FIELD OF THE INVENTION

[001] The present invention relates generally to a test device. The invention provides a test device and methods of use for determining the presence, potency, integrity, or quantity of a biologic.

BACKGROUND OF THE DISCLOSURE

[002] Onabotulinumtoxin A (BOTOX) and other botulinum toxins are used to treat several neuromuscular conditions including strabismus, blepharospasm, and hemifacial spasm and have also seen widespread use for off label and cosmetic treatments. General detection of botulinum toxins is also relevant for biological warfare and food safety. Potency testing is essential during manufacture as well as for authentication in both forensic and point of care situations.

SUMMARY OF THE INVENTION

[003] Disclosed herein is a test device for determining the quantity of a biologic in a sample comprising (a) a sample pad for receiving the biologic, (b) a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to a conjugate reagent, and (c) a test membrane comprising at least one test line, the test line comprising an immobilized mimetope. Also disclosed herein is a method of determining the presence, quantity or potency of a biologic, the method comprising: (a) contacting the biologic with a test device, wherein the test device comprises: a sample pad for receiving the biologic, a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to a conjugate reagent, and a test membrane comprising at least one test line, the test line comprising an immobilized mimetope, (b) determining whether at least one test line undergoes a color change, (c) determining, based on the color change, the presence, quantity or potency of the biologic. Further disclosed herein is a kit comprising: (i) a test device for determining the quantity of a biologic in a sample comprising a sample pad for receiving the biologic, a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to a conjugate reagent, and a test membrane comprising at least one test line, the test line comprising an immobilized mimetope, and (ii) instructions for use thereof.

[004] Further disclosed herein is a test device for determining the quantity of a biologic in a sample comprising a conjugate reagent conjugated to the mimetope, a sample pad for receiving a mixture of the biologic and mimetope conjugated the conjugate reagent, a conjugate pad comprising one or more peptide dimers, and a test membrane comprising at least one test line, the test line comprising an immobilized antibody. Also disclosed herein is a method of determining the presence, quantity or potency of a biologic, the method comprising: (a) contacting the biologic with a test device, wherein the test device comprises: a conjugate reagent conjugated to the mimetope, a sample pad for receiving a mixture of the biologic and mimetope conjugated the conjugate reagent, a conjugate pad comprising one or more peptide dimers, and a test membrane comprising at least one test line, the test line comprising an immobilized antibody, (b) determining whether at least one test line undergoes a color change, and (c) determining, based on the color change, the presence, quantity or potency of the biologic.

BRIEF DESCRIPTION OF THE DRAWINGS

[005] FIG. 1 is an overview of one embodiment of the cleavage coupled competitive lateral flow assay (C ³ -LFA). Sample is applied to the sample pad and wicked through a conjugate pad that contains the competitive peptide dimer and colloidal gold conjugated to the antibody. The peptide mimetope dimer contains an internal recognition site for BOTOX and is rapidly cleaved if the active enzyme is present. Cleavage of the dimer produces monomer peptides with weak affinity for the antibody, allowing the antibody to bind to the mimetope coated test line. Multiple test lines allow semi -quantitative readout for the various concentrations of BOTOX that are commonly prepared from a stock lyophilized vial.

[006] FIG. 2A-B is an illustration of the various components of one embodiment of the C ³ -LFA as well as the assembly algorithm. A. shows an embodiment of the C -LFA approach is the use of dimer mimetope peptides that can be cleaved to monomers by BOTOX endopeptidase. The dimers have strong affinity for their cognate antibody but the monovalent intrinsic affinity is relatively weak. Thus when BOTOX is present and active the antibody conjugated colloidal gold is unmasked through the cleavage of the otherwise blocking dimer mimetopes. B. is a schematic of an assembled device. The backing card and cassette are not shown.

[007] FIG. 3 is an overview of one embodiment of the cleavage coupled competitive lateral flow assay (C ³ -LFA). Sample is applied to the sample pad and wicked through a conjugate pad that contains the competitive peptide dimer, rFC, and colloidal gold conjugated to the antibody. The peptide mimetope dimer is cleaved if LPS is present in the sample. Cleavage produces monomer peptides with weak affinity for the antibody, allowing antibody binding to the mimetope coated test line. Multiple test lines enable semi-quantitative readout.

[008] FIG. 4A-B is an illustration of the various components of one embodiment of the C ³ -LFA as well as the assembly algorithm. A. shows that the core of the C ³ -LFA approach is the use of dimer mimetope peptides that can be cleaved to monomers by LPS-activated rFC. The dimers have strong affinity for their cognate antibody but the monovalent intrinsic affinity is relatively weak. Thus when LPS is present and rFC is activated, the antibody conjugated colloidal gold is unmasked through the cleavage of the otherwise blocking dimer mimetopes. B. is a schematic of an assembled device. The backing card and cassette are not shown. [009] FIG. 5 is an illustration of another aspect of the C3-LFA assay. In this aspect of the assay, the antibody is coated on the test lines. The colloidal gold bound mimetope is on an albumin carrier. Anti-BSA protein is used for the control line.

[0010] FIG. 6 is an illustration of another aspect of the C3-LFA assay. In this aspect of the assay, the antibody is coated on the test lines. The colloidal gold bound mimetope is on a protein carrier. Anti-carrier protein is used for the control line.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] The gold standard for determining potency is the LD ₅₀ in mice. Moreover, Allergan (Dublin, Ireland) has developed a cell based potency assay using human neuroblastoma lines as explained in Fernandez-Salas, E., et ah, Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay. Plos One, 2012. 7(11). Similarly the EZYBOT® field test was developed by Pharmaleads (Paris, France) to detect the botulinum toxin. However, this assay is not suitable for field or point of care use.

[0012] Several limitations exist with the existing tests. The current methods for evaluating botulinum toxins require cumbersome animal or cell based assays, specialized laboratory equipment, or trained personnel to perform the tests, limiting their use outside of research laboratories. Therefore there exists a need for a simple, inexpensive, and rapid assay for point of care or forensic testing of the quality, potency and counterfeit detection of botulin toxins.

