Detection Of AIV Antibodies And Simultaneous Distinction Of Antibodies Against H5 And H7 Subtypes By Protein Microarray

AIV can infect avian and cause great economic losses,even more serious fact is that AIV can infect people,especially the huge harm and loss caused by H5 and H7 subtypes of highly pathogenic avian influenza to human beings leads to several concerns in the public,so there are many techniques for detecting AIV.As is well-known,monoclonal antibodies(MAbs)have many advantages such as higher specificity,stronger sensitivity and the unlimited supply,they are effective tools and broadly applied in different detection methods,mAbs against AIV are also frequently used in the diagnosis of AIV.In recent years,protein microarrays as an emerging technology are being developed,it is a powerful tool for high-throughput way.In order to reduce the threat of AIV to human health and property,accurate detection and judgment of avian influenza virus type is important for timely monitoring of the virus,control of virus transmission and anti-avian influenza virus vaccine research and development.According to the epidemic situation of avian influenza,the diagnosis method should be rapid,effective,high throughput,specific and convenient.In this study,based on the advantages of MAbs and the convenience of protein microarray method,a method of blocking protein microarray for detection of avian influenza antibody was established.This method can be applied to detect influenza antibodies in the serum from different species and simultaneously distinguish H5 or H7 subtypes.Serological assays allow us to determine whether susceptible species or humans have the history of infection,providing vital clues for surveillance and diagnosis of AIV.1.Preparation and identification of monoclonal antibodies against NP protein of avian influenza virusIn this study,BALB/c mice were immunized with an antigen that was purified H5N6,lymphocyte hybridoma technology was used to prepare hybridoma cells,and the positive hybridoma cells were screened by indirect ELISA,the hybridoma cells secreting anti-NP protein were obtained,named 3D9 and 2B10,respectively.The binding ability and specificity of the two MAbs to antigen were identified by indirect ELISA,western blot and indirect immunofluorescence assay.Indirect ELISA analysis showed the antibody titers in the supernatants of 3D9 and 2B10 cells was 1:1×103 and 1:1×104,respectively,while the titers in ascites reached 1:1×105 and 1:1×106,respectively;the heavy chain subtypes of 3D9 and 2B10 were IgM,with ? light chain.Western blot analysis showed that two MAbs supernatants could specifically react with different AIV at 56 KD,and specifically react with the recombinant NP protein of prokaryotic expression.Indirect immunofluorescence assay showed that two MAbs could produce green fluorescence reaction with MDCK cells infected with different AIV.The two MAbs are target at the NP protein of H5N6 AIV,and they have good specificity,conservativeness and broad-spectrum.2.Expression and purification of H5N1-HA1,H7N3-HA1 and H3N2-NP recombinant proteinsH5N1-HA1 gene was cloned into two prokaryotic expression vectors,pCold I and pGEX-4T-1.Two recombinant plasmids were respectively transformed into BL21 strain for the induced expression of the target protein,the results showed the H5N1-HA1 was successfully expressed a His-fused protein in pCold ?,and also could produce a GST-fused protein in pGEX-4T-1.At 37? or 16?,H5N1-HA1 proteins in two prokaryotic expression system were induced by IPTG of ImM,both are expressed as inclusion bodies,the His-fused HA1 protein was selected for expression for further experiments.The recombinant plasmids pCold I-H7N3-HA1 and pET-32a-H3N2-NP were obtained by our laboratory,they were transformed into E.coli BL21 strain for prokaryotic expression,at the IPTG concentration of 1mM,induced pCold I-H7N3-HA and pET-32a-H3N2-NP at 16?,and obtained H7N3-HA1 protein in the inclusion body and AIV-NP partially expressed in the supernatant.The three proteins purified recombinant proteins for highly purified protein,using monoclonal antibodies to western blot experiments,the results analysis found that three recombinant proteins have good specificity,which provides high-quality antigens for the detection of avian influenza antibody blocking protein microarray.3.Construction and optimization of protein microarray detection methods for AIV and H5,H7 subtypes antibodiesIn this study,in order to achieve the differential diagnosis and epidemic surveillance of avian influenza and H5 and H7 subtypes,a novel inhibition protein microarray system as a new was developed,which could not only determine the presence of avian influenza antibodies in animals of different species,but also distinguish the H5 and H7 subtypes antibodies of avian influenza virus,this is a new method for diagnosis of avian influenza.In the protein microarray,the optimal purified antigens and mAbs concentrations were identified.In order to obtain the high intensity chemiluminescence signal which is prone to observe and calculate the inhibition percentage,concentrations of 0.2mg/mL H5 and H7 antigens and concentrations of 0.05mg/mL NP antigen were determined to be optimal;and the optimal dilutions of the H5,H7 and NP mAbs were 1:2000,1:2000 and 1:320000;the best dilution for serum samples was 1:32.Then the cut-off value were determined,they were 40%,50%,30%inhibition for the H5 antibody detection,50%,50%,20%for NP antibody detection and 40%,50%,40%for H7 antibody detection in chicken,peacock,duck sera respectively.The microarray was tested using positive sera of other avian disease viruses,showing no cross-reaction.We observed that the serum with known hemagglutination inhibition test(HI)titers were incubated in protein microarray,results indicated that the detection limit of the H5 and H7 antibodies were both 1 HI titers in the protein microarray.When HI titers of serum was titrated based on H5 and H7 subtype positive,the detection limit of the NP antibodies were both 0.5 HI titers.The protein microarray was compared with commercial AIV antibodies test enzyme-linked immunosorbent assay(ELISA)kit(IDEXX)or HI,the 55 chicken serum samples were detected,the results showed the diagnostic sensitivity and specificity of the detection of H5 and NP antibodies in the protein microarray were 100%,and the diagnostic sensitivity and specificity of the detection of H7 antibodies were 100%and 97.06%,respectively.The 20 ducks and 20 peacocks serum samples were incubated in the protein microarray,compared with HI,the detection of H5 antibodies in the microarray showed 100%coincidence ratio in peacock and duck,while the H7 displayed 100%in peacock and 90%in duck.The method developed in this study has the advantages of high throughput,accuracy,specificity and sensitivity,it is of great value for the serological diagnosis and epidemiological investigation of AIV and the monitoring of vaccination efficiency.More importantly,this approach has the potential to replace complex HI experiments that can identify the H5 and H7 subtypes of AIV-moreover,the approach lays a foundation for the subsequent simultaneously identifying more AIV subtypes and detecting other pathogens.