Process for preparation of high molecular weight cell-associated protein of Campylobacter pylori and use for serological detection of Campylobacter pylori infection

Abstract

An antigen for the detection of Campylobacter pylori infections and an assay for the serological detection of Campylobacter pylori. The antigen includes high molecular weight cell-associated proteins purified from Campylobacter pylori. The antigen can be used in a variety of assays including radioimmunoassay, ELISA, latex agglutination, complement fixation, and indirect hemagglutination. Furthermore, the antigens can be combined with a solid support in kit form.

What is claimed is: 
     
       1. A composition comprising substantially purified antigens from the high molecular weight cell-associated proteins of Campylobacter pylori, said antigens: having a molecular weight of about 300,000 to 700,000 daltons as determined by agarose A-5 m column;   having a PI on isolectric focusing of about 5.9 to 6.3;   having urease activity.Iadd.; .Iaddend.   being soluble in PBS and Tris-chloride buffers;   being derived from the outer surface of the membrane of Campylobacter pylori; and   being solubilized from the outer surface of the membrane with n-octyl-glucoside.   
     
     
       2. A serological assay for the detection of Campylobacter pylori infection, comprising, combining the antigens of claim 1 with a serum sample to be tested according to a method selected from the group consisting of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, indirect hemagglutination and latex agglutination wherein measurement of an antigen-antibody complex is proportional to the amount of Campylobacter pylori in the serum sample. 
     
     
       3. The serological assay of claim 2, wherein said method is the enzyme-linked immunosorbent assay, which includes the steps of: adding serum sample to an antigen immobilized on a solid phase support;   incubating the mixture of serum sample and immobilized support to form an antigen-antibody complex;   adding enzyme-conjugated anti-human IgG to said antigen-antibody complex;   incubating the antigen-antibody complex and enzyme-conjugated anti-human IgG mixture to form an antigen-antibody-enzyme-conjugated anti-human IgG complex;   adding substrate to the antigen-antibody-enzyme-conjugated anti-human IgG complex; and   measuring the product or the change in the substrate to determine the amount of said antibody; wherein the product or change in the substrate measured is proportional to the amount of Campylobacter pylori in the serum sample.   
     
     
       4. The serological assay of claim 3, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and beta galactosidase. 
     
     
       5. A method of monitoring the treatment of Campylobacter pylori infection, comprising, collecting serial serum samples from the treated subject and repeating the steps of claim 3 on each sample. 
     
     
       6. The serological assay of claim 2, wherein said method is the radioimmunoassay, which includes the steps of: adding serum sample to a well coated with antigen;   incubating said serum sample in said coated well to form an antigen-antibody complex;   adding radioactive labeled anti-human IgG;   incubating the mixture of the antigen-antibody complex and anti-human IgG to form an antigen-antibody-anti-human IgG complex; and   measuring the amount of radioactivity bound in the antigen-antibody-anti-human IgG complex; wherein the amount of radioactivity bound is proportional to the amount of Campylobacter pylori in the serum sample.   
     
     
       7. The serological assay of claim 2, wherein said method is latex agglutination, which includes the steps of: adding serum sample to latex beads coated with antigen;   incubating the serum sample and coated latex beads; and   measuring the degree of clumping; wherein the degree of clumping is proportional to the amount of Campylobacter pylori in the serum sample.   
     
     
       8. A kit for determining the presence of Campylobacter pylori antibody, comprising, a container having the antigens of claim 1 immobilized on a solid phase support. 
     
     
       9. The kit of claim 8, further comprising, .Iadd.a container having .Iaddend.false negative controls and .[.a control having.]. false positive controls. .Iadd. 
     
     
       10.  A composition comprising substantially purified antigens from the high molecular weight cell-associated proteins of Campylobacter pylori, said antigens: having a molecular weight of about 300,000 to 700,000 daltons as determined by agarose A-5 m column;   having a PI on isolectric focusing of about 5.9 to 6.3;   having urease activity;   being soluble in PBS and tris-chloride buffers;   being derived from the outer surface of the membrane of Campylobacter pylori; and   being capable of being solubilized from the outer surface of the membrane with n-octyl-glucose. .Iaddend. .Iadd.   
     
     
       11.  A serological assay for the detection of Campylobacter pylori infection, comprising, combining the antigens of claim 10 with a serum sample to be tested according to a method selected from the group consisting of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, indirect hemagglutination and latex agglutination wherein measurement of an antigen-antibody complex is proportional to the amount of Campylobacter pylori in the serum sample. .Iaddend. .Iadd.12. The serological assay of claim 11, wherein said method is the enzyme-linked immunosorbent assay, which includes the steps of: adding serum sample to an antigen immobilized on a solid phase support;   incubating the mixture of serum sample and immobilized support to form an antigen-antibody complex;   adding enzyme-conjugated anti-human IgG to said antigen-antibody complex;   inhibiting the antigen-antibody complex and enzyme-conjugated anti-human IgG mixture to form an antigen-antibody-enzyme-conjugated anti-human IgG complex;   adding substrate to the antigen-antibody-enzyme-conjugated anti-human IgG complex; and   measuring the product or the change in the substrate to determine the amount of said antibody; wherein the product or change in the substrate measured is proportional to the amount of Campylobacter pylori in the serum sample. .Iaddend. .Iadd.13. The serological assay of claim 12, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and beta galactosidase. .Iaddend. .Iadd.14. A method of monitoring the treatment of Campylobacter pylori infection, comprising, collecting serial serum samples from the treated subject and repeating the steps of claim 12 on each sample. .Iaddend. .Iadd.15. The serological assay of claim 11, wherein said method is the radioimmunoassay, which includes the steps of:   adding serum sample to a well coated with antigen;   incubating said serum sample in said coated well to form an antigen-antibody complex;   adding radioactive labeled anti-human IgG;   incubating the mixture of the antigen-antibody complex and anti-human IgG to form an antigen-antibody-anti-human IgG complex; and   measuring the amount of radioactivity bound in the antigen-antibody-anti-human IgG complex; wherein the amount of radioactivity bound is proportional to the amount of Campylobacter pylori in the serum sample. .Iaddend. .Iadd.16. The serological assay of claim 11, wherein said method is latex agglutination, which includes the steps of:   adding serum sample to latex beads coated with antigen;   incubating the serum sample and coated latex beads; and   measuring the degree of clumping; wherein the degree of clumping is proportional to the amount of Campylobacter pylori in the serum sample.   
     
     
        .Iaddend. .Iadd.17.  A kit for determining the presence of Campylobacter pylori antibody, comprising, a container having the antigens of claim 10 immobilized on a solid phase support. .Iaddend. .Iadd.18. The kit of claim 17, further comprising, a container having false negative controls and false positive controls. .Iaddend.