小鼠血清中弓形虫Peroxiredoxin抗原间接ELISA检测方法的建立及其血清流行病学应用
发布时间：2018-08-31 14:30
【摘要】： 实验目的 建立小鼠血清中弓形虫Peroxiredoxin抗原的间接酶联免疫吸附试验(ELISA)检测方法。用此法检测人血清中弓形虫Peroxiredoxin抗原,进行人群弓形虫病血清流行病学调查。 实验方法 第一部分：应用棋盘滴定方法选择适宜的被检血清浓度、抗体浓度及酶标二抗浓度。观察封闭液对实验结果的影响,并绘制蛋白定量标准曲线。对30份感染弓形虫小鼠血清及30份正常小鼠血清进行间接ELISA检测,评价该方法的检测效果。测定其敏感度、特异性及重复性。 第二部分：采用整群抽样的研究方法,在神池县城和神池农村,各抽取一个居民小组居民作为研究对象。采集1021份空腹静脉晨血,用间接ELISA法检测人血清中弓形虫Peroxiredoxin抗原。 实验结果 被检血清最适包被浓度为1：50,大鼠抗弓形虫血清最佳稀释度为1：100(实际效价1：128),辣根酶标记山羊抗大鼠IgG的最适稀释度为1：4000。封闭液对实验结果的影响不大。该方法敏感度高、重复性好、特异性强,检测30份弓形虫感染小鼠血清的检出率为100%。 不同年龄组人群血清弓形虫IgG抗体阳性率不同,差异无统计学意义(χ2趋势=4.195,P=0.522)。不同性别人群血清弓形虫IgG抗体阳性率不同,差别无统计学意义(χ2=0.000,P=0.990)。不同文化程度人群血清弓形虫IgG抗体阳性率不同,差异无统计学意义(χ2趋势=6.322，P=0.097)。不同职业人群血清弓形虫IgG抗体阳性率不同,差异无统计学意义(χ2趋势=8.584,P=0.284)。调查1021人中,未养犬和猫者弓形虫IgG抗体阳性率低于养犬者和养猫者,差异有统计学意义(χ2=13.157,P=0.004)。有吃生鸡蛋习惯者弓形虫IgG抗体阳性率高于无吃生鸡蛋习惯者,差异有统计学意义(χ2=8.417,P=0.004,)。经常生吃蔬菜和不洁瓜果者弓形虫IgG抗体阳性率高于偶而有该习惯者和无此习惯者,差异有统计学意义(χ2=12.201,P=0.002)。调查1021人中,加工生肉与熟食所用刀板经常不分者血清弓形虫IgG抗体阳性率高于偶尔不分者和从不混同使用者,差异有统计学意义(χ2=9.767,P=0.008)。经常接触泥土者弓形虫IgG抗体阳性率高于偶而接触和从未接触泥土者,差异有统计学意义(χ2=13.753,P=0.001)。饭前便后洗手做到每次或经常洗者弓形虫IgG抗体阳性率低于偶尔或从不洗者,差异有统计学意义(χ2=43.125,P0.001)。特殊人群弓形虫IgG抗体阳性率分别为：临床免疫功能低下者为20.00%,献血员17.85%,屠宰人员和生肉销售者27.78%,其中屠宰人员和生肉销售者弓形虫IgG抗体阳性率最高,差异无统计学意义(χ2=1.012,P=0.603)。 结论 建立了小鼠血清中弓形虫Prx抗原的间接ELISA法。该方法敏感、特异、稳定,可适用于弓形虫病的诊断及流行病学调查。成功应用ELISA法检测人血清中Peroxiredoxin抗原并应用于血清流行病学研究。
[Abstract]:Objective to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for detection of Toxoplasma gondii Peroxiredoxin antigen in serum of mice. The Peroxiredoxin antigen of Toxoplasma gondii in human serum was detected by this method, and the seroepidemiology of Toxoplasma gondii was investigated. The first part: chessboard titration was used to select the suitable serum concentration, antibody concentration and enzyme labeled second antibody concentration. The effect of blocking solution on the experimental results was observed and the quantitative standard curve of protein was drawn. Indirect ELISA detection was performed in 30 sera of Toxoplasma gondii infected mice and 30 sera of normal mice. Its sensitivity, specificity and repeatability were determined. The second part: adopting cluster sampling method, selecting one resident group resident in Shenchi County and Shenchi Countryside as the research object. 1021 fasting venous morning blood samples were collected for detection of Toxoplasma gondii Peroxiredoxin antigen by indirect ELISA method. The results showed that the optimal coating concentration of the tested serum was 1: 50, the optimal dilution of rat serum against Toxoplasma gondii was 1: 100 (actual value 1: 128), and the optimal dilution of horseradish labeled goat anti-rat IgG was 1: 4000. The seal fluid has little effect on the experimental results. The method was sensitive, reproducible and specific. The detection rate of 30 sera from mice infected with Toxoplasma gondii was 100. The positive rate of serum Toxoplasma IgG antibody in different age groups was different, and the difference was not statistically significant (蠂 ~ 2 trend: 4.195 / P ~ (0.522). The positive rate of Toxoplasma gondii IgG antibody was different in different sex population, and the difference was not statistically significant (蠂 ~ 2 ~ (0.000) P ~ (0.990). The positive rate of Toxoplasma gondii IgG antibody was different among people with different education level, and the difference was not statistically significant (蠂 ~ 2 trend was 6.322%). The positive rate of serum Toxoplasma IgG antibody in different occupational groups was different, and the difference was not statistically significant (蠂 2 trend was 8.584%, P < 0.284). The positive rate of IgG antibody to Toxoplasma gondii was significantly lower in dogs and cats than that in dogs and cats (蠂 ~ 2 ~ 2 ~ (13.157) P ~ (0. 