Detection of Norwalk Virus Antibodies and Antigen with a Biotin-

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1985, p /85/ $02.00/0 Copyright 1985, American Society for Microbiology Vol. 22, No. 2 Detection of Norwalk Virus Antibodies and Antigen with a Biotin- Avidin Immunoassay G. WILLIAM GARY, JR.,* JONATHAN E. KAPLAN, SARAH E. STINE, AND LARRY J. ANDERSON Division of Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 4 February 1985/Accepted 29 April 1985 Biotin-avidin immunoassays (BAIs) were developed to detect Norwalk virus antigen and to measure Norwalk virus antibody. The BAI detected Norwalk virus infections by a fourfold titer rise in antibody in sera or by antigen in stool, with a sensitivity similar to or greater than that of the radioimmunoassay (RIA), and the BAI appeared to be more sensitive than the RIA for detecting antibody in single serum specimens. The BAI antigen test detected Norwalk antigen in all stools that were positive by the RIA, and in two stools negative by the RIA. Of 106 serum pairs tested by both the RIA and BAI, 94 demonstrated seroconversion, or lack of seroconversion, in both assays; 12 gave discordant results. Titers by the BAI generally exceeded those by the RIA by two- or fourfold. The BAI had advantages over the RIA in that it had added sensitivity in the detection of Norwalk antibody, was less time consuming, did not require frequent labeling of antibody, and did not have the problems associated with the use of isotopes. Norwalk virus is the prototype of 27-nm viruses associated with gastroenteritis, and it was discovered by studies of specimens collected during an outbreak of gastroenteritis in an elementary school in Norwalk, Ohio (1). Dolin et al. (3) successfully transmitted the disease through two serial passages in human volunteers by the oral administration of a fecal filtrate derived from the stool specimen of a secondary case in the Ohio outbreak. Kapikian et al. (10) visualized a 27-nm, viruslike particle in a stool filtrate prepared from a second-passage diarrheal stool from a human volunteer. They used this stool as antigen to detect Norwalk virus antibody by immune electron microscopy and observed a serological response to the 27-nm particle in naturally and experimentally induced illness. In 1978, Kapikian et al. (9) developed an immune adherence hemagglutination assay for detecting antibody to Norwalk virus, and shortly thereafter, Greenberg et al. developed a solid-phase radioimmunoassay (RIA) for detecting Norwalk virus antigens and a blocking RIA for detecting antibodies (7). In both RIAs the detector antibody is directly labeled, which enables the one good source of Norwalk virus antibody, human convalescentphase serum, to be used as both capture and detector antibody. The RIA proved to be the most sensitive test for antigen and antibody detection. The antibody RIA also conserved antigen, making it a practical test for epidemiological studies of Norwalk virus infections. The antibody RIA now has been used in a number of studies to study the prevalance of Norwalk virus antibody and to investigate outbreaks of acute infectious nonbacterial gastrointestinal illness believed to have been caused by this virus (5, 6, 11). Although the RIA proved to be a sensitive and reliable test for detecting antibodies to Norwalk virus, it requires 6 days to perform and uses radioisotope labeling with the attendant cost, hazards, and disposal problems. These problems with the RIA led us to develop an assay based on another system for directly labeling antibodies, the biotin-avidin immunoassay (BAI) system, and to compare this to the standard RIA for the detection of Norwalk antibody and antigen. * Corresponding author. MATERIALS AND METHODS Norwalk antibody and antigen. Stool and serum specimens were collected during a human volunteer study of Norwalk virus infection at the Medical School, University of Texas Health Science Center at Houston. The specimens were used to prepare antigens, coating antibody, and immunoglobulin G (IgG) for the RIA and BAI. Stool suspensions were made as 5% suspensions in veal infusion broth containing 0.5% bovine serum albumin (BSA). The suspensions were mixed vigorously on a vortex-type mixer for approximately 2 min and centrifuged at 1,000 x g for 20 min at 4 C, and the supernatant fluid was removed for use as antigen. Convalescent-phase serum specimens from ill volunteers were assayed by the