乙型肝炎病毒标志物时间分辨荧光免疫分析法的研究
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摘要
标记免疫分析技术是一大类高灵敏度、高特异性检测技术的总称，因具有许多独特的优点，广泛应用于基础医学和临床各领域。时间分辨荧光免疫分析技术(TRFIA)是当前标记免疫分析研究应用领域的热点之一，具有高灵敏性和高特异性，用于生物分子的超微量检测。乙型病毒性肝炎(简称乙型肝炎)是由乙型肝炎病毒(简称乙肝病毒)引起的肝脏炎性损害，是我国当前流行最广泛、危害最严重的一种传染病。定量检测乙型肝炎病毒血清学标志物的免疫诊断方法是预防和治疗乙型肝炎的前提，同时也能动态观测和监视病情。我们应用TRFIA建立了乙型肝炎病毒五项血清学标志物的时间分辨荧光免疫分析法，经初步的临床应用，得到较为满意的结果。
通过实验研究，以Eu-DTTA为示踪物，得到如下结论：
1．以夹心法建立了乙型肝炎病毒表面抗原时间分辨免疫荧光分析法(HBsAg-TRFIA)、乙型肝炎病毒表面抗体时间分辨免疫荧光分析法(HBsAb-TRFIA)、乙型肝炎病毒e抗原时间分辨免疫荧光分析法(HBeAg-TRFIA)，所建立的标准曲线分析范围分别为0-300ng／ml、0-1280mIU／ml、0-16NCU／ml，分析灵敏度为0.05ng／ml、0.44mIU／ml、0.03NCU／ml。检测参考标准品的批内变异系数均小于10％，与IRMA同时定量检测多份血清样本，相关系数高达95％。
2．以中和抑制法建立了乙型肝炎病毒e抗体时间分辨荧光免疫分析法(HBeAb-TRFIA)和乙型肝炎病毒核心抗体时间分辨荧光免疫分析法(HBcAb-TRFIA)，标准曲线分析范围分别为0-16NCU／ml和0-8NCU／ml。检测参考标准品的批内变异系数均小于10％，与IRMA同时定量检测多份血清样品，灵敏度分别为92.8％和93.3％。
总之，我们利用Eu-DTTA为标记物建立的乙型肝炎病毒五项血清学标志物时间分辨荧光免疫分析法分析范围宽，灵敏度高，操作简便，具有优越的定量检测能力，十分适合临床推广应用。
Labeled immunoassay are the total conception of a series of highly sensitive and specific examine techniques, and have been widely applied in the basic medicine and clinic field for their special advantages. Time-resolved Fluorescence Immunoassay (TRFIA) has become one of the hotspots in the field of labeled immunoassay application. Hepatitis B is a damage of hepar by the Hepatitis B virus .Now it is a epidemic disease in our country .
The following results have been obtained from experiments:
l.To establish a two-site time-resolved fluoroimmunoassay(TRPIA) of hepatitis B virus surface antigen(HBsAg) hepatitis B virus surface antibody(HBsAb) and hepatitis B virus e antigen(HBeAg) based on the direct sandwich technique.The assay ranges of standard curves were 0-300ng/ml 0-1280mIU/mland 0-16NCU/ml,respectively, the assay sensitivities were 0.05ng/ml 0.44mIU/mland 0.03NCU/ml The within-run coefficient variations for standard samples were less than 10%. Compared this method with radioimmunoassay,they were interrelathed with each other Repression coefficient was 95%.
2.To establish a neutraJizing-inhabition time-resolved fluoroimmunoassay(TRFIA) of hepatitis B virus e antibody(HBeAb) and hepatitis B virus core antibody(HBcAb) based on the competion .The assay ranges of standard cures were 0-16NCU/mland 0-8NCU/ml.The within-run coefficient variations for standard samples were less than 10%. Compared this method with radioimmunoassay,the sensitivities were 92.8% and 93.3%.
In conclusion, the diagnostic methods we developed for immunological markers of hepatitis B virus by using time-resolved fluoroimmunoassay, Eu-DTTA as label, have wide assay ranges, high sensitivity and specificity. They would have widely application in the further research work and clinical diagnosis.
通过实验研究，以Eu-DTTA为示踪物，得到如下结论：
1．以夹心法建立了乙型肝炎病毒表面抗原时间分辨免疫荧光分析法(HBsAg-TRFIA)、乙型肝炎病毒表面抗体时间分辨免疫荧光分析法(HBsAb-TRFIA)、乙型肝炎病毒e抗原时间分辨免疫荧光分析法(HBeAg-TRFIA)，所建立的标准曲线分析范围分别为0-300ng／ml、0-1280mIU／ml、0-16NCU／ml，分析灵敏度为0.05ng／ml、0.44mIU／ml、0.03NCU／ml。检测参考标准品的批内变异系数均小于10％，与IRMA同时定量检测多份血清样本，相关系数高达95％。
2．以中和抑制法建立了乙型肝炎病毒e抗体时间分辨荧光免疫分析法(HBeAb-TRFIA)和乙型肝炎病毒核心抗体时间分辨荧光免疫分析法(HBcAb-TRFIA)，标准曲线分析范围分别为0-16NCU／ml和0-8NCU／ml。检测参考标准品的批内变异系数均小于10％，与IRMA同时定量检测多份血清样品，灵敏度分别为92.8％和93.3％。
总之，我们利用Eu-DTTA为标记物建立的乙型肝炎病毒五项血清学标志物时间分辨荧光免疫分析法分析范围宽，灵敏度高，操作简便，具有优越的定量检测能力，十分适合临床推广应用。
Labeled immunoassay are the total conception of a series of highly sensitive and specific examine techniques, and have been widely applied in the basic medicine and clinic field for their special advantages. Time-resolved Fluorescence Immunoassay (TRFIA) has become one of the hotspots in the field of labeled immunoassay application. Hepatitis B is a damage of hepar by the Hepatitis B virus .Now it is a epidemic disease in our country .
