Enzyme-Linked Immunosorbent Assay and Comparison with

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p /87/ $02.00/0 Vol. 25, No. 1 Typing of Herpes Simplex Virus by Capture Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay and Comparison with Restriction Endonuclease Analysis and Immunofluorescence Method Using Monoclonal Antibodies LATA S. NERURKAR,* NANCY R. MILLER, MAMI NAMBA, MARTA MONZON, GENE BRASHEARS, GAIL SCHERBA, AND JOHN L. SEVER Infectious Diseases Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland Received 28 May 1986/Accepted 22 September 1986 A sensitive enzyme-linked immunosorbent capture assay using biotin and streptavidin (capture B/SA ELISA) was developed using type-specific monoclonal antibodies for typing of herpes simplex virus. Rabbit anti-herpes simplex virus immunoglobulin G was used as the capturing antibody, and biotin-linked type-1-specific mouse monoclonal antibody or rabbit type-1- or type-2-specific polyclonal antibody served as the detecting antibody. The captured antigen was detected by an ELISA with alkaline phosphatase-conjugated streptavidin, which reacted with biotin molecules on the detector antibody. The capture B/SA ELISA was compared with other methods for efficiency and reliability in typing. Results obtained by restriction endonuclease digestion of the radiolabeled viral genome were used to determine the type (1 or 2) of clinical isolates. These results were then used as a reference for determining the accuracy of the capture B/SA ELISA, as well as that of the immunofluorescence method, both of which are easily adaptable for use in the clinical laboratory. The three methods were in perfect agreement. It was determined that both the capture B/SA ELISA and the immunofluorescence method using monoclonal antibodies provided typing results with 100% specificity and 100% sensitivity and thus were accurate and reliable. However, the ELISA was the method of choice because of its simplicity, rapidity, and use of nonradioisotopic reagents. Herpes simplex virus (HSV) is a widespread infectious agent in the human population. After initial (primary) infection, the virus enters a latent, asymptomatic state, in which it is dormant in the nervous system. Recurrent infections at the same or adjacent sites occur when the nerve is triggered. Stress or hormonal or immunological changes and some physiological conditions may stimulate replication of the virus, which is then mobilized back to the target tissue. The severity of HSV infections varies widely, ranging from asymptomatic infection to an often fatal herpes encephalitis. The frequency and severity of recurrences also vary to a great extent. Considerable differences between the two antigenic types (1 and 2) of HSV are recognized, especially in their susceptibility to antiviral drugs (5, 6) and the site and rate of recurrence (25). This has made the typing of HSV a valuable tool for a better understanding of the disease. In this report, we describe the typing of clinical isolates by a capture enzyme-linked immunosorbent assay using biotin and streptavidin (capture B/SA ELISA) with both polyclonal and monoclonal antibodies and compare the sensitivity and specificity of this system with those of restriction endonuclease analysis and the immunofluorescence assay using monoclonal antibodies. Restriction enzyme analysis provides accurate typing information because the two prototype (1 and 2) viruses have distinct restriction endonuclease sites (i.e., produce different sizes of DNA fragments after enzyme cleavage), especially with enzymes KpnI and HpaI (14). There is also enough individual variation within the type 1 and 2 groups that * Corresponding author. 128 analysis of the genome can make it possible to determine whether a second site of infection is the result of the spread of the same virus or of transmission of a different virus from another individual. However, the method is timeconsuming, involves the use of radioisotopes, and is not well suited to a clinical laboratory. There is a distinct need for accurate and less expensive or less elaborate technology for routine typing. MATERIALS AND METHODS Clinical specimen collection. Clinical specimens were collected from patients by rubbing lesions, in oral or genital areas, or genital areas of people suspecting infections with sterile cotton swabs. The swabs were transferred in 3 ml of Hanks balanced salt solution containing streptomycin, penicillin, acromycin, mycostatin, and gentamicin (henceforth referred to as collection medium), refrigerated before