两种鉴别诊断恶性疟和间日疟免疫层析试条检测效果评价

Evaluation on two colloid gold labeled strip tests for rapid detection and dif- ferentiation of Plasmodium vivax and P. falciparum

目的对两种快速、简便能区分诊断恶性疟和间日疟的胶体金免疫层析试条的检测效果进行评价。方法筛选基于恶性疟原虫富组蛋白Ⅱ和恶性疟原虫乳酸脱氢酶制备的单克隆抗体对，采用柠檬酸三钠还原法制备胶体金颗粒，标记筛选到的单克隆抗体C6E9和G4C9，并将其同时吸附于样品垫；将单克隆抗体B2G10（针对恶性疟原虫与间日疟原虫）和C6H11（只针对恶性疟原虫）分别划线包被于同一硝酸纤维素膜适当位置，制成免疫层析检测试条。用该试条检测疫区非疟疾发热病人血样（120份）和内脏利什曼病患者血样（20份）以评价其特异性，检测确诊的疟疾患者血样（间日疟105份，恶性疟95份）以评价其敏感性。均用单盲法检测，同时用基于恶性疟原虫乳酸脱氢酶制备的单克隆抗体对制备的试条进行平行检测。结果检测120份疫区非疟疾发热病人血样和20份内脏利什曼病患者血样，有138份显示为阴性，特异性为98．6％，其中20份内脏利什曼病患者血样全部为阴性。检测200份疟疾患者血样，阳性190份，敏感性为95．0％，其中间日疟检出率为9313％（98／105），恶性疟检出率为96．8％（92／95）。基于恶性疟原虫乳酸脱氢酶制备的单克隆抗体对制备的试条检测的敏感性和特异性分别为93．5％（187／200）和95．7％（134／140），与本研究制备的基于恶性疟原虫富组蛋白Ⅱ和恶性疟原虫乳酸脱氢酶制备的单克隆抗体对的试条比较两者敏感性（χ2=0．42，P〉0．05）和特异性（χ2=1．09，P〉0．05）的差异均无统计学意义。结论研制出的基于恶性疟原虫富组蛋白Ⅱ和恶性疟原虫乳酸脱氢酶制备的单克隆抗体对的快速诊断疟疾胶体金免疫层析试条敏感性、特异性均较高。

Objective To evaluate the effect of two gold immunochromatographic strip tests for detection and differentiation of Plasmodium vivax and P. falciparum. Methods The monoelonal antibodies were screened for detection and differentiation of P. vivax and P. falciparum based on the P falciparum histidinerich protein Ⅱ （HRP-Ⅱ ） and P. falciparum lactate dehydrogenase （LDH）. Firstly, the monoclonal antibodies, C6E9 and G4C9, were conjugated with colloid gold as detecting reagent. Secondly, the monoclonal antibodies B2G10 （targeting on P. vivax/P, faciparum） and C6H11 （only targeting on P. falciparum） were immobilized on same nitrocellulose in proper position. Blood samples from 120 febrile patients without malaria infection from endemic area of malaria and 20 patients with visceral leishmaniasis were used for evaluating the specificity of the tests. Blood samples of malaria patients（ 105 with P. vivax and 95 with P.falciparum） were used for evaluating the sensitivity of the tests. The results of our immunoehromatographic strip were compared with that of the immunochromatographic strip based on the monoelonal antibodies of LDH. Results Only two samples outof 120 febrile patients and 20 patients with visceral leishmaniasis showed false positive reaction with a specificity of 98.6%（138/140）, moreover, all the 20 samples from patients with visceral leishmaniasis were nega- tive. Two-hundred blood samples of malaria patients showed a sensitivity of 95.0%（190/200） totally, and sen- sitivities of 93.3%（98/105） and 96.8% （92/95） for patients infected with P. vivax or P. falciparum, respectively. The sensitivity and specificity of the immunochromatographic strip based on the monoclonal antibodies of LDH were 93.5% （187/200） and 95.7% （134/140）, respectively. Both sensitivity and specificity showed no statistical difference between the immunochromatographic strip based on the monoclonal antibodies of LDH and the immunochromatographic strip based on the monoclonal antibodies of HRP-Ⅱ and LDH. Conclusion The immunochromatographic strip test based on the monoclonal antibodies of HRP- Ⅱ and LDH is a sensitive, specific, simple and rapid assay for malaria diagnosis.