Multiple hybrid immunoassay

Abstract

The invention relates to compositions and methods for the immunoassay of an analyte of interest. The analyte is detected in an immunoassay using three or more antibodies, wherein each antibody specifically binds to a different epitope on the analyte. When the analyte of interest in a clinical marker for an acute disease, the detection of the analyte by immunoassay is a diagnosis of the occurrence of the disease.

What is claimed is:  
     
         1 . An immunoassay composition for detecting an analyte comprising at least three different antibodies, the at least three different antibodies being capable of binding to at least three different epitopes on an analyte, in which at least two of the at least three different antibodies are capable of binding to at least two different epitopes on a subform of the analyte, and in which at least one of the at least three different epitopes on the analyte is unavailable for binding on the subform.  
     
     
         2 . An immunoassay composition for detecting an analyte comprising m different antibodies, 
 (a) in which at least n of the m different antibodies are capable of binding to n different epitopes on an analyte, and    (b) in which no more than n−1 of the n different epitopes on the analyte are available for binding on a subform of the analyte, 
 provided that m and n are greater than or equal to 3, and m is greater than or equal to n.  
   
     
     
         3 . The immunoassay composition of  claim 1  in which the analyte comprises TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, or combinations thereof.  
     
     
         4 . The immunoassay composition of  claim 1  in which at least one of the at least three antibodies comprises a full length antibody, a single-chain antibody, or an antibody fragment.  
     
     
         5 . The immunoassay composition of  claim 4  in which the antibody fragment comprises a Fab fragment.  
     
     
         6 . The immunoassay composition of  claim 1  in which the analyte is cTnI.  
     
     
         7 . The immunoassay composition of  claim 1  which further comprises a common member conjugated to at least one of the at least three antibodies.  
     
     
         8 . The immunoassay composition of  claim 7  in which the at least one of the at least three antibodies is covalently attached to the common member.  
     
     
         9 . The immunoassay composition of  claim 8  in which the covalent attachment comprises a crosslinker, which is derived from a crosslinking agent.  
     
     
         10 . The immunoassay composition of  claim 9  in which the crosslinking agent comprises succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy[6-amidocaproate], glutaraldehyde, adipic acid dihydrazide, bis-diazotized benzidine, 1,4-butane diglycidyl ether, bis-maleimido hexane, sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, or N-hydroxysuccinimidyl 4-azidosalicylic acid.  
     
     
         11 . The immunoassay composition of  claim 7  in which the common member is a microparticle.  
     
     
         12 . The immunoassay composition of  claim 11  in which the microparticle comprises latex beads, polystyrene beads, polystyrene/acrylic acid beads, or combinations thereof.  
     
     
         13 . The immunoassay composition of  claim 7  in which the common member comprises a signal-generating element.  
     
     
         14 . The immunoassay composition of  claim 13  in which the signal-generating element comprises a radiolabel, metal particle, fluorescent dye, chromogenic dye, labeled protein, enzyme, or combinations thereof.  
     
     
         15 . The immunoassay composition of  claim 14  in which the enzyme comprises peroxidase, glucose oxidase, phenol oxidase, β-galactosidase, alkaline phosphatase, or combinations thereof.  
     
     
         16 . The immunoassay composition of  claim 13  in which the molar ratio of the at least one of the at least three antibodies conjugated to the signal-generating element is from about 1:1 to about 10:1.  
     
     
         17 . The immunoassay composition of  claim 16  in which the molar ratio of the at least one of the at least three antibodies conjugated to the signal-generating element is about 3:1.  
     
     
         18 . The immunoassay composition of  claim 13  in which at least two of the at least three antibodies are conjugated to the signal-generating element.  
     
     
         19 . The immunoassay composition of  claim 18 , in which the molar ratio of the at least two of the at least three antibodies conjugated to the signal-generating element is from 1:1 to 10:1.  
     
     
         20 . An immunoassay composition for detecting an analyte comprising four different antibodies, the four different antibodies being capable of binding to four different epitopes on an analyte, in which at least one of four different epitopes on the analyte is unavailable for binding on a subform of the analyte, in which a first and second of the four different antibodies are conjugated to a surface, in which a third and fourth of the four different antibodies are each conjugated to an enzyme, in which at least one of the first and second antibodies is capable of binding to at least one epitope on the subform of the analyte, and in which at least one of the third and fourth antibodies is capable of binding to at least one epitope on the subform.  
     
     
         21 . An immunoassay composition for detecting an analyte comprising m different antibodies, 
 (a) in which n of the m different antibodies are capable of binding to n different epitopes on an analyte,    (b) in which no more than n−1 of the n different epitopes on the analyte are available for binding on a subform of the analyte,    (c) in which p of the n different antibodies are conjugated to a surface,    (d) in which p of the n different antibodies are each conjugated to an enzyme,    (e) in which at least p−1 of the p antibodies in step (c) is capable of binding to at least p−1 different epitopes on the subform of the analyte, and    (f) in which at least p−1 of the p antibodies in step (d), is capable of binding to at least p−1 different epitopes on the subform of the analyte. 
 provided that m and n are 4, and that p is 2.  
   
     
     
         22 . An immunoassay device comprising one or more sensing elements and at least one surface on which an immunoassay can be conducted for detecting an analyte, which at least one surface comprises at least two different antibodies, the at least two different antibodies being capable of binding to at least two different epitopes on the analyte, in which at least one of the at least two different antibodies is capable of binding to at least one epitope on a subform of the analyte, in which at least one of the at least two different epitopes on the analyte is unavailable for binding on the subform, and in which the at least two different antibodies are bound to the at least one surface.  
     
