Immunoglobulin M Antibody Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of St. Louis Encephalitis

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1984, p /84/1784-7$2./ Copyright D 1984, American Society for Microbiology Vol. 2, No. 4 Immunoglobulin M Antibody Capture Enyme-Linked Immunosorbent Assay for Diagnosis of St. Louis Encephalitis T. P. MONATH,* R. R. NYSTROM, R. E. BAILEY, C. H. CALISHER, AND D. J. MUTH Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Fort Collins, Colorado 8522 Received 21 March 1984/Accepted 9 July 1984 Sera from patients ith St. Louis encephalitis ere tested ith an immunoglobulin M (IgM) antibody capture enyme immunoassay (MAC ELISA). The assay used five reagents: antihuman IgM, test serum, sucrose-acetone-extracted mouse brain antigen, broadly cross-reactive flavivirus monoclonal antibody conjugated to alkaline phosphatase, and substrate (p-nitrophenyl phosphate). MAC ELISA endpoint titers correlated (r =.893) ith the absorbance value of a 1:1 dilution of patient serum. Significant (fourfold or greater) changes in the endpoint titers beteen paired sera corresponded to a critical ratio (ratio of absorbance values at the 1:1 dilution) of.1.3. IgM antibodies ere detected in 71% of patients bled at to 3 days after the onset of illness, in 99% bled at 4 to 21 days, and in 91% bled at 22 to 67 days. Thereafter, the IgM seropositivity rate declined; hoever, 29% of sera ere still positive at 115 to 251 days after the onset of illness. MAC ELISA titers ere significantly correlated ith hemagglutination inhibition (r =.258) and neutraliation (r =.711) titers. Because IgM antibodies appeared early and aned rapidly, a diagnosis as made on the basis of a decrease in titer more often by MAC ELISA than by hemagglutination inhibition, complement fixation, or neutraliation tests. IgM antibodies generally shoed a high degree of specificity; heterologous cross-reactions ere, hoever, present in 4 of 14 sera examined. The MAC ELISA is useful for the rapid, early diagnosis of St. Louis encephalitis. St. Louis encephalitis (SLE) is the most important epidemic arboviral disease in the United States (15). From the public health point of vie, rapid diagnosis of SLE is important for surveillance and vector control strategies. Because attempts to isolate virus from patients are rarely successful, diagnosis relies on serological tests. The classic techniques of hemagglutination inhibition (HI), complement fixation (CF), and neutraliation (N) are useful but have a number of drabacks (3). Although HI and N antibodies appear early during the course of infection, they are also long lasting. Demonstration of HI or N antibody in a single serum sample may provide a presumptive diagnosis, but often one cannot distinguish beteen remote and recent infections; tests of paired serum samples are necessary, causing delays in diagnosis. The presence of CF antibody is useful as an indication of recent infection, but CF antibody appears rather late (2 to 4 eeks) after the onset of illness and is not of value for early diagnosis. More important, up to 15% of infected persons never develop detectable CF antibodies, and up to 3% of those ho do develop lo-titer antibodies (3). In recent years, immunoassays have been applied for the serodiagnosis of arbovirus infections, including a number of flaviviruses: Japanese encephalitis (1, 2, 12), SLE (2), tickborne encephalitis (9, 1, 18), and yello fever (6). Techniques for measuring immunoglobulin M (IgM) antibodies, in particular by antibody capture enyme immunoassay (ELISA) (1, 7, 1, 11, 16-19), have proved especially useful. In this paper, e report the application of an IgM antibody capture ELISA (MAC ELISA) to the serodiagnosis of SLE. The test uses a monoclonal antibody-enyme conjugate. The chronology of IgM antibody responses and correlations ith HI, CF, and N tests are described. * Corresponding author. 784 MATERIALS AND METHODS Human sera. Sera from patients ith clinical encephalitis during the SLE epidemics of 1974 through 1975 in Tennessee, Arkansas, and Alabama ere obtained from the serum bank of the Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, Colo. All sera tested by MAC ELISA ere from patients ith serologically diagnosed SLE, as determined by conventional (HI, CF, and N) serological tests of paired sera. The criteria used for serological diagnosis have been previously described (3). The sera had been stored for 8 to 9 years at - 2 C, during L) 64r r = CRITICAL RATIO FIG. 1. Correlation beteen the fold change in endpoint ELISA titers and critical ratios (at the 1:1 dilution) of paired sera from 26 SLE patients. A significant (fourfold or greater) change in the endpoint titer corresponded to a critical ratio of.1.3.

