino or acylpiperidino, benzoxazolium, benzthiazolium or benzamidazoliu .

The symbol X ^" represents a counterion which can include, alone or in combination, moieties such as halide, i.e., fluoride, chloride, bromide or iodide, sulfate, alkylsulfonate, e.g., methylsulfonate, arylsulfonate, e.g., p-toluenesulfonate, substituted arylsulfonate, e.g., anilinonaphthylenesulfonate (various isomers) , diphenylanthracenesulfonate, perchlorate, alkanoate, e.g., acetate, arylcarboxylate, e.g., fluorescein or fluorescein derivatives, benzoheterocyclic arylcarboxylate, e.g., 7- diethylamino-4-cyanocoumarin-3-carboxylate, organic dianions such as p-terephthalate may also be represented by X ^' .

The symbol n represents a number such that the molecular weight of such poly(vinylbenzyl quaternary salts) will range from about 500 to about 500,000 (weight average), and preferably from about 20,000 to about 70,000, as determined by intrinsic viscosity or LALLS techniques.

Methods for the preparation of these polymers, related copolymers and the related starting materials where M is nitrogen are disclosed in G. D. Jones et al. Journal of Polymer Science. 25 _r 201, 1958; in U.S. Patents 2,780,604; 3,178,396; 3,770,439; 4,308,335; 4,340,522; 4,424,326 and German Offenlegunsschrift 2,447,611.

The symbol M may also represent phosphorous or sulfur whereupon the corresponding sulfonium or phosphoniu

polymers have been described in the prior art: U.S. Patents 3,236,820 and 3,065,272.

Methods of preparation of the polymers of this invention are set forth in the referenced U.S. Patents, as well as U.S. Application Serial No. 07/811,620 and do not constitute any aspect of this invention, per se.

Copolymers containing 2 or more different pendant onium groups may also be utilized in the invention described herein.

The symbols X, M' , R ¹ ', R ² ', R ³ ' are as described above for X, M, R ¹ -R ³ . The symbols Y and Z represent the mole fraction of the individual monomers comprising the copolymer. The symbols Y and Z may thus individually vary from .01 to .99, with the sum always equalling one.

As preferred moieties, M is N or P, and R ¹ -R ³ are individually, independently, alkyl, cycloalkyl, polycycloalkyl (e.g. adamantane) aralkyl or aryl, having 1 to 20 carbon atoms, unsubstituted or further substituted with hydroxyl, amino, amido, ureido groups, or combine to form via a spiro linkage to the M atom a heterocyclic

(aromatic, aliphatic or mixed, optionally including other N, S or 0 heteroatoms) onium moiety.

Applicants have discovered that poly(vinylbenzyl- tributylammonium chloride) , alone, gives unpredictably superior enhancement in combination with a chemiluminescent assay based on the use of a 1,2-dioxetane reporter molecule, as described above. In point of fact, the improvement in chemiluminescent signal obtained by the addition of TBQ, alone, was generally on the order of at least twice the improvement obtained through addition of the next best enhancement agent, BDMQ. For completeness, TBQ was also evaluated, alone, versus cetyltrimethylammoniura bromide, another art recognized enhancement agent. Evaluation of TBQ Alone as an Enhancement Aσent

Protocol for the evaluation of various enhancers

The enhancer samples were prepared from stock solutions in a 0.1 diethanolamine, ImM MgCl, pH 9.5 buffer. CSPD or AMPPD was added to 0.5 ml of an enhancer solution (final dioxetane concentration, 0.4 mM) . The background was recorded at room temperature in a Berthold 952T luminometer, 10 and 60 minutes after the dioxetane addition. Subsequently, alkaline phosphatase was added (final enxyme concentration, 9.35 x 10 ^"H M) , and the chemiluminescent signal was measured at 10, 30 and 60 (not for all samples) minutes as a 5 sec. integral.

All buffers contained 1 mM MgCl _z

**The half-time to plateau was determined as follows: 0.4 mM AMPPD in the desired buffer was preincubated at 37°C or 30°C and alkaline phosphatase was then added to each tube (final concentration 4.1 x 10 ^'13 M) . The tube was inserted into a Turner model 20-E Luminometer and the half-time to steady state light emission was calculated.

TABLE 1

HALF-LIFE OF AMPPD Effect of CTAB, TBQ, BDMQ, pH and Temperature

30°C, 0.1 M Sodium Carbonate

30°c, 0.75 M Sodium Carbonate

37°C, 0.1 M Sodium Carbonate

37°C, 0.75 M Sodium Carbonate

TABLE 2

CHEMILUMINESCENCE OF AMPPD

SIGNAL AND BACKGROUND

Effect of CTAB, TBQ, BDMQ, pH and Temperature

30°C, 0.1 M Sodium Carbonate

30°C, 0.75 M Sodium Carbonate

37°C, 0.1 M Sodium Carbonate

37°C, 0.75 M Sodium Carbonate

As can be seen from the foregoing enhancement analysis, TBQ offers sharp improvements in chemiluminescent enhancement, and elevated S/N, as compared with the dioxetane alone, or with other art-recognized enhancement agents. Thus, the addition to the sample of TBQ, alone, provides substantial enhancement of the chemiluminescent signal and S/N values obtained. In point of fact, Applicants have found that the addition of amounts as low as below 0.005 percent down to 0.001 percent, by weight, based on the sample volume, of TBQ results in an enhancement of the chemiluminescent signal 4-5 fold over the signal obtained from the dioxetane without any enhancer.

