Process for the demonstration and determination of reaction components having specific binding affinity for each other

Abstract

The present invention relates to a process for the determination of a component of the reaction between a specific binding protein and the substance being specifically bound by such a protein comprising reacting the component to be determined with its binding partner in an insolubilized form, separating the solid phase of the reaction mixture from the liquid phase, reacting the solid phase with a determined amount of a coupling product of the substance to be determined with an enzyme, and finally determining the enzyme activity of the liquid or solid phase of the reaction mixture obtained. .Badd.

We claim: 
     
       1. Process for the demonstration and determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance, and a protein capable of binding said bindable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein, comprising the steps of: a. providing a given quantity of the binding partner of the component to be determined, which binding partner is in an insoluble form;   b. reacting the component to be determined with its binding partner to form a .Badd.first .Baddend.reaction mixture having a .Badd.first .Baddend.solid phase and a .Badd.first .Baddend.liquid phase;   c. separating the .Badd.first .Baddend.solid phase from the .Badd.first .Baddend.liquid phase;   d. adding to said .Badd.first .Baddend.solid phase a given quantity of a coupling product .[..[.of.]..]. .Badd.formed by coupling .Baddend.the component to be determined with an enzyme, .[..[.and.]..]. .Badd.which quantity of coupling product is at least sufficient to react with all of the binding partner of the component to be determined, to form a second reaction mixture having a second solid phase and a second liquid phase.Baddend.;   .Badd.e. separating the second solid phase from the second liquid phase; and .Baddend.   .[..[.e.]..]. .Badd.f. .Baddend.determining the enzyme activity of the .Badd.second .Baddend.solid phase which is a measure of the quantity of reaction component to be determined.   
     
     
       2. Process for the demonstration and determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten and a low molecular substance, and a protein capable of binding said bindable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein, comprising the steps of: a. providing a given quantity of the binding partner of the component to be determined in soluble form;   .Iadd.b. reacting the component to be determined with its binding partner; .Iaddend.   .[.b..]. .Iadd.c. .Iaddend.adding a given quantity of an antibody against said binding partner, which antibody is an insoluble form, to form a .Badd.first .Baddend.reaction mixture having a .Badd.first .Baddend.solid phase and a .Badd.first .Baddend.liquid phase;   .[.c..]. .Iadd.d. .Iaddend.separating the .Badd.first .Baddend.solid phase from the .Badd.first .Baddend.liquid phase;   .[.d..]. .Iadd.e. .Iaddend.adding to said .Badd.first .Baddend.solid phase a given quantity of a coupling product .[..[.of.]..]. .Badd.formed by coupling .Baddend.the component to be determined with an enzyme, .[..[.and.]..]. .Badd.which quantity of coupling product is at least immunochemically equivalent to the quantity, provided in step c, of the insolubilized antibody against said binding partner, to form a second reaction mixture having a second solid phase and a second liquid phase.Baddend.;   .Badd.f. separating the second solid phase from the second liquid phase; and .Baddend.   .[.e..]. .[..[.f..]..]. .Badd.g. .Baddend.determining the enzyme activity of either the .Badd.second .Baddend.liquid phase .[..[.of.]..]. .Badd.or .Baddend.the .Badd.second .Baddend.solid phase, which activity is a measure of the quantity of the reaction component to be determined.   
     
     
       3. The process of claim 1 in which the enzyme is an oxido-reductase. 4. The 
     
     
        process of claim 2 in which the enzyme is an oxido-reductase. .Iadd.5. Process for the demonstration and determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance, and a protein capable of binding said bindable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein, comprising the steps of: a. providing a given quantity of the binding partner of the component to be determined, which binding partner is in an insoluble form;   b. reacting the component to be determined with its binding partner to form a .Iaddend..Badd.first .Baddend..Iadd.reaction mixture having a .Iaddend..Badd.first .Baddend..Iadd.solid phase and a .Iaddend..Badd.first .Baddend..Iadd.liquid phase;   c. separating the .Iaddend..Badd.first .Baddend..Iadd.solid phase from the .Iaddend..Badd.first .Baddend..Iadd.liquid phase;   d. adding to said .Iaddend..Badd.first .Baddend..Iadd.solid phase a given quantity of a coupling product obtained by binding .Iaddend..Badd.to an enzyme .Baddend..Iadd.a substance capable of reacting specifically with one of the .Iaddend..[..[.reaction components, to an enzyme.]..]. .Badd.reactants of step b, which quantity of coupling product is at least immunochemically equivalent to the quantity, provided in step a, of the insolubilized binding partner of the component to be determined, to form a second reaction mixture having a second solid phase and a second liquid phase.Baddend..Iadd.; .Iaddend..[..[.and.]..].   e. separating the second solid phase from the second liquid phase; and   .[..[.e..]..]. .Badd.f. .Baddend..Iadd.determining the enzyme activity of the .Iaddend..Badd.second .Baddend..Iadd.solid phase which is a measure of   
     
     
        the quantity of reaction component to be determined. .Iaddend. .Iadd.6. Process for the demonstration and determination of a component of the reaction between a bindable substance selected from the group consisting of an antigen, a hapten, and a low molecular substance, and a protein capable of binding said bindable substance specifically, said protein being selected from the group consisting of an antibody and a specific binding protein, comprising the steps of: a. providing a given quantity of the binding partner of the component to be determined in a soluble form;   b. reacting the component to be determined with its binding partner;   c. adding a given quantity of antibody against said binding partner, which antibody is in an insoluble form, to form a .Iaddend..Badd.first .Baddend..Iadd.reaction mixture having a .Iaddend..Badd.first .Baddend..Iadd.solid phase and a .Iaddend..Badd.first .Baddend..Iadd.liquid phase;   d. separating the .Iaddend..Badd.first .Baddend..Iadd.solid phase from the .Iaddend..Badd.first .Baddend..Iadd.liquid phase;   e. adding to said .Iaddend..Badd.first .Baddend..Iadd.solid phase a given quantity of a coupling product obtained by binding .Iaddend..Badd.to an enzyme .Baddend..Iadd.a substance capable of reacting specifically with one of the .Iaddend..[..[.reaction components, to an enzyme.]..]. .Badd.reactants of step b, which quantity of coupling product is at least immunochemically equivalent to the quantity, provided in step c, of the insolubilized antibody against said binding partner, to form a second reaction mixture having a second solid phase and a second liquid phase.Baddend..Iadd.; .Iaddend..[..[.and.]..].   f. separating the second solid phase from the second liquid phase; and   .[..[.f..]..]. .Badd.g. .Baddend..Iadd.determining the enzyme activity of either the .Iaddend..Badd.second .Baddend..Iadd.liquid phase or the .Iaddend..Badd.second .Baddend..Iadd.solid phase, which activity is a measure of the quantity of the reaction component to be determined. .Iaddend. 7. .Badd.The process of claim 5 wherein the coupling product is obtained by binding to an enzyme the binding partner of the component to   
     
     
        be determined. .Baddend. 8.  .Badd.The process in claim 5 wherein the quantity, provided in step a, of the binding partner of the component to be determined is at least sufficient to react with all of the component to be determined. .Baddend. 9. .Badd.The process of claim 6, wherein the coupling product is obtained by binding to an enzyme the binding partner 
     
     
        of the component to be determined. .Baddend. 10.  .Badd.The process of claim 6 wherein the quantity, provided in step a, of the binding partner of the component to be determined is at least sufficient to react with all of the component to be determined, and wherein the quantity, provided in step c, of the insolubilized antibody against said binding partner is at least sufficient to react with all of said binding partner. .Baddend.