T. pyogenes PLO蛋白单克隆抗体的制备与AC-ELISA方法的建立

Preparation of monoclonal antibodies to T. pyogenes PLO protein and development of an AC-ELISA detection method

为了建立可以定量检测化脓隐秘杆菌(T.pyogenes)培养上清液中PLO蛋白含量的方法,试验首先制备针对毒力蛋白PLO分子第4结构域(D4)的单克隆抗体,然后应用筛选出的最佳捕获抗体,并结合辣根过氧化物酶(HRP)标记的靶向PLO分子第2结构域(D2)的单克隆抗体(HRP-AP-1A3)建立一种用于检测PLO蛋白含量的抗原捕获ELISA(AC-ELISA)方法,计算该方法的检测线,选择8种非T.pyogenes菌株验证该方法的特异性,绘制AC-ELISA方法的标准曲线,并应用该方法定量检测T.pyogenes HLJ-0912菌株培养上清液中wtPLO蛋白含量。结果表明:通过制备获得的5种单克隆抗体2G3、3G10、4C9、3C8、3C7对PLO蛋白均具有良好的特异性,其中单克隆抗体2G3、3G10、4C9、3C8是IgG2b亚型,均具有较强的抗溶血活性,靶向表位1(VEAGEATGLAWDPWWT);而单克隆抗体3C7是IgG1亚型,抗溶血活性非常弱,靶向表位2(KGTTLNPWVEDNVKS)。筛选出的最佳捕获抗体为单克隆抗体2G3,建立的AC-ELISA方法的最低检测限(OD_(450)值)为0.19。应用建立的AC-ELISA方法检测T.pyogenes HLJ-0912菌株的培养上清液为阳性,OD_(450)值为0.21;检测8种非T.pyogenes菌株的培养上清液均为阴性,OD_(450)值<0.19。标准曲线的方程为y=809.04x^(2)-85.695x,相关系数(R^(2))为0.999,拟合度较高。经AC-ELISA方法测定,在10,12,14小时时采集的T.pyogenes菌株培养上清液的OD_(450)值均高于最低检测限,培养上清液中的wtPLO蛋白含量分别为416.67,499.43,272.57 ng/mL。说明试验成功制备了针对PLO分子第4结构域的单克隆抗体,建立的AC-ELISA方法具有良好的特异性。

In order to establish a method for the quantitative determination of PLO protein content in culture supernatants of Trueperella pyogenes(T. pyogenes), a monoclonal antibody targeting the structural domain 4(D4) of the virulence protein PLO molecule was first prepared and then the best capture antibody screened was applied to establish an AC-ELISA method for the detection of PLO content in combination with a horseradish peroxidase(HRP)-labeled monoclonal antibody(HRP-AP-1 A3) targeting the structural domain 2(D2) of the PLO molecule. The detection limits of the method were determined;the specificity of the method was verified by selecting eight non-T. pyogenes strains;the standard curve of the AC-ELISA method was plotted, and the method was applied to quantify the wtPLO protein content in the culture supernatant of T. pyogenes HLJ-0912 strain. The results showed that five monoclonal antibodies 2 G3, 3 G10, 4 C9, 3 C8 and 3 C7 were obtained by preparation, all with good specificity for PLO protein, among which monoclonal antibodies 2 G3, 3 G10, 4 C9 and 3 C8 were IgG2 b isoform, all with strong anti-hemolytic activity and targeting epitope Ⅰ(VEAGEATGLAWDPWWT);while monoclonal antibody 3 C7 was an IgG1 isoform with very weak anti-hemolytic activity and targeting epitope Ⅱ(KGTTLNPWVEDNVKS). The best capture antibody was screened as monoclonal antibody 2 G3, and the lowest detection limit(OD_(450) value) of the established AC-ELISA method was 0.19. The culture supernatant of T. pyogenes HLJ-0912 strain was positive with an OD_(450) value of 0.21 when applied to the established AC-ELISA method. The culture supernatants of eight non-T. pyogenes strains were negative with an OD_(450) value < 0.19. The equation of the standard curve was y=809.04x^(2)-85.695x with a correlation coefficient(R^(2)) of 0.999, which was a good fit. The OD_(450) values of the culture supernatants of T. pyogenes strains collected at 10, 12 and 14 h were higher than the lowest detection limit as determined by AC-ELISA method, and the wtPLO contents in the culture supernatants were 416.67, 499.43 and 272.57 ng/mL, respectively. The results suggested that the experiment successfully prepared monoclonal antibody against the structural domain 4 of PLO molecule and the established AC-ELISA method had good specificity.