004). The positive rate of IgG antibody of Toxoplasma gondii was significantly higher in those with eating uncooked egg habit than that without eating raw egg habit (蠂 ~ 2 = 8.417P ~ (0.004), P ~ (0.004). The positive rate of IgG antibody of Toxoplasma gondii was higher in those who often ate raw vegetables and unclean fruits than that in those who had the habit and those who did not (蠂 2 / 12.201P0. 002). The positive rate of serum Toxoplasma gondii IgG antibody was significantly higher in those who were not divided between raw meat and cooked food (蠂 ~ (2) 9.767) (蠂 ~ (2) 9.767) and never mixed with users (蠂 ~ (2) 9.767 P ~ (0.008). The positive rate of IgG antibody of Toxoplasma gondii exposed to soil frequently was significantly higher than that of those exposed to or never exposed to soil (蠂 2 13.753 P0. 001). The positive rate of Toxoplasma gondii IgG antibody in the patients who wash their hands before and after meals was lower than that in those who occasionally or never washed (蠂 2: 43.125, P 0.001). The positive rates of Toxoplasma gondii IgG antibody were 20.00 in patients with low clinical immune function, 17.85 in blood donors, 27.78 in slaughtermen and meat sellers, among which the positive rates of Toxoplasma gondii IgG antibody were the highest among slaughtermen and raw meat sellers (蠂 ~ 2 ~ (2) 1. 012). There was no significant difference between them (蠂 ~ 2 ~ 2 ~ (1.012) P ~ (0.603). Conclusion an indirect ELISA method for the detection of Toxoplasma gondii Prx antigen in mouse serum was established. The method is sensitive, specific and stable. It can be used for diagnosis and epidemiological investigation of toxoplasmosis. ELISA method was successfully used to detect Peroxiredoxin antigen in human serum and applied to seroepidemiology.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R181.3
本文编号：2215275
[Abstract]:Objective to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for detection of Toxoplasma gondii Peroxiredoxin antigen in serum of mice. The Peroxiredoxin antigen of Toxoplasma gondii in human serum was detected by this method, and the seroepidemiology of Toxoplasma gondii was investigated. The first part: chessboard titration was used to select the suitable serum concentration, antibody concentration and enzyme labeled second antibody concentration. The effect of blocking solution on the experimental results was observed and the quantitative standard curve of protein was drawn. Indirect ELISA detection was performed in 30 sera of Toxoplasma gondii infected mice and 30 sera of normal mice. Its sensitivity, specificity and repeatability were determined. The second part: adopting cluster sampling method, selecting one resident group resident in Shenchi County and Shenchi Countryside as the research object. 