Process and reagent for the determination of an antibody

Abstract

The present invention provides a process for the determination of an antibody by incubation with three different reagents R 1 , R 2 and R 3 , of which R.sub. and R 3 are present in liquid phase and are bindable with the antibody, R 2 is present bound to a solid phase and is bindable with R 1 and R 3 carries a label, separation of the solid phase from the liquid phase and measurement of the label in the solid phase, wherein as R 1 there is used a conjugate of a substance specifically recognized by the antibody to be determined and a reaction component of a specific binding system, as R 3 there is used a conjugate of a substance specifically recognized by the antibody to be determined and a label and as R 2 there used the other binding component of the specific binding system. The present invention also provides a composition for the determination of an antibody, wherein it contains two different soluble reagents R 1 and R 3 specifically bindable with the antibody, of which R 1 carries a reaction component of a specific binding system and R 3 carries a label and also a receptor R 2 which is present in solid phase and is bindable with the receptor R 1 , the insoluble receptor R 2 and the soluble receptor R 1 being present physically separated from one another.

We claim: 
     
       1. Process for the determination of an antibody comprising incubating an antibody containing sample with three different reagents R 1 , R 2  and R 3 , wherein R 1  and R 3  are in liquid phase and R 2  is .[.found.]. .Iadd.bound .Iaddend.to a solid phase and binds to R 1  but not R 3 , wherein R 1  comprises a conjugate of (i) a substance having at least one epitope specific for a paratope of an antibody to be determined and is specifically recognized by said antibody to be determined and (ii) a first reaction component of a two part, specific binding system, R 3  comprises a conjugate of (i) a substance having at least one epitope which binds to the same paratope of the antibody to be determined as does R 1  and is specifically recognized by said antibody to be determined and (ii) a label, wherein R 1  and R 3  compete for binding to the two identical paratopes of the antibody to be determined, and R 2  comprises a second reaction component of said two part, specific binding system, so as to bind R 1 , complexes of R 1  and antibody, and conjugates of R 1 , antibody and R 3  to said solid phase, separating liquid phase from said solid phase and measuring R 3  bound to said solid phase as a measure of antibody in said sample. 
     
     
       2. Process according to claim 1, wherein, as specific binding system, there is used biotin/avidin; biotin/streptavidin; biotin/antibiotin; hapten/antihapten; an Fcγ fragment of an antibody/antibody against the Fcγ fragment; or carbohydrate/lectin. 
     
     
       3. Process according to claim 1, wherein the reagent R 3  is labelled with an enzyme or with a fluorescing, chemiluminescing or radio-active substance. 
     
     
       4. Process according to claim 1, wherein all three reagents are added simultaneously. 
     
     
       5. Process according to claim 1, wherein the reagents R 1  and R 3  are pre-incubated with the sample and subsequently incubated with R 2 . 
     
     
       6. Process according to claim 1, wherein the amount of substance in R 1  specifically recognised by the antibody to be determined is greater than or equal to the amount of substance in R 3  specifically recognised by the antibody to be determined. 
     
     
       7. Process according to claim 6, wherein the ratio of the substance in R 1  specifically recognised by the antibody to be determined to the substance in R 3  specifically recognised by the antibody to be determined is 20 to 1:1. 
     
     
       8. Process according to claim 7, wherein the said ratio is from 5 to 1:1. 
     
     
       9. Composition useful in the determination of an antibody comprising: (a) a first soluble reagent R 1  which comprises a conjugate of (i) a substance having at least one epitope specific for a paratope of an antibody to be determined and is specifically recognized by said antibody to be determined, and (ii) a first reaction component of a two part, specific binding system;   (b) a second soluble reagent R 3  which comprises a conjugate of (i) a substance having at least one epitope which binds to the same paratope of the antibody to be determined as does R 1  and is specifically recognized by said antibody to be determined and (ii) a label; and   (c) a solid phase bound reagent R 2  which comprises a second reaction component of said two part, specific binding system wherein R 1  and R 2  are physically separated from each other.   
     
     
       10. Composition according to claim 9, wherein the reagent R 2  or the reagent R 1  are present impregnated on a carrier material, each physically separated from one another. .Iadd. 
     
     
       11.  Process of claim 1, wherein said antibody specifically binds to hepatitis B surface antigen, R 1  is a conjugate of biotin and hepatitis B surface antigen, R 2  comprises streptavidin, and R 3  is a conjugate of hepatitis B surface antigen and peroxidase. .Iaddend. .Iadd.12. Process of claim 11, wherein R 2  further comprises thermo bovine serum albumin. .Iaddend. .Iadd.13. Composition of claim 9, wherein R 1  is a conjugate of biotin and hepatitis B surface antigen, R 2  comprises streptavidin, and R 3  is a conjugate of hepatitis B surface antigen and peroxidase. .Iaddend. .Iadd.14. Composition of claim 13, wherein R 2  is bound to a test tube. .Iaddend. .Iadd.15. Composition of claim 13, wherein R 2  further comprises thermo bovine serum albumin. .Iaddend.