Addition of Enhancement Agent and Enhancement Additive

The polymeric quaternary salts that constitute the enhancement agents of this invention enhance chemiluminescence in a protic environment by forming hydrophobic regions in which the dioxetane moiety, resulting from the enzyme as the oxyanion, and the subsequently formed excited state are sequestered. By providing a hydrophobic region, light-quenching water reactions are reduced or avoided all together, resulting in an overall improvement of chemiluminescence. Applicants have discovered that use of an enhancement additive, in addition to the polymeric quaternary salt enhancement agents of the claimed invention, further enhances the observed chemiluminescent signal, and improves S/N values. The enhancement additives of the claimed invention show marked improvement, in connection with the enhancement agents, at very low levels. The addition of as much as 0.005 percent, by weight, based on the sample volume, of an enhancement additive further improves the strength of the chemiluminescent signal obtained by use of the enhancement agent, while not increasing the background levels. As with the enhancement agents, the additives can be used in amounts as high as practically useful. In general, amounts greater than 50 percent, by weight, based on the sample impede assay performance. Accordingly, inclusion of both the enhancement agent, and the enhancement additive, in amounts each of 0.005 percent, by weight, based on the sample volume, up to and including a total of 50 percent, by weight, based on the sample volume, of both, are within

the scope of this invention. The total of agent and additive should not exceed 50 percent.

The function of the enhancement additive is to improve the ability of the enhancement agent to form hydrophobic regions in which the dioxetane oxyanion and the resulting emitter can be sequestered, permitting decomposition and chemiluminescence in the absence of water, and therefore, reducing light-quenching reactions caused thereby. The enhancement additives can be drawn from any of a wide variety of compounds. In general, the enhancement additives may, but need not necessarily, enhance the chemiluminescent signal obtained, although not nearly so strongly as the enhancement agents of this invention. Thus, the addition of conventional surfactants, largely detergents, improves the ability of the enhancement agent to form a hydrophobic region which is relatively stable. These surfactants may be cationic, anionic, zwitterionic or neutral. Another class of enhancement additives also active at very low concentrations are conventional solvents, including a wide variety of alcohols. Another conventional solvent, turpentine, is also useful in this role.

A fourth effective class of enhancement additives are non-quaternary water-soluble polymers, such as poly(2- ethyl-Z-oxazoline) , (PolyOx) . While these polymers themselves may induce limited enhancement of the chemiluminescent signal without increase in background noise, when added in conjunction with the polymeric

quaternary onium salt enhancement agents of this invention, sharp improvements in the chemiluminescent signal observed are obtained.

To demonstrate the improvement obtained by the addition of an enhancement additive to the enhancement agent, buffered dioxetane solutions were provided with TBQ as well as sodium dodecylbenzenesulfonate, a surfactant, poly(2-ethyl-2-oxazoline) and octyl-3-glucoside. The results, set forth below in Tables 3 and 4, are obtained with CSPD and enhancer compositions, as indicated.

ABBREVIATIONS USED IN TABLES TBQ polyvinylbenzyltributylquaternary ammonium chloride SDBS sodium dodecylbenzenesulfonate PolyOx poly(2-ethyl-2-oxazoline) , MW 500,000 OcGluc octyl-β-glucoside

IΔB E

Polyσi Dafarganti

Stl 10 S/U 10' SN

63 64 6 2262 2704 26 2512 2flfi3 27

116 1.11 13 2229 2913 291 2652 3511 34 1292 1616 16 2070 2508 24 2015 2655 25 2479 3122 29

63 177 3.1 1299 3256 53 1099 2361 321 1190 2362 29 1712 4026 66 1567 3419 50 3529 6704 95 2410 4171 50

As is clearly reflected in the foregoing results, the addition of a variety of enhancement additives to the TBQ enhancement agent, in varying amounts, substantially improves chemiluminescent signal and S/N.

Further improvements in chemiluminescent signal and S/N can be obtained by combining two or more additives.

As reflected in the foregoing results (Table 4) , although polyox yields some nominal improvement in an enhanced signal, as an enhancement additive in connection with TBQ, improved results are obtained. It should be noted that these results were obtained according to the protocol set forth above with regard to the assessment of TBQ, using AMPPD as a dioxetane.

Improvements in enhancement are obtained by the addition of TBQ, alone, or in particular, together with an enhancement additive, independent of the dioxetane identity, provided the dioxetane is a chemiluminescent one of the type suitable for use as described above. Thus, TBQ is used efficaciously in connection with both CSPD and AMPPD. Further, as noted, enhancement additives can be used in connection with TBQ to further improve both signal strength, and S/N values. One particular enhancement additive giving excellent results in connection with TBQ and CSPD is available from DuPont corporation, under the trademark "Zelec", sold as an anti-static agent. The composition of Zelec is believed to be maintained as a trade secret, that includes turpentine, N-octylalcohol, water, isopropyl alcohol, sodium acetate, sodium oleyl

sulfate, and TS compound or compounds identified as NJ Trade Secret Registry Number 00850201001-5400P. The Material Safety Data Sheet for the Zelec anti-stat is identified by No. 5909PP. As set forth herein below, combinations of Zelec and TBQ, or TBQ copolymers, give substantial improvements and results. AMPPD and CSPD alone, as well as together with BDMQ as an enhancement agent, or in the alternative, Zelec without an enhancement agent, are included for purposes of comparison.

