GB2047889A - Detection of antibodies to chlormydia trachomatis having an immunofluorescence test - Google Patents

Abstract

Elemental bodies derived from strains of predetermined immunotypes of C. trachomatis are isolated, stabilized with formaldehyde and utilized as antigen for the determination of antibody immunotypes present in suspected sources of antibody to C. trachomatis in a microimmunofluorescence test.

Description

SPECIFICATION Immunofluorescence test and substance for use therein This invention relates to immunofluorescent test methods and to substances used therein.

Chlamydia trachomatis organisms are the etiological agents for a number of human ocular-genitai diseases, such as ocular trachoma, lymphogranuloma venereum (LGV) and non-gonococcal urethritis (NGU). The most important of these diseases in the United States is NGU. The annual incidence of NGU exceeds that of gonorrhea and it is estimated that there occur more than 2,000,000 cases of NGU yearly in the United States along.

2. Description of the Prior Art The most sensitive and specific serology of Chlamydia trachomatis has been the indirect microimmunofluorescence antibody test (micro-IF) of Wang and Grayston (J. Infect. Dis 130 388 (1974)), currently used in several major chlamydial research laboratories throughout the world. However, due to the difficulty of the test, it has not yet been incorporated as a diagnostic procedure in general clinical laboratories. The standard micro-lF test requires inclusion of 1 5 C. trachomatis immunotype elementary body (EB) antigens.

In the method of Wang and Grayston the antigen was derived from yolk sac culture. The EBs themselves were not isolated and used per se in the test, but utilized in conjunction with the yolk sac suspension. Technically, the micro-lF requires either the intact morphology or structure of the organism or a substrate for identification of the immunofluorescent reaction. However, if an intact form of the organism could be made readily available which would be capable of providing cross-reactivity to antisera of each of the immunotypes, this antigenic form could provide the sensitivity needed. This would be especially desirable if the somewhat difficult egg yolk sac culture could be avoided.

Christophersen and Manire (J. Immunol. 103, 1085 (1969)), working with C. Psittaci observed that the two developmental forms of chlamydiae, i.e., reticulated bodies (RBs) and elemental bodies (EBs) have distinct characteristics. RB is non-infectious and does not contain the characteristic chlamydial "toxins", EB is infectious and contains the "toxins". Wang observed an exact correlation of the specificity of the mouse toxicity prevention test (Wang, et al. 1 963. Classification of trachoma virus strains by protection of mice from toxic death. J. Immunol. 90: 849-856.) and the micro-lF (Wang, et al. 1 971. A micro immunofluorescence method. Study of antibody response to TRIC organisms in mice, p. 273-288. In R. L.Nichols (3d.), Trachoma and related disorders caused by chlamydial agents.

Excerpta Medica, Amsterdam.). This observation indicated that the type-specific antigens might be the same as the "toxins" in the more mature EB form.

SUMMARY OF THE INVENTION It has been found that EB derived from strains having antigenic reaction to antibodies to strains from each of the known immunotypes of C. trachomatis used as antigen in the micro-lF test crossreacted with antibodies to each of the known C. trachomatis immunotypes. Furthermore, the use of EBs rather than EBs absorbed on yolk sacs residues makes for easier reading in the fluorescent antibody microscope. In addition, the antigen when stabilized can be stored at 40C for at least 1 year, making the EB antigen extremely useful in the serological screening of sera for chlamydial antibodies even in laboratories not specialized in chlamydia research.

By utilizing IgM of IgG antihuman antibodies the test is further refined since IgM antibodies will only react in the case of inactive infection while igG antibody will react when any antibody to the EB antigen was present in the serum.

Antigen preparations have been made for each of the 1 5 immunotypes, formalinized, and stored at 40C for at least 10 months. They have been successfully used for immunotyping of new strains and for detection of type-specific antibodies in serum and secretions including, by specific immunoglobulin classes, IgM, IgG, and Secretory-lgA.

The preparation of formalinized antigens from HeLa 229 grown organisms further simplified the C.

trachomatis micro-lF test. The ability to successfuily stabilize the type specific surface antigen with formalin without ioss of specificity or sensitivity provides the opportunity for reference laboratory or commercial distribution of the antigens. Provision of micro-lF antigen will greatly increase the availabiiity of laboratory diagnosis of C. trachomatis infection. The use of formalinized organisms also eliminates the hazard of laboratory infection.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The C. trachomatis strains utilized in the preparation of the reticulated body which is the subject of the present invention are grown in a tissue culture suitably in HeLa 229 monolayers in accordance with the method of Kuo, etai(Growth of trachoma organisms in HeLa 229 cell culture, p.328-336. In D.