1021 fasting venous morning blood samples were collected for detection of Toxoplasma gondii Peroxiredoxin antigen by indirect ELISA method. The results showed that the optimal coating concentration of the tested serum was 1: 50, the optimal dilution of rat serum against Toxoplasma gondii was 1: 100 (actual value 1: 128), and the optimal dilution of horseradish labeled goat anti-rat IgG was 1: 4000. The seal fluid has little effect on the experimental results. The method was sensitive, reproducible and specific. The detection rate of 30 sera from mice infected with Toxoplasma gondii was 100. The positive rate of serum Toxoplasma IgG antibody in different age groups was different, and the difference was not statistically significant (蠂 ~ 2 trend: 4.195 / P ~ (0.522). The positive rate of Toxoplasma gondii IgG antibody was different in different sex population, and the difference was not statistically significant (蠂 ~ 2 ~ (0.000) P ~ (0.990). The positive rate of Toxoplasma gondii IgG antibody was different among people with different education level, and the difference was not statistically significant (蠂 ~ 2 trend was 6.322%). The positive rate of serum Toxoplasma IgG antibody in different occupational groups was different, and the difference was not statistically significant (蠂 2 trend was 8.584%, P < 0.284). The positive rate of IgG antibody to Toxoplasma gondii was significantly lower in dogs and cats than that in dogs and cats (蠂 ~ 2 ~ 2 ~ (13.157) P ~ (0. 004). The positive rate of IgG antibody of Toxoplasma gondii was significantly higher in those with eating uncooked egg habit than that without eating raw egg habit (蠂 ~ 2 = 8.417P ~ (0.004), P ~ (0.004). The positive rate of IgG antibody of Toxoplasma gondii was higher in those who often ate raw vegetables and unclean fruits than that in those who had the habit and those who did not (蠂 2 / 12.201P0. 002). The positive rate of serum Toxoplasma gondii IgG antibody was significantly higher in those who were not divided between raw meat and cooked food (蠂 ~ (2) 9.767) (蠂 ~ (2) 9.767) and never mixed with users (蠂 ~ (2) 9.767 P ~ (0.008). The positive rate of IgG antibody of Toxoplasma gondii exposed to soil frequently was significantly higher than that of those exposed to or never exposed to soil (蠂 2 13.753 P0. 001). The positive rate of Toxoplasma gondii IgG antibody in the patients who wash their hands before and after meals was lower than that in those who occasionally or never washed (蠂 2: 43.125, P 0.001). The positive rates of Toxoplasma gondii IgG antibody were 20.00 in patients with low clinical immune function, 17.85 in blood donors, 27.78 in slaughtermen and meat sellers, among which the positive rates of Toxoplasma gondii IgG antibody were the highest among slaughtermen and raw meat sellers (蠂 ~ 2 ~ (2) 1. 012). There was no significant difference between them (蠂 ~ 2 ~ 2 ~ (1.012) P ~ (0.603). Conclusion an indirect ELISA method for the detection of Toxoplasma gondii Prx antigen in mouse serum was established. The method is sensitive, specific and stable. It can be used for diagnosis and epidemiological investigation of toxoplasmosis. ELISA method was successfully used to detect Peroxiredoxin antigen in human serum and applied to seroepidemiology.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R181.3
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