单克隆抗体检测广州管圆线虫循环抗原的研究

Detection of Angiostrongylus cantonensis circulating antigen by monoclonal antibodies

目的制备抗广州管圆线虫成虫可溶性抗原单克隆抗体,用于广州管圆线虫循环抗原(CAg)的检测。方法广州管圆线虫成虫可溶性抗原免疫BALB/c小鼠,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,用酶联免疫吸附试验(ELISA)和免疫印迹试验对所获得的细胞克隆进行筛选和鉴定,双抗体(单抗3F1和4H2)夹心ELISA法检测实验感染广州管圆线虫大鼠、小鼠和广州管圆线虫病人血清中的CAg。结果获得了3株稳定分泌抗广州管圆线虫成虫可溶性抗原单抗的杂交瘤细胞株,经鉴定2株为IgG1(3F1、4H2),1株为IgM(2A2),杂交瘤细胞培养上清效价分别为1∶25600、1∶25600和1∶12800,诱生腹水ELISA效价分别为1∶80000、1∶80000和1∶40000。3株单抗均识别广州管圆线虫成虫相对分子量约为15000的蛋白。实验感染广州管圆线虫大鼠、小鼠血清CAg检出率分别为84.2%(48/57)和87.2%(41/47),广州管圆线虫病人血清CAg检出率为86.4%(19/22),与日本血吸虫、囊虫、肺吸虫、旋毛虫病人血清无交叉反应。实验感染小鼠血清CAg第2周即呈阳性反应,第4周A450值达到高峰。结论建立了敏感性高、特异性强的3F1、4H2双抗体夹心ELISA检测广州管圆线虫循环抗原法,可用于广州管圆线虫病的临床诊断、疗效考核和流行病学调查。

Objective To prepare monoclonal antibodies （McAbs） against soluble antigens of adult worms of Angiostrongylus cantonensis （A. cantonensis ） on the purpose to detect CAg of A. cantonensis. Methods Female BALB/c mice were immunized with soluble antigens of adult worms of A. cantonensis and the spleen cells were fused with myeloma SP2/O cells. The hybridoma cell strains were screened by enzyme- linked immunosorbent assay（ELISA） and Western blotting. Two McAbs （3F1 and 4H2 ） were applied to detect the CAg in the sera of rats and mice infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA. Results Three McAbs against A. cantonensis adult were obtained. Two McAbs（3F1,4H2） were identified as IgG1 and one McAb（2A2） was identified as IgM. The titers of culture fluid and ascites was 1：25 600,1 ： 25 600,1 ： 12 800 and 1 ： 80 000, 1 ： 80 000,1 ： 40 000 respectively. Western blotting results showed three McAb could be used to identify 15 000 protein of adult worms of A. cantonensis. The detection rates of the CAg in the sera of infected rats and mice were 84. 2% （48/57） and 87. 2% （41/47） respectively. The detection rate of the CAg in the sera of angiostrongyfiasis patients was 86.4% （19/22）, and no cross reactions with sera from patients with schistosomiasis, cysticercosis cellulosae, paragonimiasis and trichinellosis were oberserved. The CAg in the sera from mice examined at different periods after infection revealed positive 2 week after inoculation and the titer of CAg peaked 4 week after inoculation. Conclusion A new method of sandwith ELISA with high sensitivity and specificity to detect the serum A. cantonensis CAg has been obtained, it could be applicable to the diagnosis, observation of curative effect and epidemiology of angiostrongyliasis.