Passive Hemagglutination Test for Detection of Antibody to Gonococcal Ribosomal Antigen in Sera from Patients with

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1982, p Vol. 15, No /82/ $02.00/0 Pssive Hemgglutintion Test for Detection of Antibody to Gonococcl Ribosoml Antigen in Ser from Ptients with Asymptomtic Gonorrhe EIJI KITA* AND SHUZO KASHIBA Deprtment of Bcteriology, Nr Medicl College, 840 Shijyocho, Kshihr City, Nr, Jpn 634 Received 22 June 1981/Accepted 20 November 1981 Ribosoml frctions were obtined from culture of type 2 Neisseri gonorrhoee strin P-17 which ws isolted from ptient with n cute gonococcl infection; these frctions were purified to eliminte the components of the outer membrne complex by ffinity chromtogrphy (Sephrose-nti-outer membrne complex ntibody conjugtes were used s the solid immunosorbent), nd the resulting preprtion ws designted the purified ribosoml frction. The purified ribosoml frction ws used to detect ntibody ctivity in ser obtined from culture-positive symptomtic crriers nd helthy controls by pssive hemgglutintion test. This pssive hemgglutintion test hd specificity of 100% for both sexes nd sensitivities of 99.4 nd 88.2% for femle nd mle crriers, respectively, when n ntibody titer of more thn 1:3 ws defined s bnorml. Absorption of the ser with nongonococcl orgnisms did not ffect the ntibody ctivity, nd no significnt difference in ntigenicity mong vrious N. gonorrhoee strins ws observed in ribosoml frctions. An enzyme-linked immunosorbent ssy ws lso used to mesure the reltive mounts of specific ntibodies to the purified ribosoml frction, nd this ssy reveled tht the nti-purified ribosoml frction ntibodies were immunoglobulin G. During the pst 10 yers, there hs been incresing interest in serologicl methods for the dignosis of gonococcl infections. A vriety of serologicl techniques hve been described, including the complement fixtion test (4, 26), floccultion (18, 31), direct hemgglutintion (9, 19, 32), nd the indirect immunofluorescence test (2, 34, 35). Recently, rdioimmunossy (1, 21) nd n indirect hemgglutintion test (25) in which gonococcl pilus ntigen is used to detect gonococcl ntibodies hve been described, but the results of these tests hve been vrible becuse of lck of sufficient sensitivity or specificity. Except for the tests bsed on purified pili, the existing serologicl tests use either whole gonococci or chemiclly undefined extrcts s ntigens. All of these tests give some nonspecific results becuse of cross-rections with ntibodies to other Neisseri species nd orgnisms shring ntigens with the gonococci. Another problem is tht these tests cnnot differentite current infection from pst infection. However, serologicl tests might be of some help in performing epidemiologicl surveys to detect symptomtic crriers of Neisseri gonorrhoee. Furthermore, the importnce of serologicl studies is emphsized by recent demonstrtions tht symptomtic gonococcl infections re common in men s well s in women (13) nd tht mle symptomtic crriers re more common thn generlly supposed (22). We hve studied the immunologicl ctivities of the ribosoml frction of Slmonell typhimurium nd hve shown tht circulting ntibodies to the ribosoml frction, which could be demonstrted by pssive hemgglutintion ssy (PHA), exist in the ser of mice immunized with the ribosoml frction (16). Therefore, we isolted the ribosoml frction from N. gonorrhoee nd used it for PHA ntigen. In this pper the preliminry findings of n investigtion into the