Enzymatic immunological method for determination of antigens and antibodies

Abstract

The present invention relates.Iadd., inter alia, .Iaddend.to improvements in the sandwich technique for the determination of a component of an antigen-antibody reaction in a liquid sample to be tested, utilizing as reagents (a) one component of said reaction bound to the surface of a water-insoluble, water-insuspensible, solid carrier, and (b) a component having the same immunological properties covalently linked to an enzyme. .Iadd.If and only if the component of the reagent that is water-insoluble and water-insuspensible has the same immunochemical properties as the component to be determined, is a predetermined amount of a binding partner for said first reagent (e.g., the reagent that is water-insoluble and water-insuspensible) added to the liquid sample. .Iaddend.The liquid sample is contacted and incubated with the reagent(s) to form a reaction mixture, the enzyme activity of either the liquid or solid phase of which is a measure of the presence and quantity of the component to be determined. The method is especially useful for diagnostic testing for hepatitis or rubella antibodies.

What is claimed is: .[. 
     
       1.  A method for the detection and determination of a component of an antigen-antibody reaction in a liquid sample containing the component to be determined, comprising the steps of: a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody bound to the surface of a water-insoluble, water-insuspensible, solid carrier;   b. providing a given amount of a second reagent consisting of a component having the same immunological properties as the component in the said first reagent covalently linked to an enzyme, and also providing a predetermined amount of the binding partner for the component to be determined, when the component in said liquid sample and the component bound to said solid carrier have the same immuno-chemical properties;   c. contacting a given quantity of said liquid sample with said reagents forming a reaction mixture having a solid phase and a liquid phase; and   d. determining the enzyme activity of either the solid or the liquid phase which is a measure of the presence and quantity of the component to be determined..]. .[.2. A method for determining the presence of a component of an antigen-antibody reaction in a liquid sample containing the component to be determined comprising the steps of:   a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody bound to the surface of water-insoluble, water-insuspensible, solid carrier, and also providing a predetermined amount of the binding partner for the component to be determined when the component in said liquid sample and the component bound to said solid carrier have the same immunochemical properties;   b. contacting and incubating a given quantity of said liquid sample with said first reagent;   c. washing said solid carrier;   d. providing a given quantity of a substance having the same immunological properties as the component previously bound to the solid carrier, said substance being covalently linked to an enzyme;   e. contacting and incubating the solid phase from step (c) with said enzyme-linked substance;   f. washing the solid carrer; and   g. determining the enzyme activity substance bound to the solid phase, which is a measure of the presence and quantity of the component to be determined..]. .[.3. The method of claim 2 wherein said liquid sample and the binding partner of the component to be determined are preincubated with each other, before contacting the sample with said solid carrier..].   
     
     
        .[.4.  A method for the detection and determination of a component in the reaction of a hepatitis antigen and the associated antibody in a liquid sample containing said component, comprising the steps of: a. providing a given quantity of a reagent consisting of one component of said reaction selected from the group consisting of said antigen and said antibody bound to a water-insoluble, water-insuspensible, solid carrier;   b. contacting and incubating said liquid sample with said solid carrier of step (a) to form a reaction mixture;   c. providing a given quantity of a substance having the same immunological properties as the component bound to said solid carrier, said substance being covalently linked to an enzyme, and also providing a predetermined amount of the binding partner for the component to be determined when the component in said liquid sample and the component bound to said solid carrier have the same immunochemical properties;   d. contacting and incubating the reaction mixture with said enzyme-linked substance; and p1 e. determining the enzyme activity of substance bound to the solid phase, which is a measure of the presence and quantity of the component to be determined..]. .[.5. The method of claim 4 in which said hepatitis antigen is hepatitis B surface antigen, and the antibody is its associated antibody..]. .[.6. The method of claim 4 in which said solid carrier is washed after the incubation of the liquid sample with the immunological component bound to the solid carrier..]. .[.7. A diagnostic pack for the detection and determination of a component of an antigen-antibody reaction selected from the group consisting of an antigen and an antibody, consisting of:   a. one of said components coupled to a water-insoluble, water-insuspensible, solid carrier;   b. a substance having the same immunological properties as the component in (a) covalently linked to an enzyme; and   c. the binding partner of the component to be determined if the component to be determined has the same immunological properties as the component in (a)..]. .[.8. A diagnostic pack for the detection and determination of hepatitis B consisting of:   a. the antibody against Hepatitis B surface antigen coupled to a water-insoluble, water-insuspensible, solid carrier;   b. said antibody covalently linked to an enzyme; and   c. Hepatitis B surface antigen in case the component to be determined is   
     
