JOURNAL OF CLINICAL MICROBIOLOGY, June 1994, P. 1419-14260095-1137/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Enzyme Immunoassay with Enhanced Specificity for Detectionof Antibodies to Chlamydia trachomatis

J. M. OSSEWAARDE,l* A. DE VRIES,1 J. A. R. VAN DEN HOEK,2 AND A. M. vAN LOON'

Laboratory of Virology, National Institute of Public Health and Environmental Protection, Bilthoven,'and Municipal Health Service, Amsterdam,2 The Netherlands

Received 16 November 1993/Returned for modification 24 January 1994/Accepted 25 February 1994

Two different methods for preventing the binding of cross-reacting antibodies to the genus-reactivechlamydial lipopolysaccharide (LPS) were used to improve the specificity of an enzyme immunoassay for thedetermination of antibodies to Chiamydia trachomatis. Coated elementary bodies were treated with eithersodium periodate, to oxidize the antigenic sites of the LPS, or Triton X-100, to extract the LPS. By using thesenew enzyme immunoassays, the standard enzyme immunoassay, and the whole inclusion fluorescence (WIF)assay, antibodies to C. trachomatis were determined in sera from different groups of patients and controls.Paired serum samples from patients with culture-proven urogenital C. trachomatis infections showed similarresponses in all three assays. Paired serum samples from patients with Chlamydia psittaci infections showedsimilar .responses in the WIF assay and the standard enzyme immunoassay, whereas significantly reducedtiters were obtained in the enzyme immunoassays with treated antigen, especially in the convalescent-phaseserum samples. Serum samples from patients with symptoms suggestive of infection with C. trachomatis,pregnant women, and blood donors were evaluated by all three types of assays. Eighty percent of the significantreductions in immunoglobulin G (IgG), IgA, and IgM titers were observed in sera with WIF assay titers in thelower classes (IgG, 1:s256; IgA, 1:s32; IgM, 1:.16). From these results we conclude that oxidation of theantigen by sodium periodate is a simple and effective method of producing an enzyme immunoassay withenhanced specificity that could be useful for diagnostic purposes and seroepidemiological studies.

Chiamydia trachomatis is considered one of the major causesof sexually transmitted diseases and, as such, is a major publichealth problem (48). Although knowledge of the impact of C.trachomatis infections in The Netherlands is consideredpatchy, the problem is generally believed to be of the samemagnitude as in many other countries (45). Public healthmeasures to control sexually transmitted disease are based on,among others, epidemiological and clinical studies. Since thesymptoms of C. trachomatis infection are not specific, labora-tory methods are required for a definitive diagnosis of C.trachomatis infections. These methods include several directand indirect techniques (2). Direct techniques are the methodsof choice for diagnosing most C. trachomatis infections. Isola-tion in cell culture is considered the "gold standard," althoughrecently introduced assays based on PCR have been shown tobe more sensitive (31). Serological methods are recommendedonly for use in assisting in the diagnosis of complications likesalpingitis or perihepatitis or to be used as a tool in (sero)epi-demiological studies. The complement fixation test (CFT) was

the first available assay for the detection of antibodies toChlamydia psittaci (3). Five years later, antigen preparationtechniques also became available for C. trachomatis (36). CFTis still routinely performed for diagnostic purposes in manylaboratories. However, the lack of sensitivity and specificitylimits the use of CFT in seroprevalence studies. Two differentimmunofluorescence assays have been developed to improveon the CFT: (i) the microimmunofluorescence assay (MIFassay [47]) with purified elementary bodies and (ii) the wholeinclusion fluorescence assay (WIF assay [39]). Since the fluo-rescence of the inclusions used in the WIF assay is easier to

* Corresponding author. Mailing address: Laboratory of Virology,RIVM, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Phone:+31 30 743942. Fax: +31 30 281168. Electronic mail address:

ossewaarde@rivm.nl.