RIA for determination of their antibody titers to Norwalk virus. One serum specimen with a high titer (1:6,400) was selected for use as coating antibody in the RIA and BAI. A second convalescent-phase serum specimen was selected from a different volunteer with a high titer (1:25,600); this specimen was used to prepare anti-norwalk IgG for labeling with 1251 for the RIA, or with biotin for the BAI, as detector antibody. The IgG was prepared from the serum by a single precipitation with saturated ammonium sulfate, followed by ion-exchange chromatography on DE-52 (DEAE-cellulose; Whatman, Maidstone, England). In brief, the serum sample was mixed with an equal volume of saturated ammonium sulfate; the resulting precipitate was separated by centrifugation and suspended in 0.01 M phosphate buffer (ph 8) to one-half the original volume of the serum; and this solution was dialyzed overnight against phosphate buffer. To further purify the IgG, this solution was applied to a DE-52 column and eluted with phosphate buffer. The fractions from the first peak (containing the IgG) were pooled, and the concentration was adjusted to 1 mg of protein per ml of buffer. Biotinylation. IgG, prepared as described above, was dialyzed overnight at 4 C against 0.1 M NaHCO3 (ph 8), after which the protein was adjusted to 1 mg/ml. A 1.4-mg volume of biotin (n-biotinyl-w-aminocaproic acid-nhydroxysuccinimide ester) (EAB-406; Enzo Biochem Inc., New York) was dissolved in 1 ml of dimethylsulfoxide, and 200,ul of the biotin preparation was added to each milliliter 274

2 VOL. 22, 1985 DETECTION OF NORWALK VIRUS ANTIBODIES AND ANTIGEN E E - o CD 1' _.400-,j 0 Co Qa300 - n I I I I Reciprocal of Endpoint Dilution (Log2 Scale) FIG. 1. Plot of values from the Norwalk BAI of four representative serum pairs from a presumed Norwalk viral gastroenteritis outbreak. Endpoint titers of sera are: 4152 acute (4152A), 1:200; 4152 convalescent (4152C), 1:400; 4153A, <1:50; 4153C, 1:100; 4154A, 1:50; 4154C, 1:800; 4155A, 1:100; 4155C, 1:200. Points below the line represent dilutions that contained antibody sufficient to block 50%0 of the biotyinylated detecting antibody. Downloaded from of IgG, allowed to react for 4 h at room temperature while rotating slowly on a laboratory rotator, and then dialyzed overnight at 4 C against 0.01 M phosphate-buffered saline (PBS; ph 7.2). BAI. The BAI was performed in polyvinyl chloride microtiter V plates ( ; Dynatech Laboratories, Inc., Alexandria, Va.). Optimal dilutions of reagents were determined by block titrations. For both the antigen and antibody assays, plates were coated with a portion (75 pi per well) of a 1:5,000 dilution of a human volunteer serum specimen (as noted above) diluted in PBS (ph 7.2); the plates were incubated overnight at room temperature when the test was to continue the next day, and if not, the plates were stored at 4 C. The plates then were washed twice with PBS containing 0.05% Tween 20 [polyoxyethylene(20)sorbitan monolaurate] (PBS/T) and postcoated by filling the wells with PBS containing 1% BSA (PBS-BSA) and incubating for 1 h at 37 C. The plates were washed three times with PBS/T. Fifty microliters of a 1:160 dilution of Norwalk antigen, prepared as described above, was added to each well and incubated for 2 h at 37 C. The plates were washed three times with PBSIT, and 50 1ul of the appropriate dilutions of the serum samples, or diluent, was added to each well and incubated overnight at room temperature. Then, without aspirating the contents of the wells, 75,ul of the biotinylated anti-norwalk IgG (diluted 1:100 in PBS/T with 10% normal goat serum and 0.5% gelatin) was added and incubated for 1 h at 37 C. The plates were washed three times with PBS/T, and 75 pul of a 1:10,000 dilution of peroxidase-labeled avidin (A-3151; Sigma Chemical Co., St. Louis, Mo.) was added and incubated for 20 min at 37 C. The plates then were washed three times with PBS/T, and 125 pui of substrate (0.4 mg of o-phenylenediamine dihydrochloride [OPD] per ml; Aldrich Chemical Co., Inc., Milwaukee, Wis.)