     
        the antibody against Hepatitis antigen..]. .Iadd.9.  A method for the detection and determination of an antibody in a liquid sample containing said antibody to be detected and to be determined, comprising the steps of: a. providing a given quantity of an antigen to said antibody, said antigen bound to the surface of a water-insoluble, water-insuspensible, solid carrier;   b. contacting and incubating a given quantity of said liquid sample having the antibody to be detected and determined with bound antigen of step (a), forming a reaction mixture having a solid phase and a liquid phase;   c. separating the solid phase from the liquid phase;   d. contacting and incubating with said solid phase a given quantity of a coupling product obtained by covalently binding a component having the same immunological properties as said antigen, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to said antigen provided in step (a), in order to form a second solid phase and a second liquid phase; and   e. detecting and determining the enzyme activity of either the second solid or the second liquid phase of step (d) after separating the solid and liquid phases formed thereby, which detection and determination is a measure of the presence and quantity of said antibody to be detected and to be determined..Iaddend. .Iadd.10. The method of claim 9, wherein the quantity of said bound antigen in step (a) is at least sufficient to react with all of the antibody to be determined..Iaddend. .Iadd.11. The method of claim 9, wherein an additional step of separating the solid phase from the liquid phase takes place between steps (d) and (e)..Iaddend. .Iadd.12. The method of claim 11, wherein both separation steps are accomplished by   
     
     
        washing the solid carrier..Iaddend. .Iadd.13.  A method for the detection and determination of a component of an antigen-antibody reaction in a liquid sample containing said component to be detected and determined, comprising the steps of: a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody bound to the surface of a water-insoluble, water-insuspensible, solid carrier with the proviso that said first reagent has the same immunochemical properties as the component to be detected and determined;   b. providing a predetermined amount of a binding partner for said first reagent, which amount is less than or is immunochemically equivalent to the given quantity provided in step (a) of the insolubilized component,   c. contacting and incubating a given quantity of said liquid sample having the component to be detected and determined with said reagents of step (a) and step (b), forming a reaction mixture having a solid phase and a liquid phase;   d. separating the solid phase from the liquid phase;   e. contacting and incubating with said solid phase a given quantity of a coupling product obtained by covalently binding a component having the same immunological properties as the first reagent, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to the first reagent provided in step (a), in order to form a second solid phase and a second liquid phase; and   f. detecting and determining the enzyme activity of either the second solid phase or the second liquid phase of step (e) after separating the solid and liquid phases formed thereby, which detection and determination is a measure of the presence and quantity of the component to be detected and to be determined. .Iaddend. .Iadd.14. The method of claim 13, wherein said liquid sample and the binding partner of the component to be determined are preincubated with each other before contacting the sample with said   
     
     
        solid carrier..Iaddend. .Iadd.15.  A method for the detection and determination of an antibody in a liquid sample containing the antibody to be detected and determined, comprising the steps of: a. providing a given quantity of an antigen to said antibody, which antigen is bound to the surface of a water-insoluble, water-insuspensible, solid carrier;   b. contacting and incubating a given quantity of said liquid sample having said antibody to be detected and to be determined with said bound antigen of step (a), forming a reaction mixture having a solid phase and a liquid phase;   c. washing said solid carrier to separate the solid phase from the liquid phase;   d. contacting and incubating with said solid phase a given quantity of a coupling product, obtained by binding covalently a component having the same immunological properties as the antigen previously bound to the solid carrier, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to said antigen provided in step (a) to form a second solid and second liquid phase;   e. washing the solid carrier to separate the solid phase from the liquid phase of step (d); and   f. detecting and determining the enzyme activity of the solid phase of step (d) after said washing, which detection and determination is a measure of the presence and quantity of said antibody to be detected and to be determined..Iaddend. .Iadd.16. The method of claim 15, wherein said liquid sample and the binding partner of the antibody to be determined are preincubated with each other, before contacting the sample with said solid   
     
     
        carrier..Iaddend. .Iadd.17.  A method for the detection and determination of a component of an antigen-antibody reaction in a liquid sample containing said component to be detected and determined, comprising the steps of: a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody, which component is bound to the surface of a water-insoluble, water-insuspensible, solid carrier, with the proviso that said first reagent has the same immunochemical properties as the component to be detected and determined;   b. providing a predetermined amount of a binding partner for the first reagent, which amount is less than or is immunochemically equivalent to the given quantity provided in step (a) of the insolubilized component,   c. contacting and incubating a given quantity of said liquid sample having the component to be detected and to be determined with said first reagent of step (a) and step (b), forming a reaction mixture having a solid phase and a liquid phase;   d. washing said solid carrier to separate the solid phase from the liquid phase;   e. contacting and incubating with said solid phase a given quantity of a coupling product, obtained by binding covalently a component having the same immunological properties as the component previously bound to the solid carrier, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to the component of the first reagent provided in step (a) to form a second solid and second liquid phase;   f. washing the solid carrier to separate the solid phase from the liquid phase of step (e); and   g. detecting and determining the enzyme activity of the solid phase of step (e) after said washing, which detection and determination is a measure of the presence and quantity of the component to be detected and to be   
     