read than that of elementary bodies, the WIF assay is preferredby many laboratories for use in routine diagnosis (38, 39, 46).It is, however, considered less specific than the MIF assay (32).To allow handling of larger numbers of samples and to providemore objective reading of the results, several enzyme immu-noassays have been developed. Partially purified elementarybodies, purified major outer membrane protein, chlamydiallipopolysaccharide (LPS), or LPS from the Re mutant ofSalmonella spp. have all been used as antigens (8, 9, 18, 20, 34,44). In general, these enzyme immunoassays are consideredvery sensitive, but their specificities are variable and depend on

the antigen source. Genus-specific epitopes are present on themajor outer membrane protein, the 60-kDa proteins, and theLPS (24). The highest specificity is probably obtained withpurified species-specific antigens (34), and the lowest specific-ity is probably obtained with the genus-specific LPS as antigen.Removal of the genus-specific epitopes on the LPS by oxida-tion or detergent extraction probably results in a more specificantigen (17, 29).Ehzyme immunoassays have many advantages over immu-

nofluorescence assays, for example, the objective reading ofthe results and the possibility for automation. Recently, we

developed an enzyme immunoassay in which the genus-specificepitopes on the LPS are removed by either oxidation withsodium periodate or extraction with Triton X-100 (29). Herewe report our evaluation of that enzyme immunoassay fordiagnostic and epidemiological purposes by using sera fromdifferent groups of patients and controls.

MATERIALS AND METIIODS

Study groups. Sera were collected from five different groupsof subjects: (i) 11 patients with culture-proven urogenital C.trachomatis infection (paired serum samples), (ii) 10 patientswith C. psittaci infection (paired sertim samples), (iii) 50

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patients with symptoms suggestive of infection with C. tracho-matis (single serum samples), (iv) 54 healthy pregnant women(single serum samples), and (v) 50 blood donors (single serumsamples). The sera from subjects in group i were collectedfrom patients attending an outpatient clinic for sexually trans-mitted diseases. The sera from subjects in groups ii, iii, and ivwere sent to our laboratory for diagnostic or screening pur-poses. Group iii consisted of patients with symptoms ofuncomplicated urogenital infection (n = 6), ascending infec-tion (n = 17), infant pneumonia (n = 10), inguinal lymphad-enopathy (n = 6), (reactive) arthritis (n = 8), and nonspecificsystemic symptoms (n = 3). The sera from the blood donors(25 males and 25 females) were provided by the blood bank ofthe Red Cross, Utrecht, The Netherlands.WIF assay. C. trachomatis 434-B, serovar L2, and an avian

strain of C. psittaci were inoculated onto 1-day-old DEAE-dextran-pretreated monolayers of HeLa 229 cells in six-wellmicrotiter plates. The plates were centrifuged at 4,800 x g for1 h and 25°C and were incubated overnight at 37°C and 5%CO2. The next day, the cells were carefully trypsinized and 20,ul of a suspension containing 50,000 cells was spotted into eachwell of a microscope slide and incubated for 1 day at 37°C in5% CO2. The slides were then fixed in methanol and werestored at -70°C until needed. To inactivate human immuno-deficiency virus (37), all serum samples were pretreated with0.5% Nonidet P-40 (NP-40) by mixing nine parts of serum withone part of 5% NP-40 in 0.01 M phosphate-buffered saline(PBS; pH 7.2). Before determining the presence of specificimmunoglobulin G (IgG) antibodies, the sera were treatedwith a brain-liver-egg yolk suspension in order to removenonspecific staining factors (12). Before testing for specificIgM or IgA antibodies, the sera were additionally treated withsheep anti-human IgG Fc fragment to prevent the interferenceof rheumatoid factor or a high titer of specific IgG antibodies(12). Fluorescein-labeled sheep anti-human IgG and IgM wereobtained from the Department of Immunochemistry of theNational Institute of Public Health and Environmental Protec-tion. Fluorescein-labeled rabbit anti-human IgA was pur-chased from Dako (Glostrup, Denmark). The sera were di-luted in Veronal buffer with 0.5% bovine serum albumin (BSA;Organon Teknika, Boxtel, The Netherlands). All sera weretested in twofold dilution series starting at 1:8. The last dilutionyielding specific fluorescence was considered the titer.Enzyme immunoassays. Antigen of C. trachomatis serovar