Hobson and K. K. Holmes (ed.), Nongonococcal urethritis and related infections. ASM Publications, Washington, DC.). In this method, one-day old HeLa cell monolayers are inoculated with about 10 inclusion forming units/cell of organisms. The infected cells are harvested in the late stages of growth to assure a substantial predominance of EB over RB form. Such harvesting should suitably be done where the culture shows more than 80% infectivity, suitably at about 72 hours post infection. The cells are then disrupted for a very brief period suitably about 30 seconds by sonication and subjected to one cycle of differential contrifugation. The pellet is suspended in phosphate buffered saline and resonicated for about 10 minutes to destroy any remaining RBs.

The thus obtained pellet is then layered on a suitable high-density medium suitably renografin preferably 30% renografin and centrifuged. This centrifugation serves to retain low molecular weight components. The resultant pellet containing the desired high molecular weight component is resuspended in a suitable buffer and recentrifuged to remove residual renografin. The residual pellet is resuspended in buffer and in the preferred embodiment of this invention, stabilized.

Such stabilization may be carried out by treatment with formaldehyde suitably formalin or with gluteraldehyde both in saline solution. The thus stabilized EB bodies may be stored for a substantial period of time at reduced temperatures suitably at about 40C. In order to improve EB adhesion to the substrate in the micro-lF test normal yolk sac suspension in formalin/PBS may be added, however, such addition restricts the shelf life of the EB antigen to 2-3 weeks at 4"C.

The micro-lF test utilizing the EB is substantially that disclosed by Wang (Wang, et awl, 1963.

Classification of trachoma virus strains by protection of mice from toxic death. J. Immunol.

90:849-856.) as improved by Wang and Grayston (J. Infect. Dis. 130:388 at 389.) in this test there is prepared a substrate, suitably a clean microscope slide. A guidance template indicating test loci is placed under the slide and samples of the antigen (that is to say the EB samples) are placed on the slide by suitable means for example a pen point on those areas indicated by the template. The slides are the air-dried and the antigen affixed upon the substrate suitably by immersion in acetone for ten to fifteen minutes at ambient temperatures.

The sample material suspected of containing antibodies to C. trachomatis is then subjected to serial dilution in the usual manner. These sources may be animal sera suitably mouse or rabbit sera or, in the expected application of this test, may be human sources suitably human sera, tears or endocervical secretions. The test samples are placed upon the antigen dots with a bacteriologic loop.

The slides are incubated in a moist environment suitably at about 37OC for about 30 minutes and the slides washed gently preferably with phosphate buffered saline to remove the unreacted suspected antibody source material.

The substrate is then treated with anti (source materials species) antibodies coupled with the fluorescent materials thus where the antibody source material is the mouse then it can be used, for example, goat anti-mouse gammaglobulin conjugated with, for example, fluorescein isothiocyanate.

Where the source material is human there may be utilized similarly coupled goat anti-human gammaglobulin. It will be clear to those skilled in the art what type of anti-species antibodies should be utilized.

In a particuiarly preferred modification of the invention, where the antibody source material is human there is utilized a fluorescent conjugate of anti-human IgM antibody and anti-human IgG antibody. Since IgM is only present in an active infection this mode of proceeding provides a method of determining whether the antibody source material comes from an active or a passive infection.

The conjugate solution is applied to the slides with a bacteriologic loop in the manner described above and the substrate incubated, rinse, washed and air-dried. The slides are then examined under a source of UV light. The presence of fluorescence indicating the presence of antibody to C. trachomatis in the test samples.