use of this ribosoml frction in PHA to detect ntibodies to microsoml frctions of N. gonorrhoee in symptomtic gonococcl ptients re reported. In ddition, the enzyme-linked immunosorbent ssy (ELISA) ws used to mesure the reltive mounts of specific ntibodies ginst the ribosoml frction. MATERIALS AND METHODS Popultion. Ser were obtined from symptomtic nd symptomtic persons. An symptomtic crrier ws ptient who hd no clinicl symptoms but hd positive culture. A symptomtic cse ws culture- or smer-positive ptient with clinicl symptoms. Specimens were tken from the cervix of ech femle 668

2 VOL. 15, 1982 ptient nd from within the urethrl cnl of ech mle ptient for culture nd Grm stining. Some ptients were followed up from n erlier ttck of gonorrhe. Norml control ser were obtined from helthy students to whom the purpose of the investigtion hd been explined. Bcteri nd culture. N. gonorrhoee ws obtined from fresh ptient isoltes nd ws identified by Grm stining, culturl chrcteristics, oxidse rectivity, nd sugr fermenttions. Pilited gonococci (strin P- 17, isolted in our lbortory) hving P+ or P"+ phenotypes were used throughout the preprtion of the ribosoml frction. Bcteri were cultured on solid medium (GC gr bse) supplemented with 1% IsoVitleX. Cultivtion of gonococci in 1 liter of liquid medium ws ccomplished by using 5-liter fermenttion bottle. The medium in the bottle ws identicl to the solid medium except tht strch, 1% (wt/vol) glucose, nd 0.1% (wt/vol) sodium bicrbonte were dded. The culture ws grown t 37 C with continuous revolution fter the culture bottle ws tightly cpped with rubber stopper. Preprtion of ntigens. (i) Crude OMC. Outer membrne complex (OMC) ws prepred s described by Zollinger et l. (36). Briefly, 50 g of wet pcked cells suspended in TSE buffer (0.05 M Tris-hydrochloride, 0.15 M NCI, 0.01 M EDTA, ph 7.4) ws incubted t 60 C for 30 min nd subjected to mild shering in n Omnimixer, nd then the OMC ws seprted by centrifugtion. The OMC sedimented by centrifugtion t 100,000 x g ws suspended in wter nd lyophilized. A portion of this lyophilized OMC ws suspended in 2 ml of 25% (wt/wt) sucrose solution in TSE buffer to concentrtion of 2 mg of outer membrne mteril per ml nd ws pplied to discontinuous grdient prepred by lyering sequentilly 2.0-ml portions of sucrose solutions rnging in concentrtion from 65 to 25% (wt/wt) in nitrocellulose centrifuge tubes. Smples were centrifuged in n RPS 27-2 rotor (Hitchi Koki Co., Ltd.) for 18 h t 280,000 x g to isopycnic conditions. The tube contents were frctionted by puncturing the bottoms of the tubes nd collecting drops; the bsorption of UV light t 280 nm ws mesured for the frctions, nd single bnd ws obtined nd used s n ntigen for immuniztion. (li) Antiserum to OMC. Rbbits were immunized with OMC emulsified in Freund incomplete djuvnt. Ech rbbit received 0.25 ml of the emulsion in ech footpd nd 1 ml injected subcutneously in the npe of the neck. The rbbits were boosted subcutneously three times every week fter immuniztion. The -yglobulin frction of the ntiserum ws prepred by precipittion with N2SO4, using the method of Kekwick (15). Specific rbbit nti-omc ntibodies were obtined from the y-globulin frction by using btch dsorption t ph 6.8 on DEAE-cellulose (Brown Co.) nd gel filtrtion on Sephdex G-200 (Phrmci Fine Chemicls, Uppsl, Sweden). During immunoelectrophoresis, this rbbit immunoglobulin G (IgG) formed one single precipittion rc when it ws ssyed with sheep ntiserum ginst whole rbbit serum. Frctions