L2 and control antigen of noninfected HeLa 229 cells wereprepared as described previously (29), with minor modifica-tions. In short, C. trachomatis was inoculated onto 1-day-oldDEAE-dextran-pretreated monolayers of HeLa 229 cells insix-well microtiter plates. The plates were centrifuged at 4,800x g for 1 h and 25°C and were incubated for 3 days at 37°C in5% CO2. The cells were then scraped from the surface of thewells, and the antigen was partially purified by sonication,differential centrifugation, and centrifugation through a layerof 35% sodium diatrizoate (Sigma, St. Louis, Mo.). Theantigen was stored in aliquots of 50 [LI at -70°C (30).Polyvinylchloride microtiter plates (Falcon; Becton Dickinson,Oxnard, Calif.) were coated overnight with antigen or controlantigen. The coated antigen was subsequently treated eitherwith 1% sodium periodate in 0.01 M phosphate-bufferedphysiological saline (pH 6.0) or with 1% Triton X-100-5 mMEDTA in PBS as described previously (29). To inactivatehuman immunodeficiency virus (1), all serum samples werepretreated by mixing 95 ,ul of serum with 5 RI of 3-propiolac-tone prediluted 1:20 in PBS and incubating the mixture at 4°Covernight; this was followed by incubation for I h at 37°C tohydrolyze the remaining 3-propiolactone molecules. The sera

were then diluted in PBS with 0.5% Tween 20 (Sigma), 0.5%gelatin (Sigma), and 1% BSA and were tested in twofolddilution series starting at 1:100. Peroxidase-labeled rabbitanti-human IgG and IgA were purchased from Dako, andaffinity-purified peroxidase-labeled goat anti-human IgM waspurchased from Cappel (Organon Teknika). The last dilutionwith an absorption above the corresponding cutoff curve wasconsidered the titer. The cutoff curves were constructed foreach enzyme immunoassay separately by joining the calculatedcutoff values at each dilution. The cutoff values were calculatedby adding two times the standard deviation to the mean of theabsorbance values of 75 serum samples that were all negativein the immunofluorescence test for specific IgG, IgA, and IgMantibodies to C. trachomatis. IgM-positive samples were testedby a latex agglutination assay (Behring, Marburg, Germany)for the presence of rheumatoid factor.

Statistical analysis. The relationships between the titersobtained in the immunofluorescence tests and the enzymeimmunoassays were analyzed by calculating the Spearman rankorder correlation coefficient of the titers. Before analyzing thefrequency distribution of IgG titers obtained in the differentgroups of sera, the results were reduced to four classes of IgGtiters (immunofluorescence, <8, 8 to 32, 64 to 256, and -512;enzyme immunoassay, <100, 100 to 400, 800 to 3,200, and-6,400). The significance of the difference in frequency distri-butions was tested by the chi-square test. A P value of less than0.05 was considered significant in all of the analyses.

RESULTS

Analysis of antibody frequency distributions. The frequencydistributions of IgG, IgA, and IgM antibody titers in sera fromall five groups were determined. The results of antibodydeterminations in the WIF assays are given in Table 1. Theresults of IgG antibody determinations in the enzyme immu-noassays with untreated and sodium periodate-treated anti-gens are given in Table 2. The results obtained in the enzymeimmunoassay with Triton X-100-treated antigen were verysimilar to those obtained in the enzyme immunoassay withsodium periodate-treated antigen. The results of IgA and IgMantibody determinations in the enzyme immunoassays and theWIF assay were similar to those of IgG antibody determina-tions, but the number of positive results was lower.

Analysis of group i patients. With one exception, sera fromall patients in group i had C. trachomatis-specific antibodies atthe time of the first visit. In addition, sera from two patientshad IgM antibodies detectable in the WIF assay and serumfrom one of them also had IgM antibodies detectable in theenzyme immunoassays. A paired serum sample from anotherpatient showed a significant fourfold rise in titer for both IgGand IgA antibodies in the WIF assay and the enzyme immu-noassays. Two acute-phase serum samples and none of theconvalescent-phase serum samples showed a reduction inenzyme immunoassay titer after treatment of the antigen withsodium periodate or Triton X-100. Spearman rank ordercorrelations between titers obtained in the WIF assay and theenzyme immunoassays calculated for sera from both the acutephase and the convalescence phase are listed in Table 3.Comparison of the frequency distribution of IgG titers of thesera from the acute phase with that of the sera from theconvalescent phase showed no significant differences (Table 4).Figure 1 shows a typical example of the effect of antigentreatment on the dose-response curve from a convalescent-phase serum sample from a patient with C. trachomatis infec-tion.

