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Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections

Denise A. Martin, David A. Muth, Teresa Brown, Alison J. Johnson, Nick Karabatsos and John T. Roehrig J. Clin. Microbiol. 2000, 38(5):1823.

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. (2). TERESA BROWN. but they also use purified virus as antigen. thawed once. Phone: (970) 221-6445. This approach has resulted in a reliable.O. Louis encephalitis virus (SLE). we have reduced the number of antigens necessary for testing. p.6) was used as coating buffer.00ϩ0 Vol. These new tests use a defined set of antigens. While many separate IgM ELISAs have been developed for arboviruses. 13. these tests are not well standardized (1. National Center for Infectious Diseases. hemagglutination-inhibition test (10). and LAC strain Original. pH 9.S. 38. which limits test specificity. With a blind-coded human serum panel. Because of the wide variety of viruses.org/ on April 12. as previously described (2). National Center for Infectious Diseases. Division of Vector-Borne Infectious Diseases (DVBID).015 M sodium carbonate. arboviral diagnostic serology is complex. Many of these viruses are considered emerging or reemerging infectious diseases that can be readily transported from one area of the world to another. P. Carbonate-bicarbonate buffer (0. AND JOHN T. St. IgM appears early in infection. These assays use polyclonal antiviral antibody as a detector. or La Crosse encephalitis virus (LAC) in a previously performed serologic test. and aliquots were stored at Ϫ70°C long-term. Md. 14). Furthermore. by grouping antigens geographically and utilizing known serological cross-reactivities. 14). 1823 Downloaded from http://jcm. rises rapidly in the disease course. 15. implemented.035 M sodium bicarbonate.GOV. Flaviviridae. personal communication). The serum dilution PRNT was performed with Vero cells. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody. which is impractical for all but the largest reference laboratories that prepare these specialized reagents routinely (4–7.asm. which consists of specimens sent to DVBID for arboviral diagnostic testing. 5 Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections DENISE A. have been inconsistent. serum bank. This test was a modification of the assay previously reported by Beaty et al. PRNT. and Bunyaviridae. Public Health Service. 11. Most medically important arthropod-borne viruses (arboviruses) belong to three virus families: the Togaviridae. No. The IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) was designed specifically to detect IgM antibody. U. May 2000. Box 2087. MUTH. Fax: (970) 221-6476. usually expressed as a positiveto-negative (P/N) ratio. Serum specimens were obtained from the Centers for Disease Control and Prevention. and held at 4°C thereafter. and readily transferable system for monitoring arbovirus disease.. Fort Collins. and complement fixation test (8) were still widely used (M. 0. E-mail: DZM9 @CDC. the criteria that indicate positive reactions. Inc. CO 80522. due to poorly timed specimens. and low reactivities were chosen. 1823–1826 0095-1137/00/$04. 2014 by guest been extensively evaluated (7. Colorado 80522 Received 3 August 1999/Returned for modification 1 November 1999/Accepted 8 March 2000 Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Sera were selected on the basis of a positive result to either eastern equine encephalitis virus (EEE). Centers for Disease Control and Prevention. Mailing address: Division of Vector-Borne Infectious Diseases. plaque-reduction neutralization test (PRNT) (2. SLE strain TBH-28. Centers for Disease Control and Prevention. 19) but has relied on results from an earlier study (21). For the MAC-ELISA standardization. tailored to the geographic origin of the specimen. MATERIALS AND METHODS Human serum. The method is readily standardizable. Finally. this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.0 as a positive cutoff value. and are often poor measures of the early antibody immunoglobulin M (IgM). which is a valuable tool for rapid diagnosis of acute viral infections. Fort Collins. using a commercial source of anti-human IgM capture antibody and broadly reactive antiviral monoclonal antibody (MAb)-enzyme conjugates as detectors. Using prototype viruses from each of these families and a panel of well-characterized human sera. The following viruses were used to represent the three viral genera in all tests: EEE strain NJ/60. JOHNSON. using a positive-to-negative ratio of >2. These polyclonal antibody detectors vary in batch-to-batch potency and can be quite virus cross-reactive. ALISON J. while maintaining adequate detection sensitivity. Bunning. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae. Many of these tests are technically demanding. Rockville. 22 positive and 13 negative sera were used for EEE. Department of Health and Human Services.) was used as capture antibody. MARTIN.* DAVID A. NICK KARABATSOS. Flaviviridae. medium. 20). Virus isolation is rarely a viable option even in epidemic situations. we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family.JOURNAL OF CLINICAL MICROBIOLOGY. ROEHRIG Division of Vector-Borne Infectious Diseases. Furthermore. Positive serum specimens with high. which is impractical for multiple agents (4–7. 17). and validated a standardized MAC-ELISA for rapid screening of human serum samples for various arboviruses. making them difficult to apply reproducibly. Most use a commercial source of anti-human IgM as capture antibody. A recent survey of diagnostic laboratories in the United States that perform arboviral testing determined that the indirect immunofluorescence assay (2). and 18 positive and 9 negative sera were used for LAC. We have developed. 24 positive and 12 negative sera were used for SLE. Endpoints were determined at a 90% plaque-reduction level. and Bunyaviridae. the selection of the screening serum dilution has not * Corresponding author. obtaining results from these tests may take several days and require paired serum samples or live cell culture. Goat anti-human IgM (PerImmune. and is usually less virus cross-reactive than IgG (16). Furthermore. rapid. 11. MAC-ELISA.