[0013] Similarly, bacterial endotoxins, namely lipopolysaccharides (LPS), are common contaminants of many pharmaceutical, medical device, and food products that can elicit strong immune responses in people and animals, sometimes resulting in septic shock. As such, monitoring for endotoxins in medical and food products is essential. Current methods for detecting endotoxins are complex, expensive, and difficult to standardize. The standard method, the Limulus Amebocyte Lysate (LAL) assay, uses the endotoxin-induced clotting of an extract from horseshoe crab blood cells as a highly sensitive detector of endotoxin. However, the assay is cumbersome and difficult to standardize. Moreover there is growing pressure on horseshoe crab populations as the worldwide demand for the assay has surged. Another assay for detecting LPS is the PYROGENE™ assay developed by Lonza (Basel, Switzerland). That assay uses a recombinant form of the first enzyme in the limulus clotting cascade, Factor C (rFC), and a substrate that, when cleaved by active rFC, produces a fluorescent readout. The ENDOLISA® assay developed by Hyglos GmbH (Bernried am Starnberger See, Germany) utilizes a solid phase ELISA method in which an LPS-specific phage protein is pre-coated on the wells of a microplate and LPS in test samples is detected by addition of rFC and a fluorogenic substrate. Similarly, InvivoGen (San Diego, CA) has introduced a distinct cell-based colorimetric approach for detection and quantitation of endotoxin in biological samples that uses HEK cells engineered to express human TLR4 and an NF-KB-inducible secreted alkaline phosphatase reporter gene.

[0014] Despite these improvements, these assays still require a wet lab, specialized laboratory equipment, complex set-up and analysis, and trained personnel. Therefore, there remains a need to develop a simple, inexpensive, and rapid assay that verifies the presence, potency, and integrity of a biologic such as BOTOX or LPS in the sample and that is amenable for use at any point in the supply chain, including bedside.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise stated, the following terms used in this application, including the specification and claims, have definitions given below.

[0016] The term "biologic" as used herein refers to any product or preparation obtained from living organisms, or a bioengineered agent produced in a living organism. In some embodiments, the biologic is capable of participating in a chemical or enzymatic process wherein the biologic cleaves a bond or dissociates an otherwise stable interaction. In some embodiments, the biologic comprises one or more of the following: proteases, endonucleases, cellulolytic enzymes, glycosidases, and toxins. In some embodiments, the toxin is a neurotoxin. Examples of neurotoxins include, but are not limited to, lead, ethanol (drinking alcohol), glutamate, nitric oxide (NO), botulinum toxin (e.g. Botox), tetanus toxin, and tetrodotoxin. In a preferred embodiment, the botulinum toxin is Onabotulinumtoxin A (BOTOX). In other embodiments, the toxin is an exotoxin. In some embodiments, the exotoxin is botulinum toxin produced by Clostridium botulinum, or Coryne bacterium diphtheriae toxin produced during diphtheria, or tetanospasmin produced by Clostridium tetani. In other embodiments, the toxin is an endotoxin. As used herein, the terms "Lipopolysaccharide (LPS)," "Lipoglycan" and "endotoxin" holds the same meaning and they are used interchangeably.

[0017] The term "protein" as used herein refers to at least one sequence of amino acids linked by sequential peptide bonds, and is generally synonymous with "polypeptide." In one embodiment, the amino acid sequence is one that occurs in nature. In other embodiments, the amino acid sequence is engineered by humans. The term protein includes, but is not limited to, proteins having pharmaceutical, diagnostic, agricultural, and/or any of a variety of other properties that are useful in commercial, experimental, and/or other applications. In some embodiments, the protein is a protein therapeutic. The term "protein therapeutic" (or "therapeutic protein") as used herein contemplates a protein that has a biological effect in the body, or on a region in the body on which it directly acts, or on a region of the body on which it remotely acts via intermediates, etc. Examples of therapeutic proteins are, but are not limited to, pharmaceutically or commercially relevant enzymes, receptors, receptor fusions, soluble receptors, soluble receptor fusions, antibodies (e.g., monoclonal and/or polyclonal antibodies), antigen-binding fragments of an antibody, Fc fusion proteins, SMIPs5 cytokines, hormones, regulatory factors, growth factors, coagulation / clotting factors, and antigen-binding agents. The above list of proteins is merely of examples found in nature, and is not intended to be a limiting recitation.

[0018] The term "antibody" as used herein contemplates a polypeptide or a protein complex that specifically binds an epitope of an antigen or mimetope thereof. An antibody includes an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, and single-chain antibodies. In some embodiments, an antibody is referred to as an immunoglobulin and include the various classes and isotypes, such as IgA (IgAl and IgA2), IgD, IgE, IgM, and IgG (IgGl, IgG3 and IgG4) ect. In some embodiments, the antibody is polyclonal or monoclonal. In some embodiments, the antibody is from any origin, such as mouse, rabbit, pig, dog, human, or other mammals, including a chimeric antibody thereof. In some embodiments, the antibody is humanized.

