Analysis of Western Blotting (Immunoblotting) Technique in

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1 JOURNAL OF CLINICAL MICROBIOLOcY, Mar p /94/$04.00+() Copyright American Society for Microbiology Vol. 32. No. 3 Analysis of Western Blotting (Immunoblotting) Technique in Diagnosis of Congenital Syphilis M. P. MEYER,12* T. EDDY,2 AND R. E. BAUGHN' Departments of Paediatrics and Child Health' and Clinical Science and Immunology, ' University of Cape Towni, Cape Town, South Africa, and the Department of Microbiology and Immuinology, Baylor College of Medicine and the Veterans Affairs Medical Center, Houston, Texas Received 12 July 1993/Returned for modification 7 September 1993/Accepted 7 December 1993 The diagnosis of congenital syphilis in apparently healthy infants continues to be problematic. Immunoglobulin M antibodies specific for a subset of Treponema pallidum antigens have been detected by Western blotting (immunoblotting). In the present study we investigated the sensitivity and specificity of this method. We tested 26 infants aged 0 to 4 months who fulfilled the accepted criteria for the diagnosis of congenital syphilis. There were 14 symptomatic infants. Sera from 13 of these infants were positive for the 47-kDa treponemal antigen (92% sensitivity). The remaining 12 infants were clinically asymptomatic when tested at birth but subsequently displayed features consistent with the disease. Reactive blots (antibodies to the 47- and/or the 15-kDa antigens) were noted in 10 of the 12 infants (83% sensitivity). Thirty infants whose mothers had syphilis were monitored and shown to be uninfected. Nonreactive blots were seen in sera from 27 infants, while sera from 3 older infants had false-positive tests (90% specificity). The Western blotting technique is sensitive (even in the diagnosis of clinically inapparent cases) and, in the absence of immunoglobulin M rheumatoid factor, is a useful confirmatory test for congenital syphilis. Despite the resurgence of congenital syphilis and increased awareness of the condition, laboratory diagnosis remains difficult (7). Part of the problem arises because the pathogen, Treponema pallidlum subsp. pallidum, is not easily identified. In addition, the standard serological tests are less useful in newborns because of immunoglobulin G (IgG) transfer across the placenta. IgM tests which depend on the infant's response (e.g., the fluorescent treponemal antibody absorption test [FTA-ABS] and investigations such as radiographs of the long bones) have reduced sensitivities in asymptomatic patients (9, 1 1). Consequently, newer tests including Western blotting (immunoblotting) have been developed, and a sensitivity of 92% in diagnosing symptomatic congenital syphilis has been reported (10). However, the majority of newborns who are subsequently proven to have congenital syphilis are asymptomatic at birth, and the sensitivity of the procedure in this group is unknown. Here we report our experience with the test and include results for at-risk infants in whom the diagnosis of congenital syphilis was established at follow-up. MATERIALS AND METHODS Background. The study was performed on infants born in the Peninsula Maternal and Neonatal Service as well as those presenting to the Red Cross War Memorial Children's Hospital in Cape Town, South Africa. During the time of the investigation the hospital policy was similar to that proposed by Kaufman et al. (8) and the World Health Organization (15); i.e., patients with clinical signs compatible with the diagnosis of congenital syphilis were treated, while apparently healthy infants were monitored and serial Venereal Disease Research Laboratory (VDRL) titers were determined. Those with a titer that showed a fourfold rise or whose sera did not become negative by 6 months were considered to be infected and were * Corresponding author. Mailing address: Neonatal Medicine, Old Main Building H46, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa. 629 treated with procaine penicillin at 50,000 U/kg of body weight per day for 10 days. Informed consent for the study was obtained from the parents, and the protocol was approved by the Ethics and Research Committee of the University of Cape Town. We enrolled infants in the first 4 months of life, and the infants described here were examined clinically at birth and were monitored by one of the authors (M.P.M.). The patients with congenital syphilis fulfilled the criteria suggested by Kaufman et al. (8) and were divided into the following two groups: those with clinical signs (symptomatic) and those who were clinically asymptomatic (regardless of laboratory test results). This approach was followed because in South