侵袭性曲霉感染特异性抗体的筛选及其初步临床应用研究

Identification and priliminary application of monoclonal antibodies against inrisive Aspergillus

目的 筛选出可用于侵袭性曲霉感染实验室早期诊断的特异性单克隆抗体.方法 应用单克隆抗体技术制备针对不同曲霉抗原的特异性抗曲霉单克隆抗体,根据单抗识别表位不同,初步筛选出两组灵敏度高、特异性强的单抗组合,并分别建立了双抗体夹心ELISA检测法.通过检测常见临床和环境分离曲霉株、马尔尼菲青霉及念珠菌培养液,感染动物模型标本,临床标本,初步评估检测方法的敏感性和特异性.并应用WB对抗体所识别靶标进行初步鉴定.结果 获得32株稳定分泌曲霉单抗的杂交瘤细胞株,建立了2种双抗体夹心ELISA法,第1种方法可识别环境和临床分离的19种曲霉.第2种方法仅特异性识别临床和环境分离的烟曲霉,与其他曲霉抗原无交叉.对同一种烟曲霉培养液而言,第1种方法可检测稀释度为第2种方法的10倍.WB分析显示该组单抗识别的靶标为相对分子质量在25 000～75 000之间的甘露聚糖蛋白.结论 本研究获得了可用于侵袭性曲霉感染实验室诊断的特异性单克隆抗体,单抗识别的抗原为相对分子质量在25 000～75 000之间的甘露聚糖蛋白.该抗原是侵袭性曲霉感染早期诊断的潜在标志物.

Objective To screen monoclonal antibodies （mAbs） for early diagnosis of invisive Aspergillus. Methods Monoclonal antibodies against different antigens of Aspergillus fumigatus were produced. The two pairs of combinations of monoclonal antibodies were selected accoring the distinct epitopes and double-antibody sandwich ELISA based on mAbs above were established. The sensitivity and specificity of the methods were analyzed by detecting culture supernatants of clinical isolates and environmental isolatesof Aspergillus. spp, Penicillium Marneffei, Candidas, and serum from animal models and patients. The epitopes recognized by mAbs were identified by immunobotting. Results A total of 32 hybridoma cell lines that stably produced MAbs were obtained. Two double- antibody sandwich ELISAs were established. One method was specific for 19 clinical isolates and environmental isolates of Aspergillus. spp, whereas the other one was specific for the clinical and environmental isolates of Aspergillus fumigatus without cross-reation with other Aspergillus. spp. For the same kind of medium of Aspergillus fumigatus, the sensitivity of the first method was 10 fold higher than the second method. Conclusions The specific mAbs for early diagnosis of invisive Aspergillus were obtained. Antigen recognized by the specific mAbs was mannoprotein with molecular weights of approximately 25 000-75 000. This antigen was potential early diagnostic marker for invasive Aspergillus.