应用重组抗原(rSAG1)检测IgM抗体诊断弓形虫病的研究

DETECTION OF IgM ANTIBODY WITH RECOMBINANT ANTIGEN rSAG1 FOR TOXOPLASMOSIS DIAGNOSIS

目的 以刚地弓形虫 RH株重组主要表面抗原 1作为检测抗原 ,建立检测弓形虫 Ig M抗体的 r SAG1- EL ISA。方法 用 Ni2 +螯合柱纯化重组抗原 r SAG1;以不同浓度的 r SAG1的包被聚苯乙烯酶标条 ,阳性和阴性混合血清分别作 1∶ 10 0和 1∶ 2 0 0稀释 ,以辣根过氧化物酶标记的羊抗人Ig M为二抗 ,采用正交试验确定 r SAG1- EL ISA的最佳检测条件 ;按其最佳检测条件分别对弓形虫Ig M阳性和阴性混合血清重复测 2 0次进行精确度的检测 ;采用抗体抑制试验检测其特异性 ;同时对用进口试剂盒筛得的 35份弓形虫 Ig M阳性血清和 5 7份弓形虫 Ig M阴性血清进行检测。结果 制备的 r SAG1重组蛋白纯度在 90 %以上 ;r SAG1- EL ISA的最佳检测条件为 :r SAG1的包被浓度为2 .5μg/ m l,血清稀释度 1∶ 10 0 ,酶标记的羊抗人 Ig M 1∶ 4 0 0 0稀释 ;用 r SAG1- EL ISA对混合弓形虫 Ig M阳性和阴性血清的重复检测表明 :Ig M阳性混合血清检测值的变异系数 (CV值 )为 13.8% ,Ig M阴性混合血清的 CV值为 7.7% ;灵敏度检测表明血清稀释度在 1∶ 5 0～ 1∶ 2 0 0均可检出阳性 ;特异性试验的抑制率为 6 2 .0 % ;r SAG1- EL ISA与进口试剂盒的总符合率为 88.0 % ,其中阳性和阴性符合率分别为 82 .9% (2 9/ 35 )和 91.2 % (5 2 /

Objective To establish rSAG1-IgM-ELISA with purified rSAG1 fusion protein for immunodiagnosis of toxoplasmosis. Methods The rSAG1 fusion protein was purified by Ni 2+ column. The ELISA plate was coated with different concentrations of rSAG1, reacted with pooled positive and negtive human sera. Goat anti-human IgM conjugated to horseradish peroxidase was used as the second antibody. The appropriate detecting condition of the rSAG1-IgM-ELISA assay was determined by orthogonal experiment. The reproducibility, sensitivity and specificity of the assay were assessed. Thirty-five IgM-positive and 57 IgM-negative human sera detected by the imported IgM-ELISA kit were detected with the rSAG1-IgM-ELISA. Results The purity of rSAG1 was above 90%. The appropriate detecting condition was that the coated rSAG1 was 2 5 μg/ml, the human serum was in 1∶100 dilution, and the second antibody was in 1∶4000 dilution. The coefficient of variation (CV) value of IgM-positive and IgM-negative pooled sera were 13 8% and 7 7% respectively. The inhibition rate of the assay was 62 0% The positive correspondence rate and negative correspondence rate were 82 9% (29/35) and 91 2% (52/57) respectively,the total correspondence rate was 88 0%, compared with the imported IgM-ELISA kit. Conclusions The rSAG1-IgM-ELISA has high sensitivity and specificity, and good correspondence rate with the imported IgM-ELISA kit. It indicates that rSAG1-IgM-ELISA has potential value for early diagnosis of toxoplasmosis.