Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP - PubMed (original) (raw)

Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP

Noritoshi Kitamoto et al. J Clin Microbiol. 2002 Jul.

Abstract

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family CALICIVIRIDAE: The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.

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Figures

FIG. 1.

FIG. 1.

Western blot of polypeptides from VLPs reacting with group A MAbs. The results shown are for MAbs NV23, NV37, F8, and F120 and VLPs rSV, rNV, rSeV, rCV, rFUV, rSMA, rGV, rKAV, rNAV, rCHV, and rUEV. Proteins were boiled for 2 min and lysed with sample buffer (about 1 to 2 μg/well), and SDS-polyacrylamide gel electrophoresis (PAGE) was conducted in 10% polyacrylamide gels. The separated proteins were transferred to nitrocellulose sheets, which were incubated overnight at room temperature with the indicated MAbs (at a 1:1,000 dilution of ascites) and then incubated with HRPO-conjugated goat anti-mouse IgG, IgM, and IgA for 1 h at 37°C and treated with substrate (4-chloro-1-naphthol). Calibration kits were used for molecular weight determination (lane M); numbers on the left side indicate the apparent molecular weights (103). These MAbs reacted with all VLPs of the NLV genus whose major polypeptide had an apparent molecular weight of 58,000 but not with rSV. Oligomers or possible breakdown bands (short arrows) were seen in some VLP preparations.

FIG. 2.

FIG. 2.

Western blot of polypeptides from VLPs reacting with group B (panel A) and C (panel B) MAbs. Proteins were boiled for 2 min and lysed (about 1 to 2 μg/well), and SDS-PAGE was conducted in 10% polyacrylamide gels. Each MAb (ascites) was diluted to a level of 1:1,000. (A) The group B NV51 and NV3912 MAbs reacted with all VLPs of genogroup I of genus NLVs and with the major polypeptide whose apparent molecular weight was 58,000 but not with genogroup II or rSV proteins. (B) The group C NS14 and NS28 MAbs reacted with all VLPs of genogroup II of genus NLVs and with the major polypeptide whose apparent molecular weight was 58,000 but not with genogroup I or rSV proteins. Oligomers or breakdown products (short arrows) were seen.

FIG. 3.

FIG. 3.

Western blot of polypeptides from VLPs reacting with a subset of group D and E MAbs. The results for MAbs NV5610 and SV137 are shown. Proteins (about 1 to 2 μg/well) were boiled for 2 min or not boiled (lanes marked with asterisks). SDS-PAGE was conducted in 10% polyacrylamide gels. Each MAb (ascites) was diluted to a level of 1:1,000. The NV5610 MAb reacted only with rNV and rSeV of the NLV genus and detected higher-molecular-weight forms only when the protein was not boiled. The SV137 MAbs reacted only with rSV, detecting high-molecular-weight forms whether the protein was boiled or not boiled, but did not react with proteins of other VLPs.

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