Composition for homogeneous multiplexed microparticle-based assay

Abstract

A method and composition for the purpose of detecting and measuring analytes, such as antibodies, which are capable of binding with certain binding partners such as antigens. A homogenous assay is performed in the presence of free unbound antibodies. Such a homogeneous assay testing for specific antibodies is herein possible by defining of test subsets of microparticles having specific antigens thereon which are capable of binding with specific target antibodies. The microparticle suspension also includes at least two calibration subsets of microparticles having a binding partner thereon with at least two known levels of concentration which is capable of binding with human antibodies for the purpose of assay calibration. A verification subset of microparticles is included with another binding partner thereon at a known concentration, capable of binding with anti-human antibodies. This suspension is incubated with a human sample and then is incubated with a tagging component which is measurable for calibration and verification and finally for determining the concentration of target antibodies bound to the test subsets. A separate verification subset of microparticles is included for verifying the calibration and for yielding a correction factor therefore. A complete calibration calculation is performed for each sample being tested. Preferably, the attaching component of the tagging component is the same molecule as the first binding partner material positioned on the calibration subsets of microparticles.

1 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample comprising: 
 A. a multiplexed suspension of microparticles including a plurality of sets thereof for detection, calibration and calibration verification, said suspension including: 
 (1) at least one test subset of microparticles including an antigen positioned thereon capable of binding to specific antibodies found present in the human serum sample in order to detect the presence and concentration of specific antibodies therewithin;  
 (2) at least one calibration subset of microparticles including a first binding partner positioned thereon at known concentrations wherein the known concentration level for each calibration subset is different from other subsets thereof, said first binding partner being capable of binding with human antibodies found in the human serum sample for facilitating calibration of the immunoassay;  
 (3) at least one verification subset of microparticles including a second binding partner thereon at a known concentration to facilitate verification of the calibration provided by said calibration subsets of microparticles; and  
   B. a tagging component adapted for mixing with the multiplexed suspension of microparticles after exposure thereof to a human serum sample, said tagging component being capable of binding with any microparticles of said test subset having specific antibodies bound thereto during exposure to the human serum sample to indicate the amount of specific antibodies bound thereto, said tagging component also being capable of binding with any microparticles of said calibration subsets having human antibodies captured thereto during exposure thereof to the human serum sample for calibration of the immunoassay, said tagging component also being capable of binding with the second binding partner located on said verification subset of microparticles to facilitate verification of the calibration provided by said calibration subsets of microparticles.    
   
   
       2 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said first binding partner comprises an anti-human antibody capable of binding to human antibody present within the human serum sample.  
   
   
       3 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said first binding partner comprises an anti-human immunoglobulin type-G antibody capable of binding to any human immunoglobulin type-G antibody present within the human serum sample.  
   
   
       4 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said first binding partner includes a goat antibody capable of binding to any human antibody present within the human serum sample.  
   
   
       5 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said second binding partner includes a human antibody at a known concentration which is capable of binding to said tagging component to facilitate verification of calibration of the immunoassay provided by said calibration subsets of microparticles.  
   
   
       6 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said second binding partner includes a human immunoglobulin type-G antibody at a known concentration which is capable of binding to said tagging component to facilitate verification of calibration of the immunoassay provided by said calibration subsets of microparticles.  
   
   
       7 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said tagging component includes: 
 A. an attaching component capable of binding to a human antibody; and    B. an indicating component capable of detection to indicate the presence and concentration of said tagging component and material bound with respect thereto, said indicating component being bound to said attaching component such that binding of the attaching component to human antibody will also attach said indicating component thereto in order to facilitate detection and measurement of human antibody present.    
   
   
       8 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 7  wherein said attaching component and said indicating component are conjugated with respect to one another.  
   
   
       9 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 7  wherein said indicating component comprises phycoerythrin.  
   
   
       10 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 7  wherein said attaching component comprises an anti-human antibody.  
   
   
       11 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 10  wherein said anti-human antibody comprises an anti-human immunoglobulin type-G antibody.  
   
   
       12 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 11  wherein said anti-human antibody comprises a goat antibody.  
   
   
       13 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 7  wherein said first binding partner positioned on said calibration subsets of microparticles has the same binding characteristics as said attaching component of said tagging component.  
   
   
       14 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 13  wherein said first binding partner positioned on said calibration subsets of microparticles and said attaching component of said tagging component each comprise the same anti-human antibody.  
   
   
       15 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 13  wherein said first binding partner positioned on said calibration subsets of microparticles and said attaching component of said tagging component each comprise goat antibody.  
   
