Ngu J L, Nkenfou C, Capuli E, McMoli T E, Perler F, Mbwagbor J, Tume C, Nlatte O B, Donfack J, Asonganyi T
Immunology-Biotechnology Laboratories, Faculty of Medicine and Biomedical Sciences, University of Yaoundé, Cameroun.
Trop Med Int Health. 1998 May;3(5):339-48.
Sensitive, specific and low-cost diagnostic tests for onchocerciasis are indispensable for monitoring the efficacy of control programs, as well as for preventing blindness (when the tests are combined with efficacious chemotherapy. Three new tests to detect Onchocerca-specific antigens in tears, dermal fluid and urine employ antibodies to O. volvulus-specific recombinant proteins, Oncho-C27 and OvD3B, encoded by genes within the immunodominant Onchocerca OV 33-3 gene family, and expressed in yeast and in E. coli, respectively. In these assays, Onchocerca-specific antigens in test samples are bound onto a solid surface and revealed using appropriate enzyme-labelled antibodies. Proteins in the samples are first transferred to Hybond-N + membrane disks or nitrocellulose paper using either a transblot or a dotblot machine, and then reacted with specific O. volvulus antibodies. Bound antibodies are revealed with species-specific peroxidase-labelled antibodies and peroxidase substrate. Positive tests give a brown colour. In one of the two assays developed to detect Onchocerca antigens in tears, the sensitivity was enhanced by first adsorbing the specific antibodies onto the membrane surface in order to immobilize and concentrate the Onchocerca-specific antigen molecules on the membrane. The specificity of the recombinant proteins for Onchocerca volvulus had been verified by ELISA, classical Western blot and modified DSIA. The tests are a dipstick immunobinding assay for ocular microfilariae (DSIA), a transblot immunobinding assay for the detection of skin microfilariae (TADA) and a dot-blot immunobinding assay for detecting urinary microfilariae and their antigens (DIA). Their specificity and sensitivity were evaluated in the field on 110 subjects with proven ocular microfilariae, 130 subjects with clinical and parasitological evidence of onchocerciasis, 25 subjects infected with other helminths and 120 normal controls. The minimal detection limits of Oncho-C27 protein by DSIA, TADA and DIA were 500 ng/ml, 154 ng/ml and 508 ng/ml, respectively By contrast, their sensitivities were: 100% for DSIA and 82.5% for TADA employed on samples of tears; 97% for TADA skin test and 96% for DIA used on urine samples.
盘尾丝虫病的灵敏、特异且低成本的诊断测试对于监测控制项目的效果以及预防失明（当测试与有效的化疗相结合时）而言不可或缺。三种用于检测眼泪、皮肤渗出液和尿液中盘尾丝虫特异性抗原的新测试采用了针对旋盘尾丝虫特异性重组蛋白Oncho - C27和OvD3B的抗体，这些重组蛋白分别由免疫显性盘尾丝虫OV 33 - 3基因家族中的基因编码，并分别在酵母和大肠杆菌中表达。在这些检测中，测试样品中的盘尾丝虫特异性抗原被结合到固体表面，并使用适当的酶标记抗体进行显色。样品中的蛋白质首先使用转印或点印机转移到Hybond - N +膜盘或硝酸纤维素纸上，然后与特异性旋盘尾丝虫抗体反应。结合的抗体用物种特异性过氧化物酶标记抗体和过氧化物酶底物显色。阳性测试呈现棕色。在开发的两种检测眼泪中盘尾丝虫抗原的测试之一中，通过首先将特异性抗体吸附到膜表面，以固定和浓缩膜上的盘尾丝虫特异性抗原分子，从而提高了灵敏度。重组蛋白对旋盘尾丝虫的特异性已通过酶联免疫吸附测定（ELISA）、经典蛋白质印迹法和改良的双位点免疫放射分析（DSIA）得到验证。这些测试分别是用于检测眼内微丝蚴的试纸条免疫结合测定（DSIA）、用于检测皮肤微丝蚴的转印免疫结合测定（TADA）以及用于检测尿液微丝蚴及其抗原的点印迹免疫结合测定（DIA）。在现场对110名经证实有眼内微丝蚴的受试者、130名有盘尾丝虫病临床和寄生虫学证据的受试者、25名感染其他蠕虫的受试者以及120名正常对照进行了它们的特异性和灵敏度评估。DSIA、TADA和DIA对Oncho - C27蛋白的最低检测限分别为500 ng/ml、154 ng/ml和508 ng/ml。相比之下，它们的灵敏度分别为：用于眼泪样本的DSIA为100%，TADA为82.5%；用于皮肤样本的TADA为97%，用于尿液样本的DIA为96%。
