双抗原夹心ELISA法检测马尔尼菲青霉Mp1p抗体

摘要

目的 建立一种检测马尔尼菲青霉特异性抗体的双抗原夹心ELISA方法.方法 利用毕赤酵母系统表达重组马尔尼菲青霉特异性甘露糖蛋白Mp1p,并利用改良过碘酸钠法标记Mp1p,经棋盘滴定法建立一种可检测马尔尼菲青霉Mp1p特异性抗体的双抗原夹心ELISA法,并检测100例健康人群对照血清、21例血培养确诊其他真菌感染病人血清和15例血培养确诊马尔尼菲青霉病人血清,联合本实验室前期建立的马尔尼菲青霉抗原检测方法评价其临床应用价值.结果 成功建立一种检测马尔尼菲青霉Mp1p特异性抗体的双抗原夹心ELISA法,经健康人群对照及其他真菌感染病人血清评价特异度为100％(121/121),检测15例马尔尼菲青霉病人血清,Mp1p特异性抗体2例阳性,Mp1p特异性抗原12例阳性,Mp1p抗体与抗原联合检测可明显提高灵敏度,达到93.3％(14/15).结论 双抗原夹心ELISA法检测马尔尼菲青霉Mp1p特异性抗体具有高度的特异性,联合抗原检测可提高马尔尼菲青霉感染诊断率.%Objective To establish an immunological method for detecting antibodies of Penicillium marneffei. Methods The recombinant Mplp protein of Penicillium marneffei was expressed in Pichia pastoris and labeled with HRP (Mp1p-HRP) with a modified sodium periodate method. A double-antigen sandwich enzyme-linked immunosorbant assay (ELISA) was established by determining the optimal coating concentration of Mplp protein and the concentration of the detecting protein Mplp-HRP. The sensitivity and specificity of the assay was evaluated by detecting Mplp antibodies in 100 serum samples from healthy donors, 15 samples from culture-confirmed penicilliosis patients, and 21 samples from patients with culture-confirmed other fungal infections. Results A double-antigen sandwich ELISA was successfully established for detecting Mplp-specific antibody. The specificity of the assay was 100% (121/121) for detecting Mplp-specific antibody in the sera from healthy donors and patients with other fungal infection. The detection results of the 15 serum samples from patients with culture-confirmed penicilliosis showed positivity for Mplp antibody in 2 samples and Mplp antigen positivity in 12 samples; combining the detection results of Mplp antigen and antibody obviously increased the diagnostic sensitivity to 93.3% (14/15). Conclusion The double-antigen sandwich ELISA shows a high specificity in detecting Mplp-specific antibody, and simultaneous detection of Mplp antigen and antibody can increase the diagnostic sensitivity for penicilliosis.