Generation of Synthetic Peptide-Specific Antibody for the Development of A Southern Rice Black-Streaked Dwarf Virus Diagnostic Test

Abstract

Southern rice black-streaked dwarf virus (SRBSDV) causes severe epidemical disease on rice with the infected area up to millions of hectares in South China and North and Central of Vietnam. So far, there are no effective, cheap, quick, and practicable methods for diagnosing SRBSDV. The conventional RT-PCR technique is the most popular method for detecting SRBSDV with high accuracy. However, it is hard to apply this method for large-scale SDBSDV diagnosis because of the requirements of expensive reagents and instruments, as well as complex procedures. Meanwhile, SRBSDV diagnostic techniques based on antigen detection have outstanding advantages due to their low cost, easy manipulation, and wide application possibility. Today, there are still no commercially available specific antibodies to SRBSDV. In a previous study, to develop the SRBSDV diagnostic technique by the ELISA technique, a SRBSDV specific antibody was generated by a recombinant P10 envelope protein (66kDa), which has a titer of 1:5,000. In this study, we continued to study the production of SRBSDV specific polyclonal antibodies from small antigen–rich peptides from the SRBSDV P10 envelope protein. The resulting purified antibody can specifically bind to the P10 protein and at the diluted concentration of 1:100,000 it can detect SRBSDV in infected rice samples via the dot-blot technique. Our research results open up new opportunities for proactive antibodies to develop a SRBSDV membrane rapid diagnostic kit.

Development of a Dot-Elisa Assay for Diagnosis of Southern Rice Black-Streaked Dwarf Disease in the Field

“…Four antigens, namely purified P10 protein and P10.1, P10.2, and P10.3 synthetic peptides obtained from previous studies (Hanh et al, 2015;Phuong et al, 2020;Hanh et al, 2021a;Hanh et al, 2021b) were supplied by the Molecular Pathology Department (Agricultural Genetics Institute).…”

“…Anti-SRBSDV antisera were produced using P10 protein and P10.1, P10.2, and P10.3 peptides as antigens for immunization in mice as previously described (Hanh et al, 2015;Phuong et al, 2020;Hanh et al, 2021a;Hanh et al, 2021b) with several minor modifications for the P10 antigen. The four achieved antisera were diluted at serial concentrations from 1:5000 to 1:500000 and used for the dot-ELISA assay to detect the purified recombinant SRBSDV P10 protein (50 ng/dot) (Tam et al, 2013).…”

“…Optimization and standardization of reagents and test procedures for serodiagnosis of viral diseases are vital for assay validity, reproducibility, and quality control (Michael et al, 1984). In previous studies, we produced anti-SRBSDV antibodies by immunizing mice with either recombinant protein or synthetic peptides that were derived from the P10 coat protein of SRBSDV (Hanh et al, 2015;Phuong et al, 2020;Hanh et al, 2021a;Hanh et al, 2021b) but the titers of these antibodies were not high enough to apply in a practical virus diagnostic assay. This may have been the result of the immunization method with an inadequate amount of injected antigens.…”