Comparison of Signal to Noise Levels

Time, minutes

CSPD BACKGROUND KINETICS IN ZELEC/TBQ MIXTURES

Tlme, min

R U, 5 sec

□ r 3- • 3— c c _r a u o o c

S3

6fSZ0/f6SO/13d

CSPD S/N IN ZELEC/TBQ MIXTURES

1ΔBLE

Buffer: 0.1 M dielhanolamlne. 1 mM MgCl _2, pH 9.5

Dioxetane concentration: 0.4 mM

Alkaline phosphatase concentration: 9.35 X 10 ^H M

Luminometer: Berthold 952T

Data: 5 second RLU

GQmι-iτι>ι4-ion3 of Enhancement Acrent ana Enhancement Additive

As noted previously, TBQ, alone, gives impressive improvements in observed chemiluminescence. TBQ in conjunction with an enhancement additive which improves the ability of TBQ to form hydrophobic regions in which chemiluminescent dioxetane species are sequestered further improves both chemiluminescent signal enhancement, and S/N ratio. It would of course be less desirable to improve the signal strength, if noise increased as well. The foregoing data clearly demonstrates that noise is maintained at a low level, when using commercially available dioxetanes, available from Tropix, Bedford, Mass. The improvements are far beyond those that could be expected by reason of the addition of TBQ alone, or the addition of the enhancement additive, alone, the additive occasionally having some, but generally modest, enhancement effects in the absence of the polymeric quaternary onium salt enhancement agent of the claimed invention.

To further demonstrate the improvements obtainable by using the polymeric quaternary onium salt enhancement agents of the claimed invention in combination with enhancement additives which can improve the ability of the enhancement agent to form hydrophobic regions in which chemiluminescent species are sequestered, a wide variety of combinations of enhancement agent, and one or more enhancement additives were prepared and evaluated for chemiluminescence. These enhancement agents are drawn from a wide variety of compound classes, including surfactants,

conventional solvents, and water-soluble polymers. The results are reflected on the following pages.

The following method was used to evaluate various enhancer systems. Samples (0.5 ml) of each enhancer formulations were prepared from concentrated stock solutions. The buffer used was 0.1 M diethanolamine, 1 mM MgCl ₂ , pH 9.5. After the enhancer formulation was prepared, CSPD was added (final dioxetane concentration 0.04 mM) . The background was then measured at room temperature in a Berthold 952T luminometer at approximately 10 minutes after substrate addition and at 1 hour. Next, alkaline phosphatase was added (final concentration, 9.35 X 10 ^*u M) and the chemiluminescent signal (5 second RLU) was measured at 10, 30 and 60 (sometimes) minutes.

Abbreviations used in tables:

AMPA-8 aminomethylatθd polyacrylamide, 8 % solids

AMPA-5 aminomθthylatθd polyacrylamide, 5 % solids

AMPA-3 aminomethylated polyacrylamide, 3 % solids

BDMCAC benzyldimethylcetyl ammonium chloride

BDMDAB bβnzyldimelhyldodecy! ammonium bromide

BDMTDAC benzyldimethyltetradecyt ammonium chloride

BDMQ poly(vinylbenzyldimelhylbenzyl ammonium chloride)

BDMQ TBQ BDMQ copolymerized with TBQ

Benz-PEI benzylated polyethylenimine (MW 70.000)

BOP benzotriazol- 1 -yloxytris-(dimemylamino)phosphonium hexafluorophospt ιate

CHAPS (3 l(3-cholamidopropyl)-dimelhylammonio|- 1 propanesulfonale)

CHAPSO (3-((3-cholamidopropyl)-dimethylammonio|-2 hydroxy- 1 -propanesullonate)

CTAB cetyl trimethyl ammonium bromide

DMQ-TEQ poly(vinylbenzyldimethyldodecyl ammonium chloride) copolymerized with poly(vinylbenzyltriethylammonium chloride)

HDTBPB hθxadecyltributyl phosphonium bromide

OcGluc octyl |l-glucoside

P-DMDMPC poly( 1 , 1 -dimethyl-3,5-dimethylene pipe idmium chloride)

PEI polyethylenimine

PEO polyethylene oxide) (MW 7.000.000)

PO23LE polyoxyelhylene-23-lauryl ether

PolyOx poly(2-elhyl-2-oxazoline)

PPG polypropylene glycol

PTHF poly(tetrahydroluran) (MW 250)

PVA polyvinyl alcohol

PVEE poly(vinyl ethyl ether)

PVME polyjviπyl melhyl elher)

SBS sodium benzyl εullate

SDS sodium dodecyl sullate

SDBS sodium dodecylbenzenesulfonate

TBQ polyvinylbenzyllributyl ammonium chloride

TBQ/THQ TBQ copolymerized wil poly(vinylbenzyllιιl.eχyl ammonium chloiide)