[0019] Examples of antibodies include, but are not limited to, bevacizumab, trastuzumab, rituximab, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, brentuximab vedotin, canakinumab, cetuximab, certolizumab pegol, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, raxibacumab, tocilizumab, tositumomab and ustekinumab. Other examples of antibodies include, but are not limited to, 3F8, abagovomab, abatacept, acz885, adecatumumab, afelimomab, aflibercept, afutuzumab, alacizumab, altumomab, anatumomab, anrukinzumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, bavituximab, bectumomab, belatacept, bertilimumab, besilesomab, biciromab, bivatuzumab, blinatumomab, cantuzumab, capromab, catumaxomab, cedelizumab, citatuzumab, cixutumumab, clenoliximab, cntol275 (ustekinumab), cntol48 (golimumab), conatumumab, dacetuzumab, detumomab, dorlimomab, dorlixizumab, ecromeximab, edobacomab, edrecolomab, efungumab, elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab, etanercept, etaracizumab, exbivirumab, fanolesomab, faralimomab, felvizumab, figitumumab, fontolizumab, foravirumab, galiximab, gantenerumab, gavilimomab, gomiliximab, ibalizumab, igovomab, imciromab, inolimomab, inotuzumab ozogamicin, iratumumab, keliximab, labetuzumab, lebrilizumab, lemalesomab, lerdelimumab, lexatumumab, libivirurnab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab, myo-029, nacolomab, naptumomab, nebacumab, necitumumab, nerelimomab, nimotuzumab, nofetumomab, ocrelizumab, odulimomab, oportuzumab, oregovomab, otelixizumab, pagibaximab, panobacumab, pascolizumab, pemtumomab, pertuzumab, pexelizumab, pintumomab, priliximab, pritumumab, pro- 140, rafivirumab, ramucirumab, regavirumab, reslizumab, rilonacept, robatumumab, rovelizumab, rozrolimupab, ruplizumab, satumomab, sevirumab, sibrotuzumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulurnab, sulesomab, tacatuzumab, tadocizumab, talizumab, tanezumab, tapliturnomab, tefibazumab, telimomab, tenatumomab, teneliximab, teplizumab, tgnl412, ticilimumab (tremelimumab), tigatuzumab, tnx-355 (ibalizumab), tnx-650, tnx-901 (talizumab), toralizumab, tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, vapaliximab, vedolizumab, veltuzumab, vepalimomab, visilizumab, volociximab, votumumab, zalutumumab, zanolimumab, ziralimumab, and zolimomab.

[0020] The term "membrane" as used herein refers to materials such as woven mesh or cellulose filters, glass fiber, mixed glass fiber and cellulose, synthetic fiber, mixed fiber, surface modified plastic (polyester, polypropylene, or polyethylene), nitrocellulose, or graded density polyethersulfone (PES). In some embodiments, the membranes are nitrocellulose membranes. In some embodiments, the membranes is chosen based on its pore size and flow rate. In some embodiments, the flow rate is a slower flow rate, for example a capillary flow time of about 120 sec/4 cm or greater. In some embodiments, the optimal membrane is determined empirically by testing several commercially available membranes. In some embodiments test lines are striped onto the membrane using an automated programmable dispenser. Membranes are typically blocked to reduce non-specific binding. In some embodiments, blocking the membrane further slows the flow rate.

[0021] The term "sample pad" as used herein generally refers to a hydrophilic element, such as a membrane that receives the biologic. In some embodiments, the sample pad is part of the conjugate pad, or a discrete pad positioned downstream of, and in fluid communication with, the conjugate pad. In some embodiments, the sample pad is useful for promoting the even and controlled distribution of the biologic onto the conjugate pad, controlling the rate at which the biologic enters the conjugate pad, or preventing flooding of the test device. In some embodiments, the sample pad receives a particular amount of the biologic. In some embodiments, the sample pad is pretreated and blocked. In some embodiments, surfactants are included in the sample pad to improve flow and subsequent release of biologies and other agents from the conjugate pad. [0022] In some embodiments, the sample pad is of the same or different material as the conjugate pad. In some embodiments, the sample pad is impregnated with agents to influence the flow rate of the sample, including, for example, surfactants or protein detergents, viscosity enhancers, signal enhancers, or buffer salts. In some embodiments, chemical agents are added to the sample pad to prevent the biologic from binding nonspecifically to any downstream materials or to modify the chemical nature of the biologic. Examples of chemical agents that are optionally added include, but are not limited to, albumin (e.g., BSA), sodium dodecyl sulfate (SDS), and Tween 20®. In some embodiments, the chemical agent is added to promote resolubilization of the conjugate, reduce nonspecific binding of the conjugate, and/or minimize adsorption of the analyte to the membrane. In some embodiments, the sample pad further comprises a buffer, pH calibrator, peptide, or antibody.

[0023] The term "conjugate pad" refers to a hydrophilic element, such as a membrane, comprising a conjugate reagent. In some embodiments, the conjugate reagent is a solid support. In certain of these embodiments, the solid support comprises a metal. In further embodiments, the conjugate reagent is colloidal gold. In other embodiments, the conjugate reagent comprises latex particles, enzymes, colored dyes, or paramagnetic or fluorescent particles. In some embodiments, the conjugate pad acts to ensure uniform transfer of the detectable marker and the antibody onto the test membrane.

[0024] In some embodiments, the conjugate pad is part of the sample pad, or a discrete pad positioned upstream of, and in fluid communication with the sample pad. In some embodiments, the conjugate pad is optimized for parameters such as flow rate, release characteristics, and stability. In some embodiments, the conjugate pad is pretreated with blocking and stabilizing buffers to improve features such as flow rate, release characteristics, and stability.

[0025] In some embodiments, the conjugate pad comprises a peptide dimer bound to antibody conjugated with the conjugate reagent. In further embodiments, the conjugate pad further comprises Factor C. In some embodiments, the Factor C is recombinant Factor C (rFC). Factor C is a serine protease zymogen coagulation factor that initiates the Limulous Amebocyte Lysate (LAL) clotting reaction in the presence of even trace amounts of LPS. In some embodiments, the colloidal gold is prepared by reduction of aqueous HAuC . 40 nm colloidal gold has a cherry red appearance. The lateral flow assay described herein utilizes a conjugate reagent conjugated antibody to achieve sensitive and selective detection of biological materials. Antibodies are conjugated to the conjugate reagent particles, in some embodiments, using known methods. In some embodiments, the conjugate reagent conjugates are dried onto the pretreated pads, and the conjugate reagent conjugate returns to solution without losing activity or specificity when it encounter the biologic sample. [0026] The term "mimetope" as used herein contemplates a compound that is recognized by the same binding molecule, such as an antibody, as a particular epitope but which has a different composition from the epitope. In one embodiment, the binding molecule is an antibody which recognizes (i.e., binds to) an epitope comprising a linear sequence of amino acids. A mimetope of this epitope comprises a different linear sequence of amino acids but which is still recognized by the same antibody. In one embodiment, mimetope includes a peptide epitope that is able to mimic the ability of an epitope to bind to an antibody.

