Monoclonal Antibody-based Serological Detection Techniques for Tomato black ring virus
LIU Li1, HE Wan-Qin2, YU Cui3, WU Jian-Xiang2,*
1 The Department of Applied Engineering, Zhejiang Economic and Trade Polytechnic, Hangzhou 310018, China; 2 State Key Laboratory of Rice Biology/Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China; 3 Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Customs, Shanghai 200135, China
Abstract：Tomato black ring virus (TBRV) is a kind of harmful organisms that can infect a wide range of economically important herbaceous and woody species. TBRV now is a port quarantine pest in many countries including China. Strengthening the inspection and quarantine of TBRV at ports is the key to prevent TBRV from invading China. Serological assays are well known to be quick, simple, low-cost and high-throughput techniques for plant viral detection. The aim of present study was to develop serological techniques for TBRV detection using the prepared specific monoclonal antibody (MAb) against TBRV, which could serve TBRV detection, inspection and quarantine at ports. The purified TBRV virions were used as the immune antigen to immunize BALB/c female mice (Mus musculus). Four hybridoma cell lines (10A8, 21A8, 21B8 and 8D3) secreting anti-TBRV MAbs were successfully prepared by fusing mouse myeloma cells (Sp2/0) with splenocytes from the immunized BALB/c mouse, screening hybridomas and antibodies, and cloning cells. The hybridomas were intraperitoneally injected into pristine-primed BALB/c mice, and about 20 mL ascetic fluids contained MAbs for each hybridoma cell line was obtained. The titers of all 4 MAbs in ascitic fluids determined by an indirect enzyme-linked immunosorbent assay (indirect-ELISA) were up to 10-7. Isotypes and subclasses of all these 4 MAbs were determined to be IgG1, κ light chain using a commercial MAb isotyped kit. The IgG yields of 4 MAbs in ascetic fluids ranged from 5.44 to 12.93 mg/mL. Western blot assay of MAb specificity showed that all 4 MAbs had a specific immune reaction with the approximately 43 kD subunit of TBRV capsid protein in infected plant tissues, but had a negative immune reaction with healthy plant tissues. Dot enzyme-linked immunosorbent assay (Dot-ELISA) and Tissue print enzyme-linked immunosorbent assay (Tissue print-ELISA) serological approaches for TBRV detection were developed using the created MAbs as primary antibodies. Both developed Dot-ELISA and Tissue print-ELISA could specifically detect TBRV in infected plants, while all the detection with healthy plants and the plants infected by Tobacco ringspot virus (TRSV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tomato yellow leaf curl virus (TYLCV) respectively showed negative reactions. Furthermore, established Dot-ELISA serological approaches based on MAbs 21B8, 8D3, 10A8 and 21A8 could reliably and effectively detect TBRV in infected tomato leaf crude extracts diluted up to 1∶10 240, 1∶10 240, 1∶5 120 and 1∶5 120, respectively. The above results indicated that the 4 created MAbs and the newly established Dot-ELISA and Tissue print-ELISA serological approaches had very high specificity and sensitivity for TBRV detection in plants, and could be accurately and effectively used for the inspection and quarantine of TBRV at ports and the detection and diagnosis of TBRV in field plants.
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