TPP/TBQ poly(vinylbenzyllriphenylphosphonum. chloiide) copolymerized with T BQ

TPP/BDMQ poly(vinylbenzyltriphenylphosphonium chloride) copolymerized wilh BDMQ

TABLE 6

CSPD

Signal _SZH

Additive Concentration Noise 10 min 30 pιin 60 min 10 min 30 min 60 min

1. none 282 17417 17705

2. BDMQ 1 mg/ml 536 369711 419173

3. TBQ 1 mg/ml 662 873852 1116942

4. Zelec DP 2.5 μl/ml 508 1563198 3836680 TBQ 1 mg/ml

5. polyvinyl alcohol 0.1 mg/ml 539 1004417 1281062

TBQ 1 mg/ml

6. isopropanol 1 mg/ml 574 962628 1271409 TBQ 1 mg/ml

7. 2-octanσl 10 mg/ml 568 1036203 1362463 TBQ 1 mg/ml

8. Zelec DP 2.5 μl/ml 927 837481 2586843 SDS 1 mg/ml TBQ 1 mg/ml

9. Zelec DP 2.5 μl/ml 726 735959 21 18539 SDS 1 mg/ml BDMQ 1 mg/ml

10. Zelec DP 2 5 μl/ml 853 594827 1586672 Tween-20 5 mg/ml TBQ 1 mg/ml

1 1 . Zelec DP 2.5 μl/ml 1 129 545747 1 289720 483 1 142 Tweβn-20 0 01 mg/ml BDMQ 1 mg/ml

TABLE 6 CONTINUED

12. Zelec DP 2.5 μl/ml 1112 545295 1222221 490 1099 Tween-20 0.05 mg/ml BDMQ 1 mg/ml

13. Zelec DP 2.5 μl/ml 1338 957409 2375419 716 1775 Tween-20 0.01 mg/ml

TBQ 1 mg/ml

14. Zelec DP 2.5 μl/ml 1097 969399 2345374 884 2138 Tween-20 0.05 mg/ml

TBQ 1 mg/ml

15. TBQ/THQ 1 mg/ml 9997 5429086 8543357 543 855 (35 mole% THQ)

CTAB 0.05 mg/ml

16. isopropanol 10 mg/ml 340 435740 533074 1282 1568 BDMQ 1 mg/ml

17.2-octanol 10mg/ml 365 405753 510671 1112 1399 BDMQ 1 mg/ml

18. ZelecDP 3 μl/ml 10851 5605794 9637819 517 888 TBQ/THQ 1 mg/ml (35 mole% THQ)

19. AMPA-8 0.5μl/ml 385 297926 400787 774 1041 BDMQ 1 mg/ml

20. AMPA-8 05 μl/ml 677 790317 1109404 1167 1639

TBQ 1 mg/ml

21. AMPA-5 05μl/ml 398 530827 624527 1336 1569 BDMQ 1 mg/ml

22. AMPA-5 05μl/ml 859 1006579 1289099 1172 1501 TBQ 1 mg/ml

TABLE6 CONTINUED

23. AMPA-3 0.5 μl/ml 564 194022 251987 BDMQ 1 mg/ml

24. AMPA-3 0.5 μl/ml 796 757153 1021267 TBQ 1 mg/ml

25. TPP TBQ 2.5 mg/ml 407 9440 183054

26. TPP/BMQ 2.5 mg/ml 361 164641 201410

27. TBQ/THQ 1 mg/ml 4436 1501695 3853047 Tween-20 5 mg/ml

28. Zelec DP 2.5 μl/ml 8332 669331110215623 803 1226 TBQ/THQ 1 mg/ml OJ

(35 mole% THQ) Tween-20 1 mg/ml Oi

29. Zelec DP 2.5 μl/ml 729 138425 332143 190 456 Tween-20 2.5 mg/ml TBQ/THQ 1 mg/ml (35 mole% THQ) CTAB 0.25 mg/ml

30. Zelec DP 2.5 μl/ml 402 633521 1675565 1576 4168 TritonXIOO 0.625 mg/ml

TBQ 1 mg/ml

TABLE6 CONTINUED

34. TBQ/THQ 0.5 mg/ml 1574 5084709 6077275 (21 mole% THQ)

35. Benz-PEI 0.5 mg/ml 504 667698 151051 BDMQ 1 mg/ml

36. Lanoquat 0.5 mg/ml 401 222433 359929 BDMQ 1 mg/ml

37. Benz-PEI 1 mg/ml 858 745446 1774863 TBQ 1 mg/ml

38. Lanoquat 0.5 mg/ml 570 600923 878859 TBQ 1 mg/ml

39. Hipotix DDD 1 mg/ml 418 346956 402685 BDMQ 1 mg/ml

40. Hipotix DDD 2.5 mg/ml 1040 760173 859718 TBQ 1 mg/ml

41. Hipolix DD-NF0.5 mg/ml 343 345329 390776 BDMQ 1 mg/ml

42. Hipolix DD-NF1 mg/ml 493 713154 854962 1447 1734 BDMQ 1 mg/ml

43.