and during transport, and frozen at -70 C if not studied immediately (24). Tissue culture determination of HSV in clinical specimens. Human foreskin fibroblast (Flow 7000; Flow Laboratories, Inc., McLean, Va.) monolayer tube cultures were inoculated with 0.2-ml volumes of clinical specimens. The specimens were absorbed on monolayers for 1 h after which fresh, complete Eagle minimum essential medium (EMEM)-2% fetal bovine serum (1.5 ml) was added. The cultures were incubated at 37 C and evaluated for cytopathic effects (CPE) for up to 7 days. Specimens with positive CPE were further passaged on Flow 7000 and rabbit kidney cell monolayers and confirmed for the presence of virus. When showing 80 to 90% CPE, the cell monolayers were scraped, properly

2 VOL. 25, 1987 resuspended in supernatant medium, and frozen at -70 C in aliquots. The negative cultures were discarded after 7 days. Capture B/SA ELISA. The capture B/SA ELISAs were performed as recently described (18). The HSV isolates grown in Flow 7000 cell cultures up to 4+ CPE were diluted 1:10 with EMEM-2% fetal bovine serum, and the ELISA reactions were run simultaneously against anti-hsv type 1 (HSV-1) and anti-hsv-2 antibody reagents. In the polyclonal antibody test, the capturing antibodies were identical to detecting antibodies, except that the latter were linked with biotin. In the monoclonal antibody test, polyclonal HSV-1 reagent was used as the capture antibody and biotinlabeled type-1-specific monoclonal reagent was used as the detector antibody. In brief, the test was performed as follows. Costar (Cambridge, Mass.) 96-well polystyrene irradiated plates were coated with 300 pil of anti-hsv-1 or anti-hsv-2 polyclonal antibody (DAKO; Accurate Chemicals, Westbury, N.Y.) diluted in sodium carbonatebicarbonate buffer (1.59 g ofna2co3, 2.93 g ofnahco3, and 0.2 g of sodium azide per liter [ph 9.6]) in a humid chamber overnight. The plates were washed three times with phosphate-buffered saline (PBS; M NaCI, M phosphate buffer [ph 7.2]; Biofluids, Rockville, Md.) containing 0.05% Tween 20 (Sigma Chemical Co., St. Louis, Mo.), 0.1% bovine serum albumin (Miles Laboratories, Inc., Elkhart, Ind.), and 0.02% sodium azide (Sigma), henceforth referred to as the ELISA wash. A 100-puI sample of diluted HSV clinical isolates, stock virus, or control medium was added to duplicate wells, and the plates were incubated at 37 C in a 5% C02 incubator for 1 to 2 h. After the plates were washed three times with the ELISA wash, pretitrated biotinlinked polyclonal or monoclonal antibody diluted in PBS containing 1% bovine serum ablumin, 0.02% sodium azide, and 0.05% Tween 20 was added (100 pil per well). After 1 h of incubation at 37 C, the plates were washed three times with the ELISA wash. Pretitrated streptavidin-alkaline phosphatase conjugate (100 pil per well) was then added, and incubation was continued at 37 C for 30 min. The unbound enzyme conjugate was removed by washing three times with the ELISA wash, and 100 pil of substrate p-nitrophenyl phosphate (1 mg/ml; Sigma) in 0.5 M diethanolamine buffer (ph 9.8; containing 100 mg of MgCl2 6H20 and 200 mg of sodium azide per liter) was then added. The reaction was stopped after 10 min by adding 3 M NaOH (50 pil per well). The A405 measurements were made with Dynatech ELISA reader model MR580. The ratio of A405 using polyclonal anti-hsv-1 and anti-hsv-2 reagents was determined. A ratio of >1.0 was indicative of HSV-1, and <1.0 was indicative of HSV-2. In the monoclonal antibody test, positive-negative cutoff values were established as described before (18), and HSV types were identified. HSV tissue culture on Lab-Tek chamber slides. Human foreskin fibroblast cell cultures (Flow 7000) were grown on eight-well Lab-Tek chamber slides (Miles Scientific, Div. Miles Laboratories, Inc., Naperville, 111.) as described previously (17). In brief, the chamber slides were first rinsed and incubated overnight with EMEM containing 10% fetal bovine serum to remove any toxic products (which were occasionally seen in certain batches of these chambers). Approximately 3 x 104 to 4 x 104 cells were seeded in each well and allowed to grow for 24 to 48 h. Monolayers were initially infected with 0.2-ml volumes of HSV-containing clinical specimens and laboratory HSV isolates for various times and at various concentrations to optimize the test conditions. All specimens (100 to 200 pul) were then inoculated in duplicate. Two wells of each chamber slide were HSV TYPING BY BIOTIN-STREPTAVIDIN ELISA 129 mock infected or infected with