[0027] The terms "polypeptide" and "peptide" are used broadly to refer to molecules or macromolecules comprising linear polymers of natural or synthetic amino acids. In some embodiments, polypeptides are derived naturally or synthetically by standard methods. While the terms polypeptide and peptide are synonymous, the term polypeptide, as used herein, generally refers to molecules of greater than 40 amino acids, while the term peptide generally refers to molecules of 2 to 40 amino acids.

[0028] The term "test line" as contemplated herein, refers to a band or zone on the test membrane that contains at least one mimetope peptide. The mimetope peptide is usually immobilized in a band or zone such that after reaction with the antibody-detectable marker complex, the band or zone produces an observable or measurable signal reflecting the presence or amount of biologic present in the sample. As a non-limiting example, if the conjugate pad contains the rituximab antibody conjugated to colloidal gold, then at least one test line will contain a mimetope peptide sequence specific for rituximab immobilized on the test membrane. In some embodiments, the test membrane contains one, two, three, four, or more test lines. In some embodiments, the test lines contain the same or different immobilized mimetopes. In some embodiments, more than one test line is applied for multianalyte testing or for semi -quantitative evaluation.

[0029] In some embodiments, the test line further comprises Bovine Serum Albumin (BSA). In one embodiment, the mimetope peptides are synthesized and attached to BSA. In certain embodiments, the mimetope-bound BSA is prepared by a commercial vendor. The carrier BSA improves the adsorption of the peptide onto the test line. In some embodiments, the test lines are coated with different concentrations of the mimetope peptides. In some embodiments, the coating concentration for the two or more test lines is used for the semi -quantitative feature of the C ³ -LFA.

[0030] In another embodiment, the test line comprises the antibody specific for the mimetope peptide. In some of these embodiments, the mimetope is conjugated to the conjugate reagent and the antibody on the test line recognizes and binds to the mimetope. [0031] The term "control line" as used herein contemplates a band or zone on the test membrane used for, but not limited to, confirming negative test results, for comparison with at least one test line, determining incorrect biologic, inactive biologic, wrong biologic, or concluding failed experiments.

[0032] The term "wicking pad" as used herein contemplates an absorbent pad attached at the distal side of the test device. The wicking pad aids to maintain the flow of liquid to the end of the strip. In some embodiments, the wicking pad comprises, but not limited to, an absorbent material, cellulose filter, or any other material which acts to maintain the flow of liquid to the end of the strip.

[0033] It is the general object of the present disclosure to provide a novel lateral flow assay to determine the presence, absence, or potency of a specific biologic and/or to visually quantify or semi-quantify an amount of the specific biologic. It is also a general object of the present disclosure to provide devices, methods, and kits which are used to visually determine the presence or absence of a specific biologic and/or to visually quantify or semi-quantify an amount of a specific biologic.

TEST DEVICE

[0034] In one aspect, disclosed herein is a test device for determining the quantity of a biologic in a sample, the test device comprising a sample pad for receiving the biologic, a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to a conjugate reagent, and a test membrane comprising at least one test line, the test line comprising an immobilized mimetope. In some embodiments, the conjugate reagent is a solid support. In certain of these embodiments, the solid support comprises a metal. In further embodiments, the conjugate reagent is colloidal gold.

[0035] In some embodiments, the sample pad of the test device receives the biologic in a fluid sample. The sample as used herein refers to an aqueous or non-aqueous fluid, or organic or nonorganic fluid, or biological or non-biological fluid. In some embodiments, the non-biological fluid is a pharmaceutical composition. In some embodiments, the non-biological fluid is a liquid waste. In some embodiments, the liquid waste is organic liquid waste. In some embodiments, the liquid waste is inorganic liquid waste.

[0036] In one embodiment, the biologic is introduced to the sample pad using a dipstick format and contacting one end of the test device with the polypeptide. In another embodiment, the biologic is introduced onto the sample pad using an applicator such as a pipette, a syringe, a dropper, a spray, and others known technologies. The amount of biologic received on the sample pad is preferably between about 1 and 1000 μΐ., preferably between 1 and 200 μΐ., more preferably between about 3 and 100 μΐ,, and most preferably between about 5 and 50 μΐ,. [0037] In one embodiment, the sample pad of the test device further comprises a buffer for pH stabilization, a pH calibrator, a surfactant to guarantee a uniform wetting, a stabilizing polymer, and a blocker.

[0038] In some embodiments, the conjugate reagent present in the conjugate pad is conjugated to a polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. The antibody conjugated to the conjugate reagent binds to a peptide dimer. In some embodiments, the peptide comprises of two mimetope peptides linked via a third peptide.

[0039] In one embodiment of the present disclosure, the test device includes qualitative readout (e.g., presence/absence of a specific biologic). Semi-quantitative or quantitative results are also contemplated. In some embodiments, semi-quantitative or quantitative results is achieved by including a series of test lines of mimetopes of varying concentrations. In some embodiments, the test device of the present disclosure detects active biologic of at least about 30, 40, 50, 60, 70, 80, 90, or 95% activity. In certain embodiments, evaluation of the signal intensity is also performed. In some embodiments, the signal is digitized and evaluated using a flatbed scanner or a CCD camera and appropriate software. In some embodiments, sensitivity is increased by use of enhancement agents such as, for example, a silver enhancer. More sensitive chemiluminescent or fluorescent labels are optionally used to increase the sensitivity as well. Free mimetope peptide, to compete for binding with the protein, is optionally included in the conjugate pad to threshold the assay, when decreased sensitivity is desired. In some embodiments, sensitivity is also controlled by adjusting, for example, the bed volume of the membrane, dimensions of the test membrane, porosity of the test membrane, position and width of the test line and control line.

