猫弓形虫感染ELISA和胶体金试纸检测方法建立与初步应用

发布时间：2018-04-10 01:30

本文选题：弓形虫　切入点：间接ELISA　出处：《吉林大学》2017年硕士论文

【摘要】：弓形虫是一种全球性分布的人畜共患寄生虫,全世界每年约有190,100人感染弓形虫病。该病的宿主较为广泛,可感染所有温血动物及部分变温动物。健康人群呈隐性感染,一般不出现症状,但艾滋病人及一些患有免疫缺陷类疾病的患者症状较明显。猫既是弓形虫的终末宿主又是中间宿主。人类主要通过摄入被污染的或未煮熟的含有包囊的食物感染。处于妊娠期的人类及动物感染后常表现为妊娠终止,出现流产、产死胎、木乃伊胎、弱子等症状。目前弓形虫的诊断方法主要包括:病原学诊断、分子生物学诊断、免疫学诊断等。弓形虫表面抗原(SAG)基因家族成员较多。主要包括SAG1、SAG2、SAG3三种。SAG3存在于弓形虫侵入宿主细胞的所有阶段,大小在43KDa。家族成员中与SAG3最为相似的是SAG1。SAG1是速殖子的特异性抗原,其免疫原性较好,广泛的应用于核酸疫苗的研究,被认为是弓形虫疫苗制备的首选。SAG3和SAG1具有相同结构和功能,目前对于SAG1的报道有很多,但是关于SAG3的报道较少,因此,本研究选择SAG3进行猫弓形虫感染检测方法的建立。猫弓形虫感染间接ELISA检测方法的建立本研究选用纯化后的弓形虫表面抗原SAG3作为包被抗原,优化条件后进行ELISA检测方法的建立。最后确定抗原的最佳包被量为0.75μg/孔;待检血清的最佳稀释比例为1:800;HRP标记山羊抗猫Ig G最适稀释比例为1:5000;敏感度可达到1:12800;选取猫杯状病毒标准血清和猫传染性胃肠炎病毒标准血清进行特异性试验,结果未有交叉反应出现,本试验方法特异性较好;选取不同生产日期的96孔检测板进行批间和批内试验,结果显示:批间试验和批内试验的变异系数均小于6%。对临床中96份猫血清样品进行检测,结果与商品化间接血凝试剂盒进行对比,结果显示:间接ELISA阳性率为61.46%(59/96),间接血凝试剂盒的阳性率为56.25%(54/96)。猫弓形虫感染Dot-ELISA检测方法的建立本研究利用纯化后的弓形虫SAG3蛋白来进行包被,选取硝酸纤维素膜(NC)膜作为固相载体来进行试验。最终确定本研究方法中待检血清的稀释倍数达到1:1600时为最佳;HRP标记山羊抗猫Ig G最佳稀释度为1:5000;抗原的最佳包被量0.125μg/片;敏感性可达到1:12800;特异性试验证实,本方法与猫杯状病毒标准血清和猫传染性胃肠炎病毒标准血清均无交叉反应。对临床中96份猫血清样品进行检测并与商品化的间接血凝试剂盒进行对比。结果显示,Dot-ELISA试验的阳性率为62.5%(52/96),间接血凝试剂盒的阳性率为56.25%(54/96)。猫弓形虫感染免疫胶体金试纸条的研制本研究选取柠檬酸三钠还原氯金酸来制备胶体金溶液。以金颗粒标记金黄色葡萄球菌A蛋白(SPA)和抗SPA的二抗来组建猫弓形虫病胶体金试纸条。当每毫升胶体金溶液中加入3μL K2CO3(0.2mol/L)混匀后的p H值为最佳;金标SPA的最佳标记量为6μg/m L,检测线(T)质控线(C)最佳包被量分别为0.5mg/m L和1mg/m L。对试纸条进行性能试验,敏感性可达1:640,通过检测猫杯状病毒标准血清和猫传染性胃肠炎病毒标准血清均未出现交叉反应,选取不同批次的试纸进行重复性试验后,重复性较好。对不同储存条件下(室温、4℃、37℃)及不同保存期(1d、7d、30d、60d、90d、120d)的试纸条进行检测,结果显示4℃保存的效果较好,可达到120d。室温加有干燥剂的试纸条保存相对较好,保存期可达到90d(可见轻微条带)。37℃保存的试纸条效果相对较差,保存期在90d时已经失效。对临床中96份猫血清样品进行检测并与商品化的间接血凝试剂盒进行对比。结果显示:免疫胶体金检测方法的阳性率为57.29%(52/96),间接血凝试剂盒的阳性率为56.25%(54/96)。
[Abstract]:Toxoplasma is a global distribution of zoonotic parasites, about 190100 people infected with toxoplasmosis worldwide each year. The host disease widely, can infect all warm blooded animal and animal health. Temperature populations showed a recessive infection, generally do not have symptoms, but some people suffering from AIDS and immunodeficiency disease symptoms than patients the cat is obvious. The end host is the intermediate host of Toxoplasma gondii. Humans mainly through ingestion of contaminated or undercooked food containing cysts in human and animal infection. Infection in pregnancy usually presents after termination of pregnancy, miscarriage, stillbirth, fetal mummies, weak current diagnostic methods and other symptoms. Toxoplasma mainly includes: etiologic diagnosis, molecular diagnosis, immunological diagnosis. Surface antigens of Toxoplasma gondii (SAG) gene family members more. Including SAG1, SAG2, SAG3 three.SAG3 There are in all stages of Toxoplasma gondii invade host cells, in the size of a member of the 43KDa. family, and SAG3 is most similar to SAG1.SAG1 is the specific antigen of tachyzoites, its good immunogenicity, widely used in the research of nucleic acid vaccine, is believed to be the first choice of.SAG3 and SAG1 of Toxoplasma gondii vaccine prepared with the same the structure and function of the SAG1 report that there are many, but few reports about the SAG3, therefore, this study established SAG3 cat Toxoplasma infection detection method. Indirect ELISA method for detection of Toxoplasma infection in cats established in the study of surface antigen SAG3 of Toxoplasma gondii purified as antigen to establish ELISA detection method for optimizing conditions. Finally, the best coating