欧亚类禽H1N1猪流感病毒HA蛋白单克隆抗体的制备及竞争ELISA检测方法的建立

Preparation of monoclonal antibody against Eurasian avian-like H1N1 swine influenza virus HA protein and development of competitive ELISA method

为建立一种快速检测欧亚类禽H1N1猪流感病毒(EA H1N1 SIV)抗体的检测方法,本研究以EA H1N1 SIV代表株A/Swine/Liaoning/445/2018(LN445)灭活的全病毒免疫BALB/c小鼠,通过HI试验筛选得到7株HA蛋白单克隆抗体(MAb),分别命名为1B3、2B12、2E1、3B4、3F6、3G5、3H12。抗体亚类鉴定结果显示,HA蛋白MAb除3B4重链为IgG1之外,其余均为IgG2a,7株MAb轻链均为κ链。间接免疫荧光试验结果显示,7株HA蛋白MAb均可以与EA H1N1 SIV发生特异性反应。采用纯化的EA H1N1 SIV(LN445)灭活全病毒作为包被抗原,以EA H1N1 SIV SPF猪阳性血清作为待检血清,选取HA蛋白MAb 2B12作为竞争抗体,经条件优化后初步建立了检测EA H1N1 SIV抗体的竞争ELISA方法。特异性试验结果显示EA H1N1 SIV的阳性血清对2B12 MAb具有良好的阻断作用,而甲型H1N1 SIV、H3N2 SIV、猪圆环病毒2型(PCV2)、轮状病毒(RV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、非洲猪瘟病毒(ASFV)、猪瘟病毒(CSFV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)和肠致病性大肠杆菌(EPEC)阳性血清对其均无阻断作用,表明该方法的特异性较强;敏感性试验结果显示,该方法对HI效价为1:10的EA H1N1 SIV阳性血清检测仍为阳性结果,敏感性较高;重复性试验结果显示,该方法批内、批间变异系数均小于10%,重复性较好。利用本研究建立的竞争ELISA方法与GB/T 27535-2011猪流感HI抗体检测方法同时对100份临床猪血清样品进行检测,结果显示该方法与国标HI检测方法总体符合率为81%,表明该方法的准确性较高。本研究为EA H1N1 SIV抗体检测和流行病学调查提供了有效的检测手段。

To establish a rapid diagnostic method for the detection of Eurasian avian-like H1N1 swine influenza virus(EA H1N1 SIV) antibodies, BALB/c mice were immunized with inactivated whole virus originated from EA H1N1 SIV representative strain A/Swine/Liaoning/445/2018(LN445), and seven HA protein monoclonal antibodies(MAb) were selected, designated as 1B3,2B12, 2E1, 3B4, 3F6, 3G5, and 3H12, respectively. The results of antibody subclass identification showed that the heavy chains of the MAbs are IgG2a except for that of 3B4, which is IgG1, while the light chains of all seven MAbs are κ chain. The results of indirect immunofluorescence assay showed that all seven HA protein MAbs could specifically react with EA SIV. A competitive ELISA was preliminarily established for detection of EA H1N1 SIV antibody, with purified EA H1N1 SIV(LN445) inactivated whole virus as coating antigen, EA H1N1 SIV positive SPF swine serum as positive serum, and MAb 2B12 as competitive antibody.The results of specificity test showed that the positive serum of EA H1N1 SIV has a good blocking effect against 2B12MAb, while the positive sera of classic H1N1 SIV, H3N2 SIV, porcine circovirus 2(PCV2), rotavirus(RV), porcine epidemic diarrhea virus(PEDV), transmissible gastroenteritis virus(TGEV), African swine fever virus(ASFV), classical swine fever virus(CSFV), porcine reproductive respiratory syndrome virus(PRRSV) and enteropathogenic Escherichia coli(EPEC) have no blocking effect, indicating the good specificity of the established method;the sensitivity test results showed that diluted EA H1N1 SIV positive serum with hemagglutination inhibition titer of 1:10 is still detectable by this method, indicating the good sensitivity;the repeatability test results showed that the intra-batch and inter-batch coefficients of variation of this method were less than 10%;100 clinical serum samples were tested with the competitive ELISA method established in this study and GB/T 27535-2011 swine influenza HI antibody detection method. The results showed that the overall coincidence rate between this method and the standard HI detection method was 81%, indicating the good accuracy of this method. This study provides an effective detection method for EA H1N1 SIV antibody surveillance.