胶体金免疫比色法测定血清h-FABP水平的初步研究
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摘要
目的初步建立一种基于胶体金均相免疫比色技术定量检测血清心脏型脂肪酸结合蛋白(hFABP)的方法。方法依据均相比色原理定量测定样本中抗原或抗体水平的免疫学检测技术原理,制备直径为80nm左右的胶体金颗粒并标记抗体,利用胶体金吸附标记物在540、660nm处吸收峰的变化初步建立用于检测血清h-FABP水平的方法。结果制备的胶体金颗粒液相分布均匀,制备的胶体金颗粒标记抗体呈均相分布,符合检测实验要求。通过实验确定反应体系组成为标本用量为30μL,h-FABP抗体标记量为100μg/100mL胶体金溶液,反应时间为10min,反应温度为37℃,以实验确定h-FABP≤3.6ng/mL为界限值,对临床标本进行初步分析并与免疫比浊的检测结果比较,符合率达95.09%。结论初步完成了h-FABP的胶体金免疫比色法的建立,与成熟的免疫比浊方法检测结果具有一定的符合性,有必要进行更深一步的研究。
Objective To establish a colloidal gold colorimetric assay method for detection of hard-type fatty acid binding protein(h-FABP)in serum.Methods According the principle of homogeneous colorimetry for quantitative determination of antigen or antibody in a sample,based on the changes of absorption peak of colloidal gold adsorption marker at 540 and 660 nm,we established a method for detecting h-FABP in serum.Results The colloidal gold particles were well distributed,and the prepared antibodies labeled colloidal gold particles were homogeneous distribution,which were suitable for the requirements of the test.In the preliminary analysis of the clinical specimens,compared with the Immune Turbidimetry method,the coincidence rate was95.09%.Conclusion A colloidal gold colorimetric assay method for h-FABP in the human serum had been established preliminarily,the results were certain consistent with those obtained by Immune Turbidimetry method,and can be further applied to clinical detection.
Objective To establish a colloidal gold colorimetric assay method for detection of hard-type fatty acid binding protein(h-FABP)in serum.Methods According the principle of homogeneous colorimetry for quantitative determination of antigen or antibody in a sample,based on the changes of absorption peak of colloidal gold adsorption marker at 540 and 660 nm,we established a method for detecting h-FABP in serum.Results The colloidal gold particles were well distributed,and the prepared antibodies labeled colloidal gold particles were homogeneous distribution,which were suitable for the requirements of the test.In the preliminary analysis of the clinical specimens,compared with the Immune Turbidimetry method,the coincidence rate was95.09%.Conclusion A colloidal gold colorimetric assay method for h-FABP in the human serum had been established preliminarily,the results were certain consistent with those obtained by Immune Turbidimetry method,and can be further applied to clinical detection.
引文
[1] OCKNER R K,MANNING J A,POPPENHAUSEN R B,et al.A binding protein for fatty acids in cytosol of intestinal mucosa,liver,myocardium,and other tissues[J].Science,1972,177(443):56-58.
[2] FURUHASHI M,HOTAMISLIGIL G S.Fatty acidbinding proteins:role in metabolic diseases and potential as drug targets[J].Nat Rev Drug Discov,2008,7(6):489-503.
[3] YOUNG J M,PICKERING J W,GEORGE P M,et al.Heart fatty acid binding protein and cardiac tropnin:development of an optimal rule-out strategy for acute myocardial infarction[J].BMC Emerg Med,2016,16(1):34.
[4] VUPPUTURI A,SEKHAR S,KRISHNAN S,et al.Heart-type fatty acid-binding protein(H-FABP)as an early diagnostic biomarker in patients with acute chest pain[J].Indian Heart J,2015,67(6):538-542.
[5]刘相应,唐良秋,徐新,等.心型脂肪酸结合蛋白在急性心肌梗死中的应用[J].中国心血管研究杂志,2015,13(7):588-592.
[6] PYATI A K,DEVARANAVADAGI B B,SAJJANNAR S L,et al.Heart-type fatty acid-binding protein,in early detection of acute myocardial infarction:comparison with CK-MB,troponin I and myoglobin[J].Indian J Clin Biochem,2016,31(4):439-445.
[7]尚红,王毓三,申子瑜.全国临床检验操作规程[M].4版.北京:人民卫生出版社,2015.
[8] LEUVERING J H,THAL P J,VAN DER WAARTM,et al.Sol particle immunoassay(SPIA)[J].J Immunoassay,1980,1(1):77-91.
[9]程黎明,朱晓华,朱耀武,等.液相免疫胶体金技术用于检测血浆半胱氨酸蛋白酶抑制剂C的方法学评价[J].检验医学,2010,25(3):214-218.
[10]王会中,徐荣.定量免疫分析技术的应用现状与展望[J].中华检验医学杂志,2017,40(6):478-480.
[11]张莹,冯海,吴秋平,等.胶体金免疫层析技术的研究进展[J].现代生物医学进展,2015,15(12):2349-2351.
[12]戴婉如,陈发林,吴来州.免疫胶体金技术测定血清胱抑素C及其对早期肾损害的诊断价值[J].检验医学与临床,2009,6(5):336-337.