Africa the availability of diagnostic tests at birth is variable and the usefulness of some tests, e.g., the cerebrospinal fluid VDRL in asymptomatic infants, is debatable (5, 14). Patients. Fourteen infants presented with clinical signs compatible with symptomatic congenital syphilis. Their mothers were asymptomatic and had VDRL titers ranging from 1:16 to 1:512; all had a positive T pallidlunm hemagglutination (TPHA) test result. The mothers had not received antenatal therapy for syphilis because of failure to attend antenatal clinics or because of enrollment in an antenatal clinic late in their pregnancies. Congenital syphilis was diagnosed at birth in 12 infants, at age 2 weeks in 1 infant, and at 10 weeks in another infant. The commonest findings were hepatosplenomegaly (14 infants), bone changes (13 infants), edema (8 infants and skin rashes (6 infants). Twelve infants had no physical features of disease at birth. Their mothers were untreated or inadequately treated in the last month of pregnancy. The maternal VDRL titers varied from 1:2 to 1:512, and the TPHA test was positive in all cases. Four of the infected infants had abnormal long-bone X rays and were treated in the first few days of life; 8 infants had at least a fourfold increase in VDRL titer by 4 months of age. Five of the eight infants developed snuffles and organomegaly, in addition to the rise in VDRL titer. These 12 infants have been described in a publication which evaluated the FTA-ABS

2 630 MEYER ET AL. (IgM) test (12). Sera which were obtained from the infants at birth were divided into aliquots and were stored at - 80 C until use; repeated freezing and thawing was avoided. Thirty infants were uninfected, even though they were born to seropositive mothers with untreated or inadequately treated syphilis. These babies were examined clinically at birth, and a VDRL titer and long-bone X rays were obtained. At follow-up, the VDRL titer became negative and there were no clinical signs of congenital syphilis. Twenty-one of the serum specimens tested were specimens obtained at birth; the other nine serum specimens were from infants aged 6 weeks to 4 months. In addition, sera from 10 healthy infants who, together with their mothers, had negative serology for syphilis (VDRL and TPHA tests) were subjected to Western blotting. Pooled serum from 10 adults with secondary or early latent syphilis was used as a positive control. Western blots. A suspension of T pallidum antigens was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using a 4% stacking gel and a 12% separating gel as described previously (1). Antigens and low-molecularweight standards (Bio-Rad) were then electrophoretically transferred to nitrocellulose paper. Strips of nitrocellulose were cut and blocked for 1 h with 5% fetal calf serum. Test serum (1:100) in Tris-buffered saline (TSB; ph 7.4) with 0.05% Tween 20 (TSB-T) was reacted with the strips for 16 h at room temperature. After three washes with TSB-T, a mouse anti-human IgM monoclonal antibody (p. chain specific; kindly provided by J. Conradie, Natal Blood Transfusion Service, Durban, South Africa) diluted 1:1,000 in TSB was added. After washing, a biotinylated rabbit anti-mouse antibody (1:1,000 in TSB) and then streptavidin-biotin horseradish peroxidase complex (Strept ABComplex HRP; Dako, Glostrup, Denmark) were added. Diaminobenzene (25 mg/ml) with 3% cobalt chloride and hydrogen peroxide was used to develop the blots. The molecular weights of the T. pallidum antigens were determined as described previously (10); the identification of antigens was assisted by silver staining of T. pallidum gels and by reacting nitrocellulose strips with pooled serum from adults with recent syphilis and pooled IgG from rabbits infected with T. pallidum. IgM fractionation. IgM fractions were prepared by passing 100 p.1 of serum over an ion-exchange column (Quick Sep, System II; Isolab). The eluted IgM was reconcentrated to ca. 60 I.1 with the Centricon 30 microconcentrator system [Amicon]). The sample was then subjected to high-pressure liquid chromatography (HPLC) by using a size exclusion column (Biosep SEC S3000 with dimensions of 300 by 7.8 mm; Phenomonex). The mobile phase was 20 mm sodium phosphate with 0.1 M sodium sulfate, and the flow rate was 0.5 mvmin. The IgM fractions were identified by timing the appearance of IgM standards and by a dot blot method. For the latter, 2 p.1 of the HPLC fraction was applied to nitrocellulose paper. Peroxidase-labelled antibodies to human IgM and IgG (Cappel, Cochranville, Pa.) at dilutions of 1:1,000 and 1:2,000, respectively, were added; this was followed by the addition of substrate (a 3-mg/ml solution of 4-chloro-1-naphthol in