-0.5% H202 in citrate phosphate buffer (ph 5.5) was added to each well and incubated for 40 min in the dark at room temperature. The reaction was stopped with 25 pul of 3.5 M HCI, and absorbance at 490 nm was read on a Dynatech Microelisa Auto Reader MR580. The reader was blanked on a well containing substrate and HCI. One Norwalk antibody-positive serum specimen was included in each BAI as a control to establish reproducibility. In each test, the titer of this specimen had to be within twofold of the mean for the test to be accepted as valid. The blocking titer of serum antibody was determined from serial twofold dilutions with two wells per dilution. The endpoint was taken as the highest dilution that gave an absorbance of c50% of the maximum absorbance reading (Fig. 1). Maximum absorbance was taken as the mean of five wells with diluent in place of the serum samples. Background absorbance was taken as the mean of three wells with control antigen in place of Norwalk antigen and diluent in place of the serum sample and was subtracted from the absorbance readings before the calculations were done. If after the initial testing of a serum pair it was found that any serum antibody titer exceeded the endpoint in the test, this serum was retested at a higher dilution, unless a fourfold or greater rise in titer was demonstrated in the first test. The BAI for antigen detection was adapted from the antibody test. Half of each plate was coated with a 1:5,000 dilution of the preinfection serum counterpart (Norwalk RIA antibody titer, 1:400) of the postinfection serum (Norwalk on October 31, 2018 by guest

3 276 GARY ET AL. RIA antibody titer, 1:6,400) used as coating antibody in the antibody assay described above; the other half of the plate was coated with a 1:5,000 dilution of the postinfection serum with high-titer antibodies to Norwalk virus. The plate was postcoated with 1% BSA and washed. After the postcoating and a wash step, 50,ul of 5% suspensions of the stool samples to be tested were placed in each of four wells coated with preinfection antibody and in each of four wells coated with postinfection antibody. The plate was incubated for 2 h at 37 C and washed twice with PBS/T. A 75-pul volume of the biotinylated anti-norwalk IgG was added, and the remaining steps were carried out as in the antibody assay. If the mean absorbance of any stool suspension was (the limit of the spectrophotometer), it was diluted 1:2 or 1:20 in PBS- BSA and retested. In the BAI antigen test, the mean absorbance values of the four wells initially coated with postinfection antibody were divided by the like values from the wells coated with the preinfection antibody, giving a positive/negative (P/N) ratio. As described in the paper of Greenberg et al. (7), we used a P/N of.2 as indicative of the presence of Norwalk antigen. RIA. The RIA used in this study was also performed in polyvinyl chloride microtiter V plates by the standard method as described by Greenberg et al. (7). The tests for antigen and antibody detection were performed in a fashion identical to that for the corresponding BAI tests except that the detection antibody was labeled with 125I, counts were measured in a gamma counter (as counts per minute), and incubation times were longer, as described by Greenberg et al. (7). Increased incubation times were with the 1% BSA postcoat (overnight), antigen (overnight), and labeled IgG (4 h). In addition, counting of residual radioactivity in the gamma counter required 5 h for à typical test, and, of TABLE 1. Comparison of the BAI and RIA for detecting Norwalk antigen in selected stool suspensions from human volunteers Specimen BAI" RIA' volunteer (day)a A490-P A490-N P/N cpm-p cpm-n P/N 1A (0) B (+1) C (+5) A (0) B (+2) C (+3) A (0)d ,640 5, B (+1)" ,888 4, C (+2)e ,662 8, D (+3)e ,056 5, E (+5) ,597 2, A (+2) B (+3) ,647 1, C (+4)" ,854 2, SA (+1)d ,554 2, SB (+1ly The values within parentheses are the number of days relative to the day that the volunteer was given the inoculum. A490-P, A4w for wells containing postinoculation coating antibody; A4M-N, A4W for wells containing preinoculation çoating antibody. cpm-p, Counts per minute for wells containing postinoculation coating antibody; cpm-n, counts per minute for wells containing preinoculation coating antibody. " The absorbance readings on the original suspension were out of range; the suspension was diluted further to 1:2. "'The absorbance readings on the original suspension were out of range; the suspension was diluted further to 1:20. f Collected 1 hour after specimen SA. J. CLIN. MICROBIOL. TABLE 2. Comparison of results from Norwalk serology by the BAI and RIA BIA results RIA results Positive Negativç Positive 40 4 Negative 8 54 a Positive, Serum pairs with fourfold or greater rise in antibody titer between acute- and convalescent-phase specimens; Negative, serum pairs with less than a fourfold rise in antibody titer between acute- and convalescent-phase specimens. necessity, was performed after working hours with data not available until the next morning. Clinical specimens. Serum specimens for this study were selected from outbreak specimens of presumed viral gastroenteritis previously tested for antibody to Norwalk virus by the RIA. Those selected included sera from outbreaks with previously demonstrated seroconversions to Norwalk virus by the RIA, outbreaks without demonstrated seroconversions to Norwalk virus by the RIA, and one outbreak of Campylobacter spp. infection. Stool specimens for antigen detection testing came either from the human volunteer study or from outbreaks of Norwalk gastroenteritis. Stool specimens from the volunteer study included both pre- and postinfection specimens. Outbreak stool specimens were either from field cases that had fourfold rises in Norwalk antibody or from outbreaks where other cases had fourfold rises. RESULTS The BAI and the RIA gave similar results for detecting Norwalk antigen in fecal specimens from the human volunteer study (Table 1) and from outbreaks of Norwalk antibody-positive gastroenteritis. Two specimens from the volunteer study were positive (P/N -2) by both tests, and two were positive by the BAI and negative by the RIA. The remainder were negative by both tests. In those tests that were positive, P/N values were generally higher in the BAI than in the RIA. Among the 24 outbreak specimens, 1 was positive in both tests and the others were negative in both tests. A total of 106 serum pairs from gastroenteritis outbreaks were tested for Norwalk antibody by the BAI and RIA (Table 2). The BAI antibody test appeared to be more sensitive than the RIA test for detecting the presence of Norwalk antibody. For 110 of 212 sera the BAI titer was fourfold or more higher than the RIA titer, for 4$ sera the BAI titer was twofold higher, for 52 sera the titers were equal, and for 2 sera the titers were higher by the RIA. A total of 45 sera had no detectable antibody by the RIA but had antibody by the BAI (for purposes of comparing negative titers in one assay with positive titers in the other assay, the negative titers were assigned a titer one dilution lower than the lowest screening dilution), 26 sera had no antibody in either test, and one had antibody by the RIA and none by the BAI. Although the antibody titers were higher by the BAI, results for detecting a fourfold rise in Norwalk antibody were similar (Table 2). Results from 18 serum pairs were chosen to illustrate the titers detected by the two tests (Table 3); two specimens (no and 3914) demonstrated fourfold or greater rises by the BAI but not by the RIA. Two other specimens (no and 3913) demonstrated fourfold or

4 VOL. 22, 1985 DETECTION OF NORWALK VIRUS ANTIBODIES AND ANTIGEN 277 TABLE 3. Comparison of Norwalk serum antibody titers in selected serum pairs as determined by BAI and RIA Titer' Serum pair no. BAI RIA Acute Convalescent Acute Convalescent ,600 6, , ,400 3, , ,400 12,800 1,600 3, < <50 < , , < < , , <50 50 < <50.1,600 <50.1, <50 <50 <50 < , <50 < , ,200 <50 <50 Reciprocal of endpoint dilution. greater rises by the RIA but not by the BAI. No Norwalk antibody titer rises were detected by either assay in the six serum pairs from the nonviral gastroenteritis Campylobacter spp. outbreak. DISCUSSION Progress in the study of Norwalk virus has been limited by the inability to grow the virus in cell culture, lack of an efficient animal model, limited supply of reagents for diagnostic testing, and lack of a rapid diagnostic test. Since the agent was first identified in 1972 (10), the most sensitive method for diagnosing infection has been detecting a fourfold rise in antibody titer between acute- and convalescentphase sera. In contrast, virus or antigen has been detected infrequently in field specimens (11; unpublished data). The blocking RIA proved to be the best of the tests developed and has been used extensively for studying outbreaks of gastroenteritis and the prevalence of Norwalk virus antibodies (5, 6, 11). The BAI system provides an alternative way to directly label antibodies and, therefore, to replace the radiolabel with biotin and use an enzyme detection system. The BAI system has been used successfully by a number of investigators in enzyme immunoassays, including the initial studies by Guesdon et al. (8) and subsequent studies on detecting hepatitis B surface antigen antibody (12), human IgE (13), and herpes simplex types 1 and 2 antigens in clinical specimens (2). In these studies the BAIs were at least as sensitive as standard enzyme immunoassays or RIAs and were less time consuming to complete. The BAI for Norwalk antigen that we developed appears to be at least as sensitive and possibly more sensitive than the corresponding RIA tests. The BAI antigen test detected all stools positive by the RIA and two stools positive for Norwalk virus not detected by the RIA; in thôse specimens positive by both assays, the P/Ns were higher by the BAI. The specimens positive by the BAI but negative by the RIA are probably true-positives since both specimens came from experimentally inoculated volunteers at the time when excretion of virus normally occurs in Norwalk infections. Preinoculation or earlier postinoculation specimens from the same volunteers were negative. As indicated above, detection of antigen or virus in field specimens has been infrequent. Although the BAI antigen test gave excellent results with volunteer specimens, its applicability to field specimens will have to be determined by examining a large number of specimens over a period of time. BAI antigen tests with rotavirus-positive stools and with stools from volunteers inoculated with the Snow Mountain agent (4) were negative by our criteria, indicating a lack of crossing activity with these viruses (data not presented). The RIA and BAI gave identical results for the presence of a fourfold titer rise for 94 of 106 serum pairs; 9 serum pairs that gave discordant results came from Norwalk virusconfirmed outbreaks. The other serum pairs came from an outbreak in which only one of seven serum pairs tested demonstrated a fourfold increase in Norwalk antibody titer. For seven of the eight serum pairs positive by the BAI and negative by the RIA, the convalescent specimen was either negative or positive only at the lowest test dilution by the RIA, and therefore, the lack of a fourfold rise probably resulted from the RIA being less sensitive. Of the four pairs of sera with a fourfold titer rise by the RIA but not by the BAI, two had twofold titer rises by the BAI and two had stable titers. Although the BAI antibody test was not substantially better in detecting Norwalk virus infection by showing a fourfold titer rise, it appeared to be more sensitive for detecting antibody, often giving a two- to fourfold higher titer in a single serum specimen than did the RIA antibody test. This added sensitivity may make the BAI antibody test a better indicator of the prevalence of Norwalk virus antibodies and, therefore, the rate of previous Norwalk virus infections in different populations. This apparent increased sensitivity for single serum specimens may be related to the difference in measuring endpoints by absorbance rather than by counts per minute as in the RIA. The principle advantages of the BAI, however, are time and safety. The BAI for antibody can be performed in 3 days instead of 6 days and does not require frequent labeling of antibody. '25I-labeled Norwalk antibody has a shelf life of 2 weeks, whereas the biotinylated antibody has a shelf life at -20 C of at least 3 months (14). The BAI also does not require the use of radioisotopes with their attendant cost, hazard, and disposal problems. Further, the BAI should be easier to automate, as it can be read by automated spectrophotometers and does not require the cutting of plates and counting of radioactivity in individual wells. The BAI for antibody and antigen detection should prove to be a valuable tool for studying Norwalk and Norwalk-like virus infections. ACKNOWLEDGMENTS We thank Bruce Keswick, Philip Johnson, Sandy Secor, and Herbert DuPont of the Medical School, University of Texas Health Science Center at Houston, for their considerable effort in the performance of a human Norwalk volunteer study that provided much of the material used as reagents in this study. We also thank Nancy Nowak and Neil Blacklow of the University of Massachusetts Medical School, Worcester, for the initial testing of some stool and serum specimens. LITERATURE CITED 1. Adler, J. L., and R. Zickl Winter vomiting disease. J. Infect. Dis. 119: Adler-Storthz, K., C. Kendall, R. C. Kennedy, R. D. Henkel, and

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