GILBERT REISING, JOHN D. SCHMALE, DAN G. DANIELSSON,1 AND JAMES D. THAYER2 .... Danielsson, D. G., J. D. Schmale, W. L. Peacock, Jr., and.
APPLIED MICROBIOLOGY, Sept. 1969, p. 337-339 Copyright ( 1969 American Society for Microbiology
Vol. 18, No. 3 Printed in U.S.A.
Reactivity of Two Selected Antigens of Neisseria gonorrhoeae GILBERT REISING, JOHN D. SCHMALE, DAN G. DANIELSSON,1 AND JAMES D. THAYER2 Venereal Disease Research Laboratory, National Communicable Disease Center, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia 30333
Received for publication 28 May 1969
Two antigen preparations, the soluble antigen and a fraction 1 thereof, isolated in the course of a systematic study of the various antigens of the virulent gonococcus, have been investigated for their ability to serve as antigens for the detection of antibody in patients infected with the gonococcus. The soluble antigen was reactive with 88.2% of the sera from infected females, and fraction 1 was reactive with 71.6% of the sera. Of sera from infected males, only 27.6% reacted with the soluble antigen and only 20.4% with fraction 1. Of sera from individuals presumed free of gonococcal infection, approximately 4% reacted with the soluble antigen; none reacted with fraction 1. This study suggests that these antigens might be adaptable to the detection of human gonococcal antibody, especially in the female.
The systematic study of the various antigens of the gonococcus was undertaken before attempting to develop a serologic test for gonococcal infection. We decided to use complement fixation as one of several research tools to detect immune response against various antigens. This technique can be used to detect immune reactions when other manifestations of antigenantibody combination, such as precipitation or agglutination, are absent. Complement fixation is particularly valuable in comparing a variety of antigens since either soluble or insoluble antigens may be used (5). The intent in using the complement-fixation procedure was to gain antigen reactivity data which might be used in future work to adapt antigens to serological procedures less cumbersome than the complement-fixation test. In a previous paper, the isolation of a fraction 1 antigen (Fl) from gonococcal soluble antigen (SA) was reported (1). The present paper reports the use of SA and F1 to determine the reactivity of these two antigenic preparations in the human immune response to gonococcal infections.
MATERIALS AND METHODS Source of sera. All gonococcal patient sera were obtained from a local venereal disease clinic. Blood (10 ml) was drawn from patients who fulfilled the following criteria: Males were diagnosed as having gonococIPresent address: Department of Clinical Bacteriology, Regional Hospital, Orebro, Sweden. 2 Deceased.
cal infection if they had a typical history of urethritis with dysuria and purulent discharge with typical oxidase-positive colonies of gram-negative diplococci cultured on Thayer-Martin medium (6). Females were diagnosed as having gonococcal infection if cervical cultures on Thayer-Martin medium revealed the presence of typical oxidase-positive colonies of gram-negative diplococci. All cultures were incubated for 18 hr at 37 C with CO2 enrichment. Blood donors were selected from a group presumed free of gonococcal infection. Sera were procured from 50 males and 58 females between the ages of 18 and 60 years. Antigen preparation. A strain of virulent Neisseria gonorrhoeae, designated F62 of colonial morphology type 1 (2), was used for the SA and F1 preparation. The bacteria were grown on GC Medium Base (Difco) plus a defined supplement (2). The cultured organisms were harvested into distilled water and passed through a Ribi cell fractionator as previously described (4). The SA was separated from the insoluble cell wall material by centrifugation at 36,000 X g for 30 min, and the SA was lyophilized. The preparation of the F1 is reported in detail elsewhere (1). Briefly, the preparation involves passage of gonococcal SA through Sephadex G-200 followed by collection and concentration of the various fractions. The first half of the first peak eluted had been shown to react with sera from certain patients with gonococcal infection. This antigenic fraction has been designated the Fl. Complement-fixation test. The Laboratory Branch Complement-Fixation test was used throughout this study (7). The antigens were dissolved and diluted in Veronal buffered diluent. Diluted antigens were titrated for the complement-fixation test by initially using goat antiserum against the organism causing gonorrhea. As soon as large quantities of high-titer (1: 32 or higher)
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human antigonococcal sera were accumulated, pools were used for antigen titrations. The end point of human-serum titrations was taken as the highest dilution showing 30% or less hemolysis.
RESULTS
None of the 106 sera from the blood donor group reacted with the Ft. Two of the male sera (4%) and two of the female sera (3.3%) re58
481
(2)E
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FIG. 1. Complement-fixation reactions in sera from 106 blood donors. D NONREACTIVE L
REACTIVE WITH
SOLUBLE ANTIGEN FRACTIONS I ANTIGEN
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6' 6 TOTAL 1-2
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FIG. 2. Complement-fixation reactions in sera from 98 culturally positive males.
TOTAL 1-2
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1-32 1-64 1-128
FIG. 3. Complement-fixation reactions in sera from 102 culturally positive females.
