Development And Preliminary Application Of Indirect Enzyme-linked Immunosorbent Assay Test Kit For Detecting Antibody Against Bovine Leukemia Virus

Enzootic bovine leucosis(EBL) is a disease of adult cattle caused by the retrovirus, bovine leukemia virus(BLV),characterized either by persistent lymphocytosis or lymphosarcoma formation.EBL occurs worldwide in cattle-raising countries,and the prevalence of BLV infection or EBL was 30%～50%in some herds of our country, indicating it was the most important disease of bovine in China.The antibody against BLV test kits were expensive from abroad,therefore,it is critical for development of BLV antibody test kit independently for prevention and treatment of EBL.Based on the results of preliminary test of ours,an indirect enzyme-linked immunosorbent assay for detecting antibody against BLV was developed by using expressed protien gp51 as coating antigen.The results are as follow:1.Preparation and purification of expressed protien gp51 of BLV.The recombinant gp51 protein was produced at 37℃in cultures grown at log phase by adding IPTG.The rgp51 was purified using a Ni²⁺-NTA column.The column was washed and the protein was eluted.The concentration of rgp51 protein was 0.216mg/mL by ultraviolet light spectrophotometer.2.Preparation and evaluation of the kit.The indirect ELISA kit for detecting antibody against BLV was developed by using expressed protein gp51 as coating antigen. The main component of the kit are one 96-well plate coated by gp51 protein, HRP-labeled rabbit anti-bovine IgG,the standard positive/negative serum.The specificity,sensitivity,reproducibility and keeping time of the kit were tested and the results demonstrated that the kit has high specificity,sensitivity,reproducibility and long keeping time,and no cross reaction with Infectious rhinotracheitis,Bluetongue,Bovine viral diarrhea,and Akabane disease positive sera.The valence of reference sera tested was 1:256～1:512.The sensitivity of the kit was 4～8 fold of the AGIDT,the coefficient of variance among and between batch were less than 10%and 15% respectively,stable results can be obtained from kits stored at -20℃for at least six months and stored at 4℃for one month.The kit was compared side by side with BLV ELISA kit produced by France Synbiotics Company,the results showed that the sensitivity,specificity and coincidence were 75%,96.1%and 94.6%respectively.3.The kit were used to test 164 sera from cow in JiNan city,ShanDong province and 398 sera from Australia import cow,6.10%and 4.77%of them were positive respectively.All results demonstrated that gp51-ELISA kit was high specificity,good sensitivity and easy manipulation,which provided an available technique for detection and serological survey of EBL.The kit is effective and cheap,it refer to dairy farm, veterinary station,shambles and seaport practical,and it also provide to investigate epidemic status of EBL in China.