2 VOL. 2, 1984 ELISA FOR DIAGNOSIS OF ST. LOUIS ENCEPHALITIS 785 A. MAC ELISA If) i *[ 4. *.:... [. 3. " -* ll I. -- * % a. 4 I,1 44. U / ( B. HEMAGGLUTINATION- INHIBITION S C lr 32 I 8C I- 2 * o"* * *-m. S... * *O < % % C. COMPLEMENT- FIXATION 64r 4 * - 32p 16F 8 4 % <8 -, _ v....* S *, Wh D. NEUTRALIZATION 2,48r * % t. 2 < FIG. 2. Results of MAC ELISA and conventional serological tests of sera from SLE patients. hich time they had been thaed one to to times for ously described (13). The TBH-28 strain of SLE virus as testing. used for all serological tests, including MAC ELISA. HI, CF, and N tests. HI tests ere done by the method of MAC ELISA reagents. Goat antihuman IgM as obtained Clarke and Casals (5) adapted to a microtiter technique. The from Cappel Laboratories. Antigens, prepared as sucrose- CF test used as that of Casey (4). Serum dilution plaque acetone extracts of infected suckling mouse brains (5), ere reduction N tests ere performed in Vero cells, as previ- obtained from the Division of Vector-Borne Viral Diseases

3 TABLE 1. Serological responses to SLE infection Patient Patient Days post Titer MAC ELISA Titer Critical onset HCFNP/N"1 change ratio 8 1 <1 b < Increase , , Decrease ,28 2, < Decrease Increase Increase , <1 < Increase , Decrease Decrease , <8 <1 2.7 Increase < Increase , , < Decrease , Decrease < Decrease , ,28 <8 5, Decrease , < Increase , , < Increase < Increase < Increase <8 1, Decrease <8 1, Decrease < Decrease Decrease < Decrease , Decrease <8 1, <8 2, Decrease < < Decrease < a P/N = [absorbance value of test (P) serum]/[absorbance value of negative (N) control serum]. b -, Not tested. 786

4 VOL. 2, 1984 ELISA FOR DIAGNOSIS OF ST. LOUIS ENCEPHALITIS 787 It) r-l a. a--- CL) cn r-- N. Cl) I - 4. a. ccl cf) I.5 A * S S ~~~ a edi Mdin r =.258 a W i i i *.5 a, I I HI (log) 5 5r B 4.5 F 3.5 F 2.5 F I.5F.51 C I.5 a * Median \ / = r= -.12 * v : CF(!og) Teen ere added in 5-,ul volumes to duplicate ells and incubated for 1 h at 37 C. Plates ere ashed as described above. Controls in each test included one knon positive and to knon negative human sera and normal saline. (iii) Adding antigen. SLE antigen (5,ul of a 1:32 dilution in PBS-1% Teen) as added to each ell. Heterologous flavivirus antigens used in some tests are described belo. Antigens ere titrated before use in a simple sandich ELISA ith hyperimmune mouse ascitic fluid, antigen serially diluted tofold, and 6B6C-1-alkaline phosphatase conjugate. The dilution chosen for use as fourfold loer than the endpoint, thus providing an excess of antigen. Antigen as incubated overnight at 4 C. Plates ere ashed as described above. (iv) Adding conjugate. Monoclonal antibody 6B6C-1 conjugated to alkaline phosphatase as diluted 1:64 in PBS-1% Teen, and 5,ul as added to each ell. Plates ere incubated for 1 h at 37 C and ashed six times in PBS-.5% Teen and to times in PBS. (v) Adding substrate. We added 1 RI1 of p-nitrophenyl phosphate (3 mg/ml in 1. M Tris [ph 8.]; Sigma Chemical Co.) to each ell. Plates ere incubated at room temperature for 1 to 15 min, the reaction as stopped by the addition of 5,u1 of 3 N NaOH, and absorbance values at 45 nm (A45) ere determined ith a TiterTek Multiskan apparatus (Dynatech Laboratories, Inc.). Mean absorbance values for duplicate ells of each dilution of test (P) serum ere divided by mean values of the corresponding dilution of negative (N) control serum. A P/N ratio of -2. as considered positive. C. RESULTS r.711 MAC ELISA titer. Previous ork from this laboratory has shon that ELISA endpoint titers are highly correlated (r =.9) ith the absorbance value of a 1:1 dilution of patient serum (17). We determined endpoint IgM antibody titers of paired sera from 26 SLE patients. The fold change in the endpoint titer as compared ith the ratio of the convalescent-phase MAC ELISA value at the 1:1 serum dilution (A45) to the value for the acute specimen. In the case of a * falling titer, the ratio of early to late convalescent specimens -,,,,, as used. Analysis of variance shoed a coefficient of.5! correlation (r) of.893 (Fig. 1). A critical ratio (14) of 1.3 N ( log ) corresponded to a fourfold or greater change in the endpoint titer. In subsequent tests, sera ere tested at to dilutions FIG. 3. C.orrelations beteen MAC ELISA and conventional only (1:1 and 1:1,). For paired samples, critical ratios serological t( ests of sera from SLE patients. ere