contining IgG were concentrted to 50 mg/ml by ultrfiltrtion nd used for conjugtion to Sephrose. (iii) Preprtion of Sephrose-nti-OMC ntibody conjugtes. Sephrose-nti-OMC ntibody conjugtes were used s the solid immunosorbent to obtin PHA FOR GONOCOCCAL ANTIBODY 669 purified ribosoml frction (PRF) tht ws free from contmintion by OMC ntigens. The specific rbbit nti-omc ntibodies were conjugted directly to cynogen bromide-ctivted Sephrose 4B (Phrmci) by the method of Cutrecss (3). The coupling ws crried out with 5 mg of protein per ml t ph 10 nd 4 C for 16 h. Sephrose ws ctivted with 100 mg of CNBr per ml of pcked Sephrose 4B. (iv) Purifiction of the ribosoml frction by ffinity chromtogrphy. The ntigens tht cross-rected with nti-omc ntibodies were removed from the ribosoml frction by pssing this mteril over 15-ml column contining conjugted Sephrose 4B. Elution ws crried out with 0.1 M Tris-hydrochloride buffer (ph 8.2), nd the opticl densities of 3-ml frctions were mesured t 260 nm. A single pek ws obtined, concentrted to 1 ml, nd used s the PRF. The PRF ws pooled nd used s the ntigen for the serologicl study. Anti-PRF ntiserum prepred in rbbits did not rect with lipopolyscchride or the OMC preprtion in the immunodiffusion test. PHA. The ser of persons with symptomtic or symptomtic gonococcl infections were exmined for the presence of ntibodies ginst the gonococcl PRF by using PHA. A modifiction of the originl PHA method of Stvitsky (28) ws used. For the PHA, 10 volumes of 1.0%o wshed sheep erythrocytes ws mixed with 3 volumes of 2.5% glutrldehyde nd 1 volume of the PRF (30,ug of protein per ml), nd the preprtion ws incubted for 1 h t room temperture. The sheep erythrocytes were wshed in phosphte-buffered sline (PBS) three times nd treted with 1 M glycine buffer (ph 7.2) overnight, fter which the cells were wshed in PBS nd suspended to finl concentrtion of 1% in 1% norml humn serum in PBS. The PHA ws performed in microtiter pltes contining ml of PBS plus norml humn serum, test serum, nd sensitized sheep erythrocytes. Agglutintion titers were determined fter overnight incubtion t 4 C. ELISA procedure. The ELISA ws performed s described by Engvll et l. (6-8). Disposble polystyrene tubes (13 by 100 mm; Flcon Plstics, Oxnrd, Clif.) contining 1 ml of the PRF in 0.05 M crbonte buffer (ph 9.6) (coting buffer) were incubted for 3 h t 37 C. The optiml working concentrtion of the PRF for use s the coting ntigen ws determined by the ELISA rection, using positive control serum of known titer. The optiml concentrtion turned out to be 15 p.g of protein per ml. The PRF ws diluted to concentrtion of 15 jig of protein per ml of coting buffer. Excess ntigen ws removed, nd the tubes were wshed three times with PBS contining 0.05% Tween 20 nd 0.02% sodium zide (PBS-Tween). The test ser were diluted 1:1,000 in PBS-Tween, nd 0.5-ml portions were dded to the tubes coted with PRF nd to uncoted tubes. Anti-humn IgG (4.4 mg) nd nti-humn IgM (3.2 mg; Miles Lbortories, Ltd., Slough, Englnd) were conjugted to lkline phosphtse (type VI; Sigm Chemicl Co., St. Louis, Mo.) in 2:5 rtio, s described by Voller et l. (30). After the tubes were incubted t room temperture for 2 h on horizontl rotry shker, they were wshed four times with PBS- Tween, nd 0.5-ml portions of lkline phosphtseconjugted nti-humn IgG or nti-humn IgM diluted