APPL. M ICROBIOL.
acted with the SA at titers from 1:2 to 1:8. These reactions are shown in Fig. 1. The test results of the sera from 98 males and 102 females with gonococcal infection are shown in Fig. 2 and 3. Of the 98 samples of sera from male patients, 27 or 27.6% reacted in the complement-fixation test at a titer of 1:2 or greater with the SA, and 20 or 20.4% reacted with the FH. F1 reactors gave the same or higher titer with the SA. The reactivity of the sera from female patients was higher with both antigens. Ninety of the 102 (88.2%) reacted with the SA, and 73 (71.6%) reacted with the F1 (Fig. 3). All those that reacted with the F1 also reacted with the SA.
DISCUSSION In a preliminary study of the antigens important in human response to a gonococcal infection we have studied a serological reactivity of gonococcal SA and an extract of the SA. With the sera from the group presumed free of gonococcal infection, there was a small percentage of reactivity at low dilutions with the SA only. These reactions may be due to a crossreaction with antibodies from other members of the Neisseria genus or other organisms outside the genus. There is also a possibility that reac-tions are due to an inapparent infection or a previously treated infection with the antibody titer near the end of its decline. Whatever the reason, the number of reactions are small. The percentage of female or male sera, or both, that reacted with the SA was greater than the percentage that reacted with FH. Approximately one half of those that reacted with SA, but not with F1, did so only at a 1:2 titer; whereas in the remainder, the reactivity occurred at higher dilutions. Although reactivity at a 1:2 dilution may represent experimental error, it may also be due to cross-reactions with antibodies against other organisms; or it may be due to reactivity with antigens other than the FH. There are several possibilities for the wide discrepancy in the serological detection rate between males and females. First, it may be related to the duration of infection. Since infected males as a group are more symptomatic, they tend to seek treatment earlier. Thus, their decreased exposure time to the gonococcus may account for their lower detectable immune response. Second, infection may be handled differently by the immune mechanism in the two sexes. In the infected male there is generally a purulent urethral discharge, whereas in the asymptomatic infected female the infection usually resides in the cervix. Third, the males may give a greater response against an antigen not
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now in our present fractions, or they may re- edge that IgA is not fixed by complement, a spond with an antibody that is not complement specific test such as an immunofluorescent profixing, such as IgA. cedure using anti-IgA should be investigated Even though the complement-fixation test is to see if the number of infected male reactors used only as a research tool in this study, the could be increased over that obtained with the results can be compared to other reports in the complement-fixation procedure. literature (3). The male detection rates were LITERATURE CMD 10%, 50%, 17%, and 21%; and the female deD. G., J. D. Schmale, W. L. Peacock, Jr., and Danielsson, 1. were and 50%. 26%, 41%, 37%, tection rates J. D. Thayer. 1969. Antigens of Neisseria gonorrhoeae: In the present study the male detection rates of Characterization by gel filtration, complement fixation, 20.4% with F1 and 27.6% with SA, and female and agar-gel-diffusion of antigens of gonococcal protoplasm. J. Bacteriol. 97:1012-1017. detection rates of 71.5% with F1 and 88.2% with SA give a favorable impression of the ability 2. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I. of these antigens to detect gonococcal antibody. Virulence gentically linked to clonal variation. J. Bacteriol. Both the gonococcal SA and F1, thereof, 85:1274-1279. would be very useful antigens to employ in the 3. Magnusson, B., and J. Kjellander. 1965. Gonococcal complement-fixation test in complicated and uncomplicated gonordevelopment of a convenient serologic procedure rhoea. Brit. J. Vener. Dis. 41:127-131. for the detection of gonococcal antibodies in 4. Martin, J. E., Jr., W. L. Peacock, Jr., G. Reising, D. S. Kelthe case of the asymptomatic female. The SA logg, Jr., E. Ribi, and J. D. Thayer. 1969. Preparation of cell walls and protoplasm of Neisseria with the Ribi cell would have a slight edge over the F1 by showing fractionator. J. BacterioL 97:1009-1011. more reactors. This would be done with the M. M. 1961. Complement and complement-fixation, awareness that reactors to the SA might be 5. Mayer, p. 133-240. In E. A. Kabat and M. M. Mayer (ed.), Excaused by antibodies to organisms with somewhat perimental immunochemistry, 2nd ed. Charles C Thomas, Publisher, Springfield, Ill. similar antigenic makeup. Thus, such reactions J. D., and J. E. Martin, Jr. 1964. A selective medium would indicate a closer look at the patient. 6. Thayer, for the cultivation of N. gonorrhoeae and N. meningitidis. From the detection rates with males, it is eviPublic Health Rep. 79:49-57. dent that if the present antigen cannot be altered 7. U.S. Department of Health, Education, and Welfare. 1965. Public Health Monograph No. 74, Public Health Publicato increase the number of reactors, another tion No. 1228. Standardized diagnostic complement fixation be should other sought antigens antigen or method and adaptation to micro test. U.S. Government Printing Office, Washington, D.C. which would bring this about. With the knowl-