[0040] In one embodiment, the conjugate pad of the test device comprises a detectable marker. In another embodiment, the detectable marker in the conjugate pad is capable of binding the biologic that the sample pad receives. In one embodiment, the conjugate pad acts to ensure uniform transfer of the detectable marker and the biologic onto the test membrane. In one embodiment, the detectable marker comprises, but is not limited to, particles, luminescent labels, calorimetric labels, fluorescent labels, chemical labels, enzymes, radioactive labels, metal colloids, and chemiluminescent labels. In one embodiment, gold colloidal spheres are used. In another embodiment, other metal sols and latex microparticles are used as well. In other embodiments, photostable, color tunable nanoparticles such as carbon, selenium, or quantum dots are used as detectable markers. These detectable markers provide colorimetric indicators for reporting whether the target molecule is present. The size of the detectable markers are related to the porosity of the membrane. The markers are preferably sufficiently small to be transported along the membrane by the capillary action of the fluid. In one embodiment, the amount of detectable marker present varies depending on the size and composition of the detectable marker, the composition of the membrane, and the level of sensitivity of the assay. The detectable marker will bind to the antibody to form an antibody-detectable marker complex. In one embodiment, the detectable marker comprises gold colloidal spheres. In another embodiment, the detectable marker comprises a secondary antibody. In some embodiments, the secondary antibody is conjugated to gold.

[0041] In one embodiment, the test membrane in the test device comprises at least one test line and at least one control line. In another embodiment, the test membrane comprises two or more test lines. In another embodiment, at least one test line comprises an immobilized mimetope. In another embodiment, each test line contains an immobilized mimetope different from another test line. In some embodiments, the immobilized mimetope mimics the native antigen epitope recognized by an antibody.

[0042] In some embodiments, the at least one test line is upstream of at least one control line. In other embodiments, the at least one test line is downstream of at least one control line.

[0043] In one embodiment, at least one control line comprises a light chain antibody. In some embodiments, the light chain antibody is an anti-kappa light chain antibody. In another embodiment, the light chain antibody is an anti-lambda light chain antibody. In other embodiments, the light chain antibody is a known light chain antibody.

[0044] In one embodiment, the at least one control line of the disclosed test membrane comprises a secondary antibody that binds directly to the antibody of the conjugate reagent such as, for example, anti-IgG. In some of these embodiments, the anti-IgG acts as a non-specific control and turns positive regardless of the presence or absence of biologic within the test sample. In some embodiments, the test device consists of only one control line. In one embodiment, the test device of the present disclosure consists of two control lines. In another embodiment, the test device of the present disclosure consists of three control lines. It is also contemplated that the test device consists of more than three control lines. In some embodiments, the control lines of the test device contain different densities of coating. In some embodiments, the test device of the present disclosure comprises a first control line comprising a first density of coating, and a second control line comprising 1/10 the concentration of the first density of coating. In some embodiments, the test device of the present disclosure comprises a first control line comprising a first density of coating, a second control line comprising 1/2 the concentration of the first density of coating, and a third control line comprising 1/10 the concentration of the first density of coating.

[0045] In some embodiments, the test device further comprises a wicking pad. CLEAVAGE COUPLED COMPETITIVE LATERAL FLOW ASSAY (C3-LFA)

[0046] The C3-LFA leverages the difference in monoclonal antibody binding of monomer and dimer peptides to create a cleavage coupled competitive lateral flow assay.

[0047] Monoclonal antibodies bind to mimetope dimers more strongly as compared to a single mimetope. This difference in avidity between a single mimetope and a dimer has been exploited for monoclonal antibody detection by fluorescence resonance energy transfer (FRET) using fluorophore tagged oligonucleotide conjugates that hybridize and stabilize the dimer form, as explained in Tian, L. and T. Heyduk, Antigen peptide-based immunosensors for rapid detection of antibodies and antigens. Anal Chem, 2009. 81(13): p. 5218-25. C3-LFA leverages this difference in binding affinity to detect and quantify biologies. When a specific biologic is present in the sample, the high avidity dimer mimetopes are cleaved. The conjugate reagent conjugated antibody has a lower binding affinity for the cleaved monomelic peptide. The conjugate reagent conjugated antibody ascends chromographically to the mimetope coated test strip. If the antibody is unmasked due to the presence of the biologic, the antibody binds to the mimetope present in the test strip, thereby giving a positive test readout. If the antibody is bound to the peptide dimer due to the absence of the biologic or presence of the inactive biologic, then there is a negative test readout.

[0048] In some embodiments, if the biologic of interest is not sufficient to cleave enough dimer peptide as the sample flows through the lateral flow device, or if release of the dimer from the conjugate pad proves difficult, the sample is premixed with the dimer peptide prior to introduction onto the lateral flow device, with the time of pre-incubation calibrated for the putative activity of the sample. In one embodiment, the biologic is a clinical BOTOX preparation. In another embodiment, the biologic is LPS.

[0049] The activity in clinical BOTOX preparations is generally sufficient to cleave enough dimer peptide as the sample flows through the lateral flow device. If this is not the case, or if release of the dimer from the conjugate pad proves difficult, the sample is premixed with the dimer peptide prior to introduction onto the lateral flow device, with the time of pre-incubation calibrated for the putative activity of the sample.

[0050] The present disclosure provides an example of the translation of monoclonal antibody- mimetope pairs into an in vitro assay for enzyme activity. By coupling the convenience and simplicity of lateral flow with the signal amplification intrinsic in enzymatic assays, the C3 -LFA exceeds the sensitivity of conventional LFAs while avoiding the complex instrumentation required for conventional colorimetric or fluorescent enzyme activity assays.

[0051] The C3-LFA assay will be better appreciated when illustrated by the following two exemplary embodiments, BOTOX and Lipopolysaccharide (LPS). BOTOX

[0052] In one embodiment, the test device described herein determines the presence, integrity, potency, or quantity of the biologic Onabotulinumtoxin A (BOTOX). An overview of the C3- LFA as it applies to this embodiment is described in FIG. 1 and FIG. 2. Sample, containing a biologic, is applied to the sample pad. The biologic is wicked through a conjugate pad that contains the competitive peptide dimer and the conjugate reagent conjugated to the monoclonal antibody.