The following results have been obtained from experiments:
l.To establish a two-site time-resolved fluoroimmunoassay(TRPIA) of hepatitis B virus surface antigen(HBsAg) hepatitis B virus surface antibody(HBsAb) and hepatitis B virus e antigen(HBeAg) based on the direct sandwich technique.The assay ranges of standard curves were 0-300ng/ml 0-1280mIU/mland 0-16NCU/ml,respectively, the assay sensitivities were 0.05ng/ml 0.44mIU/mland 0.03NCU/ml The within-run coefficient variations for standard samples were less than 10%. Compared this method with radioimmunoassay,they were interrelathed with each other Repression coefficient was 95%.
2.To establish a neutraJizing-inhabition time-resolved fluoroimmunoassay(TRFIA) of hepatitis B virus e antibody(HBeAb) and hepatitis B virus core antibody(HBcAb) based on the competion .The assay ranges of standard cures were 0-16NCU/mland 0-8NCU/ml.The within-run coefficient variations for standard samples were less than 10%. Compared this method with radioimmunoassay,the sensitivities were 92.8% and 93.3%.
In conclusion, the diagnostic methods we developed for immunological markers of hepatitis B virus by using time-resolved fluoroimmunoassay, Eu-DTTA as label, have wide assay ranges, high sensitivity and specificity. They would have widely application in the further research work and clinical diagnosis.
引文
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[2] 吴健民主编 《临床化学自动化免疫分析》，科学出版社，2000
[3] 陶义训主编《免疫学和免疫学检验》，人民卫生出版社，2001
[4] Yallow RS, Berson SA.Immunoassay of adogenous plasma insuin in man. J. Clin. Invest 1960; 39: 1157～1175
[5] Engvall E, Perlman P. Enzyme-linked immuosorbent assay (ELISA): Quantitative assay of immunoglobulin G. Immunochem, 1971, 8: 871
[6] Tijssen P. Practice and Theory of Enzyme Immunoassay. Amsterdam, New York, Oxford: Elsever Science Publishers, 1985
[7] Portstmann T, Kiessig ST. Enzyme Imrriunoassay Techniques, An Overview. J immunol Methods, 1992; 150: 5
[8] Hemmila I, Dakubu S, Mukukala VM, et al. Europuim as a label in time-resolved fluoroimmunoassay. Anal Biochem 1984; 137: 335-34
[9] Soini E, Kojola H. Time-resolved fluorometer for lanthanide chelates: a new generation on nonisotolpic immunoassays. Clin. Chem, 1983, 29: 65～68
[10] 李振甲，陈泮藻，高平等编著《时间分辨荧光分析技术与应用》，科学出版社，1996
[11] 常文保，王敏灿，张柏林等．稀土螯合物探针及其在时间分辨荧光免疫分析中的应用，大学化学，1997，1：1～6
[12] 秦秋平．多标记时间分辨荧光免疫分析及其应用．标记免疫分析与临床[M]．1994，4：233～236
[13] Hemmila I,Holttinen S, Pettersson K, et al.Double-label time-resolved immunofluoromerry of lutropin and follitropin in serum. Clin Chem 1987; 33: 2281～2283
[14] Pettersson K, Alfthan H, Stenman U H, et al. Simultaneous assay of α-gryoptein and free subunit of human chorionic gonasotropin by dual-label time-resolved immunolfuorometric assay. Clin Chem 1993; 39: 2084～2809
[15] Xu YY, pettersson K Blomberg K, et al. Simutaneois quadruple-label fluorometric immunoassay of thyroid-stimulating hormone, 17 α-hydroxyprogesterone,immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots. Clin Chem, 1992; 38: 2038～2043
[16] Ito K, Oda M, Tsuji A, et al. Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay. Journal of pharmaceutical and biomedical analysis, 1999, 20(1-2): 169～178
[17] Kimura H, Mukaida M, Wang G, et al. Dual-label time-resolved fluoroimmunoassay of psychopharmaceuticals and stimulants in serum. Forensic science international, 2000, 113(1-3): 345～351
[18] Vuori J, Rasi S, Takala T, et al. Dual-label time-resolved fluoroimmunoassay for simultaneous detection of myoglobin and carbonic anhydrase 111 in serum.Clin Chem1991: 37(12): 2087～2092
[19] Siitari H. Dual-label time-resolved fluoroimmunoassay for the simultaneous detection of adenovirus and rotavirus in faeces. Journal of virologicalmethods, 1990: 28(2): 179～187
[20] 张安胜，李振甲．新型时间分辨荧光免疫分析技术检测癌胚抗原．中华医学检验杂志，1991，14：90
[21] Fiet J, Giton F, Boudi A, et al. Highly sensitive and specific time-resolved fluoroimmunoassay of cyproterone acetate and free cyproterone. Jounal of steroid biochemistry and molecular biology, 2000: 75(4-5): 315～322
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