   
       16 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said multiplexed suspension of microparticles includes three calibration subsets of microparticles wherein each subset thereof includes a different distinguishable concentration of said first binding partner thereon to enhance calibration of the immunoassay.  
   
   
       17 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample as defined in  claim 1  wherein said multiplexed suspension of microparticles is adapted to be mixed with a human serum sample and incubated therewith and thereafter mixed with said tagging component and incubated therewith.  
   
   
       18 . A composition for performing a multiplexed homogeneous immunoassay for detecting antibodies to specific antigens in a human serum sample comprising: 
 A. a multiplexed suspension of microparticles including a plurality of sets thereof for detection, calibration and calibration verification, said multiplexed suspension of microparticles being initially capable of incubation with the human serum sample for facilitating immunoassay thereof, said suspension including: 
 (1) at least one test subset of microparticles including an antigen positioned thereon capable of binding to specific antibodies found present in the human serum sample in order to detect the presence and concentration of specific antibodies therewithin, each test subset of microparticles being separately identifiable;  
 (2) at least two calibration subsets of microparticles including a first binding partner comprising an anti-human immunoglobulin type-G antibody positioned thereon at a known concentration level which is different for each calibration subset of microparticles, said first binding partner being capable of binding with human antibodies found in the human serum sample for facilitating calibration of the immunoassay, each calibration subset of microparticles being separately identifiable from one another and from said test subsets thereof;  
 (3) at least one verification subset of microparticles including a second binding partner comprising a human immunoglobulin type-G antibody thereon at a known concentration to facilitate verification of the calibration provided by said calibration subsets of microparticles, each verification subset of microparticles being separately identifiable from one another and from said test subsets and calibration subsets thereof;  
   B. a tagging component adapted for mixing and incubation with the multiplexed suspension of microparticles after incubation thereof with a human serum sample, said tagging component being capable of binding with any microparticles of said test subset having specific antibodies bound thereto during exposure to the human serum sample to indicate the amount of specific antibodies bound thereto, said tagging component also being capable of binding with any microparticles of said calibration subsets having human antibodies captured thereto during exposure thereof to the human serum sample for calibration of the immunoassay, said tagging component also being capable of binding with the second binding partner located on said verification subset of microparticles to facilitate verification of the calibration provided by said calibration subsets of microparticles, said tagging component including: 
 (1) an attaching component capable of binding to a human antibody, said attaching component including an anti-human immunoglobulin type-G antibody which is the same as the anti-human immunoglobulin type-G antibody comprising said first binding partner located on said calibration subsets of microparticles; and  
 (2) an indicating component capable of detection to indicate the presence and concentration of said indicating component and material bound with respect thereto, said indicating component being conjugated to said attaching component such that binding of the attaching component to human antibody will also attach said indicating component thereto in order to facilitate detection and measurement of human antibody present.  
   
   
   
       19 . A composition for performing a multiplexed homogeneous assay for detecting bound analytes to specific analyte binding partners in a sample comprising: 
 A. a multiplexed suspension of microparticles including a plurality of sets thereof for detection, calibration and verification, said suspension including: 
 (1) at least one test subset of microparticles including an analyte binding partner positioned thereon capable of binding to specific analytes found present within the sample in order to detect the presence and concentration of specific analytes therewithin;  
 (2) at least one calibration subset of microparticles including a first binding partner positioned thereon at known concentrations wherein the known concentration level for each calibration subset is different from other subsets thereof, said first binding partner being capable of binding with general analytes found in the sample for facilitating calibration of the assay;  
 (3) at least one verification subset of microparticles including a second binding partner thereon at a known concentration to facilitate verification of the calibration provided by said calibration subsets of microparticles; and  
   B. a tagging component adapted for mixing with the multiplexed suspension of microparticles after exposure thereof to the sample, said tagging component being capable of binding with microparticles of said test subset having specific analytes bound thereto during exposure to the sample to indicate the amount of specific analyte bound thereto, said tagging component also being capable of binding with microparticles of said calibration subsets having general analytes captured thereon during exposure thereof to the sample for calibration of the assay, said tagging component also being capable of binding with the second binding partner located on said verification subset of microparticles to facilitate verification of the calibration provided by said calibration subsets of microparticles.    
   
   
       20 . A composition for performing a multiplexed homogeneous assay for detecting bound analytes to specific analyte binding partners in a sample as defined in  claim 19  wherein said analyte binding partners comprise antigens and wherein the sample being tested comprises a human serum sample and wherein the analytes comprise human antibodies and wherein said first binding partner is a anti-human antibody and wherein said second binding partner is a human antibody.