[0053] The dimer peptide comprises of two mimetope peptides linked via a peptide fragment of the SNAP-25 protein. SNAP-25 protein is the target of BOTOX endopeptidase activity. The BOTOX endopeptidase activity cleaves the dimer mimetope peptides into monomers. The monomers have a lower affinity for the monoclonal antibody. Thus, when BOTOX is present and active, the monoclonal antibody conjugated conjugate reagent is unmasked through the cleavage of the otherwise blocking dimer mimetopes.

[0054] The monoclonal antibody conjugated conjugate reagent is wicked to the test membrane containing the test lines. If BOTOX is present and active in the sample, the unmasked monoclonal antibody binds to the mimetope peptide present in the test lines, thereby giving a positive reading. If BOTOX is absent, or not active in the sample, then there is a negative reading.

[0055] In one embodiment, two test lines contain different densities of mimetope peptide and spaced by a fixed distance. This allows for a semi -quantitative readout of the amount of active BOTOX present in the sample. This is needed because BOTOX is used at different concentrations for different clinical applications, ranging from 10 to 100 units/ml for approved uses. In some embodiments, the test lines are adjusted for spacing and mimetope density such that the lowest concentration causes only the first test line to show a positive reading, and the highest concentration causes both test lines to equally develop and show positive reading. In some embodiments, the lowest concentration is 10 units/mL. In some embodiments, the highest concentration is 100 units/mL. Intermediate concentrations have distinct ratios of intensity between those two extremes. In some embodiments, the test device of the present disclosure comprises a first test line comprising a first density of coating, and a second test line comprising 1/10 the concentration of the first density of coating. In some embodiments, the test device of the present disclosure comprises a first test line comprising a first density of coating, a second test line comprising 1/2 the concentration of the first density of coating, and a third test line comprising 1/10 the concentration of the first density of coating. In some embodiments, a portable or smartphone connected LFA reader provides quantitative densitometry output. [0056] Control lines function to confirm that the test is functional or valid, independently of whether the antibody conjugate reagent binds to the mimetope peptide immobilized on the test lines.

LPS

[0057] In another embodiment, the test device described herein determines the presence, integrity, or determines the quantity of the biologic lipopolysaccharide (LPS). An overview of the C3-LFA as it applies to this embodiment is described in FIG. 3 and FIG. 4. Sample, containing a biologic, is applied to the sample pad. The biologic is wicked through a conjugate pad that contains the competitive peptide dimer, inactive recombinant Factor-C (rFC) and colloidal gold conjugated to the monoclonal antibody.

[0058] The peptide consists of two mimetope peptides linked via a third peptide sequence. In some embodiments, the third peptide is Ile-Glu-Ala-Arg. If LPS is present in the biologic, rFC is activated. Activated rFC cleaves the peptide sequence Ile-Glu-Ala-Arg. In some embodiments, the optimal peptide construct is determined based on the greatest difference in affinity between the dimer form and the cleaved monomers as well as rapid cleavage kinetics. In some embodiments, synthetic peptides that are cleaved by activated Factor-C are used, including known synthetic peptides, including, but are not limited to, Val-Pro-Arg, Leu-Gly-Asn-Lys-Val- Ser-Arg, and Ile-Thr-Thr-Val-Gly-Arg.

[0059] The peptide monomers have a lower affinity for the monoclonal antibody. Thus, when LPS is present and active, the monoclonal antibody conjugated colloidal gold is unmasked through the cleavage of the otherwise blocking dimer mimetopes.

[0060] The monoclonal antibody conjugated colloidal gold is wicked to the test lines. If LPS is present in the sample, the unmasked monoclonal antibody binds to the mimetope peptide present in the test lines, thereby giving a positive reading. If LPS is absent, then there is a negative reading.

[0061] In one embodiment, two test lines contain different densities of mimetope peptide and spaced by a fixed distance. This allows for a semi -quantitative readout of the amount of LPS present in the sample. In some embodiments, the test lines are adjusted for spacing and mimetope density such that the lowest concentration causes only the first test line to show a positive reading, and the highest concentration causes both test lines to equally develop and show positive reading. In some embodiments, the lowest concentration is 0.1 EU/mL. In some embodiments, the highest concentration is 1.0 EU/mL. Intermediate concentrations have distinct ratios of intensity between those two extremes. In some embodiments, the test device of the present disclosure comprises a first test line comprising a first density of coating, and a second test line comprising 1/10 the concentration of the first density of coating. In some embodiments, the test device of the present disclosure comprises a first test line comprising a first density of coating, a second test line comprising 1/2 the concentration of the first density of coating, and a third test line comprising 1/10 the concentration of the first density of coating. In some embodiments, a portable or smartphone connected LFA reader provides quantitative densitometry output.

[0062] Control lines function to confirm that the test is functional or valid, independently of whether the antibody conjugate reagent binds to the mimetope peptide immobilized on the test lines.

SELECTION AND OPTIMIZATION OF DIMER PEPTIDE

[0063] The process of selecting and validating peptide mimetopes for antibodies has been described previously. Table 1 shows a list of mimetope peptides that bind to antibodies such as trastuzumab and bevacizumab. In all cases the peptides are selected from a library that contains cysteines flanking the peptide mimetope sequence to increase stability of the peptide through disulfide bond formation. The use of these peptides for lateral flow assays is disclosed in the provisional patent application 61/883, 136, which is incorporated by reference in its entirety, including the drawings.

