Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles. Brief Report

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1 Arch Virol (2005) 150: DOI /s Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles Brief Report G. S. Hansman 1,2, K. Natori 1, H. Ushijima 2, K. Katayama 1, and N. Takeda 1 1 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan 2 Department of Developmental Medical Sciences, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Received December 6, 2004; accepted January 27, 2005 Published online March 17, 2005 c Springer-Verlag 2005 Summary. Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains were recently divided into five genogroups (GI to GV). We characterized novel polyclonal antibodies raised against SaV GV virus-like particles (VLPs) by Western blot analysis, and both antibody and antigen enzyme-linked immunosorbent assays (ELISAs). Our results have indicated SaV GI and GV VLPs were antigenically distinct by Western blotting and ELISAs. These reagents may be useful for genogroup specific detection of SaV. Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains can be divided into five genogroups (GI to GV) based on their capsid (VP1) sequences. Human SaV strains are thought to mostly infect infants, occasionally causing outbreaks [10, 11]. SaV strains were originally detected using electron microscopy (EM), but nowadays the most widely used method to detect SaV is reverse transcription-polymerase chain reaction (RT-PCR), which has a high sensitivity [11]. Several groups have purified native SaV particles and produced antisera against them for use in immunoassays, radioimmunoassays, and enzyme-linked immunosorbent assays (ELISAs) [7 9, 12]. SaV strains were recently divided into five genogroups (GI to GV), of which GI, GII, GIV, and GV strains infect humans, while GIII strains infect porcine species [2]. We recently reported the cross-reactivity among SaV GI, GII, and GV

2 1434 G. S. Hansman et al. using recombinant VP1 (rvp1) and hyperimmune rabbit antisera raised against GI virus-like particles (VLPs) and Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1 [4]. Western blot analysis showed that GI antisera crossreacted with GV rvp1 but not GII rvp1; GII antisera cross-reacted weakly with GI rvp1 but did not cross-react with GV rvp1; and GV antisera reacted only with GV rvp1. An antigen ELISA using rabbit and guinea pig antisera raised against GI VLPs showed that GI VLPs were antigenically distinct from GII and GV VLPs [4]. In the current study we characterized novel polyclonal antibodies raised against GV VLPs by Western blot analysis, and both antibody and antigen ELISAs. As previously described, VLPs derived from GI Mc114 strain (GenBank accession number AY237422), GII C12 strain (AY603425), and GV NK24 strain (AY646856) were prepared in insect cells, and hyperimmune rabbit and guinea pig antisera raised against GI Mc114 VLPs [4]. For this study, we developed novel hyperimmune rabbit and guinea pig antisera raised against GV NK24 VLPs using an identical method. The cross-reactivity among Mc114, C12, and NK24 rvp1 was examined by Western blotting as previously described [4]. We found both rabbit and guinea pig antisera raised against GV NK24 VLPs was specific for NK24 rvp1 and detected neither Mc114 nor C12 rvp1 (data not shown). This result was similar with our previous findings that showed hyperimmune rabbit antisera raised against E. coli-expressed NK24 N- and C-terminal VP1 was specific for NK24 rvp1 and cross-reacted with neither Mc114 nor C12 rvp1 [4]. We then analyzed the cross-reactivity between GI Mc114 and GV NK24 VLPs by an antibody ELISA, which used hyperimmune antisera raised against Mc114 and NK24 VLPs. Wells of 96-well microtiter plates (Maxisorp, Nunc, Denmark) were each coated with either 100 µl of purified Mc114 or NK24 VLPs (1.0 µg/ml in carbonate bicarbonate buffer ph 9.6) (Sigma, USA) and incubated overnight at 4 C. The wells were washed twice with PBS containing 0.1% Tween 20 (PBS-T) and then were blocked with PBS containing 5% skim milk (PBS-SM) for 1 h at room temperature. After the wells were washed twice with PBS-T, 100 µl of 2-fold diluted (either Mc114 or NK24) hyperimmune rabbit or guinea pig antisera from a starting 1:2,000 in PBS-T-SM was added to each well, and the plates were incubated for 1 h at 37 C. The wells were washed six times with PBS-T, and then 100 µl of a 1:1,000 dilution of horseradish peroxidase (HRP)- conjugated rabbit anti-guinea pig immunoglobulin G (IgG) or HRP-conjugated goat anti-rabbit IgG diluted in PBS-T-SM was added to each well. The plates were then incubated for 1 h at 37 C. The wells were washed six times with PBS-T, and then 100 µl of substrate o-phenylenediamine and H 2 O 2 was added to each well and left in the dark for 30 min at room temperature. The reaction was stopped by the addition of 50 µl of2nh 2 SO 4 to each well, and the absorbance was measured at 492 nm (A 492 ). As shown in Figure 1, the rabbit and guinea pig antisera were specific for the homologous VLPs, but slightly crossreacted with the heterologous VLPs at low dilutions. Using an OD cutoff point at 0.15, which was equal to 3 times the mean OD of preimmune serum (data

3 Sapovirus genogroup specific enzyme-linked immunosorbent assays 1435 Fig. 1. An antibody ELISA for SaV GI and GV. Wells were each coated with either 100 µl of purified SaV GI Mc114 or SaV GV NK24 VLPs. After the wells were washed hyperimmune rabbit or guinea pig antisera that were raised against either Mc114 or NK24 VLPs were used to detect the antigens. Antisera were 2-fold diluted from a starting dilution of 1:2,000 to 4,096,000 in PBS-T-SM (represented by 2 to 4,096 on the bottom axis) not shown), Mc114 and NK24 rabbit antisera detected Mc114 and NK24 VLPs until 1:1,024,000 and 1:2,048,000 dilutions, respectively, whereas, Mc114 and NK24 rabbit antisera detected NK24 and Mc114 VLPs until 1:4,000 dilutions. Likewise, Mc114 and NK24 guinea pig antisera detected Mc114 and NK24 VLPs until 1:2,048,000 and 1:2,048,000 dilutions, respectively, whereas, Mc114 and NK24 guinea pig antisera detected NK24 and Mc114 VLPs until 1:2,000 dilutions. The cross-reactivity at these low dilutions may have been attributed to baculovirus or insect cell antigens that were present in the antigen preparation for the immunization. We also analyzed the cross-reactivity among Mc114, C12, NK24 VLPs, and clinical stool specimens [3] by an antigen ELISA as previously described [4] except that in this study we used rabbit (capture) and guinea pig (detector) antisera raised against NK24 VLPs. In order to determine a significant positive, which is defined as the mean plus three standard deviations [6], 68-negative SaV clinical specimens described above were screened. Briefly, wells were coated with 100 µl of a 1:8,000 dilution of either NK24 hyperimmune rabbit (P) antiserum or

4 1436 G. S. Hansman et al. NK24 preimmune rabbit (N) serum diluted in PBS, and the plates were incubated overnight at 4 C. Wells were washed 3 times with PBS-T, and then blocked with PBS-SM for 1 h at room temperature. After washing the wells 4 times with PBS- T, 100 µl of the clinical specimens were added to duplicate hyperimmune rabbit and duplicate preimmune rabbit wells, and the plates were incubated for 1 h at 37 C. After washing the wells 4 times with PBS-T, 100 µl of a 1:8,000 dilution of NK24 hyperimmune guinea pig antiserum diluted in PBS-T-SM was added to each well, and the plates were incubated for 1 h at 37 C. The plates were then processed as described above. The mean P/N ratio of the 68-negative SaV specimens was 1.04, with a standard deviation of Therefore, a specimen with ana 492 (P N) > 0.1 and a P/N ratio >1.34 were considered significantly positive. This ELSIA was shown to be 100% specific for NK24 VLPs (P = 2.46, N = 0.05, P N = 2.41, and P/N ratio = 49) and detected neither Mc114 VLPs (P = 0.05, N = 0.05, P N = 0.0, and P/N ratio = 1) nor C12VLPs (P = 0, N = 0.05, P N = 0.0, and P/N ratio = 1). This result indicates that GI Mc114 and GII C12VLPs were antigenically distinct from GV NK24 VLPs. Our previous antigen ELISA findings showed that GI Mc114 VLPs were antigenically distinct from GII C12 and GV NK24 VLPs [4]. Finally, we examined the 80 clinical stool specimens [3] with the antigen ELISA using antisera raised against GV NK24 VLPs. All SaV and norovirus specimens were negative by the antigen ELISA (P N values were approximately = 0 and P/N ratios were approximately = 1) using antisera raised against NK24 VLPs, including the single GV SaV clinical specimen from which NK24 VLPs was prepared. This false-negative result was not unusual, since the NK24 specimen was collected 10 days after the onset of illness and was positive by nested RT-PCR but not single-round RT-PCR. Infected individuals usually excrete virus particles within the first 4 days after the onset of infection and for up to 9 days, but 50% of the patients have no detectable virus by EM 5 to 7 days after the onset of illness [1]. Further studies are clearly needed in order to determine the sensitivity of this novel GV antigen ELISA. Apart from our recent cross-reactivity study [4] and this current study, little is known about the antigenicity among SaV strains [5]. The results of this current study have confirmed that SaV GI Mc114 and GV NK24 VLPs were antigenically distinct by an antigen ELISA. This result suggests these reagents could be used for genogroup-specific detection methods. Additional screening of clinical specimens is needed to determine if these novel GV reagents can be used for antibody and antigen ELISA detection methods, however SaV GV strains have only been reported in two studies [2, 3]. Acknowledgements This work was supported by Grants-in-aid from The Ministry of Education, Culture, Sports, Science and Technology, Japan and a Grant for Research on Re-emerging Infectious Diseases from The Ministry of Health, Labor, and Welfare, Japan. We are grateful to the Japanese Monbusho for the PhD scholarship provided to Grant Hansman.

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