Preparation Of Monoclonal Antibody Against CA Protein Of Small Ruminant Lentiviruses And Establishment Of An Antigen Capture ELISA For Detection CA Protein

Small ruminant lentivirus(SRLV)is the general name of Maedi visna virus(MVV)and caprine arthritis encephalitis virus(CAEV).These two viruses mainly infect sheep or goats and spread through nasal secretions,excreta and milk,causing chronic,progressive and inflammatory pathological changes in animals.The clinical manifestations of the diseased animals are pathological changes of lung,breast,central nervous system and joint.The disease is chronic and progressive,and often results in death.The diagnosis of SRLV infection depends on laboratory tests.The nucleocapsid CA of SRLV is highly conserved among different strains.It is a group specific antigen and a target recommended by OIE to identify SRLV infection.It is often used as a serological diagnostic antigen.At present,the serological diagnosis methods based on CA protein are limited to the detection of specific antibodies of CA protein,but the direct detection of viral CA protein has not been reported.In order to establish an in vitro diagnostic method for direct detection of viral CA protein,several monoclonal antibodies against SRLV CA protein were prepared,and antigen capture ELISA was established.In this study,the gene encoding CA protein was amplified form genome of a MVV strain preserved in the laboratory by RT-PCR and cloned into the prokaryotic expression vector p ET30a.The gene was induced to express in vitro and purified by His tag.The recombinant expression plasmid of ca gene based on prokaryotic expression vector p GEX-6p-1 was constructed.After induced expression in vitro,GST-CA protein was purified by GST tag of fusion expression,and used as ELISA coating antigen to detect CA antibody in serum of immunized mice.The spleen cells of mice with high serum antibody titer were fused with mouse myeloma cell SP2/0,and the monoclonal cells were screened by limited dilution method.After three subclonal screening,23 monoclonal cells secreting anti CA protein specific antibody were obtained.Ten monoclonal cells were selected to purify the antibodies.The linear epitopes of the antibodies were identified by the truncated mutants of CA protein with different lengths expressed in vitro.Western blot analysis showed that the 3 MAbs recognized three different epitopes:⁶²NRAQKELIQGKLNEE⁷⁶,¹⁴¹LVKQKNTESYEDFIARL¹⁵⁷and ¹⁸⁷CQKQMDRVLGTRVQQATVEEK MQACRDV²¹⁴.Three antibodies G8F7,B1B12 and A8G10 with high affinity and recognizing different epitopes were selected.After pairing experiment,A8G10 was detected as capture antibody and B1B12as detection antibody for antigen capture ELISA for CA.In this AC-ELISA,coating concentration of capture antibody was 1 ng/?L and using concentration of detected antibody was 0.25 ng/?L.After detection,the 450 nm absorbance value of the method showed a good linear relationship in the range of protein standard concentration from 0.610 ng/m L to 78.125 ng/m L,the correlation coefficient R²>0.99,the minimum detection concentration was 0.610 ng/m L,the coefficient of variation of repeatability in and between batches was less than 6%,indicating that the method has a high sensitivity and a good detection linear range.SRLV Gag protein could be detected in 16 fold diluted SRLV Gag protein stock solution by this method,while Western blot could detect 64 fold diluted Gag protein stock solution.According to the protein standard curve,the concentration of secretory SRLV Gag protein stock solution was 75.734 ng/m L.In conclusion,several monoclonal antibodies against MVV CA protein were successfully prepared and an antigen capture ELISA method was established.The method is sensitive and reproducible,and can be used for the quantification of small ruminant lentivirus CA protein in laboratory.The monoclonal antibody and antigen capture ELISA obtained in this study provide technical support for the basic laboratory research of SRLV and lay a foundation for the further establishment of rapid detection method of SRLV antigen in vitro.