Comparison And Establishment Of An Indiret-Blocking ELISA For Detection Of Antibodies Against Protein M1 And NS1 Of AIV

Avian Influenza(AI) is an infectious and/or disease syndrome caused by type A avian influenza virus(AIV). In recent years, the highly pathogenic avian influenza(HPAI) was very popular in many countries, caused huge economic losses to the poultry industry, and becoming a threat to the human health in world wide. We carried out the study, because rapid detection technology of AIV is the detection method and important prerequisite for effective control of AIV.M1 protein is the type-specific protein for influenza viruses, main basis for influenza virus classification, and slowly variabilitive and highly conserved in the evolution of Influenza A virus. Therefore it can be used as detection antigen to diagnose the different subtype of AIV.The NS1 protein is the non-structural protein of influenza viruses, with highly conservatism, and strongly expressed in the early stage of viral replication, which only in the virus-infected cells, and also can induce the body to produce antibodies against the NS1 protein. Therefore NS1 protein can be used as detection antigen to distinguish whether the flocks are infected or vaccinated by NS1 antibody detection.M1 and NS1 indirect blocking ELISA were established by using a recombinant AIV M1 and NS1 protein as the detection antigen, and rabbit anti serum M1 and NS1 as the blocking antibody. Working condition of M1 indirect inhibition ELISA: the optimal concentration of M1 was 0.78μg/mL, the optimal dilution of the blocking antibody was 1:15 000; the optimal dilution of the serum was 1:10. Working condition of NS1 indirect inhibition ELISA: the optimal concentration of NS1 was 0.6μg/mL, the optimal dilution of the blocking antibody was 1:10000; the optimal dilution of the serum was 1:10.(1) M1 indirect-blocking ELISA: An assay was tested on 38 positive samples and 26 negative samples of Avian, which showed the sensitivity of this method was 97.37%, and the specificity was 96.16%. Crossreaction detection showed there's no cross-reaction with other eight kinds of Avian disease positive serum such as NDV. After detecting 268 clinical samples, the coincidence of blocking ELISA and HI was over 92%. (2)NS1 indirect-blocking ELISA: An assay was tested on 42 infected samples and 50 vaccinated samples of Avian, which showed the sensitivity of this method was 95.2%, and the specificity was 90.0%. Crossreaction detection showed there's no cross-reaction with other eight kinds of Avian disease positive serum such as NDV. The results of clinical detection showed that the negative rates of 186 samples antiserum against the inactivated vaccine, 20 samples antiserum against the recombinant live vaccine and 42 samples healthy and not vaccinated serum were 89.2%,95% and 100%. (3)The comparison of M1 and NS1 Indirect- blocking ELISA: the sensitivity of M1-ELISA was better when detecting infected serum.The specificity of NS1-ELISA was better when detecting healthy and not vaccinated serum.The results showed that: This two methods are out of bounds of the animal species and the avian flu subtype serum, could detect serum antibody levels in different subtypes of many species animals once. M1 indirect-blocking ELISA could provide an effective quantitative basis for antibody levels of poultry immune serum. And also provided a rapid and reliable laboratory detection methods to judge whether the non-immune breeding and wild poultry has been infected by AIV. While the NS1 indirect-blocking ELISA can distinguish the naturally infected flocks and the immuned flocks by NS1 antibody levels, as the basis for early infection. In clinical applications, match usage of this two methods can further distinguish natural infection of AIV, inactivated vaccine immuned and the healthy non-immune flocks. It provided a feasible serodiagnosis method for flocks purification.