3 670 KITA AND KASHIBA 1:150 were dded to the pproprite tubes. The rection mixtures were incubted for 16 h t room temperture. After nother series of three wshed with PBS-Tween, 1 mg of the substrte p-nitrophenyl phosphte disodium (Sigm) in 1 ml of 0.05 M crbonte buffer (ph 9.8) contining 10-' M MgCl2 ws dded to ech tube. The rection ws stopped by dding 1 ml of 0.2 M NOH fter 100 min of incubtion t room temperture. The color rection in ech tube ws red t 405 nm, nd the opticl densities of the uncoted tubes t 405 tim were subtrcted from those of the coted tubes contining the sme solutions. The negtive control serum ws pool of ser from helthy students, nd the positive control ws tken from long-term symptomtic crrier with positive cultures for penicillin-resistnt N. gonorrhoee. Both of these ser were diluted 1:1,000 for use. Results were expressed s described by Glynn nd Ison (12) s corrected extinction vlues (Ecr) nd were clculted s follows: Ecr = (Et - E) x [men (Ep - E.)]/[(Ep - E) on test dy] x 100, where Et, Ep, nd En re the extinction vlues (opticl density t 405 nm) of the test, positive, nd negtive control ser, respectively. The men (Ep - En) is the men difference between the positive nd negtive control vlues mesured in 20 ssys over period of months. Chemicl ssy. Protein determintions of the PRF nd OMC were performed by the method of Lowry et l. (20). The protein contents of the immunoglobulins were determined by mesuring the bsorbnce t 280 nm in 1-cm cuvette, using vlue of 13.8 s the bsorbnce of 1% solution of protein. RNA ws mesured by the orcinol method (5), nd hexose ws determined by the nthrone rection described by Roe (27). The method of Weissbch nd Hurwitz (33) s modified by Osborne (24) ws used for determintions of 2-keto-3-deoxyoctonte. Clcultions were mde by using the extinction coefficient given by Osborne (24). Corrections for interference by silic cid were mde bsed on the silic cid content of the smple, s determined by the resorcinol method of Svennerholm (29). Bovine serum lbumin (Sigm), yest RNA (Sigm), L-(+)-rhmnose (Sigm), nd 2-keto-3-deoxyoctoite (Sigm) were used s stndrds. Pieprtion of microorgnisms for bsorption studies. Cultures of Stphylococcus ureus, Escherichi coli, Neisseri meningitidis, nd Brnhmell ctrrhlis were obtined from ptients. Cells of S. ureus nd )F. coli were grown on brin hert infusion gr (Difco Lbortories, Detroit, Mich.) t 37 C for 24 h. N. meningitidis nd B. ctrrhlis were inoculted onto chocolte gr, plted, nd incubted t 37 C for 24 h. All cells were then wshed off the gr nd suspended in sterile sline. Ech suspension ws wshed three times, suspended in 20 ml of sterile sline contining 0.5% formlin, het inctivted in 65 C wter bth for 60 min, nd checked for vibility by plting on the growth medium. Ech suspension ws centrifuged t 4,000 rpm for 20 min, nd the pcked cells were suspended in 20 ml of sterile sline nd used for the bsorption test. Absorption of the symptomtic ptient ser ws done by the method of Gines nd Lndy (10). Briefly, the ser (0.6 ml) were mixed with 0.1 ml of pcked cells, incubted t 37 C for 1 h, nd then kept t 4 C overnight. After centrifugtion t 800 x g for 30 min, the bsorbed ser were removed nd tested for nti- J. CLIN. MICROBIOL. PRF ntibody ctivity in duplicte. The titers of ntibodies ginst the PRFs prepred from four different strins of N. gonorrhoee (P-4 nd P-5 isolted in the Philippines, N-1 nd N-3 isolted in Jpn) nd from N. meningitidis nd B. ctrrhlis were lso determined in ser of symptomtic ptients by the method described bove. Sttistics. Chi-squre nlysis ws used to determine the sttisticl significnce of individul fctors in