基于人工多表位抗原的恶性疟疾抗血清筛查方法的建立  

Development of a method for screening of antisera agaisnt Plasmodium falciparum malaria based on artificial multi-epitope antigen

作　　者：姚美雪 邓唯唯[1] 张国强[2] 张佳佳[1] 王恒[1] 

机构地区：[1]中国医学科学院基础医学研究所病原系寄生虫研究室,北京100005 [2]北京生物制品研究所有限责任公司,北京101100

出　　处：《中国生物制品学杂志》2016年第9期967-972,共6页Chinese Journal of Biologicals

基　　金：国家传染病科技重大专项(2012ZX10004220)

摘　　要：目的建立基于人工多表位抗原的恶性疟疾抗血清筛查方法。方法以人工多表位抗原M.RCAg-1（malaria random constructed antigen-1）和M.RCAg-3（malaria random constructed antigen-3）作为检测蛋白,建立间接ELISA法和双抗原夹心ELISA法,并对两种方法的抗原包被量、一抗稀释度、封闭液、孵育时间及显色时间进行优化。用两种方法检测恶性疟疾病人和正常人血清抗体水平,比较其敏感度,选出最佳检测方法,并检验其特异性和精密性。结果间接ELISA法最佳检测条件：以0.1μg/孔浓度的抗原包被酶标板120 min,用3%羊血清封闭120 min,加入一抗（1∶200稀释）孵育120 min,加入二抗（1∶20 000稀释）孵育60 min,TMB显色10 min;双抗原夹心ELISA法最佳检测条件：一抗稀释度为1∶20,二抗稀释度为1∶100,其他同间接ELISA法。经敏感性比较,确定以M.RCAg-1为抗原的间接ELISA法为最佳检测方法,且该抗原对间日疟血清抗体无明显交叉反应,该方法的批内和批间精密性试验变异系数（CV）为3.05%-5.82%和4.75%-8.54%,均〈10%。结论单独包被M.RCAg-1的间接ELISA法的检出率高,特异性和精密性良好,操作简便,可用于疫区恶性疟疾的血清流行病学筛查。Objective To develop a method for screening of antisera against Plasmodium falciparum malaria based on artificial multi-epitope antigen. Methods Indirect ELISA and double antigen sandwich ELISA were developed using malaria random constructed antigen-1（M. RCAg-1） and malaria random constructed antigen-3（M. RCAg-3） as coating antigens, of which the antigen content for coating, dilution of the first antigen, blocking solution as well as time durations for incubation and color development were optimized. The serum antibody levels of patients with P. falciparum malaria and healthy persons were determined by the two methods separately, of which the sensitivities were compared. The optimal method was screened and tested for specificity and precision. Results The condition for indirect ELISA was optimized as follows： microtiter plate was coated with antigen for 120 min, 0. 1 μg per well, and blocked with 3% goat serum for 120 min, then added with the first antigen（1 ∶ 200 diluted） and incubated for 120 min, followed by addition with the second antigen（1 ∶ 20 000 diluted）, incubation for 60 min, and color development with TMB for 10 min. However, the condition for double antigen sandwich ELISA was basically identical to that for indirect ELISA, except that the dilutions of the first and the second antigens were 1 ∶ 20 and 1 ∶ 100 respectively. The indirect ELISA using M. RACAg-1 as antigen was defined as the optimal method based on sensitivity test, which showed no cross reaction with serum antibody against Plasmodium vivax. The CVs of test results by the method in intra- and inter-assays were 3. 05% - 5. 82% and 4. 75% -8. 54% respectively, both of which were less than 10%. Conclusion Indirect ELISA method using M.RCAg-1 showed high sensitivity, specificity and precision, which was easy to handle and suitable for the seroepidemiological screening of P. falciparum malaria in epidemic area.

关 键 词：恶性疟疾 人工多表位抗原 酶联免疫吸附试验 

分 类 号：R382.31[医药卫生—医学寄生虫学] R392-33[医药卫生—基础医学]