44. Hipolix 491 0.5 mg/ml 684 291815 366201 TBQ 1 mg/ml

45. Aerotex M-3 2.5 mg/ml 449 197957 233053 BDMQ 1 mg/ml

46. Aerotex M-3 5 mg/ml 791 492405 527229 623 723 TBQ 1 mg/ml

TABLE 6

CONTINUED

47. PVEE (lo mw) 1 mg/ml 568 2372633 378368 BDMQ 1 mg/ml

48. PVEE (lo mw) 1 mg/ml 655 730024 1 151041 TBQ 1 mg/ml

49. Polyox (m 50K)1 mg/ml 574 342402 422461 BDMQ 1 mg/ml

50. Polyox (mw 50K)1 mg/ml 699 1284452 1587003 TBQ 1 mg/ml

53. PTHF (mw 250)2.5 mg/ml 700 265174 352224 379 503 BDMQ 1 mg/ml

54. PTHF (mw 250)0.5 mg/ml 758 634639 851396 837 1 123 TBQ 1 mg/ml

55. Polyox(mw 500K) 0.5 mg/ml 699 316630 385553 453 552 BDMQ 1 mg/ml

56. Polyox(mw 500K) 5 mg/ml 889 628735 822133 707 925

TBQ 1 mg/ml

329 384

698 897

TABLE 6

CONTINUED

59. CelqualH 100 0.5 mg/ml 406 284794 353576 BDMQ 1 mg/ml

60. CelqualH 100 0.5 mg/ml 719 734280 970599 TBQ 1 mg/ml

61. Celquat L200 0.5 mg/ml 499 268280 326627 BDMQ 1 mg/ml

62. Cθlquat L200 0.5 mg/ml 969 722551 928806 TBQ 1 mg/ml

63. CθlquatSC240 0.5 mg/ml 530 31 7199 382101 BDMQ 1 mg/ml

64. CθlqualSC240 1 mg/ml 804 81 1714 1030836 TBQ 1 mg/ml

65. Plurσnic122 0.5 mg/ml 608 47854 1 17319 BDMQ 1 mg/ml

66. Pluronic122 1 mg/ml 746 66584 181683 TBQ 1 mg/ml

67. Pluronic123 0.5 mg/ml 648 69686 150161 108 232 BDMQ 1 mg/ml

68. Pluronic 123 0.5 mg/ml 1453 178632 386930 123 266

TBQ 1 mg/ml

69. Pluronic150R1 0.5 mg/ml 896 156254 173414 174 194

BDMQ 1 mg/ml

70. 1996 285940 324257 143 162

71 . CHAPS 0.5 mg/ml 210 14326 42519 68 202

TABLE6 CONTINUED

72. CHAPSO 0.5 mg/ml 218 15119 42489 69 195

73. Zelec DP 2.25 μl/ml 638 741248 CHAPS 1616666 1 mg/ml 1162 2534 BDMQ 1 mg/ml

74. Zelec DP 2.25 μl/ml 858 1146672 2754178 CHAPS 1 mg/ml 1336 3210 TBQ 1 mg/ml

75. Zelec DP 2.25 μl/ml 555 718553 1530910 CHAPSO 1 mg/ml 1295 2758 BDMQ 1 mg/ml

76. Zelec DP 2.25 μl/ml 900 1090555 2453191 CHAPSO 1 mg/ml TBQ 1 mg/ml

77. Polybrene 5 mg/ml 413 19740 CHAPS 24261 5 mg/ml

78. Polybrene 5 mg/ml 2853 486059 CHAPS 705032 5 mg/ml BDMQ 1 mg/ml

79.

80. Polybrene 5 mg/ml 6348 832574 1239584 CHAPSO 5 mg/ml 131 195 BDMQ 1

81. Polybrene 0 5 mg/ml 6441 736510 897356 CHAPSO 0.5 mg/ml 114 139 BDMQ 1 mg/ml

82. BenzethoniumCI 0.

TABLE 6 CONTINUED

83. BDMQ/TBQ 1 mg/ml 568 742480 859880 1307 1514

(20% BDMQ)

84. BDMQ/TBQ 1 mg/ml 740 927108 1 1 13083 1253 1 504 (10% BDMQ)

85. Zelec DP 2.5 μl/ml 1910 899187 176851 1 471 926 BDMQ/TBQ 1 mg/ml

(20% BDMQ)

86. Zelec DP 2.5 μl/ml 8392 1057422 2177684 126 259 BDMQ/TBQ 1 mg/ml

(10% BDMQ)