methanol). In this way, IgM fractions free of IgG were obtained. The IgM level was measured by nephelometry (Beckman), and Western blotting was carried out with the same IgM concentration as was originally present in the serum. Owing to the small quantities of serum generally available, we prepared IgM fractions only in selected cases. Rheumatoid factor (RF) latex tests (Orthodiagnostics, Beerse, Belgium) were carried out at a serum dilution of 1:5. Evaluation. The sensitivity of the Western blotting proce a b c d FIG. 1. Reaction profiles in Western blots. Lane a, IgM reactivity with pooled adult serum; lane b, profile with pooled rabbit IgG; lanes c and d, IgM reactivity in sera from infants with symptomatic congenital syphilis. dure was defined as the percentage of patients with congenital syphilis (according to the criteria of Kaufman et al. [8]) with a positive blot. The specificity was defined as the percentage of uninfected infants with a nonreactive test in relation to the total number of unaffected infants (9). In the present study the infants of seropositive mothers who did not develop congenital syphilis were regarded as true negatives (9). RESULTS Symptomatic patients. The reaction profiles of pooled adult serum, pooled rabbit IgG, and sera from symptomatic infants are compared in Fig. 1. The infant sera demonstrated reactivity with treponemal antigens with molecular masses of between 110 and 15 kda (Fig. 1, lanes c and d; Fig. 2, lanes a, b, and c); on average, each serum specimen reacted with seven antigens (Table 1). The commonest responses were directed against 47- and 45-kDa antigens (13 of 14 infants) the 72-kDa protein (9 infants), a 39-kDa antigen (8 infants), and a 42-kDa component (7 infants). Clinically asymptomatic patients. Sera from the 12 infants I _ ' N- J. CLIN. MICROBIOL. a b c d e f g FIG. 2. IgM reactivity in Western blots. Lanes a to c, sera from infants with symptomatic congenital syphilis; lanes d to f, sera from infants with asymptomatic congenital syphilis; lane g, serum from an uninfected infant of a seropositive mother.

3 VOL. 32, 1994 TABLE 1. IgM reactivity to T. pallidum antigens in whole, unfractionated infant sera No. of patients Molecular mass (kda) Symptomatic Asymptomatic Uninfected, at risk (n = 14) (n = 12) (n = 30) IgM WESTERN BLOTS IN CONGENITAL SYPHILIS m 84.* a b c d e f 9 FIG. 3. IgM reactivity in Western blots. Lane a, sera from infants with symptomatic congenital syphilis; lanes b and c, sera from infants with symptomatic congenital syphilis before (lane b) and after (lane c) IgM fractionation; lanes d and e, sera from infants with asymptomatic congenital syphilis before (lane d) and after (lane e) IgM fractionation; lanes f and g, serum from an uninfected infant of a seropositive mother before (lane f) and after (lane g) IgM fractionation. in the clinically asymptomatic group demonstrated IgM reactivity against fewer treponemal antigens (average, three per patient). Sera from eight individuals had an IgM response to the 47- and 45-kDa antigens (Fig. 2, lanes d and e), whereas sera from five individuals reacted with the 84-kDa protein. In sera from two patients, there was reactivity with the 15-kDa antigen without a response to the 47- or 45-kDa protein, while an additional two individuals showed no seroreactivity to the 47- or 45-kDa antigens or lower-molecular-mass proteins. None of the serum specimens reacted with the 72-kDa antigen. Uninfected infants of seropositive mothers. Sera from the uninfected 30 infants of seropositive mothers showed reactivity against a number of treponemal antigens (Fig. 2, lane g), the most frequent being 84 kda (six infants), 39 kda (eight infants), 42 kda (five infants), and 28 or 24 kda (six infants). Of the 21 neonates, sera from 16 had no IgM reactivity or a single faint band only; sera from none of the neonates had a positive blot for the 47- or 45-kDa protein. In contrast, sera from all nine older infants (1 to 4 months of age) showed a response to one or more treponemal antigens, and sera from three infants reacted with the 47-kDa antigen (Table 1). Sera from two of these infants were RF latex test positive. Healthy infants. The serum samples from the newborns with negative serologies for syphilis showed no reaction with treponemal antigens when Western blotting was carried out. Definition of positive and negative blots. Overall, reactivity to a number of antigens (those of 72, 47, 45, 37, 33, 30, 17, and 15 kda) was more frequent (P < 0.05) in patients with congenital syphilis than in uninfected infants of seropositive mothers (Table 1). We therefore regarded blots with reactivities to any of these antigens as positive. Responses to the remaining antigens, including the proteins of 84, 42, 39, 28, and 24 kda, were nonspecific and were observed in both patients and uninfected infants and, for the purposes of the study, were regarded as a negative test result. By applying this categorization, the sensitivity of Western blotting in the diagnosis of congenital syphilis was 92% in symptomatic patients, 83% in those without physical signs, and 75% in the subgroup with normal long-bone X rays. The specificity was 90%. IgM fractionation. IgM fractions were tested in three symp- tomatic infants whose sera were RF latex test positive. One of the three was a patient whose whole serum showed no response to the 47- and 45-kDa proteins and had a negative blot with whole serum (Fig. 3, lane b). In this individual, reactivity to the 47- and 33-kDa antigens was identified when the IgM separated by chromatography was tested (Fig. 3, lane c). Results for the other two symptomatic infants showed minor differences when the patterns obtained before and after IgM fractionation were compared (Table 2). Three clinically asymptomatic infants whose IgM fractions were tested had no IgM response to the 47- or 45-kDa protein. There were few changes in the blots after the procedure, and the sensitivity of the procedure was not improved (Fig. 3, lanes d and e). The IgM reaction to the 15-kDa antigen was no longer evident in one patient (Table 2). IgM fractions were also prepared on three serum specimens from uninfected seropositive patients whose sera had false- TABLE 2. Patient group IgM reactivity to T. pallidunm antigens before and after IgM fractionation Whole serum Antigen (kda) IgM fraction Symptomatic Patient 1 110, 72, 47, 45, 42, 39, 110, 84, 72, 69, 59, 47, 37, 28 45, 42, 28 Patient 2 84, 72, 47, 45, 39, 37, 72, 47, 45, 39, 37, 33, 33, 24, 17, 15 24, 17, 15 Patient 3 110, , 61, 47, 33 Clinically inapparent Patient 4 84, Patient 5 84, 61, Patient Uninfected, at risk Patient 7 47, 39 No reactivity Patient 8 110, 84, 47, 45, 42, 39, 110, 84 35, 33, 24, 17, 15 Patient 9 72, 61, 47, 45, 42, 39 72, 61, 59

4 632 MEYER ET AL. positive blots and whose whole serum reacted with the 47-kDa antigen. When the blots were repeat tested, fewer antigens were detected and no response to the 47-kDa protein was identified (Fig. 3, lanes f and g). DISCUSSION We investigated Western blotting as a test for the diagnosis of congenital syphilis in patients with symptomatic disease, clinically inapparent syphilis, and two groups of uninfected infants. Practically, it is important that tests for congenital syphilis be able to distinguish asymptomatic infants of seropositive mothers who are infected from those who are not (9). We were able to investigate this question by monitoring at-risk infants without abnormal physical features. Overall, our findings obtained by Western blotting with whole sera confirm and extend those of previous studies (6, 10, 13). The 47- and 45-kDa antigens appear to be the major immunogens in both symptomatic and asymptomatic patients with congenital syphilis. The pattern of reactivity seen in the sera of patients presenting with clinical signs of the disease was similar to that described by other authors. Sanchez et al. (13) reported that IgM responses to treponemal antigens with molecular masses of 72, 47, 45, 42, 37, 17, and 15 kda were useful for distinguishing those infants with syphilis. We found, in addition, that reactivity to the 33- and 30-kDa proteins also detects infants congenitally infected with T. pallidum. Conversely, an IgM response to the 42-kDa antigen was nonspecific, being present in the sera of 17% of uninfected infants aged 1 to 4 months. The sensitivity and specificity of the Western blotting procedure were not increased by considering reactivity to antigens other than those with molecular masses of 47 and 15 kda. In the sera of infants with clinically inapparent disease, we noted IgM responses not only to the 47- and 45-kDa antigens but also to the 15-kDa protein (in the absence of RF). Because two of the three asymptomatic infants whose sera reacted with the 15-kDa antigen had no IgM response to the 47- or 45-kDa antigen, we suggest that the former response may occur early in the disease. On the other hand, none of the asymptomatic infants demonstrated reactivity with the 72-kDa antigen, indicating that such a response may occur relatively late in the disease. We noted a sensitivity of 75% for the demonstration of IgM by Western blotting in asymptomatic infants with normal long-bone X rays, and this is similar to the figure of 65% obtained by FTA-ABS to detect IgM in patients with delayedonset disease (9). These results should be compared with a sensitivity of 14% reported for