Preparation and Detection Application of Broad-spectrum Monoclonal Antibody Against Three Potyviruses Infecting Chinese Passionflower (Passiflora edulis)
LI Xue1, LI Jin-Hai2, WANG Lan-Hua2, QI Duo1, GUO Meng-Meng1, ZHOU Xue-Ping1, WU Jian-Xiang1,*
1 State Key Laboratory of Rice Biology/Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China; 2 Agricultural Green Development Service Center of Qiubei County, Qiubei 663200, China
Abstract：Telosma mosaic virus (TelMV), East Asian passiflora virus (EAPV), and Passion fruit severe mottle-associated virus (PFSMaV) belonging to the genus Potyvirus have seriously damaged the passionflower (Passiflora edulis) industry in China. So far, there are no effective agents for preventing and curing plant viruses. Thus, Developments of viral detection techniques are crucial for establishing scientific prevention and control system of plant viral diseases. In this study, field-collected passionflower leaves, co-infected with TelMV, EAPV and PFSMaV were used for virus particle purification. Purified and mixed virions of these 3 potyviruses were used as the immunogen to immunize 4 BALB/c mice (Mus musculus). Four hybridoma strains (11A12, 6D8, 6D12 and 17E4) secreting specific and broad-spectrum monoclonal antibodies against above 3 infecting passionflower plant potyviruses were obtained by the hybridoma technique including cell fusion, hybridoma cell screening, antibody detection, cell cloning and ascites preparation. Western blot assay showed that the 4 monoclonal antibodies could simultaneously recognize capsid proteins of TelMV, EAPV and PFSMaV. Further, the produced monoclonal antibodies was used as the detection antibodies to develop two serological assays, i.e. dot enzyme-linked immunosorbent assay (Dot-ELISA) and Tissue print-ELISA for the broad-spectrum, specific and sensitive detection of these 3 potyviruses. The two established serological methods could simply, quickly, specifically, sensitively, broadly and efficiently detect TelMV, EAPV and PFSMaV in passionflower samples. Meanwhile, there were negative reactions when they detected the diseased leaves infected with Plumpox virus (PPV), Potato virus Y (PVY), or Potato virus A (PVA), Turnip mosaic virus (TuMV), Passiflora latent virus (PLV), Cucumber mosaic virus (CMV) or uninfected passionflower leaves. Sensitivity analysis assays showed that the virus could be detected in infected plant crude extracts diluted up to 1∶20 480 (W/V, g/mL), i.e. in 0.097 6 ng virus-infected plant tissues of the absolute detection amount by the established Dot-ELISA serological assay. The detection results of field samples showed that the two developed serological assays could accurately, sensitively and broadly monitor these 3 potyviruses in passionflower samples. The coincidence rate of serological and RT-PCR detection results of field samples was 100%. This study provides technical support for the detection and diagnosis of these 3 potyviruses, production of virus-free passionflower seeds and seedlings, and the scientific prevention and control of these viral diseases.
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