87. Avitθx ML 0.5 mg/ml 1051 142640 298297 BDMQ 1 mg/ml

88. Avitθx ML 0.5 mg/ml 1318 466655 866897 TBQ 1 mg/ml

89. Polyox(500K) 0.5 mg/ml 1730 862023 1239458 CTAB 0.01 mg/ml

TBQ 1 mg/ml

90. Polyox(50K) 0.5 mg/ml 952 872307 1 183521 916 1 243 CTAB 0.01 mg/ml

TBQ 1 mg/ml

91. Agelloc B50 0.5 mg/ml 952 872307 1 183521 1961 2262 TBQ 1 mg/ml

92. Agelloc B50 0.5 mg/ml 574 308582 347312 538 605 BDMQ 1 mg/ml

93. Agelloc A50 1 mg/ml 689 1015523 1225260 1474 1 778 TBQ 1 mg/ml

TABLE 6

CONTINUED

94. Agelloc A50 0.5 mg/ml 693 309616 355165 447 513 TBQ 1 mg/ml

95. Agelloc A50HV 0.5 mg/ml 1265 1 190551 1343823 941 1062 TBQ 1 mg/ml

96. Agelloc A50HV 0.5 mg/ml 769 284170 325540 370 423 TBQ 1 mg/ml

97. SDBS 0.5 mg/ml 725 758078 2071 149 1046 2857 Tweβn-20 20 mg/ml

BDMQ 1 mg/ml

98. SDBS 0.5 mg/ml 668 638270 1763156 955 2639

Triton X-100 1 mg/ml

BDMQ 1 mg/ml

99. SDBS 0.5 mg/ml 1337 1021526 2582544 764 1932

CHAPS 1 mg/ml

BDMQ 1 mg/ml

100. SDBS 0.5 mg/ml 1476 940142 2282165 637 1546 CHAPSO 1 mg/ml

BDMQ 1 mg/ml

101. SDBS 0.5 mg/ml 1329 883203 2195667 665 1652

CTAB 0.05 mg/ml

BDMQ 1 mg/ml

102. DAXAD 1 mg/ml 866 1059832 1225160 1224 1415

TBQ 1 mg/ml

103. DAXAD 0.5 mg/ml 802 771273 833134 962 1039 BDMQ/TBQ 1 mg/ml (20% BDMQ)

104. DAXAD 0.5 mg/ml 510 301223 351656 591 690

TABLE6 CONTINUED

105. DextranSul 0 5 mg/ml 306 37132 39345 121 129 BDMQ 0.1 mg/ml

106. SDS 0.6 mg/ml 961 772634 2258788 804 2350 TWΘΘΠ-20 1 mg/ml TBQ 1 mg/ml

107. SDS 0.6 mg/ml 1100 867025 2581024 788 2346 Triton X-100 1 mg/ml TBQ 1 mg/ml

108. SDS 0.6 mg/ml 721 592457 1575837 822 2186 Tween-20 1 mg/ml BDMQ 1 mg/ml

109. SDS 0.6 mg/ml 697 412432 1143747 592 1641 Triton X-100 1 mg/ml BDMQ 1 mg/ml

1 10. SDBS 0.1 mg/ml 2097 957195 2016212 456 962 Tween-20 1 mg/ml octyl glucoside 1 mg/ml TBQ 1 mg/ml

1 1 1.

288 22720 56363 79 196

TABLE6 CONTINUED

1 13. SDBS 0.5 mg/ml 291 17291 43494 59 149 Triton-X-100 1 mg/ml CTAB 1 mg/ml TBQ 1 mg/ml

1 14. SDBS 0.5 mg/ml 231 34158 101381 148 439 Tween-20 1 mg/ml BDMCAC 1 mg/ml TBQ 1 mg/ml

1 15. SDBS 0.5 mg/ml 233 31723 90256 136 387 Trilon X-100 1 mg/ml BDMCAC 1 mg/ml TBQ 1 mg/ml

1 16. SDBS 0.1 mg/ml 878 1052025 2086077 1198 2376 TWΘΘΠ-20 1 mg/ml CTAB 0.1 mg/ml TBQ 1 mg/ml

1 17. SDBS 0.1 mg/ml 1009 1358360 2551569 1346 2529 TWΘΘΠ-20 1 mg/ml CTAB 0.01 mg/ml TBQ 1 mg/ml

1 18. CTAB 0.1 mg/ml 412 395868 714210 961 1734 Triton X-100 1 mg/ml TBQ 1 mg/ml

1 19.

