H1N1亚型猪流感病毒单克隆抗体IPMA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2019.09.024

Abstract

Abstract: This experiment was aimed to establish an immunoperoxidase monolayer assay (IPMA) screening method for the detection of anti-H1N1 subtype swine influenza virus monoclonal antibody.The specificity,sensitivity and reproducibility of the established IPMA screening method were evaluated by optimizing the reaction conditions of MDCK cells,culture time after cell inoculation,type of blocking solution,working concentration and working time.The results showed that the optimal reaction conditions for the established IPMA were MDCK cells with 10^2.63 TCID₅₀/100 μL H1N1 subtype swine influenza virus,cultured at 37℃ for 24 h,3 ‰ H₂O₂ methanol fixed at room temperature for 15 min,5% skim milk blocking for 2 h at 37℃,50 μL of hybridoma cell supernatant was used as primary antibody,incubated at 37℃ for 2 h,and goat anti-mouse HRP-IgG secondary antibody was incubated at 37℃ for 1 h.The established IPMA method could specifically detect H1N1 subtype swine influenza virus monoclonal antibodies,and did not cross-react with porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2) and swine fever virus (CSFV) positive serum.The sensitivity test results showed that 1:3 200 HI=2^-9 standard H1N1 swine positive serum could be detected;The repeatability test between inter-batch and intra-batch was good.In summary,this experiment successfully established an IPMA detection method for anti-H1N1 subtype swine influenza virus monoclonal antibody,which had strong specificity,high sensitivity and good reproducibility,and provided a simple,practical and effective detection method for the production of monoclonal antibodies against H1N1 subtype swine influenza virus.

Key words: H1N1 subtype swine influenza virus; immunoperoxidase monolayer assay (IPMA); monoclonal antibody

CLC Number:

S852.65

SHI Jianzhou, LI Qingmei, WANG Yanhong, LIU Xiao, LI Ge, GUO Junqing, DENG Ruiguang, ZHANG Gaiping. Establishment of a Method of IPMA for Detection of the Monoclonal Antibody Against H1N1 Swine Influenza Virus[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2691-2698.

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Establishment of a Method of IPMA for Detection of the Monoclonal Antibody Against H1N1 Swine Influenza Virus

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