determined by using the A45 for 1:1 dilutions. A ratio of -1.3 as interpreted as a significant change in titer. Sera from 69 SLE patients bled to 251 days after the reference c.ollection. Flavivirus group-reactive monoclonal onset of illness (serologically confirmed by conventional antibody (( 6B6C-1), a gift from John Roehrig, Division of tests) ere tested as described above; single serum samples rne Viral Diseases. as nirifipd hv S,as ere available from 18 patients, and to or more serum Vector-Bor * _%-^-_%1 *Ws XS_#sW111 TV&I" V uc4a F_ V} 1q"%. V.W protein A chromatography and conjugated to alkaline phosphatase (Sigma Chemical Co.) as described elsehere (17). MAC ELISA. (i) Coating plates. Polystyrene plates (Immulon II; Dynatech Laboratories, Inc.) ere coated ith antihuman IgM diluted 1:8 in carbonate-bicarbonate buffer (ph 9.). One hundred-microliter volumes ere added to the center 6 ells of each plate; plates ere incubated for 1 h at 37 C and 1 h at room temperature (2 to 22 C). Plates ere then emptied and ashed eight times ith phosphate-buffered saline (PBS) (ph 7.4) containing.5% Teen 2. (ii) Adding test sera. For endpoint titrations, serial tofold dilutions of serum (from 1:1 to 1:25,6) in PBS-1% Titer changes detected beteen paired sera from SLE patients by four seological tests No. (%) of patients ith titer changes detected by: Response MAC HI CF N = 59)a (n = 59)" (n = 37)" (n = 32)a TABLE 2. Increase 12 (2.3) 21 (35.6) 18 (48.5) 1 (31.3) Decrease 21 (35.6) 5 (8.5) 2 (5.4) 5 (15.6) No change 26 (44.1) 33 (55.9) 17 (46.) 17 (53.1) a n, Number of paired sera tested.

5 788 MONATH ET AL. samples ere available from 51 patients. IgM antibodies ere found in 16 of the 18 patients from hom single serum samples ere obtained and in 1 or more of the 51 patients from hom multiple serum samples ere obtained. IgM antibodies ere detected in 15 (71%) of 21 sera obtained beteen and 3 days after the onset of illness, in 66 (99%) of 67 sera taken beteen 4 and 21 days, and in 31 (91%) of 34 sera taken beteen 22 and 67 days (Fig. 2). Thereafter, the IgM seropositivity rate declined; of 14 sera taken beteen 115 and 251 days after the onset of illness, only 4 (29%) ere positive. The mean IgM antibody titer increased rapidly during the first 1 days after the onset of illness and then gradually declined. Patterns of development of HI and N antibodies ere similar during the first 3 eeks; hoever, in no case did these antibodies become undetectable over the course of the observations, and high N titers tended to persist. CF antibodies appeared late, beteen 8 and 2 days after the onset of illness; 9 (39%) of 23 sera obtained beteen 1 and 3 days after the onset of illness ere CF antibody negative. J. CLIN. MICROBIOL. IgM antibody responses shoed considerable individual variation (Table 1). Examination of these data helped to clarify our knoledge of IgM development and decay. (i) IgM antibodies appeared very early after clinical onset; hoever, the development of HI and N antibodies usually coincided ith and sometimes even preceded the development of IgM antibodies detectable by MAC ELISA (e.g., patients 19, 2, 43, and 5). (ii) Tests of paired sera obtained immediately after the onset of illness and again ca. 1 days later usually shoed a rising IgM titer (e.g., patients 8, 2, 22, 47, and 5). Hoever, this as not invariably the case, and some persons shoed antibody decay during this interval (patients 17, 35, and 43). (iii) Significant decreases in the IgM titer ere frequently observed, generally in late sera dran at intervals of eeks or months after the onset of illness (patients 53, 56, 6, 62, 66, and 67). (iv) In most cases, IgM antibodies declined to undetectable levels by 4 months after the onset of illness; hoever, a fe individuals had persisting IgM antibodies, occasionally at a high titer (patients 6 and 63). Serum onset TABLE 3. Cross-reactivity ith heterologous flavivirusa antigens in HI tests and MAC ELISA Test Titer to: SLE JE MVE YF WN HI MAC ELISA HI MAC ELISA HI >64 MAC ELISA HI MAC ELISA HI MAC ELISA HI MAC ELISA HI MAC ELISA HI >64 MAC ELISA HI MAC ELISA HI MAC ELISA HI MAC ELISA HI >64 MAC ELISA HI MAC ELISA HI MAC ELISA JE, Japanese encephalitis virus (G8924 strain); MVE, Murray Valley encephalitis virus (original strain); YF, yello fever virus (17D strain for HI tests; Asibi strain for MAC ELISA); and WN, West Nile virus (EglOl strain).