... Diluted antibody was stable for only 7 to 10 days and was discarded thereafter (personal observation). f Only along the eastern coast of the United States. TBE. The vast majority of positive serum samples were representative of a few viruses that could be localized geographically. enough cross-reactivity exists especially in the family Flaviviridae (3. West Nile. A standard five washes with a Skatron microplate washer (Skatron Instruments. Va. Japanese encephalitis. CE. b California only....b VEE. which correlated well with concurrently run PRNT for the same sera... Inc. RESULTS Determination of antigen panels.....5% Tween 20 without fetal bovine serum as recommended by Beaty et al.EEE. TABLE 1. Viral and normal antigens... and negative sera (}) are plotted..... EEE. BF. Sera and homologous positive and negative antibody controls were titrated using 10 twofold dilutions starting at 1:100 to evaluate the optimum screening dilution. saving time and resources. Commercially prepared 3... POW.. and the substrate reaction was stopped with 1 N sulfuric acid. Reactions were measured using a Bio-Rad microplate reader (Bio-Rad Laboratories. EVE. An overnight incubation at 4°C was used for the antigen step to increase test sensitivity... MAY.. YF.. and tested in triplicate by MAC-ELISA by using the 1:400 serum screening dilution. SSH... YF. Downloaded from http://jcm. Determination of the MAC-ELISA screening dilution. and LAC. DEN........ FIG. SLE.. at least three positive serum specimens were collected and analyzed using the screening MAC-ELISA. Inc.. Blocking and coating buffers and undiluted conjugates after reconstitution were stored at 4°C.org/ on April 12. The negative serum control P/N must be Ͻ2.) was used. 7) to permit the establishment of seven geographically based testing panels representing the medically important arboviruses.RR. A positive test result was obtained when the P/N of the test serum was Ͼ2.. which reduced the amount of testing needed for diagnosis...asm.. SIN...c DEN. SLE... d Florida only. Ross River..0 and the positive serum control P/N must be Ն2. YF.4 for California group viruses (a gift from George Ludwig via Barbara Israel. dengue.. When the latter criterion was not met due to nonspecific reaction with the normal mouse brain antigen. A blocking step using phosphate-buffered saline with 0..0 to 2.. Although it has been shown previously that IgM is less cross-reactive than IgG (especially for alphaviruses) (5..... Venezuelan equine encephalitis.. The following MAbs were used: 2A2C-3 for alphaviruses (15).. Titration curves for the representative antiviral serum samples are shown in Fig. JE.. Reconstituted undiluted antigen was stored at Ϫ20°C. blind coded. Figure 1 demonstrates the flat line produced by negative serum specimens even though in some cases the A450 values were sufficient for negative P/N values to be in the range of 2... SIN. MAY. Sterling.. Pa.... Mayaro. 1.d EVE.. CLIN.... MVE Asia and Middle East. DEN Africa.. All reagents were titrated individually by using a twofold dilution series that provided reagent excess in the first well of the series. DEN.c WN Eastern United States . 10G5... c Texas only. WN Australia and Oceania . CHIK. tick-borne encephalitis.. SLE..WEE.. 23). e Subtype of Venezuelan equine encephalitis virus.... 19) and the California serogroup viruses (1. yellow fever. Validation testing panels..) were incubated overnight at 4°C in a humidified container and were stable for 2 weeks.0 for the test to be valid. Md. We performed a historical analysis of arboviral testing results produced in our laboratory over the past 20 years.CHIK.SIN. Dynatech Industries... Use of MAb detectors eliminated the necessity for a secondary antibody addition.... . SLE... VEE.. Gaithersburg. Log10 serum dilution versus A450 is presented. EEE. TAH..... Tahyna.WEE.. TAH.. LAC. the result was reported as uninterpretable. Powassan...0 were chosen... SIN. Barhmah Forest. 