Epitope mapping and a cocktail of monoclonal antibodies to achieve full detection coverage of potato virus Y.

Antibody-based serological methods, including enzyme-linked immunosorbent assay (ELISA), dot immunobinding assay (DIBA) and Western blot, play an essential role in the detection of plant viruses. In general, polyclonal antibodies are the first choice for the detection of many plant viruses due to their ease of preparation; however, the coat proteins (CPs) of some viruses have the same or similar epitopes, and the polyclonal antibody of one virus may produce cross-reactions with other viruses of the same genus in routine serological detection (Mrkvová et al., 2022). Monoclonal antibodies (MAbs) have the advantage of higher specificity than polyclonal antibodies, although some MAbs can only detect partial isolates of a virus (Tian et al., 2014). Potato virus Y (PVY) is the type species of the largest plant RNA virus genus Potyvirus and causes huge economic losses to the production of several solanaceous crops including potato, pepper and tobacco (Wylie et al., 2018). Based on the recognition of different resistance genes in potatoes, PVY isolates can be divided into three main strains including N, O and C. Three MAbs for PVY, MAbs 1128, 1129 and 1130, are widely used to determine the overall incidence of PVY. MAb1128 is claimed to detect PVYN specifically and MAb1129 detects both PVYO and PVYC, whereas MAb1130 is believed to detect the above three strains of PVY. The minimal epitopes recognized by MAbs 1128, 1129 and 1130 are amino acids 25NLNKEK30, 16RPEQGSIQSNP26 and 5IDAGGS10, respectively, of PVY CP; however, 20.10%, 48.57% and 12.73% of the 369 PVY isolates analysed do not contain these epitopes (Tian et al., 2014), which will significantly influence the detection accuracy. To establish an effective detection system covering all PVY isolates, 54 MAbs for PVY were prepared and used for the identification of their minimal recognition epitopes. First, we constructed the expression vector pEHISTEV-PVY-CP to express PVY CP in Escherichia coli cells. Then, we introduced deletion to the expression vector and obtained a series of deletion mutants, Δ2–15, Δ2–30, Δ16–45, Δ31–60, Δ46–75, Δ61–90, Δ61–255, Δ226–255, Δ241–267 and Δ256–267. The recognition epitope of a PVY Mab can be deduced according to its recognition with different CP mutants. After the first round of screening, the recognition epitopes of MAbs N1, M1, M2 and C1 were mapped to the region of amino acids 2–15, 31–45, 85–98 and 256–267 of PVY CP (Figure 1a; Figures S1 and S2). To elucidate the minimal recognition epitopes of each MAb, we further deleted the amino acids one by one from both N and C termini of the region identified above. The results showed that the minimal recognition epitopes for N1, M1, M2 and C1 were mapped to 4TIDAGGSTK12, 37GTSGTHTVP45, 89QFDTWYE95 and 261LLGVKN266, respectively, of PVY CP (Figure 1b; Figure S3). Substitutions of the epitope amino acids to alanine abolished recognition of the corresponding MAbs with PVY CP (Figure S4). The recognition epitopes of N1, M1 and C1 were predicted to be located in random coils and that of M2 was located in an α-helix (Figure S5). The isoforms and subclasses of N1, M1 and M2 were identified as IgG1 with the kappa light chain, while that of Mab C1 was IgG2b with the kappa light chain (Table S1). The titers of these four MAbs were all 1 : 243 000 as detected with an indirect ELISA (Table S1). To evaluate the performance of these four MAbs, we synthesized the CP of 10 PVY isolates deposited in the GenBank. These 10 PVY isolates had one to four amino acid differences from the epitope recognized by MAb N1 but contained the epitope recognized by M1 and M2. Isolate PVY8 had one amino acid difference with the epitopes recognized by MAb C1 (Figure S6). The results showed that MAb N1 could detect six isolates excluding PVY1, PVY2, PVY5 or PVY6; M1 and M2 could detect all these 10 isolates, while MAb C1 could detect nine isolates except PVY8 (Figure 1c). We then tested the specificity of these four MAbs by using DIBA, ELISA and Western blot. These four MAbs showed positive reactions only with PVY, not with other viruses tested, including seven viruses of the genus Potyvirus, one virus of the genus Potexvirus, two viruses of the genus Carlavirus and four viruses of the genus Tobamovirus (Figure 1d–f). Sequence analysis of eight potyviruses showed that the epitopes recognized by the four MAbs were PVY-specific (Figure S7). MAbs N1, M1, M2 and C1 could detect PVY from leaf extracts diluted for 10 240, 5120, 640 and 5120 times, respectively, which meant these four MAbs had high sensitivity (Figure 1g). There are 1885 full-length PVY CP sequences available in the GenBank till 12 April 2022. We numbered these PVY isolates from 1 to 1885. A total of 739, 647 and 1639 isolates, respectively, contained the recognition epitopes of MAbs 1128, 1129 and 1130 (Tables S2 and S3). A total of 121 isolates did not contain the epitopes recognized by either of these three MAbs (Tables S2 and S3); 694, 1853, 1843 and 1851 isolates contained the recognition epitope of N1, M1, M2 and C1, respectively (Tables S2 and S4). The sequence conservation analysis of 1885 PVY CPs also showed that the recognition epitopes of M1, M2 and C1 were more conserved than that of N1 and MAb1130 (Figure S8). The number of PVY isolates that did not contain the recognition epitopes of N1, M1, M2 and C1 was 1191, 32, 42 and 34, respectively (Table S2). Because these four MAbs had different and conserved recognition epitopes, a mixture of two or three MAbs will detect more PVY isolates. The mixture of M1 and M2, M1 and C1 or M2 and C1 could separately detect 1885, 1883 or 1876 PVY isolates (Figure 1h and Table S4). MAb N1 had a narrow coverage and a mixture of N1 with M1, M2 or C1 could separately detect 1867, 1851 or 1856 isolates (Table S4), which were fewer than that detected by other MAb mixtures; therefore, N1 was not included for further consideration. The specificity analysis also showed that mixtures of M1 and M2, M1 and C1 and M2 and C1 were positive only for PVY (Figure S9). To sum up, we have mapped the recognition epitopes of four newly prepared MAbs and shown that the cocktail of M1 and M2 can accurately detect all the 1885 PVY isolates deposited in the GenBank. Besides, by analysing the amino acid sequence of its CP, we can determine whether a new PVY isolate can be detected with these four MAbs. Therefore, the cocktail of M1 and M2 will play a great role in the future quarantine and routine detection of PVY. This study has established a novel approach to improve the detection coverage of MAbs by combining MAbs that recognize different and conserved epitopes. This method can be further applied to the precise detection of other plant viruses and even animal viruses including the current pandemic and rapidly mutating SARS-CoV-2. This work was funded by the National Natural Science Foundation of China (31720103912) and ‘Taishan Scholar’ Construction Project (TS201712023). The authors declare no competing interests to disclose. C.-L. Z., Q. Z. and X.-Q. M. performed the experiments; C.G., Y.-P. T. and X.-F. Y contributed to materials; Z.-Y. Y. and X.-D. L. conceived the research; all authors analysed the data and revised the manuscript. Figure S1 Schematic diagram of PVY CP deletion mutants for mapping the recognition region of MAbs N1, M1, M2 and C1. Figure S2 Identification of recognition region in PVY CP of MAb M2. Figure S3 Schematic diagram of PVY CP deletion mutants for mapping the minimal recognition epitopes of MAbs N1 (A), M1 (B), M2 (C) and C1 (D). Figure S4 Substitutions of the amino acids in each epitope to alanine abolished the recognition of MAbs N1, M1, M2 and C1 with CP. Figure S5 The positions of the recognition epitopes of MAbs N1, M1, M2 and C1 in the three-dimensional structure of CP. Figure S6 Sequence alignment of 10 PVY CPs. Figure S7 Sequence alignment of eight potyviral CPs. Figure S8 Analysis of the conservation of epitopes recognized by MAbs 1128, 1129, 1130, N1, M1, M2 and C1 in 1885 PVY isolates. Figure S9 The specificity analysis of mixtures of MAbs M1 with M2, M1 with C1 and M2 with C1 using (A) DIBA, (B) Western blot and (C) ELISA. Table S1 Characteristics of the four MAbs. Table S2 Analysis of recognition numbers and percentage of 1885 PVY isolates by MAbs based on recognition epitopes. Table S3 Analysis of recognition of 1885 PVY isolates by MAbs 1128, 1129 and 1130 based on recognition epitopes. Table S4 Analysis of recognition of 1885 PVY isolates by MAbs N1, M1, M2 and C1 based on recognition epitopes. Table S5 The primers used in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.