reltion to serologicl results. RESULTS Purifiction nd chrcteriztion of ntigens. An nlysis of frctions obtined by isopycnic centrifugtion of OMC by bsorbnce t 280 nm reveled single bnd of turbidity t grdient density of 1.34 g/cm3 (Fig. 1). A chemicl nlysis showed tht the OMC preprtion consisted of protein nd lipopolyscchride nd tht the level of RNA contmintion ws bout 1% (Tble 1). OMC contmintion ws removed from the ribosoml frction by ffinity chromtogrphy, nd single pek of n RNA-rich frction ws obtined; this ws designted the PRF. As Tble 1 shows, the rtio of RNA to protein ws 2.14, nd lipopolyscchride contmintion my hve been miniml, lthough the ctul level of contmintion ws not cler. The PRF hd no ntigenic components tht rected with nti- OMC ntiserum, s determined by the immunodiffusion test. Correltion of culture with serology. Tble 2 shows the distribution of serologicl results in N. gonorrhoee cultures. In our preliminry investigtion the ntibody levels in serum specimens from the ptients with gonococcl infections were less thn 1:4, s determined by the PHA. Thus, bsed on these results, the optiml titer of ntibody for discriminting between culture-positive nd culture-negtive cses ws rbitrrily selected s.1:4. In women, titers of more thn 1:4 were found in both culture-positive nd culture-negtive individuls. Tble 2 shows none of the fctots tht influenced the PHA results, such s pst history nd symptomtic crrier sttus. Influence of pst gonorrhe on the serologicl test. In both sexes, pst gonorrhel history significntly influenced the results of serologicl tests (Tble 3). In femle ptients with negtive cultures, pst history of gonorrhe ws responsible for positive serologicl tests. Even in culture-positive women, the percentge of positive ser ws higher mong those individuls who hd hd gonorrhe previously. Very few men with gonorrhe were exmined in this study, but the difference in the percentge of positive ser ws remrkble when the ptients who hd hd gonorrhe nd the ptients without pst history of gonorrhe were compred.

4 VOL. 15, 1982 PHA FOR GONOCOCCAL ANTIBODY 671 E c 2. 0' 1. K s _ Bottom 1 I Frction Number FIG. 1. Density of outer membrne of strin P-17. Outer membrne preprtions were lyered onto discontinuous sucrose grdient (62 to 25%, wt/wt) nd ultrcentrifuged t 280,000 x g for 18 h to isopycnic conditions. Tble 4 shows tht pst history of gonorrhe significntly influenced the results of smer dignosis. The percentge of positive ser ws not ffected by incresing time since the lst episode of gonorrhe, s shown by the PRF PHA (Tble 5). I Top Tble 6 shows tht the nti-prf ntibodies detected in the ser of ptients with gonococcl infections were usully IgG. Serologicl test for symptomtic crriers. Figure 2 shows the ntibody titers for symptomtic crriers, s determined by the ELISA nd PHA. TABLE 1. Chemicl compositions of the OMC nd PRF prepred from N. gonorrhoee strin P-17 Prepn Chemicl composition (1Lg/mg, dry wt) RNA/protein RNA Protein Hexose 2-Keto-3-deoxyoctonte rtio OMC PRF ND 96 ND ND, Not detected. TABLE 2. Distribution of serologicl results for N. gonorrhoee cultures No. of ptients with: Group PHA titer (log2)0 Positive cultures Negtive cultures Mles -1 but <4 56(74)b 62(84).4 but <6 12(16) 4(6) 26 but <8 8(11) 3(3) -8 but < Femles.1 but <4 175(42) 71(93) -4 but <6 148(35) 2(3).6 but <8 176(42) 2(3).8 but <10 15(4) 1(1).10 4(1) 0 As determined by PHA for ntibodies to ribosoml frctions of N. gonorrhoee P-17. b The numbers in prentheses re percentges.