     
        determined..Iaddend. .Iadd.18.  A method for the detection and determination of an antibody specific to an hepatitis antigen in a liquid sample containing said antibody, comprising the steps of: a. providing a given quantity of an hepatitis antigen, which antigen is bound to a water-insoluble, water-insuspensible, solid carrier;   b. contacting and incubating said liquid sample having the antigen to be detected and determined with said bound antigen of (a) to form a reaction mixture;   c. separating the first solid phase from the first liquid phase;   d. contacting and incubating with said solid phase a given quantity of a coupling product obtained by covalently binding a component having the same immunological properties as the antigen bound to said carrier, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to the first reagent provided in step (a) to form a second solid and second liquid phase;   e. contacting and incubating the reaction mixture to form a solid phase and a liquid phase; and   f. detecting and determing the enzyme activity of the solid phase of step (d) after separating the solid and liquid phases formed therein, which detection and determination is a measure of the presence and quantity of the antibody to be detected and determined..Iaddend. .Iadd.19. The method of claim 18 in which said hepatitis antigen is hepatitis B surface antigen   
     
     
        and the antibody is its associated antibody..Iaddend. .Iadd.20.  A method of the detection and determination of a component in the reaction of a hepatitis antigen and the associated antibody specific to said antigen in a liquid sample containing said component, comprising the steps of: a. providing a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of said antigen and said antibody, which component is bound to a water-insoluble, water-insuspensible, solid carrier with the proviso that said first reagent has the same immunochemical properties as the component to be detected and determined;   b. providing a predetermined amount of a binding partner for the first reagent, which amount is less than or is immunochemically equivalent to the given quantity provided in step (a) of the insolubilized component;   c. contacting and incubating said liquid sample having the component to be detected and determined with said reagent of step (a) and step (b), to form a reaction mixture;   d. separating the first solid phase from the first liquid phase;   d. contacting and incubating with said solid phase a given quantity of a coupling product obtained by covalently binding a component having the same immunological properties as the component bound to said carrier, to an enzyme, which quantity of coupling product is at least immunochemically equivalent to the first reagent provided in step (a) to form a second solid and second liquid phase;   f. contacting and incubating the reaction mixture to form a solid phase and a liquid phase; and   g. detecting and determining the enzyme activity of the solid phase of step (e) after separating the solid and liquid phases formed therein, which detection and determination is a measure of the presence and quantity of the component to be detected and determined..Iaddend. .Iadd.21. The method of claim 20 in which said hepatitis antigen is hepatitis B surface antigen   
     
     
        and the antibody is its associated antibody..Iaddend. .Iadd.22.  A diagnostic pack for the detection and determination of a component of an antigen-antibody reaction selected from the group consisting of an antigen and an antibody, consisting of: a. a given quantity of a first reagent consisting of one component of said reaction selected from the group consisting of an antigen and an antibody bound to the surface of a water-insoluble, water-insuspensible, solid carrier;   b. a given quantity of a coupling product obtained by covalently binding a component having the same immunological properties as the component in (a) to an enzyme, which quantity of coupling product is at least immunochemically equivalent to the component of the first reagent provided in (a); and   c. a predetermined amount of a binding partner of the first reagent, which amount is less than or is immunochemically equivalent to the given   
     
     
        quantity (a) of the insolubilized component..Iaddend. .Iadd.23.  A diagnostic pack for the detection and determination of an antibody, consisting of: a. a given quantity of an antigen bound to the surface of a water-insoluble, water-insuspensible, solid carrier; and   b. a given quantity of an antigen having the same immunological properties as the antigen in (a) coupled to an enzyme, which quantity of antigen is at least immunochemically equivalent to said antigen provided in (a)..Iaddend. .Iadd.24. A diagnostic pack for the detection and determination of antibody directed against hepatitis B, consisting of:   a. a given quantity of hepatitis B surface antigen coupled to a water-insoluble, water-insuspensible, solid carrier; and   b. a given quantity of a coupling product obtained by covalently binding hepatitis B surface antigen to an enzyme, which quantity is at least immunochemically equivalent to the antigen in (a)..Iaddend.