Table 1. Mimetope peptide sequences for several monoclonal antibodies

[0064] Botox recognizes the SNAP -25 protein, and cleaves it between residues Q197 and R198. Peptides as short as 13 amino acids spanning this recognition site have been reported to be active where the Botox molecule is capable of cleaving, as shown in Feltrup, T.M. and B.R. Singh, Development of a fluorescence internal quenching correction factor to correct botulinum neurotoxin type A endopeptidase kinetics using SNAPtide. Anal Chem, 2012. 84(24): p. 10549- 53. However, longer peptides, containing up to 47 amino acids, that include the upstream a- exosite have much greater binding affinity and improved reaction kinetics as shown in Breidenbach, M.A. and A. T. Brunger, Substrate recognition strategy for botulinum neurotoxin serotype A. Nature, 2004. 432(7019): p. 925-9.

[0065] The antigen binding sites of the two arms of an antibody are roughly 10 nm apart. Thus, even the short peptides are sufficiently long to allow both mimetopes to encounter an antigen binding site on a single immunoglobulin molecule. In some embodiments, longer peptides or peptides with linkers between the mimetopes and the target sequence allows for better binding. In some embodiments, the target sequence is the SNAP-25 peptide sequence or Ile-Glu-Ala-Arg peptide sequence.

SHELF LIFE

[0066] The shelf life for a typical commerical LFA is two years. Critical components that contribute to the shelf life include the stability of the reagents, in particular antibodies and peptides, as well as the physical components of the LFA such as the various membranes and their adhesives. In some embodiments, assembled tests are stored in foil pouches with a dessicant. In some embodiments, accelerated stability testing is performed to simulate long term storage and exposure to non-optimal conditions such as non-optimal temperature, humidity, and light. In some embodiments, samples are incubated at 4 °C, room temperature, 37 °C, and 55 °C and tested at various times. One week at 55 °C is roughly equivalent to a year at room temperature. In some embodiments, humidity testing is done with both open and closed packages at 30% and 80% relative humidty at room temperture and 37 °C. In some embodiments, light exposure is done for several weeks.

[0067] In another aspect, disclosed herein is a method of determining the presence, quantity or potency of a biologic, the method comprising: (a) contacting the biologic with a test device, wherein the test device comprises: (i) a sample pad for receiving the biologic, (ii) a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to colloidal gold, and (iii) a test membrane comprising at least one test line, the test line comprising an immobilized mimetope, (b) determining whether at least one test line undergoes a color change, and (c) determining, based on the color change, the presence, quantity or potency of the biologic. In some embodiments, the test device is a lateral flow immunoassay. In some embodiments, the biologic is a protease. In some embodiments, the biologic is an endonuclease. In some embodiments, the biologic induces a chemical or enzymatic process that cleaves a bond or dissociates an otherwise stable interaction. In a preferred embodiment, the biologic is a toxin. In one embodiment, the toxin is a neurotoxin. In one embodiment, the neurotoxin is botulinum toxin. In one embodiment, the botulinum toxin is Onabotulinumtoxin A (botox). In some embodiments, the toxin is an exotoxin. In some embodiments, the exotoxin is botulinum toxin. In some embodiments, the botulinum toxin is Onabotulinumtoxin A (botox). In some embodiments, the toxin is an endotoxin. In some embodiments, the endotoxin is Lipopolysaccharide (LPS).

[0068] In some embodiments of the second aspect, the sample pad further comprises a buffer, pH calibrator, peptide, or antibody. In some embodiments, the biologic is present in a fluid. In some embodiments, the fluid is an aqueous or non-aqueous fluid, or organic or non-organic fluid, or biological or non-biological fluid. In some embodiments, the non-biological fluid is a pharmaceutical composition. In some embodiments, the non-biological fluid is liquid waste. In some embodiments, the liquid waste is organic liquid waste. In other embodiments, the liquid waste is inorganic liquid waste.

[0069] In some embodiments of the second aspect, the conjugate pad further comprises Factor C

[0070] In some embodiments of the second aspect, the test membrane comprises at least one test line and at least one control line. In some embodiments, the test membrane comprises two or more test lines. In some embodiments, the at least one test line comprises an immobilized mimetope. In some embodiments, each test line contains an immobilized mimetope different from another test line. In some embodiments, the immobilized mimetope mimics the native antigen epitope recognized by an antibody. In some embodiments, at least one test line is upstream of at least one control line. In some embodiments, the at least one test line is downstream of at least one control line. In some embodiments, the at least one control line comprises a light chain antibody. In some embodiments, the light chain antibody is an anti- kappa light chain antibody. In some embodiments, the light chain antibody is an anti-lambda light chain antibody. In some embodiments, the at least one control line comprises a secondary antibody that binds directly to the antibody conjugated to the conjugate reagent.

[0071] In some embodiments the test device further comprises a wicking pad.

[0072] In another aspect, disclosed herein is a kit comprising: (i) a test device for determining the quantity of a biologic in a sample comprising (a) a sample pad for receiving the biologic, (b) a conjugate pad comprising one or more peptide dimers and one or more antibodies conjugated to colloidal gold, and (c) a test membrane comprising at least one test line, the test line comprising an immobilized mimetope, and (ii) instructions for use thereof. In some embodiments, the kit further comprises a biologic sample. In some embodiments, the kit further comprises a sample applicator.

[0073] In another aspect, disclosed herein is a test device for determining the quantity of a biologic in a sample comprising (a) conjugating colloidal gold to the mimetope, (b) a sample pad for receiving a mixture of the biologic and mimetope conjugated colloidal gold, (c) a conjugate pad comprising one or more peptide dimers, and (d) a test membrane comprising at least one test line, the test line comprising an immobilized antibody. In some embodiments of the fourth aspect, the colloidal gold is conjugated to the mimetope on a carrier protein. In some embodiments, the carrier protein is albumin. In other embodiments, the carrier protein is KLH. In some embodiments, the conjugate pad further comprises recombinant Factor C (rFC). In some embodiments, the biologic is BOTOX. In some embodiments, the biologic is LPS. This aspect of the present disclosure is further illustrated in FIG. 5 and FIG. 6

[0074] In another aspect, disclosed herein is a method for determining the presence, quantity or potency of a biologic, the method comprising: (a) contacting the biologic with a test device, wherein the test device comprises: conjugating colloidal gold to the mimetope, a sample pad for receiving a mixture of the biologic and mimetope conjugated colloidal gold, a conjugate pad comprising one or more peptide dimers, and a test membrane comprising at least one test line, the test line comprising an immobilized antibody; (b) determining whether at least one test line undergoes a color change, and (c) determining, based on the color change, the presence, quantity or potency of the biologic. In some embodiments of the fifth aspect, the colloidal gold is conjugated to the mimetope on a carrier protein. In some embodiments, the carrier protein is albumin. In other embodiments, the carrier protein is KLH. In some embodiments, the conjugate pad further comprises recombinant Factor C (rFC). In some embodiments, the biologic is BOTOX. In some embodiments, the biologic is LPS.

