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| Production and Application of Monoclonal Antibody Against BBTV |
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Abstract Banana bunchy top virus (BBTV) is an important pathogen of banana (Musa paradisiaca), which seriously harm banana production worldwide. The purpose of this study was to develop serological methods for detecting BBTV using prepared monoclonal antibodies (MAbs) against BBTV, and to provide technical support for the diagnosis and scientific prevention and control of BBTV in fields. BBTV-infected banana plants were identified by PCR and nucleotide sequencing. A 513 bp coat protein gene (CP) was amplified from infected banana plants, which had 99% nucleotide sequence identities with Chinese isolates of BBTV in GenBank. BBTV particles with a diameter of 18 nm were obtained from BBTV-infected plants by an improved purification method. Electron microscopic observation showed that concentration of purified BBTV particles was high enough to prepare MAbs against BBTV. After cell selecting and cloning, a hybridoma cell line (22E3) which secreted a MAb against BBTV was produced by fusing mouse (Mus musculus) myeloma cells (SP2/0) with spleen cells from BALB/c immunized by purified BBTV particles. The hybridoma was injected into pristane-primed BALB/c mice to prepare the ascetic fluid, which contained the MAb. The titer of this MAb in ascites determined by an indirect-enzyme-linked immunosorbent assay (in-ELISA) is up to 10-7. Isotype and subclass of this MAb belonged to IgG1, κ light chain. The IgG yield of this MAb in the ascetic fluid was 8.32 mg/mL. Western blot analysis indicated that this MAb could specifically react with the 19.3 kD CP of BBTV in BBTV-infected leaf crude extracts, had a negative reaction with healthy banana leaf crude extracts. Using the MAb, dot-ELISA for detecting BBTV was established. Results of phalanx tests showed that the dilutions of 22E3 at 1∶5 000 and goat anti-mouse IgG conjugated with alkaline phosphatase at 1∶8 000 were suitable in dot-ELISA for BBTV detection in field samples. Specificity analysis of the dot-ELISA demonstrated that this method could strongly react with BBTV-infected banana plant tissues, not with the healthy banana plant tissues. The detection sensitivity of the dot-ELISA based on 22E3 was up to 1∶640 (W/V, g/mL). The field banana samples, collected from Guangdong, Yunnan and Hainan Provinces, were tested the presence of BBTV by developed dot-ELISA, and the detection results demonstrated that the assay could accurately and reliably detect BBTV in field banana plant samples. The anti-BBTV MAb and the developed serological dot-ELISA provide material and technology for diagnosis, production of BBTV-free seedlings and scientific prevention and control of banana bunchy top disease.
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Received: 04 August 2015
Published: 29 December 2015
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