Analysis of group ii patients. Paired serum samples from five

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TABLE 1. Frequency distribution of antibody titers determined by the WIF assay

No. of No. (%) of patients with the following reciprocal titer in WIF assay:Group of patients patients Antibody: IgG, <8; IgA IgG, 8-32; IgA IgG, 64-256; IgA or IgG,

tested or IgM, <8 or IgM, 8-32 IgM, -64 .512

Culture proven (acute) 11 IgG 1 (9) 0 (0) 6 (55) 4 (36)IgA 1 (9) 3 (27) 7 (64)IgM 9 (82) 1 (9) 1 (9)

Culture proven (convalescent) 11 IgG 1 (9) 0 (0) 5 (45) 5 (45)IgA 1 (9) 3 (27) 7 (64)IgM 9 (82) 2 (18) 0 (0)

Ornithosis (acute) 10 IgG 6 (60) 2 (20) 2 (20) 0 (0)IgA 9 (90) I (10) 0(0)IgM 10 (100) 0 (0) 0 (0)

Ornithosis (convalescent) 10 IgG 0 (0) 1 (10) 4 (40) 5 (50)IgA 1 (10) 6 (60) 3 (30)IgM 5 (50) 1 (10) 4 (40)

iii (patients with symptoms suggestive of infection) 50 IgG 19 (38) 8 (16) 10 (20) 13 (26)IgA 30 (60) 2 (4) 18 (36)IgM 45 (90) 2 (4) 3 (6)

iv (pregnant women) 54 IgG 35 (65) 7 (13) 7 (13) 5 (9)IgA 43 (80) 7 (13) 4 (7)IgM 54 (100) 0 (0) 0 (0)

v (blood donors) 50 IgG 41 (82) 6 (12) 3 (6) 0 (0)IgA 44 (88) 6 (12) 0 (0)IgM 50 (100) 0 (0) 0 (0)

patients showed a significant rise in IgG titer in the CFT, theWIF assays with either C. psittaci or C. trachomatis antigen,and the enzyme immunoassay with untreated antigen. Pairedserum samples from four patients showed a significant rise inIgG titer in three of these assays (each possible combinationonce), and paired serum samples from one patient only showed

a significant rise in IgG titer in the CFT and the WIF assay withC. psittaci antigen. There was almost complete concordancebetween the results of the IgG, IgA, and IgM determinations inthe WIF assay with C. trachomatis and C. psittaci antigens.Sodium periodate treatment of the antigen considerably re-

duced antibody titers in paired serum samples from five

TABLE 2. Frequency distribution of IgG titers determined in the enzyme immunoassays with untreated and sodium periodate-treated antigen

No. (%) of patients with the following reciprocal IgG titer in enzyme immunoassay:No. of

Group of patients patients Untreated antigen Sodium periodate-treated antigentested

<100 100-400 800-3,200 .6,400 <100 100-400 800-3,200 -6,400

Culture proven (acute) 11 1 (9) 0 (0) 2 (18) 8 (73) 1 (9) 0 (0) 4 (36) 6 (55)Culture proven (convalescent) 11 1 (9) 0 (0) 1 (9) 9 (81) 1 (9) 0 (0) 4 (36) 6 (55)

iiOrnithosis (acute) 10 3 (30) 0 (0) 5 (50) 2 (20) 7 (70) 1 (10) 1 (10) 1 (10)Ornithosis (convalescent) 10 1 (10) 0 (0) 0 (0) 9 (90) 3 (30) 0 (0) 4 (40) 3 (30)

iii (patients with symptoms suggestive 50 26 (52) 2 (4) 9 (18) 13 (26) 29 (58) 6 (12) 6 (12) 9 (18)of infection)

iv (pregnant women) 54 42 (78) 0 (0) 5 (9) 7 (13) 43 (80) 3 (6) 4 (7) 4 (7)

v (blood donors) 50 38 (76) 7 (14) 5 (10) 0 (0) 43 (86) 4 (8) 3 (6) 0 (0)

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TABLE 3. Spearman rank order correlation coefficients betweenIgG titers obtained in WIF assay and enzyme immunoassays with

different antigen treatments

Correlations between titers in WIFNo. of assays and enzyme immunoassays

Group of patients patients with antigen:tested Periodate Triton X-lt)(

Untreated treated treated

Culture proven (acute) 11 0.602 0.786" 0.747"Culture proven 11 0.609" 0.808" 0.781"

(convalescent)

Ornithosis (acute) i0 0.622 0.607 0.607Ornithosis 10 0.806" 0.104 -0.061

(convalescent)

iii (patients with symptoms 50 0.762" 0.726a 0.718"suggestive ofinfection)

iv (pregnant women) 54 0.668" 0.608" 0.606"

v (blood donors) 50 0.647" 0.301" 0.348"