[0075] In one embodiment, the disclosed test device, methods, and kits are related to determining the presence, quantity, or potency of a biologic. With their high cost, biologies such as BOTOX are extremely attractive targets for counterfeiting or illegal distribution. Furthermore, being large molecules, these biologies are temperature and light sensitive, and require proper handling and storage. With a complex pharmaceutical distribution chain making adequate oversight difficult, and increasing the likelihood that such problems will persist, it is imperative that a simple, inexpensive assay be developed to determine the integrity of a protein therapeutic prior to use.

[0076] In another embodiment, the disclosed test devices, methods, and kits are related to determining the presence or quantity of biologic such as LPS. Gram-negative bacterial endotoxins, namely lipopolysaccharide (LPS), are common contaminants of many human and animal parenteral drugs, medical device, raw materials, biological products, and food products. Endotoxins, at even picogram amounts, can elicit very strong immune responses in people and animals, and endotoxemia can result in septic shock. For drug manufacturing and production, endotoxin testing is done at multiple stages, including bulk lot release testing, final product release testing, and raw materials testing. As such, early and sensitive detection of endotoxin is vital.

[0077] The following examples are provided to further illustrate the embodiments of the present disclosure, but are not intended to limit the scope of the disclosure. Other known procedures, methodologies or techniques are alternatively used. EXAMPLE 1

[0078] Peptide constructs shown in Table 2 are evaluated for (i) the blocking ability of the dimer form, (ii) the differential blocking ability of the intact and cleaved dimer, and (iii) the reaction kinetics with BOTOX. ELISA assay is used to mimic the interactions that take place on the LFA. The intact and cleaved dimers are titrated with the free antibody, and colloidal gold bound cognate antibody. Free antibody tests the ability of the dimer to occupy both intramolecular antigen binding sites whereas the colloidal gold bound mAb can be blocked by intermolecular interactions as well as intramolecular. The difference in IC50 between dimer and cleaved forms reflect the difference in affinity between the dimer and monomer mimetopes. Reaction kinetics are assayed by incubating a 5 fold excess of the IC50 concentration of the dimer with a fixed amount of BOTOX for varying lengths of time. The time required to reduce the dimer inhibition to the IC50 value reflects the enzyme kinetics. The optimal peptide construct has the greatest differential IC50 between intact and cleaved forms and the most rapid cleavage kinetics. If a single peptide does not embody those two criteria, additional variations on the constructs include altered spacer lengths or intermediate amounts of SNAP-25 sequence. Multiple short SNAP-25 sequences are also produced.

Table 2. Peptide sequences for rituximab binding dimer

[0079] In one embodiment, a single polypeptide that consists of the mimetope peptide sequence on either side of the BOTOX cleavage motif is used. In other embodiments, alternative conjugation methods and spacings create longer peptide constructs. In one embodiment, a longer peptide construct is produced by synthesizing the individual peptide components as discrete entities and then chemically linking them through known methods, such as click chemistry. Numerous commercial custom peptide synthesis providers offer these services. In some embodiments, the mimetope peptides contain internal disulfide bonds that create a constrained peptide. In other embodiments, linear mimetopes that do not have the internal disulfides are selected as described in Jiang, B., et ah, A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2. J Biol Chem, 2005. 280(6): p. 4656-62.

EXAMPLE 2

[0080] Peptide constructs shown in Table 3 are evaluated for (i) the blocking ability of the dimer form, (ii) the differential blocking ability of the intact and cleaved dimer, and (iii) the reaction kinetics with activated rFC. ELISA assay is used to mimic the interactions that take place on the LFA. The intact and cleaved dimers are titrated with the free antibody, and colloidal gold bound cognate antibody. Free antibody tests the ability of the dimer to occupy both intramolecular antigen binding sites whereas the colloidal gold bound antibody is blocked by intermolecular interactions as well as intramolecular interactions. The difference in IC50 between dimer and cleaved forms reflect the difference in affinity between the dimer and monomer mimetopes. Reaction kinetics are assayed by incubating a 5 fold excess of the IC50 concentration of the dimer with a fixed amount of activated rFC for varying lengths of time. The time required to reduce the dimer inhibition to the IC50 value reflect the enzyme kinetics. The optimal peptide construct has the greatest differential IC50 between intact and cleaved forms and the most rapid cleavage kinetics. If a single peptide does not embody those two criteria, additional variations on the constructs include altered spacer lengths or intermediate amounts of the target sequence. Multiple short target sequences are also produced.

Table 3. Peptide sequences for rituximab binding dimer

[0081] In one embodiment, a single polypeptide that consists of the mimetope peptide sequence on either side of the rFC cleavage site is used. In other embodiments, alternative conjugation methods and spacings create longer peptide constructs. In one embodiment, a longer peptide construct is produced by synthesizing the individual peptide components as discrete entities and then chemically linking them through known methods, such as click chemistry. Numerous commercial custom peptide synthesis providers offer these services. In some embodiments, the mimetope peptides contain internal disulfide bonds that create a constrained peptide. In other embodiments, linear mimetopes that do not have the internal disulfides are selected as described in Jiang, B., et al., A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope ofHER-2. J Biol Chem, 2005. 280(6): p. 4656-62.