Preparation of a new monoclonal antibody against subgroup A of avian leukosis virus and identifying its antigenic epitope

Preparation and Biochemical Characteristics of a New IgG-Type Monoclonal Antibody against K Subgroup Avian Leukosis Virus

“…The gp85 gene is the key gene for distinguishing different avian leukemia virus subgroups , and has been used to successfully prepare ALV-A/J monoclonal antibodies as discussed in previous reports. , In this study, the gp85 sequences of ALV-A/B/J/K strains were first compared with DNASTAR software, and the specific gene fragment, which is located in the 283–592 bp of ALV-K gp85 gene (Table ), was screened as the target gene due to its low homology with ALV-A/B/J strains and a high antigenic index (data not shown); then, the target gene with the size of 310 bp was amplified from the cDNA of ALV-K-JS11C1 by PCR (Figure A, lane 5). The sequencing result shows that its sequence is completely consistent with that of the screened ALV-K-JS11C1 gene fragment in Table . Compared with the sequence obtained using the whole gp85 gene as a target gene in the previous report, this strategy greatly improved the accuracy and efficiency of preparing a specific monoclonal antibody.…”

“…The fused cells were first cultured in hypoxanthine-aminopterin-thymidine (HAT) medium for 7 days and then suspended in hypoxanthine-thymidine (HT) medium. The surviving hybridoma cells grew to cover more than 1/3 of the bottom area of each well, and their supernatants were analyzed using indirect ELISA with the purified ALV-K-JS11C1 strain applied as a coating antigen according to a published method . Serum from the inoculated mice and the supernatant of SP2/0 cells were used as positive and negative controls, respectively.…”

“…Analysis of the Spatial Epitope Recognized by the MAb. According to the previously reported methods, 23,27 the secondary structural domains (e.g., α-helix, β-folder/turn, and random coil) and the biochemical characteristics (e.g., hydrophilicity, amphipathic regions, flexible regions, surface probability, and antigenic index) of ALV-K gp85 protein fragments containing the antigenic epitope were predicted using Biological Information Analysis software; additionally, a partial gp85 protein amino acid sequence of the ALV-K-JS11C1 strain containing the antigenic epitope was sent to Swiss-Model (http://swissmodel.expasy.org/) to construct a three-dimensional (3D) model online and simultaneously used to predict the spatial conformation of the antigen epitope. 27 Biological Information Analysis of MAb's Antigenic Epitope.…”

Preparation of a novel monoclonal antibody against Avian leukosis virus subgroup J Gp85 protein and identification of its epitope

“…Thus, the monoclonal antibody might be useful for the development of a specific diagnostic method used to detect ALV-J infection and for the analysis of the interaction between the antibody and antigen. Owing to the continuous emergence of new ALV subgroups or new ALV strains, it would be better to determine the specificity of the new MAb using more ALV strains in future study ( Yan et al., 2019 ).…”

“…Unfortunately, to date, there are still no vaccines or drugs which can effectively protect against ALV-J infection ( Feng et al., 2019 ). In addition, detection of exogenous ALVs is becoming increasingly difficult due to their high variability and the continuous emergence of new subgroups or strains ( Yan et al., 2019 ). Thus, it has become a major challenge in the poultry production to control and eradicate ALV-J( Payne and Nair, 2012 ; Dai et al., 2020 ).…”