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.

Description

o---o- HM-CAP ELISA + 113 3 HM-CAP ELSIA - 3 90 ______________________________________

C. pylori infection was diagnosed as either positive or negative by histological examination and culture of biopsy material, or by the ¹3 C urea breath test.

The HM-CAP ELISA assay detected 113 of the 116 samples from individuals with C. pylori infection. This is a specificity of 97.4%. The sensitivity of the assay is determined by looking at the negative HM-CAP ELISA result. The results show 90 of 93 C. pylori negative individuals were detected. This is a sensitivity of 96.8%. The overall reliability of the test is 97.1% (203 out of 209 samples were accurately predicted). These data strongly indicate that there are few, if any, misclassifications using the ELISA assay.

EXAMPLE 2

KIT: A Kit is prepared by incubating the HM-CAP antigen on a solid phase support. The solid phase support can be any charged membrane or plastic material. The solid phase support-antigen complex can then be packaged individually or in multiple combinations. The kit can also include controls for false positives and false negatives, and reagents. The kit can be used to detect 1 sample or multiple samples.

EXAMPLE 3

Latex agglutination assay: Antibodies against C. pylori can be measured in serum sample with HM-CAP coated latex bead particles. Additionally, the presence of antigens can be measured by coating the latex particles with monospecific antibody (anti-HM-CAP). Particles of polyvinyl or toluene latex of about 0.77 micron diameter or polystyrene latex particles (beads) measuring about 0.81 to 1.77 microns can be coated with HM-CAP. About 2.0 ml of the latex particles are suspended in approximately 20 ml of distilled water, mixed and filtered through a Whatman No. 40 filter paper. After adjusting the filtrate to about 2.0 optical density at a wavelength of 640 nm in phosphate-buffered saline, pH about 7.2., or equivalent buffer, about 0.1 ml of the latex suspension is diluted with about 5.0 ml of PBS. About 0.5 of a 0.5% antigen solution is added to the diluted latex suspension. This mixture is then incubated at about 37°C for 30 minutes. The latex particles are then washed twice, each time with ten volumes of PBS. The final suspension is adjusted to an optical density of 0.3 using 0.1M glycine buffer containing 0.1% bovine serum albumin. In the assay equal volumes of coated latex particles and serum dilutions in 0.1M glycine buffer are mixed. Control tubes receive saline instead of serum. Tubes are incubated at 50°C for 2 hours, centrifuged at 15,000×g for 3 minutes and gently tapped. The degree of clumping is noted. Clumping is due to the aggregation (agglutination) of the beads via the antigen-antibody complex formation.

EXAMPLE 4

Radiommunoassay: A 96-well microtiter plate is coated with about 100 microliters of an optimum concentration of antigen. After incubation at about 37°C for about 18-24 hours, the excess binding sites in the wells are blocked with 1% BSA in PBS, or equivalent. About 100 microliters per well of appropriate dilution(s) of serum to be tested are added to duplicate wells and incubated at room temperature for about 2 hours. After washing the plates numerous times with PBST, 100 microliters per well of optimal dilution of goat or rabbit anti-human IgG, which has been labeled with 125-iodine, is added. The microliter plates are incubated for about 4 hours at room temperature, washed numerous times with PBST and air dried. The wells are counted in a gamma counter to determine the amount of radioactivity remaining in each well at the end of the test. Positive and negative sera are included on each plate as controls. The procedure is standardized to determine which values differentiate between a positive and a negative serum.

One skilled in the art will really readily appreciate the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as, those inherent therein. The methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary, and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the appended claims.

Claims

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;

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, a container having false negative controls and a control having false positive controls.

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.

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. 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. 13. The serological assay of claim 12, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase and beta galactosidase. 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. 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. 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.

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. 18. The kit of claim 17, further comprising, a container having false negative controls and false positive controls.