EXPERIMENTAL The C. trachomatis strains used include representative strains of each immunotype: A/G-1 7/OT, B,TW-5/OT, C/TW-3/0T, D/UW-3/Cx, E/UW-5/Cx, F/UW-94/Ur, G/UW-57/Cx, H/UW-43/Cx, l/UW-1 2/Ur, J/UW-36/Cx, K/UW-3 1/Cx, L1/440/Bu, L2/434/Bu, and L3/404/Bu. Most of these strains have been described in Christoffersen, G. and G. P. Manire. J. Immunol. 103:1085-1088, 1969; Wang, S. P. 1971. A micro immunofluorescence method. Study of antibody response to TRIC organisms in mice, p. 273-288. In R. L. Nichols (ed.), Trachoma and related disorders caused by chlamydial agents. Excerpta Medica, Amsterdam; Wang, S. P. et al. J. Immunol. 90:849-856. 1963; Thomas, B. J. et al. J. Clin. Microbiol. 4:6-10. 1 976. The cross reactivity of these strains with antibodies to representative strains of each immunotype is set forth in Table 1 following: TABLE 1 Designation of Equivalent (Cross Reactive) Immunntypes: Representative strains Strains at ATCC Strains available at WHO indicated in the (Dr. D.A.Stevens) Trachoma Reference Laboratory* application A/G-17/OT A/HAR-13(or Eg-2)/OT VR 571 A/HAR-1(or SA-1)/OT B/TW-5/OT B/HAR-36/OT VR 573 B/TW-1/OT Ba/AP-2/OT Ba/AP-2/OT VR 347 Ba/AP-2/OT C/TW-3/OT C/TW-3/OT VR 578 C/TW-3/OT C/PK-2/OT VR 576 D/UW-3/Cx D/UWr3/Cx VR 885 D/lC-Cal-8/ON E/UW-5/Cx E/Bour/OT VR 348 E/Bour/OT F/UW-94/Ur F/lC < al-3/ON VR 346 F/lC-Cal-3/ON G/UW-57-/Cx G/UW-57/Cx VR 878 G/392/OC H/UW-43/Cx H/UW-43/Cx VR 879 H/471/Cx l/UW-12/Ur l/UW-12/Ur VR 880 1/870/OC J/UW-36/Cx J/UW-36/Cx VR 886 J/UW-36/Cx K/UW-31/Cx K/UW-3liCx VR 887 K/UW-31/Cx L1 /440/Bubo L1 /440/Bubo L2/434/Bubo L2/JH VR 121 L2/434/Bubo L3/404/Bubo L3/404/Bubo The strain designation is indicated by:Immunotype (A,B,C, ...L3)/Strain-number/Ori The abbreviations are: OT-ocular trachoma, ON-ophthalmia neoratorum, OCecular conjuntivitis, Cx-cervix, Ur-urethra, Bubo-Iymph nodes of LGV patient.

Strains of the same immunotype are interchangabla in the micro-lF test.

* c/o Dr. J. Schachter, University of California, San Francisco, Medical Center, San Francisco, California 94122.

Growth of C. trachomatis Organisms to Yield EBs The organisms grown for 72 hours (and therefore mostly at the elementary body stage) in 32ounce bottles of HeLa 229 cell culture which show more than 80% infectivity are harvested and suspended in phosphate buffered saline, pH 7.0 (PBS). This is sonicated for 20 seconds (Biosonik III Sonicator, Bronwill Scientific, Rochester, NY) and submitted to one cycle of differential centrifugation (500 x g for 10 minutes and 30,000 x g for 20 minutes). The resulting pellet is suspended in PBS, 10 ml per culture bottle. This suspension is sonicated for 1 0 minutes prior to further purification by centrifugation (20,000 x g for 40 minutes) through a layer of 1 0 ml 30% renografin. The organisms in the resulting pellet are washed with PBS and centrifuged 30,000 x g for 20 minutes) to remove the renografin.The pellet, containing relatively purified organisms, is then resuspended in PBS containing 0.02% final concentration of freshly prepared formalin (formalin-PBS), 2 ml per bottle. A homogeneous suspension of the organisms is achieved by sonicating for a few seconds. The density of the organisms at this step is approximately 5 x 1 09 organisms/ml. The formalinized antigens are stored unfrozen at 4 C. In the foregoing procedure glutaraldehyde may be used in place of formalin.

Adhesion Enhancement of EBs To be used as test antigens in the micro-lF test, normal yolk sac (NYS) suspension in formalin-PBS is added to make the final concentration of NYS 25%. The purpose of adding NYS is to facilitate the adhesion of the organisms on microscope slide. The NYS also aids location of the antigen dot in the microscopic examination. The optimum density of organisms to be used in the micro-lF test is about 1 09 organism/ml after the addition of NYS. After addition of NYS the storage at 40C is restricted to 2-3 weeks.

