Establishment And Clinical Application Of H1 Subtype SIV Antigen Capture ELISA Method

Swine influenza（SI）is an acute respiratory infection of swine caused by swine influenza virus（SIV）.Various types of swine can be infected.The clinical manifestations are mostly high fever,cough,high incidence of his disease,but death the rate is low.When the infection is mixed with other pathogens,the condition becomes worse and the mortality rate increases,causing huge economic losses to the pig industry.People are also infected with SIV,which poses a great threat to human health.The three subtypes of SIV,H1N1,H1N2,and H3N2,are most prevalent in pigs.Among them,the Eurasian avian-like H1N1 is the predominant strain widely circulating in pigs in China,so it is necessary to strengthen the detection of H1 subtype SIV.In this study,Eurasian avian-like H1N1 SIV LN225 was used as the immunogen,and 6-week-old BALB/c mice were inoculated intraperitoneally to sensitize mouse lymphocytes.The sensitized lymphocytes in the spleen were extracted and fused with SP2/0 cells to form hybridoma cells.A total of 6 positive hybridoma cells were screened by hemagglutination inhibition experiment,named respectively 1F4,3F6,4E2,5F7,2E8 and 2E7.The results of subclass identification showed that the heavy chain of Mc Abs 3F6,5F7,2E8,2E7 was Ig G2a and the heavy chain of Mc Abs 1F4,4E2was Ig G1,and the light chain isotype of all Mc Abs wereκ.The results of indirect immunofluorescence assay showed that these six Mc Abs could specifically react with the HA protein of LN225.The cell culture supernatant and ascites HI titer of Mc Ab 1F4,3F6,4E2,5F7,2E8,2E7 were 2⁹,2⁷,2⁶,2⁷,2⁸,2⁴ and 2¹⁹,2¹⁴,2¹⁴,2¹³,2¹⁴,2¹⁴,respectively.Four high HI activity and stably anti-H1 subtype HA Mc Abs 1F4,3F6,4E2 and 2E8 were selected for the further development of diagnostic method for SIV.Using purified porcine positive serum as the capture antibody and HRP-labeled monoclonal antibody 3F6 as the detection antibody,an antigen capture ELISA method for specific detection of H1 subtype SIV was established.After optimizing the reaction conditions,the purified polyclonal antibody was coated onto the ELISA plate at 5.96μg per well;the coating conditions were 37°Cfor1 h and then 4°C overnight;5%skim milk was blocked at 37°C for 1 h;the antigen to be tested was37°C for 1 h;The labeled monoclonal antibody was diluted 1:2000 and incubated at 37°C for 0.5 h;the color was developed at room temperature for 10 min.Statistical analysis of 30 SIV-negative antigen samples by antigen capture ELISA method was carried out,and it was determined that the standard of this method was more than 0.069 for positive and less than 0.063 for negative,which was suspicious in between.There is no cross-reaction with H3,H5,H6,H9 subtypes SIV.The coefficient of variation of intra-and inter-plate detection is less than 10%.Sensitivity results show that it is at least 16 times more sensitive than conventional HA tests.The antigen capture ELISA test on 12 laboratory organs infected by the laboratory has a better agreement rate with the chicken embryo virus isolation method,but it is not as sensitive as virus isolation.Twenty-four H1N1 subtype SIV chicken embryo allantoic fluids were tested positive.The above results indicated that the established antigen capture ELISA method had good specificity and sensitivity.Using this method,a total of 546 clinical samples of swine nasal swabs collected from Jiangsu,Shandong,Tianjin,and Zhejiang were tested.A total of 3 H1 subtype SIVs were detected,indicating that the method can be used for clinical samples.The monitoring and diagnosis of H1N1subtype SIV provides a quick and easy method.