The Preparation And Development Of Avian Leukosis Reference Material Candidates Of Antigens And Antibodies

The Avian Leukosis (AL) is caused by Avian Leukosis Virus (ALV) leading to poultry tissues and organs tumors. Up to now, ALV has already been divided into 11 subgroups A-K, Subgroups A, B, C, D, E,J and K are isolated from chicken. Subgroups A-D and J, K are exogenous virus, and subgroup E, extremely widespread and low or no pathogenic in chicken, is endogenous virus.In order to establish the specifications of testing and purification of avian leukosis, in this study, reference antigens and specific reference antibodies of avian leukosis, subgroup J and subgroup A avian leukosis were prepared. These biological materials were tested and evaluated the kits in the market. Very good effect was obtained which lay foundations for further eradication of avian leukosis.1. Preparation of avian leukosis reference antigen, subgroup J and subgroup A avian leukosis reference antigen candidatesp27 gene is highly conserved gene among avian leukosis virus subgroups A, B, C, D, J and K. In this study, recombinant bacteria pGEX-6p-1-p27, which could express avian leukosis virus recombination fusion protein GST-p27, avian leukosis group specific antigen, was induced to express. And the expressed protein GST-p27 was purified. The candidate material of avian leukosis reference antigen fusion protein GST-p27 was prepared. At the same time, the strain JS09GY07 of ALV-J and the strain AH 10 of ALV-A were propagated and identified by ELISA, PCR and IFA. A series of calibrations such as purity, protein concentration, TICD50 were conducted. The materials for subpackage, physical property and capacity differential coefficient analysis, and identification of bacteria and mycoplasma contamination, homogeneity, stability were conducted. The avian leukosis reference antigen P27 and subgroup J and A avian leukosis reference antigens and their corresponding weakly positive and negative antigens were prepared. Kits testing avian leukosis antigen p27 in the market were evaluated by the avian leukosis reference antigens candidates. The results showed that not all kits in the market were qualified in detection and it should be paid much anttension in eradication of avian leukosis.2. Preparation of specific monoclonal antibodies candidates for avian leukosis and subgroup J and reference antisera candidates for subgroup J and AHybridoma cells named ALVp27-5D3 which could secreting monclonal antibody 5D3, reacting with avian leukosis group specific antigen p27, and hybridoma cells named JE9 secreting monoclonal antibody JE9, reacting with subgroup J avian leukosis specific antigen env protein were used as reference monoclonal antibodies. ALVp27-5D3 hybridoma cells and JE9 hybridoma cells were injected into Balb/c female mice at 6-8 weeks of age by intraperitoneal injection respectively.5D3 ascites and JE9 ascites were purified respectively. Avian leukosis reference antibody candidate 5D3 and subgroup J Avian leukosis reference antibody candidate JE9 were made.SPF chickens were challenged by ALV-J through intramuscular injection and boosted for 5 times. The chicken antisera candidates were made post twelve days aftere final injection. ALV-A chicken antiserum candidate was made by intraperitoneal injection with ALV-A for 6 times in the way of contacting nose and eye.Avian leukosis reference antibody candidate 5D3, subgroup J avian leukosis reference antibody candidate JE9, subgroup J avian leukosis reference antiserum candidate and subgroup A avian leukosis reference antiserum candidate were calibrated by IFAn ELISA and Western-blot, and were subpacked. And a series of physical property and capacity differential coefficient analysis, and identification of bacteria and mycoplasma contamination, protein concentration or IFA titer, and the qualifications of homogeneity, stability were conducted. Finally ALV-J and ALV-A reference antisera and specificity monoclonal antibodies were obtained.These materials of monoclonal antibodies and reference antisera were determinated the positive, weak positive and negative reference respectively. And then the materials were used to evaluate different commercial kits in the market. The results showed that not all kits in the market were qualified in detection. We should pay much attention to the different kits in eradication of avian leukosis.