120. octyl glucoside 1 mg/ml 586 726013 836153 1239 1427 1 BQ 1 mg/ml

121. Tween-20 1 mg/ml 550 371246 620097 675 1128 TBQ 1 mg/ml

519 897

67 1 1 7

124. Polyox(50K) 20 mg/ml 154 26179 47219 170 307 CTAB 0.25 mg/ml

125. Polyox(50K) 20 mg/ml 243 4951 12 723δ80 2037 2978 CTAB 0.25 mg/ml TBQ 1 mg/ml

126. Polyox(50K) 20 mg/ml 607 768812 930826 1267 1533 CTAB 0.025 mg/ml TBQ 1 mg/ml

127. Polyox(50K) 20 mg/ml 402 832297 962123 2070 2393 CTAB 0.0025 mg/ml TBQ 1 mg/ml

128. Polyox(50K) 0 5 mg/ml 391 970406 1 102900 TBQ 1 mg/ml

129. Polyox(500K) 20 mg/ml 283 42628 77784 CTAB 0.25 mg/ml

130. Polyox(500K) 20 mg/ml 275 36981 44021 CTAB 0.025 mg/ml

131. Polyox(500K) 20 mg/ml 250 40563 45500 CTAB 0.0025 mg/ml

132. Polyox(500K) 1 mg/ml 397 1527695 781 760 CTAB 0 05 mg/ml TBQ 1 mg/ml

TABLE 6 CONTINUED

133. Polyox(500K) 1 mg/ml 455 1629263 1834806 3581 4033 CTAB 0.005 mg/ml TBQ 1 mg/ml

134. Polyox(500K) 1 mg/ml 514 1635987 1823δ78 3183 3548 CTAB 0.0005 mg/ml TBQ 1 mg/ml

135. Polyox(δOOK) 20 mg/ml 623 2505788 3072392 4022 4931 TBQ 1 mg/ml

136. Polyox(500K) 20 mg/ml 432 528833 653731 669867 1 224 151 3 1551 BDMQ 1 mg/ml

137. Polyox(500K) 20 mg/ml 1 7614 2047964 4050329 1 16 230 Zelec DP 2.5 μl/ml TWΘΘΠ-20 0.625 mg/ml TBQ 1 mg/ml

138. Polyox(500K) 20 mg/ml 562 76352 229205 136 408 SDBS 0.5mg/ml

CTAB 0.75 mg/ml

Tween-20 1 mg/ml TBQ 1 mg/ml

139. Polyox(500K) 20 mg/ml 2689 474537 1267603 176 471 SDBS 0 5 mg/ml C TAB 0.5 mg/ml Tween-20 1 mg/ml 1BQ 1 mg/ml

140. Polyox(500K) 20 mg/ml 5476 675082 1535300 123 280 SDBS 0.25 mg/ml CTAB 0.3 mg/ml lween-20 1 mg/ml T BQ 1 mg/ml

TABLE6 CONTINUED

141. Polyox(δOOK) 20 mg/ml 729 393791 507399 540 696 IM NaCl in DEA

TBQ 1 mg/ml

142. Polyox(500K) 20 mg/ml 714 444112 545624 622 764 0.5M NaCI in DEA TBQ 1 mg/ml

143. Polyox(δOOK) 20 mg/ml 803 271814 311794 338 388 1M NaCI (no DEA) TBQ 1 mg/ml

144. Polyox(δOOK) 20 mg/ml 723 316032 342073 437 473 O.δM NaCI (noDEA)

TBQ 1 mg/ml

14δ. Polyox(δOOK) 20 mg/ml 810 1255809 2567260 1550 2388 SDBS 0.1 mg/ml

Chaps 1 mg/ml

Tweβn-20 1 mg/ml TBQ 1 mg/ml

146.

147.

TABLE6 CONTINUED

148. Polyox(δOOK) 20 mg/ml 2593 1240847 2249328 479 591 SDBS 0.06 mg/ml Chapso O.δ mg/ml Tweβn-20 1 mg/ml

TBQ 1 mg/ml

149. Polybrene O.δ mg/ml 42δ 979δδ0 1210691 2305 2848 TBQ 1 mg/ml

1δ0. Polyox(δOOK) 20 mg/ml 606 1106615 1378277 2187 2724 Polybrene 0.5 mg/ml TBQ 1 mg/ml

151. Polyox(δOOK) 20 mg/ml 765 995361 1202025 1301 1571 CHAPS 0.5 mg/ml Polybrene 0.5 mg/ml TBQ 1 mg/ml

152. Polyox(500K) 20 mg/ml 2024 1149546 1385744 568 685 CHAPSO 0 5 mg/ml Polybrene 0.5 mg/ml TBQ 1 mg/ml

153. Polybrene 0.5 mg/ml 567 1144898 1337005 2019 2358 CHAPS 0.5 mg/ml TBQ 1 mg/ml

154. Polybrene 0.5 mg/ml 665 1240889 1442628 1866 2169 CHAPSO 0.5 mg/ml TBQ 1 mg/ml

155. Polyox(500K) 20 mg/ml 573 766172 942897 1337 1646 DEA/Caib buller 9 5 TBQ 1 mg/ml

156. TBQ 1 mg/ml 466 320952 371408 689 797 DEA/Carb buller 9 5

TABLE6 CONTINUED

157. Polyox(500K) 20 mg/ml 531 1996396 2509803 3760 4727 TBQ 0.5 mg/ml

158. Polyox(500K) 20 mg/ml 482 1649504 2021238 3422 4193 octyl-glucoside 1 mg/ml TBQ 1 mg/ml

169. Polyox(δOOK) 20 mg/ml 262 35061 36818 134 141 octyl-glucoside 5 mg/ml

160. Polyox(500K) 20 mg/ml 604 1729994 2149082 2864 3558 phenyl-glucoside 5 mg/ml TBQ 1 mg/ml

161. Polyox(500K) 20 mg/ml 268 30705 33366 150 124 phenyl-glucoside 5 mg/ml

162. Polyox(δOOK) 20 mg/ml 562 1372766 2935468 2443 5223 octyl glucoside 2.5 mg/ml SDBS 0.25 mg/ml TWΘΘΠ-20 1 mg/ml TBQ 1 mg/ml

163. Polyox(500K) 20 mg/ml 632 1197951 2459440 1895 3892 phenyl-glucoside 2.5 mg/ml SDBS 0.25 mg/ml TWΘΘΠ-20 1 mg/ml TBQ 1 mg/ml

