小鼠血清中弓形蟲Peroxiredoxin抗原間接ELISA檢測方法的建立及其血清流行病學應用
發(fā)布時間：2018-08-31 14:30
【摘要】： 實驗目的 建立小鼠血清中弓形蟲Peroxiredoxin抗原的間接酶聯(lián)免疫吸附試驗(ELISA)檢測方法。用此法檢測人血清中弓形蟲Peroxiredoxin抗原,進行人群弓形蟲病血清流行病學調(diào)查。 實驗方法 第一部分：應用棋盤滴定方法選擇適宜的被檢血清濃度、抗體濃度及酶標二抗?jié)舛�。觀察封閉液對實驗結果的影響,并繪制蛋白定量標準曲線。對30份感染弓形蟲小鼠血清及30份正常小鼠血清進行間接ELISA檢測,評價該方法的檢測效果。測定其敏感度、特異性及重復性。 第二部分：采用整群抽樣的研究方法,在神池縣城和神池農(nóng)村,各抽取一個居民小組居民作為研究對象。采集1021份空腹靜脈晨血,用間接ELISA法檢測人血清中弓形蟲Peroxiredoxin抗原。 實驗結果 被檢血清最適包被濃度為1：50,大鼠抗弓形蟲血清最佳稀釋度為1：100(實際效價1：128),辣根酶標記山羊抗大鼠IgG的最適稀釋度為1：4000。封閉液對實驗結果的影響不大。該方法敏感度高、重復性好、特異性強,檢測30份弓形蟲感染小鼠血清的檢出率為100%。 不同年齡組人群血清弓形蟲IgG抗體陽性率不同,差異無統(tǒng)計學意義(χ2趨勢=4.195,P=0.522)。不同性別人群血清弓形蟲IgG抗體陽性率不同,差別無統(tǒng)計學意義(χ2=0.000,P=0.990)。不同文化程度人群血清弓形蟲IgG抗體陽性率不同,差異無統(tǒng)計學意義(χ2趨勢=6.322，P=0.097)。不同職業(yè)人群血清弓形蟲IgG抗體陽性率不同,差異無統(tǒng)計學意義(χ2趨勢=8.584,P=0.284)。調(diào)查1021人中,未養(yǎng)犬和貓者弓形蟲IgG抗體陽性率低于養(yǎng)犬者和養(yǎng)貓者,差異有統(tǒng)計學意義(χ2=13.157,P=0.004)。有吃生雞蛋習慣者弓形蟲IgG抗體陽性率高于無吃生雞蛋習慣者,差異有統(tǒng)計學意義(χ2=8.417,P=0.004,)。經(jīng)常生吃蔬菜和不潔瓜果者弓形蟲IgG抗體陽性率高于偶而有該習慣者和無此習慣者,差異有統(tǒng)計學意義(χ2=12.201,P=0.002)。調(diào)查1021人中,加工生肉與熟食所用刀板經(jīng)常不分者血清弓形蟲IgG抗體陽性率高于偶爾不分者和從不混同使用者,差異有統(tǒng)計學意義(χ2=9.767,P=0.008)。經(jīng)常接觸泥土者弓形蟲IgG抗體陽性率高于偶而接觸和從未接觸泥土者,差異有統(tǒng)計學意義(χ2=13.753,P=0.001)。飯前便后洗手做到每次或經(jīng)常洗者弓形蟲IgG抗體陽性率低于偶爾或從不洗者,差異有統(tǒng)計學意義(χ2=43.125,P0.001)。特殊人群弓形蟲IgG抗體陽性率分別為：臨床免疫功能低下者為20.00%,獻血員17.85%,屠宰人員和生肉銷售者27.78%,其中屠宰人員和生肉銷售者弓形蟲IgG抗體陽性率最高,差異無統(tǒng)計學意義(χ2=1.012,P=0.603)。 結論 建立了小鼠血清中弓形蟲Prx抗原的間接ELISA法。該方法敏感、特異、穩(wěn)定,可適用于弓形蟲病的診斷及流行病學調(diào)查。成功應用ELISA法檢測人血清中Peroxiredoxin抗原并應用于血清流行病學研究。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R181.3
本文編號：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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R181.3
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