Enzyme-Linked Immunosorbent Assay for Mumps and

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1980, p /80/ /05$02.00/0 Vol. 11, No. 4 Enzyme-Linked Immunosorbent Assay for Mumps and Parainfluenza Type 1 Immunoglobulin G and Immunoglobulin M Antibodies PENTTI UKKONEN,* OUTI VAISANEN, AND KARI PENTTINEN Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, SF Helsinki 29, Finland A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of mumps and parainfluenza type 1 antibodies (immunoglobulin G [IgG] and IgM classes) is described and compared with the conventional complement fixation (CF) test. A highly positive correlation was found between mumps IgG ELISA and the mumps CF test, whereas parainfluenza type 1 IgG ELISA had only a moderate positive correlation with the respective CF test. Mumps IgM antibodies could be demonstrated in all patients with serologically verified and clinically typical (parotitis, meningitis, or orchitis) mumps virus infection, but not in patients with rises in parainfluenza CF titers. Mumps IgM was already present in the acute-phase sera if they were not taken during the first 2 days after onset of disease. Mumps IgM was also found in some paired sera that were taken too late to demonstrate any significant increase in the antibody titers by CF. Therefore, mumps IgM ELISA provides an improvement over the conventional laboratory diagnosis of mumps infection, since the measurement of specific IgM antibodies in a single serum by ELISA is diagnostic, rather than the identification of a fourfold or greater rise in CF antibody titer. An unexpected finding was that parainfluenza type 1 IgM antibodies could not be demonstrated by ELISA in paired sera with rises in parainfluenza CF titers, suggesting a different antibody response from that occurring in mumps infection. The laboratory diagnosis of viral infections is conventionally based on the demonstration of rising antibody titers in paired sera by complement fixation (CF) or hemagglutination inhibition using serial serum dilutions. A more rapid diagnosis may be achieved by new sensitive methods, such as radioimmunoassay and enzyme-linked immunosorbent assay (ELISA), in which immunoglobulin M (IgM) class antibodies can be detected without separation of the IgM immunoglobulins from the serum (1, 4, 11). Improvement of the laboratory diagnosis of mumps virus infection has clinical importance, since mumps is a common cause of serous meningitis. The etiological diagnosis may help the clinician in deciding whether to treat the patient, and therefore a rapid diagnosis is needed. In addition, the conventional antibody tests often show cross-reactions between paramyxoviruses, which may cause difficulties in the serological diagnosis (2, 7, 9). We developed a solid-phase ELISA for detection of mumps and parainfluenza type 1 IgG and IgM antibodies to determine whether a more rapid and specific serological diagnosis can be obtained by this method. MATERIALS AND METHODS ELISA antigens. Virus-infected allantoic fluids (from the Central Public Laboratory, Helsinki, Finland) were used as sources of mumps and parainfluenza type 1 (Sendai) virus. Infected allantoic fluid was diluted lo-4, and 0.2 ml of the dilution was used for the inoculation of the eggs. The viruses were partially purified, as described (3): allantoic fluid was clarified by low-speed centrifugation and pelleted for 90 min at 25,000 rpm in an SW27 rotor. The pellet was suspended in phosphate-buffered saline (PBS) and sonicated before use. Clarified normal allantoic fluid served as a control antigen. Three preparations of mumps antigen were compared: crude allantoic fluid, pelleted virus, and purified virus (in a 10 to 60% sucrose gradient). The pelleted virus was found satisfactory for the ELISA test, and therefore no purification experiments were done with parainfluenza type 1 virus. Test procedure. The ELISA method described by Leinikki and Passila (5) was applied with some modifications. A nine-channel photometer with polystyrene cuvettes was used (FP-9 Analyzer, Labsystems, Helsinki, Finland). The virus suspension and the control antigen were diluted in PBS to the desired protein concentration (5 to 10 tg/ml), and 200-,ul samples of the dilution were pipetted into cuvettes. After incubation 2 h at 319

2 320 UKKONEN, VAISANEN, AND PENTTINEN J. CLIN. MICROBIOL. 37 C, the cuvettes were washed twice with PBS containing 0.05% Tween 20 and twice with distilled water. The coated cuvettes were used on the same day they were prepared. The ph of the antigen solution was not critical for successful coating of the cuvettes. PBS (ph 7.4) and 0.1 M bicarbonate buffers at ph 9.5 and 10.8 gave identical results. Overnight incubation at room temperature was not better than 2 h at 37 C. The antigen solution was used only once for coating, although it could be used several times without loss of activity. Serum samples were diluted to 1:100, 1:500, and 1: 2,500 in PBS-0.05% Tween 20. Portions (200 yl) of the sample dilutions were incubated in the cuvettes for 2 h at 37 C, after which the cuvettes were washed as before. Each sample diluted 1:100 was also delivered to a cuvette coated with the control antigen. The 1: 500 dilution was used for discrimination between positive and negative results, because many normal sera as well as sera containing high titers of rheumatoid factor gave high absorbances in IgM ELISA when tested at lower dilutions (1:10 to 1:250). True positive sera remained positive when diluted 1:500 or more. Commnercial enzyme conjugates (Orion Diagnostica, Espoo, Finland) containing alkaline phosphatase were diluted in PBS with 1% bovine serum albumin, 0.02% Tween 20, and 0.01% sodium azide. A 150-fl sample of the conjugate dilution (1:400 for anti-igg and 1:100 to 1:300 for anti-igm) was incubated overnight at room temperature, after which the cuvettes were washed as before. The substrate, 4-nitrophenylphosphate (Merck), was dissolved in diethanolamine-mgcl2 buffer (Orion, Espoo, Finland) to a concentration of 1 mg/ml. A 150- Ml sample of the substrate solution was incubated for 30 min at 37 C, after which the enzymatic reaction was stopped with 250 Al of 0.2 N sodium hydroxide. The absorbance was measured by an FP-9 Analyzer at a wave length of 405 nm. The absorbance of a blank cuvette (coated with the virus antigen but incubated with PBS-0.05% Tween 20 instead of serum samples) was automatically subtracted from the readings of the test cuvettes. The cutoff level between negative and positive scoring in IgM ELISA was defined as three standard deviations above the mean absorbance (at serum dilution 1:500) of the CF-negative serum samples. This principle could not be applied for IgG ELISA because many samples that were negative with the CF test gave moderate absorbances (0.100 to 0.300) by IgG ELISA, indicating that the ELISA test was more sensitive. An arbitrary value of was chosen for IgG ELISA because the bulk of the CF negative samples had absorbance values between and in IgG ELISA. The extent of reaction of sera with control antigen varied between and with the anti-igg conjugate, and none of them was considered positive. Two sera had a clearly positive reaction with control antigen and the anti-igm conjugate (0.599 and 0.230), and they were regarded as negative in IgM ELISA using virus antigen; the other sera reacted weakly (0.000 to 0.079) with control antigen and the anti-igm conjugate. Other methods. A standard CF test in microplates was used (10). The CF antigens were purchased from Orion Diagnostica: mumps V antigen was from allantoic fluid, parainfluenza type 1 S antigen was from chorioallantoic membrane of embryonated eggs, and parainfluenza type 3 was grown in Vero cells. Rheumatoid factor was determined by the Latex test (Hyland), and protein concentration was measured by the Lowry method adapted for the FP-9 Analyzer. Pearson's coefficient of correlation was used in comparison of the various antibody tests. Serum specimens. Paired sera from 46 patients were selected from our routine diagnostic laboratory based on their CF titers for mumps and parainfluenza types 1 and 3 antibodies. Fourteen patients had diagnostic increases in mumps CF antibodies, 12 had increases in parainfluenza antibodies, and 20 patients had no significant changes in CF titers. RESULTS Specificity of the conjugates. Four sera positive for mumps antibodies by ELISA were fractionated in sucrose gradients, and the fractions were tested for mumps IgG and IgM by ELISA. All sera gave identical results, with one peak of activity corresponding to the IgG and IgM fractions respective to the conjugates. The ELISA results of one of the fractionated sera are shown in Fig. 1. Methodological variation. To assess the accuracy of the ELISA test, three different sera (strongly positive, intermediate, and negative, quantitation according to the absorbance value) were diluted 1:500 and pipetted into 27 cuvettes. Coefficient of variation (standard deviation/ mean) ranged from 5.5 to 7.4% in the case of positive sera (Table 1). The range of absorbance values was about mean ± 10 to 15%. The variation did not increase when the dilution was made separately for each cuvette. Correlation of ELISA and CF. The absorbance values obtained by mumps IgG ELISA (at 1:500 dilution of sera) showed a highly positive correlation with the logarithms of the mumps CF titers (r = 0.90, P < 0.001) (Fig. 2A). ELISA detected 10 additional positive sera as compared to CF, whereas six sera were positive only by 06. I 53.?, o o FRACTKN NUMBER FIG. 1. ELISA of a fractionated serum positive for mumps IgG and IgM antibodies. The serum was fractionated in a sucrose gradient, and 1:100 dilutions of the fractions were tested by mumps ELISA.

3 VOL. 11, 1980 TABLE 1. ELISA FOR MUMPS AND PARAINFLUENZA ANTIBODIES 321 Technical variation of the ELISA results' Absorbance at 405 nm ELISA method Mean Maximum Minimum SD SDb/mean (%) Mumps IgG Strongly positive Intermediate Negative Mumps IgM Strongly positive Intermediate Negative Parainfluenza 1 IgG Strongly positive Intermediate Negative athree sera of different antibody concentrations were diluted 1:500 and measured simultaneously in 27 cuvettes. 'SD, Standard deviation. 1 4 E 12 Ln O 08 w E 02 Q 9 It) 0 11 V) -j L'i N z LJ -i Llz cr a A. I I../ r =0.90 ~~~~~p"~000i ' MUMPS CF (TITRE) 4-B,- >% r 0577 P' = '8 8 i PARAINFLUENZA CF (TITRE) FIG. 2. Correlation of ELISA and CF in detection of (A) mumps and (B) parainfluenza type 1 antibodies. The ELISA results are expressed as absorbances at 1:500 dilutions of sera. The straight line was calculated by the least-squares method. CF. No correlation was found between mumps IgG ELISA and parainfluenza 1 or 3 CF. Parainfluenza type 1 IgG ELISA showed a moderate positive correlation with parainfluenza type 1 CF (r = 0.57, P < 0.001) (Fig. 2B) and a fair degree of relationship with parainfluenza 3 CF (r = 0.42, P < 0.01; data not shown). A slight positive correlation was also found between parainfluenza 1 IgG ELISA and mumps CF (r = 0.26, P < 0.05, data not shown). Twenty-three additional positive sera were detected by para- E L: Uc) MUMPS PARAINFLUENZA MUMPS PARAINFLUENZA IgG IgG IgG IgG FIG. 3. Rises in absorbances by mumps and parainfluenza type 1 IgG ELISA in paired sera with diagnostic rises in the CF titers of (A) mumps or (B) parainfluenza type 1 or 3 antibodies. Three serum dilutions (1:100, 1:500, and 1:2,500) were tested by ELISA, and the results of each serum pair shown in the figure (acute- and convalescent-phase sera connected with a line) represent the serum dilution that gave the greatest difference in absorbance. influenza type 1 IgG ELISA, and only three samples negative by ELISA were positive by CF (Fig. 2B). Serological diagnosis by ELISA. All 14 patients with diagnostic rises in mumps CF titers had elevations of mumps IgG antibodies as expressed by an increase of more than in the absorbance values between the paired sera (Fig. 3A). Increases in parainfluenza type 1 IgG by ELISA were also observed in nine of these patients (Fig. 3A), whose CF titers for parainfluenza types 1 and 3 were low or negative and did not rise. Twelve patients had significant rises in parainfluenza type 1 (4 patients) and/or type 3 CF

4 322 UKKONEN, VAISANEN, AND PENTTINEN J. CLIN. MICROBIOL. titers (10 patients). By the ELISA technique, all these patients were shown to have rising parainfluenza type 1 IgG antibodies (Fig. 3B). In contrast, only one of these patients had increasing mumps IgG by ELISA (Fig. 3B). He had a very high rise in parainfluenza type 3 CF titers (64-fold) while the CF titers for mumps and parainfluenza type 1 were low. These 12 patients had low mumps CF titers, and no changes in titers were observed. Of 14 patients with significant rises in mumps CF titers, 13 were shown to have mumps IgM antibodies in one or both of the paired sera (Fig. 4A). The clinical diagnoses of the patients with mumps IgM were parotitis, orchitis, meningitis, or encephalitis, whereas the only case negative by mumps IgM ELISA had fever and stomatitis. Mumps IgM antibodies were not detected in patients with rises in parainfluenza CF titers. Ten of 13 patients already had mumps IgM antibodies in their acute-phase sera. The first In C) s t 3- A ~~B 0&~P RffUN~ 4MSF~AfLNM MUMPS PARAIWLIJENZAI MUMPS PARAlLAENZAI IgM 1gM IgM IgM FIG. 4. ELISA of mumps and parainfluenza type 1 IgM antibodies in paired sera with diagnostic rises in the CF titers for (A) mumps or (B) parainfluenza type I or 3 antibodies. The ELISA results are expressed as absorbances at 1:50() dilutions of sera, and only the higher absorbance value of each serum pair is presented in the figure. The horizontal lines show the cutoff kvel between positive and negative scoring by each assay. TABLE 2. serum samples of the three cases with mumps IgM only in the second serum specimens were taken within 2 days after onset of disease. Parainfluenza type 1 IgM antibodies could not be shown, except one borderline reading, in any of the patients, including those with rises in parainfluenza titers (Fig. 4). Some discrepant results between mumps IgM ELISA and serological diagnosis by mumps CF were observed. Sera from 17 patients were reactive in mumps IgM ELISA, but only 13 of these had significant rises in mumps CF titers. The test results of the four patients with no rises in mumps CF titers are shown in Table 2. In all cases the interval between onset of disease and the first sample was extraordinarily long, from 2 weeks to several months. Interference by the rheumatoid factor. As measured by the latex test, 42% of sera positive by mumps IgM ELISA and 31% of sera negative by mumps IgM ELISA contained rheumatoid factor. The difference was not significant. Different dilutions of a mixture containing a serum with high mumps IgG level and a serum with high titer of rheumatoid factor were tested with mumps IgM ELISA. Low dilutions (1:10 and 1: 50) were strongly reactive in mumps IgM ELISA, but with further dilution (1:250 and 1: 1,250) the absorbances rapidly declined. Therefore it was concluded that with a high test dilution of sample (1:500), false-positive IgM results due to rheumatoid factor can be avoided. DISCUSSION A new ELISA for mumps and parainfluenza type 1 antibodies (IgG and IgM classes) was described and compared with the CF test. Technically, the ELISA test is reliable: methodological variation was low (Table 1), which indicates uniform adsorption of the antigen onto Clinical and laboratory data on four patients with mumps IgM antibodies by ELISA but no rises of mumnps CF titers in paired sera (a and II) Mumps antibodies Patient Age Clinical diagnosis CF titer IgG by ELISA' IgM by ELISA' (yrs) I II I II I II 1 9 Serous meningitis, parotitis 1 month be fore study 2 31 Prolonged fever, headache, enlarge ment of lymph nodes 3 33 Chronic bronchitis parotitis 1 year be b fore study 4 28 Enlargement of lymph nodes b Absorbance (405 nm) of serum dilution 1:500. b The sera of patients 3 and 4 were fractionated by sucrose gradient, and the IgM fractions were also positive by mumps IgM ELISA.

5 VOL. 11, 1980 the cuvettes and accurate pipetting with the multichannelled micropipettes. As judged from the number of sera positive by ELISA and/or CF, mumps IgG ELISA was not much more sensitive than mumps CF (Fig. 2A). However some of the six sera positive only by CF may have been false-positive ones, as found in a previous comparison of ELISA and CF (6). Parainfluenza type 1 IgG ELISA seemed to be considerably more sensitive than CF, since 23 samples were positive only by the ELISA test. The high test dilution used in ELISA (1:500) naturally lowers the sensitivity of the assay, but on the other hand, small serum volumes are sufficient for the test, and many false-positive results by IgM ELISA can be avoided. Antigenic cross-reactions between paramyxoviruses are known to interfere with the serological diagnosis of these infections (2, 7, 9). The specificity of mumps IgG ELISA was comparable to that of CF: elevations of mumps IgG by ELISA were shown in patients with rise in mumps CF titers, but not, except in one case, in patients with rise in parainfluenza CF titers. The specificity of parainfluenza type 1 IgG ELISA seemed to be lower than that of CF, since rises in IgG ELISA were also found in most patients with increases of mumps CF antibodies (Fig. 3A). However, the observed rises by parainfluenza type 1 IgG ELISA in these cases may be explained by higher sensitivity of the ELISA test compared to that of CF (Fig. 2B). Nevertheless, for an adequate evaluation of the specificities of IgG assays, larger serum materials including serum pairs with simultaneous rises in mumps and parainfluenza CF titers are needed. Rheumatoid factor often gives false-positive results in IgM assays for which the antibody ELISA FOR MUMPS AND PARAINFLUENZA ANTIBODIES 323 classes are not separated before testing (8). In our method a high sample dilution (1:500) was used because false-positive results due to rheumatoid factor became negative when sera were sufficiently diluted. The frequency of rheumatoid factor in the present sera did not differ with respect to the results of mumps IgM ELISA. Furthermore, none of the sera was clearly positive by the parainfluenza IgM ELISA, although some of the sera had high titers of parainfluenza type 1 IgG antibodies and also contained rheumatoid factor. An unexpected finding was that parainfluenza type 1 IgM antibodies could not be demonstrated in patients with rises in parainfluenza CF antibody titers. It is possible that low titers not detectable at the test dilution may have been present. However, the more local nature of parainfluenza virus infection and reinfections (upper respiratory tract) may not necessarily elicit a similar IgM response as in the case of mumps. The ELISA test itself was likely to detect IgM, since the parainfluenza IgG antibodies were readily measurable by it, and since the test procedure and conjugates were similar to those used to detect mumps IgM. Mumps IgM ELISA proves promising. Positive results were usually obtained on the acutephase sera, which permits a more rapid diagnosis. In addition, paired sera taken too late for adequate CF diagnosis (rises in titers) may still contain specific IgM antibodies. IgM ELISA may also be used to study possible chronic forms of mumps infection with prolonged IgM response, as seen in four cases in this study (Table 2). ACKNOWLEDGMENTS This work was financially supported by a grant from the Emil Aaltonen Foundation. The technical assistance of Helena Norrila is gratefully acknowledged. L1TERATURE CITED 1. Cappel, R., F. de Cuyper, and J. de Braekeleer Rapid detection of IgG and IgM antibodies for cytomegalovirus by the enzyme linked immunosorbent assay (ELISA). Arch. Virol. 58: DeMeio, J., and D. L. Walker Demonstration of antigenic relationship between mumps virus and hemagglutinating virus of Japan. J. Immunol. 78: Jensik, S. C., and S. Silver Polypeptides of mumps virus. J. Virol. 17: Kaiimo,,K. 0. K., 0. H. Meurman, P. E. Halonen, B. R. Ziola, M. K. Viljanen, K. Granfors, and T. Toivanen Solid-phase radioimmunoassay of rubella virus immunoglobulin G and immunoglobulin M antibodies. J. Clin. Microbiol. 4: Leinikki, P., and S. Passila Solid phase antibody assay by means of enzyme conjugated to anti-immunoglobulin. J. Clin. Pathol. 29: Leinikki, P., L. Shekarchi, N. Tzan, D. L. Madden, and J. L. Sever Evaluation of enzyme-linked immunosorbent assay (ELISA) for mumps virus antibodies. Proc. Soc. Exp. Biol. Med. 160: Lennette, E. H., F. W. Jensen, R. W. Guenther, and R. L. Magoffin Serologic responses to para-influenza viruses in patients with mumps virus infection. J. Lab. Clin. Med. 61: Meurman, 0. H., and B. R. Ziola IgM-class rheumatoid factor interference in the solid-phase radioimnmunoassay of rubella specific IgM antibodies. J. Clin. Pathol. 31: Penttinen, K., and K. Cantell Parainfluenza 1 (Sendai) antibodies and mumps vaccination. Ann. Med. Exp. Fenn. 45: Riski, H., S. Pyrhonen, 0. Wager, and K. Penttinen Lack of measurable complement fixing antibodies against viral antigens. Acta Pathol. Microbiol. Scand. Sect. B 85: Vejtorp, M., E. Fan0e, and J. Leerhoy Diagnosis of postnatal rubella by the enzyme-linked immunosorbent assay for rubella IgM and IgG antibodies. Acta Pathol. Microbiol. Scand. Sect. B 87: