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Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (C1YVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of C1YVV a total of ten MAbs were obtained which reacted strongly with C1YVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five C1YVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with C1YVV, form the BYMV subgroup of potyviruses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The C1YVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five C1YVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the C1YVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of C1YVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the C1YVV coat protein. Comparison of published coat protein sequences of BYMV and C1YVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of C1YVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of C1YVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1–25 and 4–30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses. When these MAbs were screened against the intact and truncated (minus 67 amino-terminal amino acid residues) coat proteins of JGMV, the two JGMV-specific MAbs reacted only with the intact coat protein, whereas the other MAbs reacted with the intact as well as with truncated coat proteins, in Western blots. These results suggest that the epitopes for the two JGMV-specific MAbs are located in the surface-exposed amino-terminal 67 amino acid residue region and those for the cross-reactive MAbs are contained in the conserved core region of the JGMV coat protein. Screening of potyvirus MAbs against intact and truncated coat proteins thus appears to be a simple procedure to select virus-specific MAbs to potyviruses.

The rat splenocytes immunized with potato virus Y (PVYn) and ratmyeloma (IR983) were fused by PEG (M. W.1450). Three kinds of stable hybridoma cell lines secreting specific monoclonal antibodies (McAbs) were derived. One kind of the cell lines producing McAbs reacts to PVYn specifically. Another reacts to PVYo specifically. The third one reacts to both of the two strains. Tested by the methods of sandwich-ELISA and indirect-ELISA, all kinds of McAbs did not react to seven plant viruses: tobacco mosaic (TMV), cucumber mosaic (CMV), tobacco tech (TEV), alfalfa mosaic (AMV), turnip mosaic (TuMV), potato leaf roll (PLRV), potato virus X (PVX). The biological properties of the hybridoma cell lines and the McAbs were tested.

Splenocytes from BALB/c mouse immunized with potato virus X (PVX) and myeloma cells (SP/2) were fused. After cloning and three screening cycles, three hybridoma cell lines secreting strain-specific monoclonal antibodies (MCAs) against PVX were obtained. These cell lines were stable for 20 generations and after storage in frozen form (in liquid nitrogen) for one year. The chromosome numbers of the three hybridoma cell lines clustered in the 92-102 range. The MCAs all belonged to the IgG3 immunoglobulin subclass. The medium supernatant antibody titer detected by indirect ELISA was 1:320-1:640. The mouse ascites antibody titer was 1:102,400-1:204,800, which was more than 300 times the rabbit antiserum titer (1:320). The MCAs had a neutralizing effect on the antigen, with neutralization titer of 1:10(2). There were no apparent changes in antibody activity after repeated freezing/thawing cycles, ammonium sulfate precipitation, or freeze-drying.