用乳酸脱氢酶为靶分子的免疫层析技术检测恶性疟原虫

摘要

目的建立一种免疫胶体金层析(GICA)法用于恶性疟原虫的检测.方法采用杂交瘤技术制备恶性疟原虫乳酸脱氢酶(LDHpf)单抗,利用筛选出来的单抗制成诊断恶性疟原虫的免疫层析条,以镜检和PCR法为对照,用GICA条检测门诊"四热"病人血样,评价其敏感性和特异性,并对其稳定性进行了初步研究.结果筛选出4株抗LDHpf单抗,间接ELISA表明4株单抗仅与恶性疟原虫发生反应,而与间日疟原虫、红细胞蛋白、刚地弓形虫和日本血吸虫不发生交叉反应.Western-blot结果显示4株单抗能识别恶性疟原虫相对分子质量约为33000处的虫源蛋白.以镜检法和PCR法为标准,GICA条检测恶性疟原虫的敏感性分别为88.37%和86.67%,GICA与镜检法的符合率均为91.55%.结论GICA检测恶性疟原虫简易快速、灵敏度高,无需特殊仪器设备,有望成为一种恶性疟原虫快速免疫诊断试剂盒.%Objective To establish a colloid gold-immunochromatography assay (GICA) for detecting Plasmodiumfalciparum.Methods The monoclonal antibodies (mAbs) against lactate dehydrogenase ofP. falciparum (LDHpf) were screened for preparation of GICA strips. With microscopic examination of the blood smears and PCR test as control, GICAwas evaluated for its sensitivity, specificity and stability in the diagnosis of malaria in the outpatient clinics in China. Results Four hybridoma cell lines against LDHpf were prepared. Enzyme-linked immunosorbent assay demonstrated that the 4 mAbs reacted only with P. falciparum, but not with protein of normal human red cell, P. vivax, Toxoplasma gondii, or Schistosomajaponicam. All the 4mAbs recognized a 33 kD protein designated as LDHpf as shown by Western blot analysis. Compared with the microscopic examination of blood smears and PCR test, GICA had the sensitivities of 88.37% and 86.67% and the specificities of 95.65%and 97.78%, respectively. Concordance between microscopic examination and GICA for P. falciparum infection was91.55%.Conclusion GICA established in this study is simple, rapid, sensitive and specific for detecting P. falciparum, and is potentially useful in developing reagent kits for clinical use.