antigens were: 0.75 g/ hole; the optimal dilution ratio of serum samples to be tested for 1:800; HRP labeled Goat anti cat Ig G the optimal dilution ratio of 1: 5000 min; The sensitivity can reach 1:12800; selection of serum and feline calicivirus standard cat transmissible gastroenteritis virus standard serum specific test results, no cross reaction, the specific test method is better; the 96 hole detection board selects different production date of the inter and intra test results show that the coefficient of variation between batch test and in the test group were less than 6%. to detect 96 clinical samples of cat serum samples, the results and the commercialization of the indirect hemagglutination kit were compared, results showed that the positive rate of indirect ELISA was 61.46% (59/96), the positive rate of indirect hemagglutination kit was 56.25% (54/96). To establish an Dot-ELISA method for detection of Toxoplasma gondii in cats the infection on the induction of SAG3 protein after purification of Toxoplasma gondii were selected on nitrocellulose membrane (NC) membrane as the solid phase carrier to test. The final dilution ratio was determined in this study detected the blood To 1:1600 for the best; HRP labeled Goat anti cat Ig G dilution at 1:5000; the best package was 0.125 g/ antigen; sensitivity can reach 1:12800; confirmed the specificity test, serum and feline calicivirus standard and the method of cat transmissible gastroenteritis virus standard serum and no cross reaction was detected in clinical. In 96 samples of cat serum samples and indirect hemagglutination test kit and commercialization were compared. The results showed that the positive rate of Dot-ELISA test was 62.5% (52/96), the positive rate of indirect hemagglutination kit was 56.25% (54/96). The development of GICA for this study selected three citric acid sodium reducing gold chloride acid to prepare colloidal gold solution to Toxoplasma gondii infected cats. Gold particles labeling staphylococcal protein A (SPA) and anti SPA two antibody to form cat toxoplasmosis colloidal gold strip. When adding 3 L K2CO3 per milliliter of colloidal gold solution (0.2mol/ L) mixed P H value is the best; best marks the gold standard of SPA 6 g/m L (T), the detection line control line (C) the best package is respectively 0.5mg/m L and 1mg/m L. performance test was carried out on the test strip, sensitivity of 1:640 by serum cat and feline calicivirus the standard detection of infectious gastroenteritis virus standard serum showed no cross reaction test, selection of different batches of repeatability, reproducibility of different storage conditions (room temperature, 4 C, 37 C) and different storage period (1D, 7d, 30d, 60d, 90d, 120d) of the test strip test results show good preservation effect of 4 deg C, can reach 120d. at room temperature and strip desiccant remain relatively good, the preservation period can reach 90d (visible light band).37 stored at the test strip is relatively poor, the preservation period has expired at 90d. The 96 clinical samples of cat serum samples detection and commodity The indirect hemagglutination test kit was compared. The results showed that the positive rate of immunogold gold detection method was 57.29% (52/96), and the positive rate of indirect hemagglutination kit was 56.25% (54/96).

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.293

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