5 672 KITA AND KASHIBA J. CLIN. MICROBIOL. TABLE 3. Effect of pst gonorrhel history on PRF PHA ntibody titers in men nd women with cultures positive for N. gonorrhoee Mles Femles Culture Pst history No. of ptients with PHA No. of ptients with PHA titer of 21:4 titer of -1:4 Positive Positive 18/44 231/242 Positive Negtive 2/ /176 <0.001 Negtive Positive 6/9 5/46 Negtive Negtive 1/60 < /30 <0.25 Number of ptients with PHA titer of -1:4/number tested. TABLE 4. Effect of pst gonorrhel history on PRF PHA ntibody titers in men nd women with smers positive for N. gonorrhoee Mles Femles Smer Pst gonorrhe No. of ptients with PHA No. of ptients with PHA titer of -1:4 titer of -1:4 Positive Positive 10/12 54/58 Positive Negtive 2/40 < /62 <0.05 Negtive Positive 17/24 36/40 Negtive Negtive 0/60 < /60 <0.05 Number of ptients with PHA titer of -1:4/number tested. If n ntibody titer of 1:3 or higher titers ws defined s bnorml, none of our norml helthy popultion gve flse-positive results, nd 2 of 17 mle crriers nd 1 of 188 femle crriers gve flse-negtive results. Thus, the specificity ws 100% for both sexes, wheres the sensitivity ws 99.4% for femle crriers nd 88.2% for mle crriers. In norml helthy popultion, ll of the Ecr vlues were less thn 30 U, lthough the helthy individuls in our investigtion were limited popultion. If n ntibody level of 30 Ecr units ws defined s bnorml, the specificity ws 100% for the symptomtic crriers of both sexes. However, the sensitivity ws 82.3% for mle crriers nd 83.5% for femle crriers. These sensitivities were rised to more thn 98% if the bnorml rnge ws reduced to 20 Ecr units, nd the specificity ws still mintined t more thn 90%. TABLE 5. Effect of time since lst episode of gonorrhe on PRF PHA ntibody titer in symptomtic women with cultures positive for N. gonorrhoee Time since No. seropositive/totl no. lst gonorrhe More thn one (months) One pst episode pst episode 1-3 7/8 (80)0 78/78 (100) 4-6 7/7 (100) 54/54 (100) 7-9 3/3 (100) 16/16 (100) /5 (80) 7/9 (78) >12 3/3 (100) 4/5 (90) The numbers in prentheses re percentges. Absorption of symptomtic ptient ser. The ntibody ctivities in the ser of six individuls with positive cultures (femle ptients F-1, F-2, nd F-3 nd mle ptients M-1, M-2, nd M-3) were determined ginst PRF by the PHA. The ntibody titers were determined ginst ribosoml frctions prepred from four different strins of N. gonorrhoee, N. meningitidis, nd B. ctrrhlis. No significnt ntibody ctivity ginst PRFs of nongonococcl orgnisms ws observed with these ser, but high ntibody ctivities to PRFs of different gonococcl strins were detected (Tble 7). To determine the nonspecific rectivity of nti-prf ntibody ctivity s mesured by the PHA, the ser were lso bsorbed with ech of four nongonococcl orgnisms. No significnt decline in the titer of nti-prf ntibody ws observed with these bsorbed ser, wheres bsorption of ptient ser with different strins TABLE 6. Antibody specificity of ELISA for PRF ntibodies in men nd women with cultures positive for N. gonorrhoee No. of mles No. of femles Antibody with ELISA with ELISA titer of 30 Ec. units titer of 30 Ecr units IgG 66/76 (87) 344/418 (82) IgM 2/76 (3) 6/418 (1) The numbers in prentheses re percentges. Number of ptients with ELISA titer of 30 ECr units/ number tested.

6 xy -t c 4 dnl VOL. 15, 1982 PHA FOR GONOCOCCAL ANTIBODY A ss r 7- *S :13 St 6 ** *.: ti _4. S.. m':iy trin* C44 * J Er UNIT B 7. S * ~~~0 0 0,, V s0 6o A eb Etr UNIT FIG. 2. (A) Anti-PRF ntibody ctivities of ser from culture-positive, symptomtic femles (0) nd helthy femles (0). (B) Anti-PRF ntibody ctivities of ser from culture-positive symptomtic mles (-) nd helthy mles (0).