Table 2 shows the results of comparative reactivities of formalin-treated and nontreated antigens in the micro-lF tests with mouse antisera and trachoma patient sera.

Table 2 Test antigens* C/TW-3 B/TW-5 Formalin: + - + Mouse antisera Anti TW-3 128 -128 0** 0 Anti TW-5 0 0 128 128 Patient sera YB-10 128 128 0 0 YB-29 128 128 0 0 YB-31 0 0 128 128 YB-38 0 0 128 128 * Both antigens were grown in HeLa 229 cells ** 0= < 8 There was no difference in terms of specificity or sensitivity between formalin-treated and nontreated antigens.

In a separate experiment (data not shown in table), different concentrations of formalin (0.2%, 0.1%, 0.05%, 0.025%, and 0.0125%) were tested for their effect on TW-3 and Tw-5 test antigens.

No appreciable difference could be seen in the reactivities of the treated antigens during storage at 40C, in sequential follow-ups, for up to 9 months.

In order to assess the reactivity of formalin-treated HeLa-grown antigens in the micro-lF test after storage for a length of time in 40C, the stored antigens were compared with our conventional standard antigens (Thomas et al (1976) and Wang, S. P. 1971 , supra.) Table shows examples of such comparisons.

Table 3 Test antigens J/UW-36 E/UW-5 Growth: HeLa Egg HeLa Egg Formalin: + - + Storage at: 400 6 mos 850C, 4 yrs 40C, 6 mos -650C, 4 yrs Mouse antisera Anti UW-36 128 128 0* 0 Anti UW-5 0 0 64 64 * 0=Titer < 8

Claims (9)

1. A method for the serological testing of sources suspected to contain antibodies to Chlamydia trachomatis which comprises subjecting said sources to a microimmunofluorescence test employing elemental body stabilized by previous treatment with formaldehyde derived from C. trachomatis as the antigen in order to detect the formation of an antigen-antibody complex.

2. An immunofluorescence test according to claim lv for the serological testing of sources suspected to contain antibodies to Chlamydia trachomatis comprising the steps of: a) contacting an inert substrate with a predetermined amount of elemental body C. trachomatis previously stabilized with formaldehyde b) fixing a portion of said elemental body to said substrate c) removing the non-fixed elemental body from the substrate d) contacting said substrate having said elemental body fixed thereto with a sample of source material suspected to contain antibody to C. trachomatis e) removing the non-reacted antibody source material f) contacting the thus formed elemental body antigen-antibody combination with an anti(source material species) antibody having a fluorescent moiety conjugated therewith.

g) removing the unreacted anti-(source material species) antibody h) examining the substrate under ultraviolet light wherein the presence of fluorescence indicates the presence of C. trachomatis antibodies in the suspected antibody source material.

3. A method according to claim 2 wherein the source material is selected from human tears, human endocervical secretion or human serum.

4. A method according to claim 2 or claim 3 wherein the anti-(source material species) antibody is antihuman IgC antibody or antihuman IgM antibody.

5. A method according to claim 2, 3 or 4 wherein the elemental body antigen is derived from a strain having an antigenic reaction to antibodies to one of the strains designated in WHO reference as: ASA1 /OT B/iW1 C/TW8-OT D/IC-Ca 1 SON E/Bour/OT F/lC-CaI-30N G/392/OC H/471/Cx 1/870/OC JUW--36/Cx K/UW-31Cx L,/440/Bu L2/434/Bu Li404/Bu

6. Methods according to claim 1 substantially as herein described and exemplified.

7. A substance. for the detection of antibodies to predetermined strains of C. trachomatis comprising an immunologically inert substrate having elemental body of C. trachomatis previously stabilized in formaldehyde adhered thereto.

8. A substance according to claim 7 wherein the elemental body antigen is derived from a strain having an antigenic reaction to antibodies to one of the strains designated in WHO references as: ASA-1/OT B/TW1 C/rWS/OT D/lC-Ca1 SON E/Bour/OT F/IC-CaI-30N G/392/OC H/471/Cx 1/870/OC JUW--36/Cx K/UW-31Cx L,/440/Bu L2/434/Bu L3/404/Bu

9. Substances according to claim 7 substantially as herein described and exemplified.