Antigen for trachoma lymphogranuloma venereum (LGV) and non-gonococcal urethritis (NGU)

4,118,469

05/680,927

April 27, 1976

Solubilized antigens of C. trachomatis strain LGV-434 upon analysis using two-dimensional immunoelectrophoresis yielded a single antigen which was found to be consistently precipitated by sera of patients with C. trachomatis infections. This antigen as antigen-antibody complex was employed as an immunogen to prepare a rabbit monospecific antiserum to this component or antigen. This monospecific antiserum demonstrated the presence of the antigen in each of the 15 strains of C. trachomatis organisms and was non-reactive with strains of C. psittaci. The C. trachomatis specific antigen was purified by immunoadsorption chromatography using monospecific antiserum as a specific ligand covalently bound to agarose gel columns and the resulting purified antigen employed to detect antibody from the sera of lymphogranuloma venereum patients using counterimmunoelectrophoresis. When the C. trachomatis specific antigen is isotopically labeled and utilized in the highly sensitive radioimmune assay antibody to the antigen should be demonstrated and a serological test based thereon should be applicable for the serological diagnosis of non-gonococcal urethritis (NGU).

Caldwell, Harlan D. (Seattle, WA); Kuo, Cho-Chou (Seattle, WA); Kenny, George E. (Seattle, WA)

Research Corporation (New York, NY)

We claim

1. Chlamydia trachomatis specific antigen useful for the serological diagnosis of lymphogranuloma venereum, said antigen having a molecular weight of about 160,000, being stable to ribonuclease and deoxyribonuclease and at a pH in the range 2.2-10.6 but whose antigenicity is destroyed by protease treatment and heating at a temperature of 56.degree. C. for about 30 minutes.

2. Radioactive isotopically labeled Chlamydia trachomatis specific antigen of claim 1.

3. An aqueous composition comprising the antigen of claim 1, said composition having a pH in the range from about 7 to about 8.6 suitable for low temperature storage.

4. An aqueous composition comprising the antigen of claim 2, said composition having a pH in the range from about 7 to about 8.6 suitable for low temperature storage.

5. A method of preparing Chlamydia trachomatis antigen which is consistently precipitated by sera of patients with C. trachomatis infections which comprises solubilizing C. trachomatis in the presence of a non-ionic surfactant and recovering the resulting solubilized C. trachomatis antigen by immunoadsorption chromatography employing monospecific antiserum to said antigen as a specific ligand for said antigen.

6. A method in accordance with claim 5 wherein said non-ionic surfactant is a 1% (volume/volume) solution of an alkyl aryl polyether alcohol.

7. A method of isolating Chlamydia trachomatis antigen in the form of its antigen-antibody complex which comprises solubilizing C. trachomatis antigen and subjecting the resulting solubilized antigen to two-dimensional immunoelectrophoresis and recovering the antigen-antibody complex.

8. A method in accordance with claim 7 wherein said C. trachomatis antigen is solubilized by means of an non-ionic surfactant.

9. A method of preparing monospecific antiserum to antigen of Chamydia trachomatis which comprises solubilizing the antigens of C. trachomatis in the presence of a non-ionic surfactant, subjecting the resulting solubilized antigens to two-dimensional immunoelectrophoresis on agarose, thereby precipitating the C. trachomatis antigen as an antigen-antibody complex, recovering said antigen-antibody complex, introducing said resulting recovered antigen-antibody complex into rabbit, thereby causing the production of antiserum therein and recovering rabbit serum containing the resulting produced antiserum.

10. A method for the serological testing of antibody of Chlamydia trachomatis which comprises subjecting serum of suspected lymphogranuloma venereum patients to counterimmunoelectrophoresis on agarose employing the antigen of claim 1 in said counterimmunoelectrophoresis, observing during said counter immunoelectrophoresis for the presence of resulting antigen-antibody complex, and detecting said complex by staining said agarose containing said antigen-antibody complex with a suitable dye.

11. A method for the serological testing of antibody to Chlamydia trachomatis which comprises subjecting serum of suspected non-gonococcal urethritis patients to radioimmune assay employing the radioactive isotopically labeled antigen of claim 2 in order to detect formation of antigen-antibody complex.

12. A lyophilized composition containing the antigen of claim 1.

13. A lyophilized composition containing a radioactive isotopically labeled antigen of claim 2.

14. A stable composition containing the antigen of claim 1 at a concentration of about 30 .mu.g/ml.

15. An agarose coated inert substrate, the agarose coating on said substrate being provided with at least two wells cut thereinto, one of said wells having deposited therein the antigen of claim 1 and another of said wells being suitable for the placement therein of serum from a patient for the diagnosis of lymphogranuloma venereum.

16. An agarose coated inert substrate in accordance with claim 15 wherein said well has antigen deposited therein in an amount of about 60 .eta.g.

17. An agarose coated substrate in accordance with claim 15 wherein said inert substrate is a glass slide.

18. An agarose coated inert substrate in accordance with claim 15 wherein said agarose coating comprises an non-ionic detergent together with a barbital buffer, said agarose coating having been applied at a pH of about 8.6.

424/1; 23/230B; 260/112R; 424/12

G01N 3316; A61K 4300

2355676 August 1944 Rake et al.
2415234 February 1947 Bunney et al.
3646346 February 1972 Catt

Collins et al., Chemical Abstracts, vol. 72, No. 11, Mar. 16, 1970, p. 216, abstract No. 53182d.
Haider et al., Chemical Abstracts, vol. 74, No. 3, Jan. 18, 1971, p. 175, abstract No. 11472j.
Chemical Trade Names and Commercial Synonyms, Haynes, Van Nostrand Co., Inc., NY, 1955, p. 428.

edspgr.04118469