2014 by guest Coated plates (Immulon II 96-well microtiter plates.. RR. medium-titered sera (F). SSH..3Ј.. California encephalitis.. GIBCO Bethesda Research Laboratories. snowshoe hare. Seven positive sera from each virus group and seven negative sera (confirmed by MAC-ELISA and PRNT results) were selected.. JE. prepared as sucrose-acetone extracts of infected suckling mouse brains (2). POW/TBE. Testing panels Test panel Arboviruses includeda J.. Murray Valley encephalitis...8 and 1. DEN.e POW.. and used as detector antibodies. DEN.. Tick-borne encephalitis virus antigen is not available in the United States. Sindbis. MAC-ELISA titration curves.5Ј-tetramethylbenzidine (TMB-ELISA.0 and the mean of the A450 values of the test serum reacted on viral antigen was at least twice the mean of the A450 values of serum reacted on normal mouse brain antigen. SIN. DEN South America . Inc. The resultant curves were typical of ELISA endpoint titrations.. and antigen dilutions were used once and discarded. and 6B6C-1 for flaviviruses (22).1824 MARTIN ET AL... Calculation of P/N values. Calculations that were performed followed guidelines set in the work of Beaty et al. SLE. MVE.. VEE. LAC...).. Hercules...5.f WN Europe..) at an absorbance of 450 nm..5.WEE..g WN Central America and Caribbean .. Powassan virus antigen is substituted.. Calif.. To determine the optimum dilution for screening sera.. BF. VEE.. Everglades... MICROBIOL. The diluent used for test sera and antigens was phosphate-buffered saline with 0.. therefore.... University of Wisconsin) (18)..) were used except before the addition of substrate where 10 washes reduced overall background.. Reagent dilutions which resulted in optical densities between 0. For viruses in the antigen batteries other than EEE. TAH.. CE... g Powassan virus and tick-borne encephalitis virus are closely related and cross-reactive... P/N values for these sera Western United States .. (West Grove. after observing a decrease in measured absorbance (A450) values when using a 2-h antigen incubation.. MAY.. Representative specimens of high-titered sera (I). were obtained from the DVBID reference collection.. Using these batteries to screen specimens ensured the detection of virtually all the endemic arboviruses in that area of the world for those virus families.. western equine encephalitis.d. WN a Virus abbreviations: WEE... These panels are shown in Table 1. Chikungunya. DEN. Group-reactive MAbs were purified and conjugated to horseradish peroxidase by Jackson Immunological Laboratories.CHIK... low-titered sera (Œ). POW/TBE.. 1.5% Tween 20 and 5% nonfat dry milk was added to reduce overall background and increase test sensitivity. (2).. (2) with the following modifications..

Development of this adaptable and rapid MAC-ELISA for detection of medically important arboviruses in serum and CSF has made clinical diagnosis more efficient. Test validation.98 0. Chikungunya virus cross-reacted with o’nyong-nyong virus.74 1.22 1. In practice. b Boldface indicates a positive result for the correct virus.22 1.93 6.75 4.43 1. a single MAC-ELISA format incorporating various antigens gave a rapid and precise picture of the IgM antibody status of a given serum.42 1.39 0.30 1. Routinely requesting a convalescent-phase specimen in this situation alleviates this dilemma. However. Our results showed that the screening dilution (1:400) had a good correlation with true endpoint values.46 1.17 1. Moreover.600.43 1. Ensuring exact incubation times. 1:400. The occasional disparity that was noted between results of the two tests is probably explained by the fact that the antibody types detected can be different.08 1. resulting in less-frequent nonspecific reactions and removing false-positive reactions caused by rheumatoid factor (12). PCR can be used for confirmation.98 1.00 1.0.34 1. Little cross-reaction was observed within the alphaviruses.35 1.23 5.27 1.16 8. a 1:400 screening dilution generated P/N ratios in the positive range that correlated well with measured endpoint titers. requiring endpoint titrations to confirm the positive reaction (personal observation). therefore eliminating the need for endpoint titrations except for confirmation of positive serum detected by the initial screening MAC-ELISA. NEG. a negative MAC-ELISA result for a very acutephase specimen (day 0 to 7) may reflect an insufficient antibody response very early in infection rather than no infection.77 1.asm.73 1.26 0.23 1. The use of broadly group-reactive MAb conjugates in combination with virus-specific antigens creates a system in which antibody to many arboviruses within a genus can be screened for concurrently by using a single virus-adaptable procedure.59 1.01 1.60 1. negative. Using a screening dilution of 1:400 eliminates most false positives. One of 12 positive LAC antibody specimens was positive by 1:400 dilution screening and negative by endpoint titration.600 dilutions were compared to endpoint titers. Flaviviruses and California serogroup viruses exhibited extensive cross-reaction between members of each respective group.15 1.83 1. Using the MAC-ELISA in tandem with an IgG ELISA (14a) yields antibody profiles capable of identifying recent infections by separate measurement of IgM and IgG. P/N values in the range of 2.30 1. only a portion of these sera with low P/N values will fail a confirmatory neutralization test. Only 2 of 20 positive EEE antibody specimens were positive by endpoint but negative by 1:400 dilution screening.03 1.84 a Antigen for which serum tested positive in the original testing.83 12.37 0.07 6. Interpretation of MAC-ELISA results is based primarily on the timing of the sample and confirmation of those results in another test. In the absence of a convalescent-phase specimen. exploiting the early rising and rapidly declining nature of IgM antibody.25 1.55 2. However. and these are screened undiluted.70 1.95 8.04 1. Competition between IgM and IgG for antigen binding is minimized. yields results that can be compared between tests.17 1. The capture format of the MAC-ELISA eliminates potential background caused by extraneous antibody.32 0. at 1:100. Correlation of the MAC-ELISA results with the PRNT was good.13 0.10 1. reducing the occurrence of false-negative results.06 7.35 1.39 1.39 1.64 0. False-positive sera yielding an endpoint generally graph as a flat line and demonstrate a fairly stable P/N range. 38.44 1. 17.94 3. and 1:25. The detection of IgM antibody to a particular arbovirus in a cerebrospinal fluid (CSF) specimen is evidence of infection with that virus (2.91 5. MAC-ELISA screening of sera testing positive to other members of the antigen batteries showed uniform positive reactions to homologous antigens. DISCUSSION An array of antibody types and subclasses is produced by a normal host humoral response to viral infection. however. Results of the blind-coded serum testing are shown in Table 2. MAC-ELISA results in an acute specimen that remain unconfirmed by PRNT may reflect the lack of neutralizing ability of IgM produced early in infection and should not be confused with a false-positive result (7). CSF specimens are submitted. Occasionally.22 1.18 7.10 0.02 6.54 1. The 22 specimens testing positive to SLE in the screening MAC-ELISA were also positive by endpoint titration.31 2. All antibody-negative control specimens were negative by both testing procedures for all three viruses.VOL. —.58 2.74 1. Results of coded sera tested by MAC-ELISA for all virus families Serum no.0 to 3. usually under 3.org/ on April 12. The MACELISA specifically detects IgM.36 1. 1:1.15 7.96 3. IgM antibody is produced early in the immune response (16).28 2.97 4. When the worldwide antibody panels based on the various exploitable cross-reactions were used (1. we do MAC-ELISA endpoint titrations on serum specimens positive in our screening MAC-ELISA.22 1.94 1. allowing for timely diagnosis of disease.08 — 1. IgM produced early in infection does not always possess neutralizing activity (7).12 1. During the West Nile encephalitis epidemic in New York City in the Downloaded from http://jcm.95 1.06 6.60 1.96 1. A positive result by MAC-ELISA in a single acute-phase specimen is presumptive evidence of a recent infection with that arbovirus (9). especially in the critical substrate step. Using chi-square analysis for all three viruses tested.91 1.11 6. result uninterpretable because of high background.31 0.19 11.77 1.53 4. 2014 by guest . However.09 0.30 1. Antigena P/N ratio for virusb: SLE EEE LAC MAC-ELISA FOR ARBOVIRUSES 1825 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 NEG SLE EEE EEE LAC SLE NEG EEE EEE SLE NEG LAC LAC SLE EEE NEG EEE SLE LAC NEG EEE NEG NEG LAC SLE LAC LAC SLE 1. Newly emerging arboviruses and those in other virus families can be added to any battery as soon as the proper viral antigens have been developed and positive controls are obtained.0 have occasionally been determined to be false positives. italics indicate a positive result for an incorrect virus. A potentially IgM-positive acute-phase serum sample is defined for purposes of this test as serum taken at least 8 days and up to 45 days after onset of symptoms (unpublished data). 19). 2000 TABLE 2.16 4. 7. 9).

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