6 VOL. 2, 1984 Correlation beteen MAC ELISA and conventional serological tests. IgM antibody titers increased proportionately ith both HI and N antibody titers (Fig. 3). By regression analysis, significant correlations ere found beteen MAC ELISA antibody titers and HI and N antibody titers. The correlation beteen MAC ELISA and HI responses as highest during the first 3 days after the onset of illness (data not shon). There as no correlation beteen MAC ELISA and CF antibody responses. In terms of ability to confirm diagnosis by detecting a significant change in titer, all four serological tests ere comparable (Table 2); because IgM antibodies appeared early and declined relatively rapidly, diagnostic decreases in titers (critical ratio,.1.3) ere found most often by MAC ELISA than by the other tests. Heterologous cross-reactivity. Sera from 14 patients ere tested against SLE and four heterologous flavivirus antigens (Table 3). Antigens ere titrated as described above, but human sera containing IgM antibodies to the heterologous viruses ere not available as controls. IgM antibodies ere highly specific in 1 of the 14 patients tested. Cross-reactions ith antigens of heterologous flaviviruses, especially those in the same antigenic subgroup (Japanese, Murray Valley, and West Nile encephalitis viruses), ere nevertheless seen in four cases. IgM cross-reactivity did not correlate ith time after the onset of illness or ith cross-reactions in the HI test. DISCUSSION The MAC ELISA proved useful for the serodiagnosis of SLE infection. The technique avoids many problems inherent in assays that use an antigen fixed to the solid phase; such assays generally require a purified antigen and are subject to false results because of competition beteen IgG and IgM antibodies for binding to the antigen (false-negatives) and because of the presence of rheumatoid factor (false-positives). The MAC ELISA in this study used a broadly cross-reactive monoclonal antibody conjugate, hich is useful in tests for antibodies against a ide array of flavivirus antigens. Mouse brain antigen extracted ith sucrose-acetone or crude aqueous suspensions of infected mouse brain antigen may be used in this assay. Endpoint titration of serially diluted sera is unnecessary, as the antibody titer of a single sample is accurately estimated by the A45 of a 1:1 serum dilution. For paired samples tested at 1:1 dilutions, a significant change in titer corresponded to an A45 ratio (critical ratio) of.1.3. By linear regression analysis it as found that MAC ELISA titers changed as HI and N titers changed. The correlation ith HI as the highest as antibody titers increased during the early phase of infection. A significant fall in IgM titer as more often the basis for a confirmed diagnosis by MAC ELISA than by the conventional tests. This result as expected, as IgM antibodies are knon to appear early and decline rapidly. In most cases, IgM antibodies declined to undetectable levels by 4 months after the onset of illness. This provides a practical basis for the presumptive diagnosis of a recent infection by the demonstration of IgM antibodies in a single serum sample. Because IgM antibodies are found in ca. 75% of sera obtained beteen days and 3 and in nearly all sera obtained thereafter, presumptive serological diagnosis is often possible during the acute phase of illness. The rapidity of the test procedure adds to its usefulness for early diagnosis. In addition, IgM antibodies usually sho little heterologous cross-reactivity ith closely related antigens. ELISA FOR DIAGNOSIS OF ST. LOUIS ENCEPHALITIS 789 IgM antibodies ere found at least 6 months after infection in a fe persons (Fig. 2). The persistence of IgM antibodies has been reported after yello fever 17D immuniation (14) and after Japanese encephalitis (8). The persistence of Japanese encephalitis IgM antibodies has been shon to correlate ith severity of illness (8); e had insufficient information to examine this relationship in the SLE patients. Recent