laboratory-purified HSV-1 (McIntyre strain) or HSV-2 (MS strain) preparations, respectively, to have negative and positive controls on the same slide. At the end of incubation, the medium was aspirated, the slides were washed three times with PBS, the plastic chambers were removed, and the slides were fixed in chilled (4 C) acetone for 10 min. The acetone was evaporated completely, and the slides were frozen at -70 C until stained. Immunofluorescence staining of HSV with polyclonal or monoclonal antiherpesvirus antibodies. The acetone-fixed slides of HSV-infected tissue cultures prepared as described above were rinsed with PBS and blotted. They were then reacted with 50 to 100 pul of hyperimmune rabbit anti-hsv immunoglobulin G biotin conjugate and fluorescein-avidin D as described before (17, 19). The slides were then counterstained with 0.006% Evans blue, mounted in PBS:glycerol (10:90), and viewed under a fluorescence microscope by at least two persons. The same procedure was repeated for the use of monoclonal antibodies which were obtained from Ortho Diagnostics, Inc., Raritan, N.J., Electro-Nucleonics, Inc., Columbia, Md., and Biotech Laboratories, Rockville, Md. In this experiment, an indirect staining procedure was used, except with the Electro-Nucleonics reagents, which involved a direct staining reaction. The indirect procedure involved incubation with monoclonal antibodies at a dilution specified by the manufacturer, followed by fluorescein-conjugated anti-mouse immunoglobulin G. Restriction endonuclease digestion of HSV DNA. Human foreskin fibroblast cells (Flow 7000) were grown in 24-well tissue culture plates (Costar) to near confluency. They were then maintained on EMEM containing 1% fetal bovine serum (dialyzed), 2 mm glutamine, and 1,uM phosphate (EMEM-low P04) for 24 to 48 h. To inoculate the monolayers, the medium was removed, and 0.5 ml of a given viral isolate (diluted 1:10 in EMEM-low P04) was added to each well. The inoculum was adsorbed for 1 h at 37 C and washed once with EMEM-low P04. A 0.5-ml sample of the same medium was then added to each well, and incubation at 37 C was continued for 3 h. At this time, 50,uCi of 32p; (New England Nuclear Corp., Boston, Mass.) was added to each well, and incubation was continued overnight at 37 C. Viral CPE was obvious within 24 to 48 h in more than 80% of the cells. The incubation up to 72 h was necessary for a few specimens which displayed low infectivity. Infected cultures showing adequate CPE were further treated to isolate 32p_ labeled herpesviral DNA as described by Lonsdale (14). Sodium dodecyl sulfate (20%; 70 pul) was added to each well, and the contents were transferred to Microfuge (Beckman Instruments, Inc., Fullerton, Calif.) tubes. A 500-pil sample of phenol saturated with 75 mm NaCl-50 mm EDTA was added; the tubes were kept on ice and centrifuged in a Beckman Microfuge for 30 min. The aqueous layer was removed to tubes containing 25 pil of 5 M NaCl, and 1 ml of ethanol was added. The mixture was kept at -20 C overnight, and the precipitated nucleic acids were pelletted by centrifugation in a Beckman Microfuge for 30 min at 4 C. The supernatant ethanol was poured off, the tubes were inverted, and the tube walls were wiped dry. To the pellets, 100 pil of water containing 20,ug of RNase A was added, and the RNA was digested for 2 h at 37 C. This was followed by partial digestion of DNA with restriction endonucleases KpnI and HpaI for 2 h at 37 C. The reaction was stopped by adding 10 pi of 0.2 M EDTA (ph 7.5) in 50% glycerol containing bromophenol blue. The DNA digests were ana-

3 130 NERURKAR ET AL. lyzed by overnight electrophoresis (35 ma) on agarose gels (0.6%) by the method of Lonsdale (14). The gels were dried for 30 min in a vacuum and heat and exposed to Kodak XAR5 X-ray film for 2 to 18 h to visualize the sizes of the 32P-labeled DNA fragments. RESULTS Typing by capture B/SA ELISA. All 30 isolates were studied by using (i) homologous polyclonal anti-hsv-1-anti- HSV-1 and homologous anti-hsv-2-anti-hsv-2 sandwiches and (ii) a heterologous polyclonal anti-hsv-1-monoclonal anti-hsv-1 sandwich. In the former, the ratio of the A405 of the type 1 sandwich to the type 2 sandwich was obtained and denoted as the typing index. It was noted that the clinical HSV-1 isolates or laboratory-purified HSV-1 always gave higher absorbance on the homologous anti-hsv-1-anti-hsv- 1 sandwich. The reverse was true for clinical HSV-2 isolates or laboratory-purified HSV-2 using the homologous anti- HSV-2-anti-HSV-2 sandwich. The results of these experiments (Fig. 1) included comparisons of the absorbances for different isolates. The typing index for HSV-1 isolates was consistently >1.0, and for HSV-2 isolates it was consistently <1.0. The average and range values for the typing index were 1.74 and 1.12 to 3.27, respectively, for HSV-1 and 0.24 and 0.18 to 0.43, respectively, for HSV-2. It is clear based on these data that polyclonal reagents could be successfully used in the ELISA, even though they led to ambiguous results in the immunofluorescence assay. A positive identification of HSV-1 was then performed using the heterologous polyclonal anti-hsv-1-monoclonal anti-hsv-1 sandwich. The results are shown in Fig. 2. The clinical HSV-1 isolates and laboratory-purified HSV-1 showed good, positive signals, with absorbance values above the cutoff limits. The cutoff value for the identification of HSV-1 was established to be or greater based on the reactions of purified HSV-1 and HSV-2. This allowed the distinction between HSV-1 and HSV-2 to be very clear and objective. The procedure identified 12 HSV-1 isolates and 18 HSV-2 isolates among the 30 isolates studied. The results of HSV-Type 1 Isolates Capturing Ab Ant -Type 1 -Type 2 Detecting Ab Anti-Type 1 -Type 2 HSV-Type 2 Isolates / -Type 1 -Type 2 -Type 1 -Type 2 FIG. 1. Differences in A405 values using homologous polyclonal antibody sandwich for detection of HSV type in capture B/SA ELISA. 1.0 r Il. ai J. CLIN. MICROBIOL. * Type 1 Mcintyre Strain * Type 1 Isoates o Type 2 MSStrain * Type 2 Isolates 0.01 Hg FIG. 2. Identification of HSV type using monoclonal HSV-1- specific antibody in capture B/SA ELISA. Capturing antibody, Rabbit polyclonal anti-hsv-1; detecting antibody, mouse monoclonal HSV-1 specific. both ELISAs were in complete agreement, and the assays were 100% specific and sensitive when compared with the immunofluorescence assay and restriction enzyme analysis. Typing by immunofluorescence. A total of 30 HSV isolates were tested. Although they contained high-titered antibodies, the rabbit HSV-1 and HSV-2 antisera were not adequately specific to type the clinical isolates by the immunofluorescence technique. All isolates showed reaction with both reagents. On the other hand, the use of monoclonal antibodies led to a conclusive determination of the HSV type in all 30 isolates. Among the monoclonal antibodies used, the Ortho type 1 reagent gave good fluorescence. Type-specific and type-common reagents from both Electro-Nucleonics and Biotech Laboratories also gave good-quality fluorescence. All reagents, either type specific or type common, showed complete correlation. Typing by restriction endonuclease analysis. Twenty of the clinical isolates were analyzed by restriction endonuclease digestion using enzymes KpnI and HpaI. Four isolates were typed as HSV-1, and 16 were typed as HSV-2. The results were in complete concordance with those achieved with monoclonal antibodies or the capture B/SA ELISA. No mixed infections were noted, but minor intratypic variations were obvious in some isolates. DISCUSSION The availability of type-specific monoclonal antisera and their use in HSV typing by immunofluorescence assays or ELISAs have provided rapid, simple, and useful tools and greatly improved the capacity of the clinical virology laboratory for HSV serotyping. A variety of reports on these approaches have appeared (1, 2, 7, 8, 11, 21-23, 28). We recently reported a technique for identification of HSV antigen(s) using the biotin-streptavidin detection system and

4 VOL. 25, 1987 HSV TYPING BY BIOTIN-STREPTAVIDIN ELISA 131 indicated that HSV antigen detection could be performed with excellent specificity and sensitivity as compared with tissue culture identification of the virus (18). As a follow-up, we have now modified this procedure for HSV typing. Calculating the typing index in a manner similar to that described by Vestergaard and Jensen (27), this method offered results which were clear-cut in typing analysis. When the monoclonal HSV-1-specific antiserum became available, we further modified the procedure for positive identification of HSV-1. The identification of HSV-2 was done by expected lack of reactivity to the monoclonal HSV-1-specific detecting antibody. The two methods described offered identical typing sensitivities (100%) and specificities (100%). It was previously shown that a small percentage of patients have mixed HSV-1 and HSV-2 infections. A simultaneous specific monoclonal antibody test for HSV-2 is being studied in our laboratory, so that such mixed infections would not be overlooked. There have been several reports of the use of HSVspecific antibodies for detection and typing of the virus. Mills et al. (16) and Vestergaard and Jensen (27) described the use of polyclonal antibodies in serotyping HSV by the double-antibody capture technique. They successfully typed 32 and 42 clinical isolates, respectively, with typing sensitivities of 100 and 97.6%, respectively, and typing specificities of 100%. Gerna et al. (9) compared the double-antibody capture ELISA with the indirect ELISA for the typing of 42 clinical isolates and found that the procedures gave similar results and were in complete agreement with those obtained by inhibition of indirect hemagglutination test. Nilheden et al. (20) used an indirect ELISA for HSV typing. In their experiments, the isolates were grown on microtiter plates, fixed with glutaraldehyde, and reacted with type-specific monoclonal antibodies, followed by enzyme-conjugated anti-mouse serum. The procedure showed 100% typing specificity when compared with type-specific plaque staining. Ziegler et al. (30) used an indirect ELISA in which tissue culture-grown viral isolates were used as the antigen to coat ELISA plates. The antigen detection was performed by reaction with type-specific monoclonal antibodies and antimouse immunoglobulin G enzyme conjugate. Their results showed 100% typing specificity compared with nucleic acid spot hybridization. Recently, Frame et al. (7) described a method for the identification and typing of HSV by a double-antibody capture ELISA using polyclonal antisera for capture and monoclonal antibodies for detection. Their results demonstrated 100% typing specificity when compared with restriction endonuclease analysis and immunofluorescence techniques. The present report describes HSV serotyping by ELISA using for the first time a biotinstreptavidin detection system. This report also describes the use of three commercial sources of monoclonal antibodies for HSV typing. All the reagents tested displayed excellent typing sensitivity (100%) and specificity (100%), but when used as furnished by the manufacturers they did not show strong signals in the ELISA. HSV typing using monoclonal antibodies in the immunofluorescence assay has been successfully performed by several workers (1, 2, 4, 7, 8, 11, 21-23, 28) who showed high concordance with the results of one or more tests, e.g., restriction endonuclease analysis (1, 7, 11, 23, 28), susceptibility to E-5-(2-bromovinyl)-2'-deoxyuridine (2, 4), and microneutralization and reverse passive hemagglutination tests (4). Monoclonal antibodies appear to have a high degree of specificity for the diagnosis of HSV infections, as well as for the identification of HSV serotypes. This technology has proved to be effective as the HSV serotyping method. In fact, the problem of antigenic or intratypic variations and possible nonrecognition by a given monoclonal antibody does not seem to be as common or as serious (1, 7, 11, 23) as was previously speculated (22). Restriction endonuclease analysis offers information about the HSV genome as a basis for identification of the HSV type. It is a valuable tool in studies related to epidemiology and mode of transmission (3, 12, 29), intratypic variations (15), and mixed or sequential infections with different serotypes (10, 13), but elaborate laboratory procedures and the use of radioisotopes in this technique prevent it from being used in the clinical laboratory. In conclusion, the B/SA ELISA is a sensitive method for the detection of HSV antigen and can be successfully used for HSV typing in routine laboratory work. It offers an advantage over the immunofluorescence technique in that it is not a subjective determination and has the possibility of automation. The ELISA may be the method of choice in the clinical laboratory because of its rapidity, simplicity, and use of nonradioisotopic reagents (26), especially when large numbers of isolates are to be typed. ACKNOWLEDGMENTS We thank Maternity Center Associates for providing the clinical specimens, Jacob Victor (Hoffman-La Roche Inc., Nutley, N.J.) for providing biotinylated monoclonal antibodies, and Lin Aspinall for excellent editorial assistance and typing of the manuscript. LITERATURE CITED 1. Balachandran, N., B. Frame, M. Chernesky, E. Kraiselburd, Y. Kouri, D. Garcia, C. Lavery, and W. E. Rawls Identification and typing of herpes simplex viruses with monoclonal antibodies. J. Clin. Microbiol. 16: Balkovic, E. S., and G. D. 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