" Correlation coefficient significantly different from t) (P < 0.05).

patients, although they still showed a significant rise in anti-body titer. The Spearman rank order correlation coefficient ofthe sera from the convalescent phase was reduced to an

insignificant level after treatment (Table 3). Also, the differ-ence in IgG titer frequency distribution in sera from the acutephase and from the convalescent phase was statistically signif-icant by the WIF assay and the enzyme immunoassay withuntreated antigen, but not by the enzyme immunoassay witheither sodium periodate- or Triton X-100-treated antigen(Table 4). A typical example of the effect of antigen treatmenton the dose-response curve from a convalescent-phase serum

sample from a patient with C. psittaci infection is shown in Fig.2. The pattern of IgA titers was similar to that of the IgG titers,but the IgA titers were generally lower. Of the six patientswhose sera had a positive IgM titer in the WIF assay with C.psittaci, sera from five of the patients had similar IgM titers inthe WIF assay with C. trachomatis, and all five serum samplesshowed a significant reduction in IgM titer in the enzyme

immunoassay after treatment with sodium periodate or Tritonx-100.

Analysis of group iii, iv, and v patients. Sera from threepatients from group iii had a positive IgM titer (1:-64) in theWIF assay and all three enzyme immunoassays (1:-1,600), andsera from six patients had a low-positive IgM titer in at leastone of the assays. The WIF assay was negative for IgM in allsera from patients in groups iv and v, while the sera from 3members of groups iv and v showed a low IgM titer (1:.800)in all three enzyme immunoassays and sera from 14 membersof groups iv and v showed a low IgM titer in one or two of theenzyme immunoassays. All tests for rheumatoid factor were

negative. There was no difference in the results for sera frommales or females in the group of blood donors. The Spearmanrank order correlation coefficients were not changed aftertreatment of the antigen in sera from patients in groups iii andiv, while they were slightly lower after treatment of the antigenin sera from patients in group v (Table 3). The frequencydistribution of the IgG titers in sera from patients in group iiidiffered significantly from that in sera from patients in theother two groups by the WIF assay and the enzyme immuno-assay with untreated antigen. After antigen treatment, thisdifference was only significant for sera from group v (Table 4).The pattern of IgA titers was similar to that of IgG titers.

DISCUSSIONDirect detection of C. trachomatis is the diagnostic method

of choice for most patients suspected of having C. trachomatisinfection. The value of serological tests has been questionedfor the diagnosis of uncomplicated C. trachomatis infections,since high levels of antibody are associated with low predictivevalues (41) and fail to correlate with isolation of the organismor antigen detection (22). However, in a few circumscribedclinical syndromes like ascending infections (salpingitis, peri-hepatitis [23, 33, 43]), immunological complications (reactivearthritis [16]), and infant pneumonia (28, 42), the value ofserological methods, especially those that measure IgA andIgM antibodies, is comparable to or better than that of culture.For these purposes (and also to establish past infection) as wellas for epidemiological studies, sensitive and specific antibodydetection assays are required.The quality of most antibody assays is determined by the

means by which the antigen is prepared. Many epitopes existon several antigenic molecules of C. trachomatis. Theseepitopes may react at the genus, species, subspecies, or serovar

level in different antibody assays (25). The dominant genus-

reactive epitopes are located on the LPS. The influence ofantibodies to LPS on the outcome of serological assays de-pends on the assay technique, the means by which the antigenis prepared, and the reading of the result in immunofluores-

TABLE 4. Comparison of IgG titer frequency distribution in selected groups

Chi-squared value of comparison of titers obtained in (corresponding P value)Group of patients Periodate-treated Triton X-1t)t) treatedWIF assay Untreated EIA"

EIA

i(culture proven, acute and 0.20202h (0.90)392) 0.39216" (0.82195) 0.00000" (1.00000) 0.00000 (l.t)0000)convalescent)

ii (ornithosis, acute and 12.00000 (0.00738) 10.45455" (0.00537) 5.40000 (0.14474) 6.81905 (0.07789)convalescent)

iii and iv (suggestive symptoms and 8.75148 (0.03278) 8.56639 (0.03565) 5.90018 (0.11657) 7.60420 (0.05494)pregnant women)

v (suggestive symptoms and blood 25.12161 (0.00001) 19.17063 (0.00025) 13.12222 (0.00438) 17.93583 (0.00045)donors)

"EIA, enzyme immunoassay."The chi-squared statistics have two degrees of freedom; all others have three degrees of freedom.

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D 1.800- Antigen treatmenta Untreated2 1.600- -- Periodate

---------- Triton X-100c 1.400- ---------------- Cutoff

1.200

= 1.000 -;0.

0.800-

0.600

0.400 ..

0B200~~~~~~~~~~~~~~~~~~.......... .....................................-------------1 2 5 8 16 32 64 128 256 512 1024 2048

Reciprocal serum dilution (x`10-2)FIG. 1. Reactivity of a convalescent-phase serum sample from a patient with C trachomatis infection. The cutoff curve was calculated for

sodium periodate-treated antigen. The level of the curve was 0.010 optical density units higher for the untreated antigen.

fluorescence of the MIF assay requires a high degree ofexpertise, the desired specific results will be obtained only byspecialized laboratories. Enzyme immunoassays yield moreobjective results than assays based on immunofluorescence,but the method of antigen preparation is very important withregard to the specificity of the assay. The use of antigenextracted by detergent from C. trachomatis elementary bodies(8, 44) or LPS from Salmonella Re mutants (18) results ingenus-specific assays. Antigen prepared by differential centrif-

I An+;non fr..ma lenttedIteK-100

cence assays. The CFT measures predominantly genus-specificantibodies, especially when heat-treated antigen is used. Cross-reactions between different Chlamydia species are thus likelyto occur (40). LPS is also present throughout intracellularinclusions (13), and it is therefore likely that anti-LPS antibod-ies will contribute to the results of the WIF assay. Sincechlamydial inclusions contain more LPS than purified elemen-tary bodies, the WIF assay is more genus specific (10, 32) thanthe MIF assay. However, since the reading of the specific

2.200-

O 2.000-

Z 1.800-

°) 1.600-C)0 1.400-co( 1.200-

O 1.000-

0.800 -

0.600 -

0.400 -

0.200-

0.000-

FIG. 2. Reactivity of a convalescent-phase serum sample of a patient with C. psittaci infection by using C. trachomatis as the antigen. The cutoffcurve was calculated for sodium periodate-treated antigen. The level of the curve was 0.010 optical density units higher for the untreated antigen.

P%,ugIeUu ius:uuUntreati

------ Perioda\---------------Triton )

--------------cutoff

V.\

\\\~~ \

2 4 8 16 32 64 128 256 512 1024 2048

Reciprocal serum dilution (x10-2)

--Ii

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1424 OSSEWAARDE ET AL.

ugation only yields a specificity equivalent to that of the WIFassay (4, 11). Both methods lack the specificity needed todiscriminate between antibodies to C. trachomatis and those toC. pneumoniae (26, 40). The use of antigen prepared by furtherpurification through a layer of a compound like sodiumdiatrizoate (7, 9, 19, 20) improves specificity, but the presenceof LPS can still be easily demonstrated in these preparations(29). Preparation of antigen from reticulate bodies instead ofelementary bodies does not reduce the cross-reactions withother Chlamydia species (14, 27). The specificity of an enzymeimmunoassay with purified major outer membrane proteinlooks very promising (34), but the production of this type ofantigen is very laborious. Genus-specific epitopes are presenton the major outer membrane protein, the 60-kDa proteins,and the LPS (24). The genus-specific epitopes of the LPS arelocated on the three 3-deoxy-D--manno-2-octulosonic acid moi-eties (5). The exact constitution of other genus-specificepitopes is not known. As a compromise between the use ofpurified elementary bodies and purified species-specific anti-gens, we examined the possibility of removing the well-char-acterized and main cross-reacting genus-specific antigen, theLPS. We have previously shown that sodium periodate oxida-tion or detergent extraction yields an antigen that is no longercapable of binding anti-LPS monoclonal antibodies (29). Bythis approach, antibodies to C. pneumoniae could be deter-mined very specifically (17).We obtained similar results by the WIF assay and the

enzyme immunoassays with untreated as well as treated anti-gen by using serum samples from patients with culture-provenurogenital C. trachomatis infection. This indicates that C.trachomatis-specific antigens are still reactive after treatmentof the antigen with sodium periodate or Triton X-100. Theseresults are in agreement with our previous findings (29). Theresults with serum samples from patients with C. psittaciinfection were very clear-cut. The difference between theresults obtained with untreated and treated antigens fromconvalescent-phase serum samples was statistically significant,while that between the results obtained with untreated andtreated antigens from acute-phase serum samples was not. Theconvalescent-phase sera from all patients showed dramaticreductions in titer after treatment of the antigen. Therefore,this difference indicates the specificity of the antibodies. Therewas almost complete agreement between the WIF assays withC. psittaci or C. trachomatis antigen and the enzyme immuno-assay with untreated antigen, while the correlation between theWIF assay and the enzyme immunoassay disappeared com-pletely after treatment of the antigen. This indicates that boththe WIF assay and the enzyme immunoassay with untreatedantigen react predominantly in a genus-specific manner. Mosttiters, however, remained positive after treatment, and therewere still significant rises in titer. This was not completelyunexpected, since many proteins share genus-reactive epitopes(25). Therefore, one cannot rely on an assay with only oneantigen. A titer reduction after antigen treatment is a strongindicator of the existence of cross-reactions, which should beverified by using antigens from other Chlamydia species.The group of blood donors can be considered to provide a

baseline for the seroprevalence of antibodies. The sera fromgroups iii and iv represent samples that can be expected inroutine diagnostic practice. Group iii represented a controlgroup of women with proven sexual activity. Since sexualactivity is a risk factor for acquiring a chlamydial sexuallytransmitted disease, more positive samples and higher titerswere observed in this group than in the group of blood donors.The group of patients with symptoms suggestive of infectionwith C. trachomatis represents patients with a category of

disease entities for which it is difficult to detect the causativeagent. Detection of C. trachomatis itself is frequently notpossible in samples from these patients, and interpretation ofthe serological results is often difficult. A low C. trachomatisantibody titer may be caused by cross-reactions, while a highantibody titer may be due to a recent or chronic infection. Oneshould keep in mind, howevcr, that antibody titers may persistfor extended periods of time (35). Statistical analysis showedthat treatment of the antigen reduced the titers in the serafrom the group of blood donors (group v) more than it did inthe sera from the other two groups (groups iii and iv). Sincegroup v, in contrast to the other two groups (groups iii and iv),is not known to have any risk factors for C. trachomatisinfection, this reduction suggests an enhanced sensitivity of theenzyme immunoassays with treated antigens.

Detection of C. trachomati.s-specific IgM appears to have itsown problems. A weak correlation between enzyme immuno-assay and the MIF assay has been reported previously (9, 20),although the use of more specific rcagents does improve theperformance of the enzyme immunoassay (19). In our study,some samples showed weak IgM reactivity in one or two of thethree enzyme immunoassays. They could easily be distin-guished from samples with true-positive results, which werepositive in all three enzyme immunoassays. However, in viewof the possible false-positive results caused by rheumatoidfactor (19) and the possible false-negative results caused by theinterference of a high IgG titer (15), development of apL-capture enzyme immunoassay, such as for the detection ofIgM antibodies to chlamydial LPS (49), is warranted.

Since blood and blood products from human sources areknown to be a vector of communicable infectious diseases, weincluded in our assays a procedure to inactivate human immu-nodeficiency virus. All sera were treated with 0.5% NP-40before testing in the immunofluorescence assays (37). How-ever, after experiments with monoclonal antibodies, it becameevident that NP-40, even at concentrations of as low as 0.005%,can extract LPS from elementary bodies. Therefore, inactiva-tion of all sera prior to testing in the enzyme immunoassayswas carried out by using 3-propiolactone (1).Sodium periodate oxidation and extraction with Triton

X-100 represent two independent and completely differentapproaches to solving the same problem. Our results show thatthey yield approximately the same results. The enzyme immu-noassays with either treatment showed a small difference in ahomologous response and a large difference in a heterologousresponse compared with differences obtained in the enzymeimmunoassay with untreated antigen. A third approach is toblock the epitopes on the LPS with monoclonal antibodies.Although this approach improves the specificity of a commer-cial immunofluorescence test (21), steric hindrance may occur.Since detergent extraction removes some of the proteins fromthe elementary bodies (6), we prefer the enzyme immunoassaywith sodium periodate oxidation of the antigen.We conclude that our approach with sodium periodate

oxidation of the antigen is a simple and effective way ofobtaining an antibody assay with enhanced specificity forclinical purposes as well as seroepidemiological studies.

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