reports (2, 9) indicate that a MAC ELISA applied to cerebrospinal fluid permits an early diagnosis of Japanese encephalitis and tick-borne encephalitis infections. The test measures IgM antibody production locally in the brain. We have recently used the MAC ELISA for cerebrospinal fluid samples from SLE patients; preliminary results indicate that the technique is sensitive and accurate in this application also. ACKNOWLEDGMENTS We thank John Roehrig for supplying the monoclonal antibody conjugate and Wendy Vesely for technical assistance. LITERATURE CITED 1. Burke, D. S., and A. Nisalak Detection of Japanese encephalitis virus immunoglobulin M antibodies in serum by antibody capture radioimmunoassay. J. Clin. Microbiol. 14: Burke, D. S., A. Nisalak, and M. A. Ussery Antibody capture immunoassay detection of Japanese encephalitis virus immunoglobulin M and G antibodies in cerebrospinal fluid. J. Clin. Microbiol. 16: Calisher, C. H., and J. D. Poland Laboratory diagnosis, p In T. P. Monath (ed.), St. Louis encephalitis. American Public Health Association, Washington, D.C. 4. Casey, H. L Standardied diagnostic complement fixation method and adaptation to microtest. Public Health Monogr. no. 74. U.S. Government Printing Office, Washington, D.C. 5. Clarke, D. H., and J. Casals Techniques for hemagglutination and hemagglutination-inhibition ith arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7: Deubel, V., V. Mouly, J. J. Salaun, C. Adam, M. M. Diop, and J. P. Digoutte Comparison of the enyme-linked immunosorbent assay (ELISA) ith standard tests used to detect yello fever virus antibodies. Am. J. Trop. Med. Hyg. 32: Duermeyer, W., F. Wielaard, and J. Vanderveen A ne principle for the detection of specific IgM antibodies applied in an ELISA for hepatitis. Am. J. Med. Virol. 4: Edelman, R., R. V. Schneider, A. Vejjajiva, R. Pornpibul, and P. Voodhikul Persistence of virus-specific IgM and clinical recovery after Japanese encephalitis. Am. J. Trop. Med. Hyg. 25: Hein, F. X., M. Roggendorf, H. Hofmann, C. Kun, and F. Deinhardt Comparison of to different enyme immunoassays for detection of immunoglobulin M antibodies against tickborne encephalitis virus in serum and cerebrospinal fluid. J. Clin. Microbiol. 14: Hofmann, H., W. Frisch-Niggemeyer, and F. Hein Rapid diagnosis of TBE by means of enyme-linked immunosorbent assay. J. Gen. Virol. 42: Jamnback, T. L., B. J. Beaty, S. W. Hildreth, K. L. Bron, and C. B. Gunderson Capture immunoglobulin M systems for rapid diagnosis of La Crosse (California encephalitis) virus infections. J. Clin. Microbiol. 16: Konishi, E., and M. Yamaoka Enyme-linked immunosorbent assay for detection of antibodies to Japanese encephalitis virus in sine sera. Kobe J. Med. Sci. 28: Lindsey, H. S., C. H. Calisher, and J. H. Mathes Serum dilution neutraliation test for California group virus identification and serology. J. Clin. Microbiol. 4: Monath, T. P Neutraliing antibody responses in the major immunoglobulin classes to yello fever 17D vaccination

7 79 MONATH ET AL. J. CLIN. MICROBIOL. of humans. Am. J. Epidemiol. 93: Monath, T. P Arthropod-borne encephalitis in the Americas. Bull. W.H.O. 57: O'Beirne, A., R. Berofsky, and D. Fuccillo Enyme immunoassays for detecting viral infections, p In R. C. Tilton (ed.), Rapid methods and automation in microbiology. American Society for Microbiology, Washington, D.C. 17. Roehrig, J. T Development of an enyme-linked immunosorbent assay for the identification of arthropod-borne Togavirus antibodies. J. Gen. Virol. 63: Roggendorf, M., F. Hein, F. Deinhardt, and C. Kun Serologic diagnosis of acute tick-borne encephalitis by demonstration of antibodies of the IgM class. J. Med. Virol. 7: Schmit, H., V. von Deimling, and B. Fleming Detection of IgM antibodies to CMV using an enyme-labelled antigen. J. Gen. Virol. 5: Wolff, K. L., D. J. Muth, B. W. Hudson, and D. W. Trent Evaluation of the solid-phase radioimmunoassay for diagnosis of St. Louis encephalitis infection in humans. J. Clin. Microbiol. 14: