Foot-and-Mouth Disease Virus Antigen Detection Enzyme-Linked Immunosorbent Assay Using Multiserotype-Reactive Monoclonal Antibodies

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2009, p Vol. 47, No /09/$12.00 doi: /jcm Copyright 2009, American Society for Microbiology. All Rights Reserved. Foot-and-Mouth Disease Virus Antigen Detection Enzyme-Linked Immunosorbent Assay Using Multiserotype-Reactive Monoclonal Antibodies Kazuki Morioka,* Katsuhiko Fukai, Kazuo Yoshida, Reiko Yamazoe, Hiroyuki Onozato, Seiichi Ohashi, Tomoyuki Tsuda, and Kenichi Sakamoto Exotic Diseases Research Station, National Institute of Animal Health, Josuihoncho Kodaira, Tokyo , Japan Received 5 April 2009/Returned for modification 19 June 2009/Accepted 3 September 2009 Monoclonal antibody (MAb)-based sandwich direct enzyme-linked immunosorbent assay (MSD-ELISA) methods that can detect foot-and-mouth disease virus (FMDV) antigens, both multiserotype (MSD-ELISA/ MS) (for O, A, C, and Asia 1) and single-serotype (MSD-ELISA/SS) (for O, A, and Asia 1, specifically), were developed. MAb 1H5 was used as an antigen-trapping antibody that reacted with all seven serotypes of FMDV. The MAbs 71F2, 70C4, 16C6, and 7C2 were used as peroxidase-labeled detecting antibodies for multiple serotypes (O, A, C, and Asia 1), type O, type A, and type Asia 1, respectively, in both MSD-ELISA/MS and MSD-ELISA/SS. Our MSD-ELISAs showed high specificity. They produced a very low background of negative samples (buffer, plasma, and saliva) and were able to detect FMDV antigens from clinical samples (plasma and saliva), with results correlating with those of real-time reverse transcription-pcr. In terms of sensitivity, the MSD-ELISAs showed higher optical density values against each diluted serotype antigen than the indirect sandwich ELISA method, which is currently recommended in the manual of the World Organization for Animal Health. The sensitivity and specificity of the MSD-ELISAs seem to be sufficient for the antigenic diagnosis of FMDV. Foot-and-mouth disease (FMD) is caused by FMD virus (FMDV), which belongs to the genus Aphthovirus of the family Picornaviridae and consists of seven immunologically distinct serotypes: O, A, C, Asia 1, and South African Territories type 1 (SAT1), SAT2, and SAT3. FMD is one of the most highly contagious viral diseases and causes devastating economic damage in the countries affected by it. Currently, FMD is endemic or sporadic in many countries in the Asian region, where type O, A, and Asia 1 viruses are prevalent. On the other hand, type C outbreaks have been confined to a very limited area, which is why Roeder et al. suggested that international efforts be made to eradicate type C first, as described previously (P. L. Roeder and N. J. Knowles, presented at the Global Control of FMD Tools, Ideas, and Ideals conference, Erice, Italy, 14 to 17 October 2008). Outbreaks of the SAT serotypes are limited to the African continent and part of the Arabian Peninsula. Types O and A have a wide range of antigenic variations within each serotype; therefore, matching the vaccine strains to the field outbreak strains has become an important issue (10, 11, 12). The FMDV antigenic diagnostic methods mentioned in the manual of the World Organization for Animal Health (OIE) (9) are virus isolation, immunological methods i.e., indirect sandwich enzyme-linked immunosorbent assay (IS- ELISA) and the complement fixation test (3, 15) * Corresponding author. Mailing address: Exotic Diseases Research Station, National Institute of Animal Health, Josuihoncho Kodaira, Tokyo , Japan. Phone: Fax: morioka@affrc.go.jp. Published ahead of print on 16 September and nucleic acid recognition methods, such as reverse transcription (RT)-PCR and real-time RT-PCR. Although the RT-PCR and real-time RT-PCR recommended in the OIE Terrestrial Manual are sensitive and specific methods for detecting viral nucleic acids, they cannot distinguish serotypes (9). RT-PCR for serotyping has been attempted, but it does not serotype FMDV perfectly (1). Moreover, genome amplification methods have a risk of accidental genome contamination. On the other hand, ELISA is able to detect viral antigens with immunological interactions and thus is able to distinguish serotypes (15). However, the current IS-ELISA is the only antigen detection method for serotyping FMDV, but it does not have sufficient sensitivity (7, 14). In addition, the current lack of adaptability of IS-ELISA to antigenic diversity remains a problem because of the extensive antigenic diversity within the O and A serotypes (10, 11, 12). The other disadvantage of IS-ELISA is that the manual recommends sampling vesicular fluid or tissue and culture fluid, but such samples may not be available in preclinical and/or subclinical diagnoses. For instance, the cattle in the 2000 FMD outbreak in Japan did not show clear vesicles (6, 8, 16). These problems would be solved if plasma and/or saliva could be used as the sample. In this study, a monoclonal antibody (MAb) against each of the FMDV types O, A, and Asia 1 was produced, and the characteristics of these MAbs were analyzed. To solve the problems of FMDV antigenic diversity, MAbs that could detect multiple serotypes were used, along with specific MAbs that could detect only a single-serotype antigen. As a result, a monoclonal antibody-based sandwich direct ELISA method (MSD-ELISA) that can detect FMDV antigens, both multiserotype (MSD-ELISA/MS) (for O, A, C, and 3663

2 3664 MORIOKA ET AL. J. CLIN. MICROBIOL. Downloaded from Asia 1) and single serotype (MSD-ELISA/SS) (for O, A, and Asia 1 specifically), was developed. MATERIALS AND METHODS Cells and viruses. IBRS-2 and/or BHK-21 cells were maintained with Eagle s minimum essential medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) with 0.3% tryptose phosphate broth (Difco Laboratories, Detroit, MI) supplemented with 0.3 mg/ml of L-glutamine, 1.5% 7.5% NaHCO 3, and 5% fetal bovine serum (Gibco, New York, NY). A confluent cultured monolayer of IBRS-2 and/or BHK-21 cells was used for virus propagation. The virus strains FMDV O/JPN/2000 (6, 8, 16), O 1 BFS 1860, O/TAW/97 (2, 4), A 15 TAI 1/60, A 22 IRQ 24/64, C PHI 7/84, Asia 1 Shamir (ISR 3/89), and swine vesicular disease virus (SVDV) J1 (5, 17) were grown on monolayers of IBRS-2 and/or BHK-21 cells. Antigens. Eight reference FMDV- and SVDV-inactivated antigens O 1 Manisa, A 5 Allier, C 3 Resende, Asia 1 CAM 9/80, SAT1 BOT 1/68, SAT2 ZIM 5/81, SAT3 ZIM 4/81, and SVDV UKG 27/72 were used in this study. Antigens of type A May 97 were purified from the type A monovalent vaccine (Merial, Pirbright, United Kingdom) by centrifugation at 12,000 g; the clear lower phase was used. The above-mentioned viruses and inactivated antigens were used as the ELISA antigens for screening of the MAb characteristics. MAbs. MAbs against FMDV O/JPN/2000, A 15 TAI 1/60, and Asia 1 Shamir (ISR 3/89) were produced by the usual method, described in a previous report (8). Representative MAbs are shown in Fig. 1. Peroxidase conjugation with MAbs. The conjugation of peroxidase with MAbs was performed using the peroxidase-labeling kit SH (Dojindo, Ltd., Tokyo, Japan), according to the instruction manual. Peroxidase-labeled MAbs were stocked at 20 C in a 50% suspension with glycerol and used in each ELISA at FIG. 1. Characteristics of the MAbs used in this study. 600-fold dilution with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST) and 8% skim milk (Wako, Osaka, Japan) adjusted to ph 7.6 with 0.8 M NaOH (PBSTM). Direct ELISA for characterization of MAbs. Direct ELISA was performed using an anti-fmdv rabbit antiserum for each serotype and peroxidase-labeled detecting MAbs. The rabbit antisera were diluted with 0.05 M carbonate buffer (ph 9.6) (Sigma, Ayrshire, United Kingdom), and a 96-well flat-bottom plate (Immulon 2HB; Thermo Electron Corporation) was coated with 50 l/well of each antiserum at 4 C overnight. The plates were washed four times with 350 l of M PBS (ph 7.4) (Sigma). The antigens were added to the wells and incubated for 1 h at 37 C. The plates were washed four times with PBS. Fifty microliters of PBSTM containing peroxidase-labeled MAbs (see above) at a ratio of 1:600 was added to the wells and incubated for 45 min at 37 C. After the plate was washed four times, 50 l ofo-phenylenediamine H 2 O 2 substrate was added to the plates, which were then incubated in the dark for 15 min at room temperature. The reaction was stopped with 50 l of1.25mh 2 SO 4, and the optical density (OD) was measured with an ELISA reader (iems-reader; Labsystems, Helsinki, Finland) at 492 nm and 690 nm. Virus neutralization test. The virus neutralization activities of the MAbs were determined using O/JPN/2000, O 1 BFS 1860, O/TAW/97, A 15 TAI 1/60, A 22 IRQ 24/64, and Asia 1 Shamir (ISR 3/89). Virus neutralization tests were performed as follows. Briefly, the supernatant of the MAb and an equal volume of % tissue culture infectious doses of virus were mixed. The mixtures were inoculated at 37 C for 1 h, and the IBRS-2 cells suspended in medium were then added to each well. After incubation at 37 C for 4 days, the MAbs that completely inhibited the cytopathic effect were judged positive. MSD-ELISA. MAb 1H5 diluted with 0.05 M carbonate buffer (ph 9.6) was added at 50 l/well to 96-well flat-bottom plates. After being kept at 4 C over- on October 12, 2018 by guest

3 VOL. 47, 2009 FMDV ANTIGEN DETECTION ELISA 3665 FIG. 2. Results of the MSD-ELISAs against FMDVs and SVDVs. MAb 1H5 was used as a trapping antibody for all of the MSD-ELISAs. The peroxidase (Po)-labeled MAbs 71F2, 70C4, 16C6, and 7C2 were used as detecting antibodies in the MSD-ELISA/MS (for types O, A, C, and Asia 1) and MSD-ELISA/SS (for types O, A, and Asia 1). night, the plates were washed four times with 350 l of M PBS. The plates were stabilized using commercial ELISA coating stabilizer (Antigenix America Inc., New York, NY) and stored at 4 C until they were used. The samples (virus culture fluids, inactivated reference antigens, and clinical samples) were added to the wells and incubated for 1 h at 37 C. The subsequent procedures were the same as for the direct ELISA (see above). The average OD of the buffer (PBST) was subtracted from the sample OD to obtain the OD result. IS-ELISA. IS-ELISA supplied by the Institute for Animal Health, Pirbright Laboratory the FMD reference laboratory for both the OIE and the Food and Agriculture Organization of the United Nations was performed following the OIE manual (9), which states that when the sample OD minus the buffer OD is 0.1 or more the case is regarded as positive, and when it is approximately 0.1, the case is regarded as doubtful. In this study, an OD over 0.08 was regarded as positive. Clinical samples. The right fore heel bulb of each of three pigs (about 2 months old) was inoculated intradermally/subcutaneously with % tissue culture infectious doses of O/TAW/97 in 1 ml of tissue culture medium. Plasma and saliva were collected each day postinoculation (p.i.) for 8 days. Undiluted plasma and saliva three times diluted with PBS were used in this study. Negative samples. Non-FMDV-infected pigs plasma and saliva were used as control samples. The buffer ODs and negative results (plasma and saliva) were collected from all of the tests in the study, and the negative results (buffer, plasma, and saliva) collected by IS-ELISA and MSD-ELISA were analyzed statistically. Statistics. The statistical calculations of the average and standard deviations (SD) were performed using Microsoft Excel software. Real-time RT-PCR. A TaqMan probe and primers were designed for the three-dimensional region of O/TAW/97 using Primer Express (Applied Biosystems, CA). The sequences were as follows: forward primer, 5 -CGAGAACAA GCGCATTGTTG-3 ; reverse primer, 5 -TGCCTACGCAGGGCGTAA-3 ; TaqMan probe, 5-6-carboxyfluorescein-CGCAACAAGCATCATCAACACAA TTTTGAA-6-carboxytetramethylrhodamine-3. The program was 48 C for 30 min, 95 C for 10 min, and 40 cycles of 60 C for 15 s and 95 C for 1 min. Serial 10-fold-diluted O/TAW/97 from a mixture of 10 6 PFU/0.1 ml was used for the positive samples to construct the standard curve. The values were calculated based on the standard curve. RESULTS Characterization of MAbs. MAbs 1H5 (immunoglobulin G1 [IgG1]), 71F2 (IgG1), 70C4 (IgG2a), 65H6 (IgG2a), and 68F9 (IgG2a) were raised against the O/JPN/2000 strain. MAbs 11H3 (IgG1), 16C3 (IgG1), 16C6 (IgG1), and 17B2 (IgA) were raised against the A 15 TAI 1/60 strain. MAb 7C2 (IgG1) was raised against the Asia 1 Shamir strain. The MAb characteristics were analyzed by the virus neutralization test and direct ELISA using peroxidase-labeled MAbs. MAbs 1H5 and 11H3 reacted with all of the tested FMDV antigens, but 11H3 showed cross-reaction with the SVDV J1 strain. MAb 71F2 reacted with all of the tested antigens except the SAT serotypes and did not react with the SVDVs. MAbs 70C4, 65H6, and 16C6 reacted with all of the tested antigens of type O and type A and showed virus-neutralizing activity toward each serotype, as well. 68F9 reacted only with the O/JPN/2000 strain and showed virus-neutralizing activity. MAbs 16C3 and 17B2 reacted only with the A 15 TAI 1/60 strain but did not show virus-neutralizing activity. MAb 7C2 reacted with both types of Asia 1 antigens but showed slight cross-reaction with the type O antigens (Fig. 1). Construction of ELISAs. From the results of the MAb characterization, various combinations of MAbs for MSD-ELISAs were examined. MAb 1H5 was used as a trapping antibody for MSD-ELISA/MS and MSD-ELISA/SS, and 71F2, 70C4, 16C6, and 7C2 were used as peroxidase-labeled detecting antibodies in MSD-ELISAs for multiserotype (O, A, C, and Asia 1), type O, type A, and type Asia 1, respectively. MSD-ELISA/MS (1H5-71F2) could detect all 12 antigens of the four serotypes tested (types O, A, C, and Asia 1). The MSD-ELISA/SS for type O (1H5-70C4) and type A (1H5-16C6) could distinguish each serotype from the other serotypes. Although the MSD- ELISA/SS for Asia 1 (1H5-7C2) showed reactivity to homologous antigens, slight cross-reactions were shown to type O serotypes (Fig. 2). The OD results of the three ELISAs (IS- ELISA, MSD-ELISA/MS, and MSD-ELISA/SS) were compared using diluted reference FMDV-inactivated antigens. Each MSD-ELISA/SS showed the highest sensitivities to O 1 Manisa, A 5 Allier, and Asia 1 CAM 9/80. The sensitivities of MSD-ELISA/MS were higher than those of IS-ELISA against A 5 Allier, and C 3 Resende and were almost the same in the case of O 1 Manisa (Fig. 3). Negative samples. The OD values of buffer (PBST) and noninfected animal saliva and plasma were examined. The OD values of the negative samples of MSD-ELISAs were under 0.02 with tests repeated several times. On the other hand, IS-ELISA showed a high background with PBST and negative clinical samples (saliva and plasma), especially in plasma (Table 1).

4 3666 MORIOKA ET AL. J. CLIN. MICROBIOL. FIG. 3. Comparison of the ODs of the ELISAs (MSD-ELISA/MS, MSD-ELISA/SS, and IS-ELISA) using serially diluted FMDV reference antigens. Shown are the results of each ELISA against type O (O 1 Manisa) (a), type A (A 5 Allier) (b), type C (C 3 Resende) (c), and type Asia 1 (CAM 9/80) (d). Sensitivities of MSD-ELISAs. The limits of the detectable antigens dilution rates were examined. Positive lines of IS- ELISA and the MSD-ELISAs were set up with 0.08 OD and 0.04 OD (over five times the SD), respectively. The MSD- ELISAs showed high sensitivity compared to IS-ELISA. The MSD-ELISA/SS showed the highest sensitivities against all of the tested FMDV antigens. The sensitivities of MSD- ELISA/MS for types A and Asia 1 were greater than those of the IS-ELISA against the relevant antigens. The MSD- ELISA/MS for type O showed sensitivity equal to that of IS- ELISA (Table 2). Clinical samples. Three pigs (P1 to P3) were inoculated with O/TAW/97, and the saliva and plasma were collected each day after inoculation. These samples were examined by MSD- ELISAs, IS-ELISA, and real-time RT-PCR. In IS-ELISA, the plasma backgrounds were high, and the OD of all of the plasma of 0 days p.i. was over Therefore, the reliability of the data on the plasma appears to be very low. In the saliva, a positive value on the IS-ELISA was obtained with only one sample (2 days p.i.). On the other hand, both MSD-ELISA/MS OD TABLE 1. Frequencies of ODs of negative samples MSD-ELISAs and SS (for type O) could detect antigens at a rate correlated with that of real-time RT-PCR. In these clinical samples of O/TAW/97, the detection limits for the MSD-ELISA/MS and SS (for type O) were about 100 to 1,000 PFU, as determined by real-time RT-PCR results (Fig. 4). DISCUSSION FMDV types O, A, and Asia 1 are prevalent in Asian regions. For types O and A, it is very difficult to match the vaccine strains against the field strains because of their wide range of antigenicity (10, 11, 12). Although the current IS- ELISA is able to distinguish seven serotypes of the FMDV antigen or SVDV and is a useful method, it has low sensitivity (7, 14) and may have low adaptability to types O and A because of their wide range of antigenicity (10, 11, 12). On the other hand, while virus genome detection methods, such as RT-PCR and real-time RT-PCR, are the most sensitive methods, they often cannot distinguish antigen serotypes and can also be subject to cross-contamination. No. of samples MS (1H5-71F2) a SS/O (1H5-70C4) b SS/A (1H5-16C6) c SS/Asia 1 (1H5-7C2) d IS-ELISA e Buffer Plasma Saliva Buffer Plasma Saliva Buffer Plasma Saliva Buffer Plasma Saliva Buffer Plasma Saliva f a Average, ; SD, b Average, ; SD, c Average, ; SD, d Average, ; SD, e Average, ; SD, f The number of samples.

5 VOL. 47, 2009 FMDV ANTIGEN DETECTION ELISA 3667 Antigen a TABLE 2. Sensitivities of ELISAs IS-ELISA Detectable limit of antigen dilutions b MSD-ELISAs O A Asia 1 Multiserotype O/JPN/ O/TAW/ O 1 BFS O 1 Manisa 2,048 5,792 2,048 A 15 TAI A 22 IRQ A 5 Allier 64 2, A Malaysia C PHI 7/ C 3 Resende 256 2,048 Asia 1 Shamir Asia 1 CAM 9/ , a O/JPN/2000, O/TAW/97, O 1 BFS 1860, A 15 TAI, A 22 IRQ, C PHI, and Asia1 Shamir were working viruses. O 1 Manisa, A 5 Allier, C 3 Resende, and Asia 1 CAM 9/80 were inactivated antigens. A Malaysia 97 was purified from a vaccine product. b All ELISAs were performed simultaneously using the same antigen dilutions. The values are detectable maximum dilution rates. The ODs were more than 0.08 and 0.04 by IS-ELISA and MSD-ELISAs, respectively. The ODs shown are the sample ODs minus the average buffer OD. In this study, MAbs were produced against FMDV O/JPN/ 2000, A 15 TAI (1/60), and Asia 1 Shamir (ISR 3/89), and MSD-ELISAs were developed. The problem of FMDV antigenic diversity was resolved by using MAbs (1H5 and 71F2) that were able to detect multiple serotypes of FMDV. Furthermore, the specificities and sensitivities of the MSD-ELISAs were improved by using serotype-specific MAbs (70C4, 16C6, and 7C2) as antigen-detecting antibodies (Fig. 3 and Tables 1 and 2). In terms of sensitivity, MSD-ELISAs showed their superiority to IS-ELISA for the reference antigens and the clinical samples (Table 2 and Fig. 3 and 4). MSD-ELISAs showed especially high sensitivity for types A, C, and Asia 1 (Fig. 3 and Table 2). While IS-ELISA showed a high background in the plasma samples, the MSD-ELISAs, using specific MAbs, showed a very low background and were able to detect the FMDV antigens from the plasma and saliva (Table 1 and Fig. 4). The low backgrounds made it possible to set the positive line at an OD of 0.04, a value more than five times that of the SD (Table 1). Normally, vesicular fluids/tissue and culture fluids are used as the samples for IS-ELISA (9). The sensitivities and low backgrounds obtained demonstrate that the MSD- ELISAs can be used for virus detection in any kind of sample. In general, viremia tends to appear earlier than the clinical signs in FMDV (13). Therefore, it seems it would be possible to use the MSD-ELISAs for antigen detection at the preclinical and/or subclinical stage of FMDV infection using plasma and/or saliva. A comparison of the procedures of the IS-ELISA and MSD- ELISA shows the advantages of the latter. For the IS-ELISA, rabbit antisera to all serotypes of FMDV have to be coated as antigen-trapping antibodies, and the peroxidase-labeled antibody has to be used after the detection antisera have reacted (9). For the MSD-ELISAs, it is possible to coat only with MAb 1H5 (which reacts with multiple serotypes) as the antigentrapping antibody and to use the peroxidase-labeled MAbs (71F2, 70C4, 16C6, and 7C2) directly instead of the detection antisera and the peroxidase-labeled antibody. Accordingly, the MSD-ELISA can be performed more rapidly. In future studies, we intend to examine more varieties of type A strains, because type A strains have wide antigenic variations within the same serotype. In addition, these combinations of MAbs will be able to be applied to other diagnostic methods (e.g., the immunochromatography method), as well. FIG. 4. Comparison of the results of the IS-ELISA (type O) (a), MSD-ELISA/MS (b), MSD-ELISA/SS type O (c), and real-time RT-PCR (TaqMan probe method) (d) using plasma and saliva from three O/TAW/97-inoculated pigs (P1 to P3). PFU, PFU/0.1 ml.

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