     
         23 . An immunoassay device comprising one or more sensing elements and at least one surface on which an immunoassay can be conducted for detecting an analyte, 
 (a) in which at least one surface comprises m different antibodies,    (b) in which at least n of the m different antibodies are capable of binding to n different epitopes on the analyte    (c) in which no more than n−1 of the n different epitopes on the analyte are available for binding on a subform of the analyte, and    (d) in which at least n of the m different antibodies are bound to the at least one surface, 
 provided that m and n are greater than or equal to 2, and m is greater than or equal to n.  
   
     
     
         24 . The immunoassay device of claim  21 (a) in which the analyte comprises TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, or combinations thereof.  
     
     
         25 . The immunoassay device of claim  21 (a) in which the at least one surface comprises glass, semiconductor, plastic, silicon dioxide, photoformable PVA, photoformable gelatin, film forming latex, conductive metal, or combinations thereof.  
     
     
         26 . The immunoassay device of  claim 25  in which the conductive metal comprises silver, iridium, gold, platinum, or combinations thereof.  
     
     
         27 . The immunoassay device of claim  21 (a) in which at least one of the at least two antibodies is absorbed or adsorbed to the at least one surface.  
     
     
         28 . The immunoassay device of claim  21 (a) in which at least one of the at least two antibodies is covalently attached to the at least one surface.  
     
     
         29 . The immunoassay device of claim  21 (a) which further comprises a microparticle in which the at least two antibodies are bound to the microparticle and in which the microparticle is bound to the at least one surface.  
     
     
         30 . The immunoassay device of claim  21 (a) which further comprises at least two microparticles in which only one of the at least two antibodies is bound to one of the at least two microparticles and in which the at least two microparticles are bound to the at least one surface.  
     
     
         31 . The immunoassay device of claim  21 (a) which further comprises a third antibody which binds to a different epitope on the analyte and the subform of the analyte.  
     
     
         32 . The immunoassay device of claim  21 (a) in which the sensing element comprises an electrode, biosensor, field-effect transistor, surface acoustic wave device, optical wave-guide, fiber optic, cuvette, radioactivity detector, immunochromatographic device, reagents that facilitate physical, nuclear, chemical, biochemical, electrical, or optical detection, or combinations thereof.  
     
     
         33 . An immunoassay kit comprising in a suitable container, at least three different antibodies, the at least three antibodies being capable of binding to at least three different epitopes on an analyte, in which at least two of the at least three different antibodies are capable of binding to at least two different epitopes on a subform of the analyte, and in which at least one of the at least three different epitopes on the analyte is unavailable for binding on the subform.  
     
     
         34 . An immunoassay kit comprising in a suitable container m different antibodies, 
 (a) in which at least n of the m different antibodies are capable of binding to n different epitopes on an analyte, and    (b) in which no more than n−1 of the n different epitopes on the analyte are available for binding on a subform of the analyte, 
 provided that m and n are greater than or equal to 3, and m is greater than or equal to n.  
   
     
     
         35 . The immunoassay kit of  claim 33  in which the analyte comprises TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, or combinations thereof.  
     
     
         36 . A sandwich immunoassay product comprising an analyte and a subform of the analyte, in which at least three different epitopes on the analyte are available for binding by at least three different antibodies, in which at least two of the three different antibodies are bound to a different epitope on the analyte, in which two of the three different epitopes on the analyte are available for binding on the subform, in which at least two of the three different antibodies are bound to a different epitope on the subform, and in which at least one of the at least three different epitopes on the analyte is unavailable for binding on the subform.  
     
     
         37 . A sandwich immunoassay product comprising an analyte, a subform of the analyte, and at least 2 of m different antibodies, 
 (a) in which the analyte includes at least n different epitopes each capable of being bound by one of the m different antibodies,    (b) in which at least 2 of the m different antibodies are each bound to a different epitope on the analyte,    (c) in which no more than n−1 of the n different epitopes on the analyte are available for binding on a subform of the analyte, and    (d) in which at least 2 of the m different antibodies are each bound to a different epitope on the subform of the analyte, 
 provided that m and n are greater than or equal to 3, and that m can be greater than or equal to n.  
   
     
     
         38 . The sandwich immunoassay product of  claim 36  in which the analyte comprises TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, or combinations thereof.  
     
     
         39 . The sandwich immunoassay product of  claim 36  in which at least one of the at least three antibodies and least one of the at least two antibodies comprises a signal-generating element.  
     
     
         40 . A method of determining whether a patient has suffered a myocardial infarction comprising: 
 (a) applying a sample from a patient suspected of suffering a myocardial infarction to a surface to which is bound at least two antibodies, in which the at least two antibodies are capable of binding to at least two different epitopes on cTnI, in which at least one of the at least two antibodies is capable of binding to at least one different epitope on a subform of cTnI, and in which at least one epitope of cTnI is unavailable for binding on the subform;    (b) adding a reagent comprising a third antibody which binds to yet another epitope on cTnI and the subform; and    (c) determining the extent of binding of the third antibody.    
     
     
         41 . A method of determining whether a patient has suffered a myocardial infarction comprising: 
 (a) applying a sample from a patient suspected of suffering a myocardial infarction to a surface to which is bound a first antibody which is capable of binding to a first epitope on cTnI and a subform of cTnI;    (b) adding separately or together at least a second and third antibody, in which the at least second and third antibodies are capable of binding to at least two different epitopes on cTnI, in which the at least second and third antibodies are not capable of binding to the first epitope, in which at least one of the at least second and third antibodies are each capable of binding to at least one different epitope on the subform of cTnI, and in which at least one epitope of cTnI is unavailable for binding on the subform; and    (c) determining the extent of binding of the at least second and third antibodies.