Development Of Monoclonal Antibody Against A Subgroup Avian Leukemia Virus(ALV)and Difference Analysis In Clinical Detection Of ALV With Different Kits

Avian leukosis virus(ALV)is a member of the avian retrovirus family,which is closely related to a variety of avian neoplastic diseases and production performance.This virus can cause birds to form tumor and the chronic death,as well as immunosuppression,growth inhibition,and decrease of egg-laying rate,inducing huge loss to the poultry industry.ALV spreadsby both vertically and horizontally.The main method to prevent the disease is to eliminate the positive chicken and eraducate the subgroups A,B,C,D,E and J.Virus of different subgroup has different biology characters and clinical symptoms.It is a key point to choose the proper kits to detect all ALV.In this paper we developed some monoclonal antibodies against gp85 of ALV-A and the comparation of different ELISA kits for ALV.This research will provide practical value for the poultry industry.1 Preparation and identification of monoclonal antibody against A subgroup avian leukemia virusGp85,an envelope protein of ALV,which is encoded by env gene,is subgroup specific antigen.In this paperwe first designed a pair of specific primers for ALV-A,andamplified the sequence of gp85 of AH 10 through polymerase chainreaction(PCR).Then with the gp85 fragment,a prokaryotic expression plasmid PET32a-AH10-gp85 was constructed and transferred into BL21 cells.Positive expression plasmid was selected and the clone was induced by IPTG.The bacteria was harvestedand brokenby ultrasonic.After the centrifuge,the supernatant and precipitate wereanalyzed respectivelyon SDS-PAGE.The results showed the the size of target protein was about 53KD.BALB/c female mice at 6-8 weeks of age immunized with the purified protein emulsified with an appropriate adjuvant.After three immunizations,the sera of the mice were tested.Three days later post boost with thepurified protein,the mouse spleen cells were fused with myeloma cells.The positive hybridoma were screened by IFA in DF1 cells infected with AH 10 virus.After 2-3 subclones of the positive clone,five strains of monoclonal antibodies with stable secretion antibodies against ALV were finally obtained,which were named 1 ALV-A-B12,ALV-A-1C12,ALV-A-1E5,ALV-A-1F8 and ALV-A-3D11,respectively.Western-blot and IFA results showed that the monoclonal antibody ALV-A-3D11 was specific to ALV-A/B membrane protein gp85 antigen,and the othe four strains can react with not only ALV-A/Bmembrane protein but alsoJ subgroup virus.In this study,a monoclonal antibody against ALV-A/B(3D11)was successfully developed,which will provide a clinical diagnostic reagent for rapid screening of field chickens infected with A/B subgroup avian leukemia virus.2Analysis of the difference results of thesamples with different kitsThe rapid,accurate,low cost and applicable diagnostic technique for patchs of tests is an important part of prevention of avian leukosis virus.ELISA detection method is widely used in breeder eraducation because it is sensitive,fast and suitable fordetection at large scale.To analyze the difference reason between different ELISA kits for detection of avian leukosis virus P27 antigen,60 2-week-old yellow feather chickens(including 30 live chickens and 30 dead chickens)were numbered.The anal swabs,blood samples and tissue samples were collected in this study.The anal swab samples were detected with IDEXX antigen detection ELISA kit and sELISA kit respectively.Blood and tissue samples were inoculated into DF1 cells to isolate the virus.The supernatant of the DF1 cells was token for detection and identification after 7 days.The positive rate of anal swab was analyzed and compared,which was confirmed by PCR,RT-PCR and indirect immunofluorescence.The results showed that the positive detection rate of anal swab samplesbysELISA kit was 68%,but only 27%by IDEXX kit.It was interesting that all positive samples by IDEXX kit were foundin the positive samples bysELISA kit.The positive differential samples were further confirmed by sequenceanalysis.The results showed they all contained K subgroupavian leukosis virus.This result showed that the sELISA kit could detect the positive anal swab samples more sensitively and effectively,especially theK subgroup avian leukosis virus in the anal samples.