164. SDBS 0.1 mg/ml 413 1457360 2768643 3541193 3529 6704 8574 Tween-20 1 mg/ml TBQ 1 mg/ml

165. SDBS 0.1 mg/ml 414 1353062 2649803 3601796 3268 6400 8700 TWΘΘΠ-20 1 mg/ml TBQ 0.5 mg/ml

END OF TABLE 6

166. SBS 1 mg/ml 318 406862 478723 473862 1276 1506 1490 BDMQ 1 mg/ml

167. SBS 0.5mg/ml 341 950755 1115291 1107561 2788 3271 3248 TBQ 1 mg/ml

* -

TABLE 7

AMPPD

Signal SOL

Additive Concentration Noise 10 min 30 min 60 min IQ mi 30 in 6Q rpiη

601 15434 22361

1021 239467 392753

1940 529838 953548

101 12 3909328 7854723

539 19978 59936

61 1 38535 92199 1019 232240 71391 7

8. Andogen464 5 mg/ml 447 19223 63303 Triton X-100 10 mg/ml

9. Andogβn464 5 mg/ml 784 49888 167538 64 214 Triton X-100 1 mg/ml

10. Andogβn464 5 mg/ml 493 32050 107977 65 219 T ΘΘΠ-20 10 mg/ml

1 1. Andogen464 1 mg/ml 718 39265 131675 55 183

TWΘΘΠ-20 1 mg/ml

12. BDMTDAC 0.1 mg/ml 1213 94008 210047 78 173 BDMQ 1 mg/ml

13. BDMDAB 0.1 mg/ml 1366 156795 273187 1 15 200 BDMQ 1 mg/ml

TABLE 7

CONTINUED

14. Zonyl Sud. 0.1 mg/ml 1597 170683 283296 107 1 77 BDMQ 1 mg/ml

15. BDMTDAC 0.1 mg/ml 752 186443 432829 TBQ 1 mg/ml

16. BDMDAB 0.1 mg/ml 1000 379583 717087 TBQ 1 mg/ml

17. Zonyl Sud. 0.1 mg/ml 1 108 393546 734506 TBQ 1 mg/ml

18. octyl-glucosideθ.1 mg/ml 1474 167319 316876 BDMQ 1 mg/ml

19. phenyl-glucosidθθ.1 mg/ml 1663 168296 31431 101 189 BDMQ 1 mg/ml

20. octyl-glucoside 1 mg/ml 359 16800 41762 47 1 16 BDMDAB 1 mg/ml BDMQ 1 mg/ml

21. oclyl-glucosideO.δ mg/ml 1000 339007 648072 TBQ 1 mg/ml

22. phθnyl-glucosidθO.δ mg/ml 1221 269016 545848 TBQ 1 mg/ml

23. HDTBPB 0.5 mg/rnl 1412 36919 1021 72

24. Andogen464 1 mg/ml 3919 223888 68281 7 phenyl-glucoside 1 mg/ml

25. HDTBPB 0.5 mg/ml 640 40379 124470 63 195 phenyl-glucoside 1 mg/ml

26. Nonidθt P-400.1 mg/ml 1 143 1 12668 252316 99 221 BDMQ 1 mg/ml

TABLE7 CONTINUED

27. Tween-80 0.5 mg/ml 844 87136 165286 BDMQ 1 mg/ml

28. PO23LE 0.1 mg/ml 1980 130369 261388 BDMQ 1 mg/ml

822 116303 231011

1167 35861 90738

451 46605 101249

420 66398 133292 158 317

3327 464178 1059036 140 318

803 25376 411277

771 266606 446988

36. P-DMDMPC 0.5 mg/ml 1031 286017 489468 BDMQ 1 mg/ml

37. Avilex DN 5 mg/ml 372 33046 67404

38. Avitex DN 0.1 mg/ml 687 251317 418395 BDMQ 1 mg/ml

39. Avitex E 0.5 mg/ml 674 273361 442352 406 656 BDMQ 1 mg/ml

END OFTABLE7

40. DMQ-TEQ 6 mg/ml 826 198197 445467

41. TBQ/THQ 1 mg/ml 20254 2910352 6446798 (36 mole% THQ) BDMQ 1 mg/ml

42. DMQ-TEQ 5 mq/ml 706 226048 502481 BDMQ 1 mg/ml

43. TBQ THQ 1 mg/ml 20339 2415719 5398310 (36 molθ% THQ) TBQ 1 mg/ml

44. DMQ-TEQ 1 mg/ml 893 370453 735995 TBQ 1 mg/ml

45. Zelec DP 2.5 μl/ml 1916 340564 8009917 phenyl-glucoside 1 mg/ml BDMQ 1 mg/ml

46. Zelec DP 2.6 μl/ml 2244 386368 1068947 172 476 phenyl-glucoside 1 mg/ml TBQ 1 mg/ml

47.

Applicants have endeavored to illustrate their invention by extensive embodiment of possible combinations. Nonetheless, it is recognized that the possible combinations are endless, and cannot be exhaustively embodied. Given the above teaching, those of ordinary skill in the art will arrive at enhancement agents and additives not specifically exemplified in the foregoing application. The examples are not intended to be limiting, and the identification of other combinations, given the foregoing disclosure, is well within the skill of those practicing this technology without undue experimentation. Such combinations are intended to be within the scope of the invention, save as expressly limited or excluded by the claims set forth below.