Development Of Monoclonal Antibodies Against ALV-p27Antigen And Double Antibodies Sandwich ELISA For Detection Of ALV

Avian leukosis is a variety of tumor diseases caused by avian leukosis virus and avian sarcoma virus. Chickens are susceptible to infect with subgroups A, B, C, D, E and J ALV. Subgroup A, B and J are common clinical exogenous viruses, mainly produced a variety of tumor and cause death or decline of poultry industry. As the virus have a capacity of vertical transmission, the disease can cause persistent infection in the populations. So, the population cleaning is the only effective way to prevent this disease by now. Protein p27is a highly conserved group specific antigen encoded by gag gene of ALV. As the main composition of viral core capsid, we prepared monoclonal antibodies against p27and then establish an ELISA for detection of ALV.In this study, we amplified p27gene of ALV, and obtained GenBank accession number:JF911742. The PCR product of p27was cut by BαmH I and Xho I restriction enzymes and then cloned into the pET-32a (+) and pGEX-6P-1. The recombinant plasmids were transformed into BL21(DE3) and BL21strains. The positive clones were chosen to produce interest protein induced by IPTG. Then, the bacterium liquid was ultrasonic cracked after harvested, and separated to make SDS-PAGE identification. Both supernatants appeared target bands respectively, His-p27protein was43kDa, GST-p27protein was53kDa. His-p27and GST-p27proteins were purified with High-Affinity Ni-IDA resin and High-Affinity GST resin.Eight-week-old BALB/c mice were subcutaneously injected with purified GST-P27protein emulsified with Freund’s complete adjuvant. Mice were subsequently injected two more times with purified GST-p27protein mixed with Freund’s incomplete adjuvant at2-week intervals. An intraperitoneal booster of the same dose of GST-p27protein was given3days before splenocytes were collected and fused to SP2/0myeloma cells. The ELISA plates coated with purified His-p27protein were used to screen the successful integration of hybridoma. The positive holes were subcloned to obtain5hybridoma cell lines which secreted monoclonal antibody anti-p27antigen. The5hybridoma cell lines were named3G6,4A11,4C7,4C9and7F4. Each BALB/c mouse was intraperitoneal injected500,000hybridoma cells to prepare ascites. The titers of ascetic fluid3G6and4A11were1:102400,4C7,4C9and7F4were1:204800. Subclass of monoclonal antibodies3G6,4A11,4C7and4C9were IgGl,7F4was IgG2b. All these monoclonal antibodies were specific against ALV-A, ALV-B and ALV-J and didn’t have the cross reactions with other chicken viruses including NDV, IBV and REV. Western-blot and IFA approved that the monoclonal antibodies were specific against the nucleocapsid protein p27of ALV.Two monoclonal antibodies7F4and4C7for the different epitopes of p27antigen were chosen by competitive ELISA, and4C7was labeled with horseradish peroxidase after two monoclonal antibodies were purified. The optimal working concentrations of two monoclonal antibodies were determined by matrix test,7F4:1μg/ml (1:1000),4C7-HRP:0.5μg/ml (1:200). Established ELISA method were specific against ALV-A, ALV-B and ALV-J and didn’t have the cross reactions with other chicken viruses including NDV, IBV and REV. His-p27protein detection limit of the assay was16ng/ml.182samples of chicken cloacal swabs were tested by our ELISA method and the IDEXX ALV test kit in parallel. The result showed that coincidence rate between our ELISA method and the IDEXX ALV test kit was95.6%.