Western blots of sera from asymptomatic infants in the recent study of Bromberg et al. (3). The presence of positive immunofluorescence for T. pallidum on a nasal swab obtained soon after birth was regarded as presumptive evidence of congenital syphilis. However, it has not been established that all infants with such an immunofluorescence response develop systemic infection with T. pallidum. False-positive blots were found with sera from three uninfected infants aged I to 4 months, and two of the three false-positive blots were RF latex test positive. Removal of IgG eliminated the reactivity to the 47-kDa antigen, suggesting that RFs, either apparent or hidden, were responsible for the observed pattern. IgM reactivities to a number of other treponemal antigens were also noted, which is similar to the findings of Dobson et al. (6). In the latter study, five of eight newborns whose mothers were serofast (i.e., had repeated unchanging positive RPR of low titers in the light of adequate treatment for previous infection) or had a biologic falsepositive nontreponemal test were seroreactive with antigens of 190, 83, 71, 45, and 41 kda. Sanchez et al. (13) also presented results for uninfected newborns of seropositive mothers. No IgM response was present, but only IgM fractions were tested and the authors pointed out that their method may have been relatively insensitive because sera from mothers of infected infants showed no IgM reactivity. Previous reports have not presented results of Western blots of sera from older uninfected infants, but our findings are in keeping with the presence of cross-reactive epitopes on nonpathogenic treponemes. The molecular masses of the shared antigens which most frequently elicit an antibody response in adults are between 80 and 90 kda and 30 and 40 kda (2, 4). The testing of IgM fractions only did not markedly affect the results for infants with congenital syphilis. This finding is in agreement with those of earlier studies (6, 13). One infant was found to have reactivity to the 47- and 33-kDa antigens after the procedure; competitive inhibition between IgM and IgG antibodies for binding sites may have been responsible for this. Among the asymptomatic infants, the IgM response to the 15-kDa antigen was no longer apparent in the serum of one infant after fractionation. This patient was IgM RF negative, and the reason for the altered result is unclear, although IgM may have been lost during fractionation. Sanchez et al. (13) noted greater clarity with the blotting technique when the IgM fraction was tested, although no infants with delayed-onset disease were investigated. We were able to compare the results obtained by Western blotting and FTA-ABS (IgM). The latter was performed on whole serum without the removal of IgG (this significantly reduces sensitivity [12]). The fluorescence test was positive for sera from all symptomatic infants and was reactive with sera from 10 of 12 of the clinically asymptomatic infants. Of the two infants with negative tests, one also had a nonreactive blot, whereas the other had antibody to the 15-kDa antigen. The number of false-positive results was the same by both tests (10%). In the present study, the performances of the two tests were similar, although the Western blotting method may be more objective and easier to standardize. We found that the Western blotting procedure had a high degree of sensitivity, even with sera from infants with clinically inapparent disease. Nevertheless, not all patients with syphilis were identified, so that a negative pattern of reactivity cannot exclude the diagnosis. Furthermore, whereas a reactive blot is a useful indicator of congenital syphilis, false-positive results were noted in infants older than 1 month. The possibility of simplifying the procedure to a dot blot method or enzymelinked immunosorbent assay as a confirmatory test is attractive, and the most useful antigens appear to be those with molecular masses of 47 and 15 kda. ACKNOWLEDGMENTS J. CLIN. MICROB ICL. Funding was obtained from the Medical Research Council of South Africa and from the U.S. Department of Veterans Affairs. REFERENCES 1. Baughn, R. E., and D. M. Musher Isolation and preliminary characterization of circulating immune complexes from rabbits with experimental syphilis. Infect. Immun. 42: Baughn, R. E., and D. M. Musher Radioimmunoassays for the detection of antibodies to treponemal polypeptide antigens in serum. J. Clin. Microbiol. 21: Bromberg, K., S. Rawstron, and G. Tannis Diagnosis of congenital syphilis by combining Treponema pallidum-specific IgM detection with immunofluorescent antigen detection for T. palli-

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