7 674 KITA AND KASHIBA J. CLIN. MICROBIOL. TABLE 7. Cross-rectivities of ribosoml frctions prepred from other gonococcl strins nd nongonococcl bcteri to symptomtic ptient ser Anti-PRF ntibody ctivity (PHA titer) with symptomtic serum from ptient: PHA ntigen from: F-1 F-2 F-3 M-1 M-2 M-3 N. gonorrhoee P N. gonorrhoee P-S N. gonorrhoee P N. gonorrhoee N N. gonorrhoee N N. meningitidis b B. ctrrhlis 2 1 Strin P-17 ws used to prepre the PHA ntigen in this b study experiment. -, Absence of gglutintion. of N. gonorrhoee reduced the PHA titers to the level of the PRFs of strin P-17 (Tble 8). Other ntigens, such s lipopolyscchride nd the OMC, did not inhibit the humn serologicl rections if the PRF ws used s the PHA ntigen. DISCUSSION The reson tht ribosoml frctions were chosen s ntigens for evlution in this investigtion ws tht the difference in ntigenicity mong strins of N. gonorrhoee ws very smll. Surfce ntigens, such s pilus, envelope, nd OMC ntigens, vried mong strins; in ddition, it hs long been known tht mny orgnisms undergo reversible muttions between the pilited nd nonpilited phses (11). Furthermore, ribosoml frctions reportedly induce delyed-type hypersensitivity in guine pigs (14, 17). In this study, ribosoml frctions were further purified by ffinity chromtogrphy, designted s PRF, nd used s ntigens for the PHA nd ELISA. Ribosoml preprtions contined severl ntigens detected by immunodiffusion unless they were purified by ffinity chromtogrphy; these preprtions could rect to ser even if serum smple ws collected from helthy control. The protein content in the PRF ws bout 30%, but no components of the OMC could be detected by the immunodiffusion test nd gel electrophoresis fter ffinity chromtogrphy. In this study the ser were collected from symptomtic crriers, s well s from ptients with cute gonococcl infections. It ws very difficult to find symptomtic mle crriers nd collect relible informtion on pst gonorrhel history, compred with femles. When the results of serologicl exmintions were evluted, only the culture-positive cses were considered. In mles the culture-negtive, seropositive individuls represented bout 48% of the individuls tested, wheres bout 16% of the femles tested were culture negtive nd seropositive. Among the culture-negtive seropositive individuls, bout 91% of the mles nd 94% of the femles hd cute infections. Among the culture-positive seropositive individuls, the percentge of the symptomtic crriers ws bout 22% in mles nd but 45% in femles. In our preliminry investigtion, the highest titer t dy 7 fter the ppernce of clinicl symptoms of gonococcl infection ws 1:4; fter dy 7 the ntibody ctivity decresed s clinicl symptoms disppered with tretment. Even in cute cses, the ntibody titers were very high if the ptient hd hd gonococcl infections in the pst. This study ws performed to detect the symptomtic crriers, nd t the erly stges of TABLE 8. Anti-PRF ntibody ctivities of humn ser tht were untreted or bsorbed with microorgnisms Anti-PRF ntibody ctivity (PHA titer) with symptomtic serum from ptient: Ser bsorbed with: F-1 F-2 F-3 M-1 M-2 M-3 Untreted N. gonorrhoee P-17 _- N. gonorrhoee P-4 1 E. coli N. meningitidis B. ctrrhlis S. ureus _, Absence of gglutintion.