[13]KYKSE-ANDERSON J,SCHMIDT C,NORDIN G,et al.Serum cystatin C,determined by a rapid,automated particle-enhanced turbidimetric method,is a better marker than serum creatinine for glomerular filtration rate[J].Clin Chem,1994,40(10):1921-1926.
[14]FINNEY H,NEWMAN D J,GRUBER W,et al.Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems(BNA,BNⅡ)[J].Clin Chem,1997,43(6):1016-1022.
[15]肖勤,林金明.化学发光免疫分析新进展[J].分析试验室,2011,30(1):111-122.
[16]HAMZA M,DEMERDASH S,IBRAHIM M.Heart-type fatty acid-binding protein as a diagnostic biochemical marker for early detection of myocardial infarction[J].Acta Cardiol,2016,71(5):537-541.
[17]刘延青,冯长州,孙淑艳.心型脂肪酸结合蛋白在胸痛6h内诊断急性心肌梗死的meta分析[J].中国实验诊断学,2015,19(1):41-44.
[18]刘首明,夏慧,江小萍,等.急性心肌梗死患者心型脂肪酸结合蛋白水平检测及临床价值[J].实验与检验医学,2015,33(5):673-675.
[19]STIERMAIER T,DE WAHA S,FRNAU G,et al.Thrombus aspiration in patients with acute myocardial infarction:Scientific evidence and guideline recommendations[J].Herz,2016,41(7):591-598.
[2] FURUHASHI M,HOTAMISLIGIL G S.Fatty acidbinding proteins:role in metabolic diseases and potential as drug targets[J].Nat Rev Drug Discov,2008,7(6):489-503.
[3] YOUNG J M,PICKERING J W,GEORGE P M,et al.Heart fatty acid binding protein and cardiac tropnin:development of an optimal rule-out strategy for acute myocardial infarction[J].BMC Emerg Med,2016,16(1):34.
[4] VUPPUTURI A,SEKHAR S,KRISHNAN S,et al.Heart-type fatty acid-binding protein(H-FABP)as an early diagnostic biomarker in patients with acute chest pain[J].Indian Heart J,2015,67(6):538-542.
[5]刘相应,唐良秋,徐新,等.心型脂肪酸结合蛋白在急性心肌梗死中的应用[J].中国心血管研究杂志,2015,13(7):588-592.
[6] PYATI A K,DEVARANAVADAGI B B,SAJJANNAR S L,et al.Heart-type fatty acid-binding protein,in early detection of acute myocardial infarction:comparison with CK-MB,troponin I and myoglobin[J].Indian J Clin Biochem,2016,31(4):439-445.
[7]尚红,王毓三,申子瑜.全国临床检验操作规程[M].4版.北京:人民卫生出版社,2015.
[8] LEUVERING J H,THAL P J,VAN DER WAARTM,et al.Sol particle immunoassay(SPIA)[J].J Immunoassay,1980,1(1):77-91.
[9]程黎明,朱晓华,朱耀武,等.液相免疫胶体金技术用于检测血浆半胱氨酸蛋白酶抑制剂C的方法学评价[J].检验医学,2010,25(3):214-218.
[10]王会中,徐荣.定量免疫分析技术的应用现状与展望[J].中华检验医学杂志,2017,40(6):478-480.
[11]张莹,冯海,吴秋平,等.胶体金免疫层析技术的研究进展[J].现代生物医学进展,2015,15(12):2349-2351.
[12]戴婉如,陈发林,吴来州.免疫胶体金技术测定血清胱抑素C及其对早期肾损害的诊断价值[J].检验医学与临床,2009,6(5):336-337.
[13]KYKSE-ANDERSON J,SCHMIDT C,NORDIN G,et al.Serum cystatin C,determined by a rapid,automated particle-enhanced turbidimetric method,is a better marker than serum creatinine for glomerular filtration rate[J].Clin Chem,1994,40(10):1921-1926.
[14]FINNEY H,NEWMAN D J,GRUBER W,et al.Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems(BNA,BNⅡ)[J].Clin Chem,1997,43(6):1016-1022.
[15]肖勤,林金明.化学发光免疫分析新进展[J].分析试验室,2011,30(1):111-122.
[16]HAMZA M,DEMERDASH S,IBRAHIM M.Heart-type fatty acid-binding protein as a diagnostic biochemical marker for early detection of myocardial infarction[J].Acta Cardiol,2016,71(5):537-541.
[17]刘延青,冯长州,孙淑艳.心型脂肪酸结合蛋白在胸痛6h内诊断急性心肌梗死的meta分析[J].中国实验诊断学,2015,19(1):41-44.
[18]刘首明,夏慧,江小萍,等.急性心肌梗死患者心型脂肪酸结合蛋白水平检测及临床价值[J].实验与检验医学,2015,33(5):673-675.
[19]STIERMAIER T,DE WAHA S,FRNAU G,et al.Thrombus aspiration in patients with acute myocardial infarction:Scientific evidence and guideline recommendations[J].Herz,2016,41(7):591-598.