H1N1亚型猪流感病毒单克隆抗体IPMA检测方法的建立

本试验旨在建立一种针对检测抗H1N1亚型猪流感病毒单克隆抗体的免疫过氧化物酶单层细胞试验（immunoperoxidase monolayer assay，IPMA）筛选方法。通过优化MDCK细胞接毒量、细胞接毒后培养时间、封闭液的种类和工作浓度、工作时间等各个反应条件，并对建立的IPMA筛选方法的特异性、敏感性和重复性进行评价。结果显示，建立的IPMA检测方法的最优反应条件为MDCK细胞接毒10^2.63 TCID₅₀/100 μL H1N1亚型猪流感病毒，37℃培养24 h，含3‰ H₂O₂的甲醇室温固定15 min，5%脱脂乳37℃封闭2 h，50 μL杂交瘤细胞上清作为一抗，37℃孵育2 h，羊抗鼠HRP-IgG二抗37℃孵育1 h。所建立的IPMA方法能特异性地检测H1N1亚型猪流感病毒单克隆抗体，与猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）和猪瘟病毒（CSFV）阳性血清不发生交叉反应；其敏感性检测结果显示，可检测1：3 200的HI=2^-9标准H1N1猪阳性血清；批间和批内重复性试验结果较好。综上所述，本试验成功建立了抗H1N1亚型猪流感病毒单克隆抗体的IPMA检测方法，该方法特异性强、敏感性高、重复性好，为生产鉴定H1N1亚型猪流感病毒单克隆抗体提供了一种简便、实用、有效的检测手段。

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

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.