8 VOL. 1 5, 1982 this study PHA titer of more thn 1:4 ws selected rbitrrily to discriminte between seropositive nd seronegtive individuls. With this PHA titer, the percentge of flse-negtive results incresed, nd the flse-positive results could be eliminted. The results of serodignosis did not correspond well with the results of the culture nd smer dignoses. Among the symptomtic crriers with positive cultures for N. gonorrhoee, the lowest titer in the PHA ws 1:3. If n ntibody titer of 1:2 ws defined s bnorml, one femle nd two mle crriers becme flse-negtive, but none of the norml controls gve flse-positive results. It ws very difficult to nlyze cultures for N. gonorrhoee becuse there were not mny chnces to perform culture exmintion on one individul. There my hve been s mny filures in the gonococcl cultures. It my be tht the cultures would hve been positive if exmintions were repeted for the culture-negtive seropositive individuls. However, if n ntibody titer of more thn 1:3 ws defined s bnorml, the specificity ws 100% for both sexes, nd the sensitivity ws 99.4% for femle crriers nd 88.2% for mle crriers. The ELISA is very excellent method for detecting specific ntibody, nd this test ws lso used in this study to determine the ctivity nd specificity of nti- PRF ntibody in ser. If n ntibody level of 20 Ecr units ws defined s bnorml, the sensitivity ws bout 93% nd the specificity ws more thn 90%. A correltion between the PHA nd the ELISA ws not estblished in this study, but very few symptomtic crriers with less thn 20 Ecr units hd PHA titer of more thn 1:3. The ELISA reveled tht the nti-prf ntibody ws IgG. Antibodies ginst ribosoml frctions of other strins of N. gonorrhoee were detected in ptient ser, but no ribosoml frction prepred from nongonococcl orgnisms rected with ptient ser. Absorption of the ser with nongonococcl orgnisms did not ffect the ntibody ctivity in the ser of symptomtic ptients. The nongonococcl flor my hve very little influence on this serologicl dignosis, nd differences in ntigenicity mong vrious strins of N. gonorrhoee were not observed in ribosoml frctions. In our preliminry study, ntibodies to ribosoml frctions strted to increse 2 weeks fter the ppernce of clinicl symptoms in ptient hving n cute gonococcl infection. After chemotherpeutic tretment filed, the ntibody ctivities reched mximum 2 months fter infection begn nd continued for long time t significntly high titer. Antibody to the OMC ppered 1 week fter the ppernce of clinicl symptoms, nd the durtion of ntibody response could lst s long s 1 month t the high PHA FOR GONOCOCCAL ANTIBODY 675 titer either with or without tretment. Ser obtined from symptomtic ptients rected with the OMC, but the ntibody titer ws very low. Subcellulr ntigens, such s ribosoml frctions, might be relesed from orgnisms shortly fter the orgnisms re engulfed by leukocytes, nd the ntigen relese could lst until the orgnisms re completely eliminted. This could ccount for the high levels of ntibody ctivity in the ser of symptomtic crriers. Bcteril culturing is not lwys successful in detecting n symptomtic crrier becuse of the vriety of sensitivities of culture results. Considering such situtions, serologicl study with the PRF PHA might be sensitive nd specific enough to perform n epidemiologicl study on symptomtic crriers. The ELISA with PRF ntigen is lso effective, nd there hs been report tht rdioimmunossy with lbeled pilus ntigen could be useful tool in the serodignosis of symptomtic gonorrhe (23). However, the PRF PHA is more useful becuse it is convenient nd esy nd hs low cost. Athough this study ws performned with limited number of subjects, its results contin informtion which suggests tht the PRF PHA could be used s screening test for the dignosis of symptomtic gonorrhe. A study is now in progress to determine the influence of cute gonococcl infections on the PRF PHA nd to determine whether cross-recting nti-prf ntibody occurs in ptients with other types of infections. ACKNOWLEDGMENTS This work ws supported by grnt from the Ohym Helth Foundtion. We thnk T. E. Tupsi (Mkti Medicl Center, Mnil, Philippines), the stff of the Mnil Venerl Disese Control Center (Philippines), nd T. Fukud (Nr, Jpn) for llowing us ccess to their ptients nd for collecting the specimens. We thnk the ptients, students, nd others who willingly gve blood, Eriko Skshit for technicl ssistnce, nd Yoshiko Shiotni for typing the mnuscript. LITERATURE CITED 1. Buchnn, T. M., J. Swnsson, K. K. Holmes, S. J. Krus, nd E. C. 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