甲胎蛋白化学发光免疫定量分析方法的建立

东方

【摘要】  目的： 建立检测甲胎蛋白化学发光免疫定量分析方法. 方法： 采用双抗体异位点一步夹心法，以甲胎蛋白单克隆抗体作为固相包被，采用改良过碘酸钠法进行甲胎蛋白多克隆抗体与碱性磷酸酶偶联制备酶标抗体；以金刚烷胺衍生物CSPD作为底物，优化CSPD与SapphireII发光体系；用国家标准品校定定量标准品，建立了人血清AFP的化学发光免疫定量分析法. 用该法检测800份正常人血清，确定正常值参考范围，将临床血清标本100份与bayer Acs: 180全自动发光体系统进行比较. 结果： 本方法的最低检出量为2.0 ng /mL，线性范围2.0~600.0 ng/mL,批内和批间的变异率分别为6.7%和7.7%，回收率在98.2%~102.5%之间. 与CEA(3 μg/mL)的交叉≤0.15%；与铁蛋白(10 μg /mL) ≤0.04%；与人血清白蛋白（200 g/L）≤2.5×10-8，与人血清丙球蛋白（100 g/L）≤2.0×10-8，与bayer Acs:180比较相关系数r=0.994（P<0.001）. 结论： 建立的甲胎蛋白化学发光免疫分析法可常规用于临床检测.
【关键词】  甲胎蛋白类；化学发光测定法；定量分析
　　Establishment of chemiluminescent immunoassay for determination of alphafetoprotein
　　FENG YanMing, MA JunFen, WU Biao, GUO LanYing, WU YongJun, LI ZhiTao,  WU YiMing
　　1Department of Labour Health and Occupational Public Health Institution,  Zhengzhou University, Zhengzhou 450052, China, 2SinoAmerican Biology Company, Luoyang, 471003, China
　　【Abstract】 AIM:  To establish a method of chemiluminescent immunoassay for determination of alpha fetoprotein(AFP) in human serum.  METHODS: The twosite enzyme immunoassay is based on the direct sandwich technique. A monoclonal antibody was bonded to the microplate for coating and the conjugation of alkaline phosphatase to another polyclonal antibody was performed by NaIO4 method. Chemiluminescent immunoassay was established by optimizing CSPD and SapphireII luminescent system. Sensitivity, linear scope, precision, interference and recovery for the method were evaluated. The reference range was defined by testing 800 serum samples from healthy subjects.The comparison for 100 serum samples from patients was assayed by the bayer Acs:180 Automated Immunoassay system. RESULTS: The detectable minimum was 2.0 ng/mL. The linear scope was from 2.0 to 600.0 ng/mL. Average inter and intraassay were 7.7% and 6.7%, respectively. The recoveries ranged from 98.2% to 102.5%. The crossreacting rate for CEA(3 μg/mL), Ferritin(10 μg /ml), HAS(200 g/L), IgG(100 g/L) were≤0.15%, 0.04%, 2.5×10-8 and 2.0×10-8, respectively. Compared with bayer Acs:180 Automated immunoassay system, the relative coefficient was 0.994（P<0.001）. CONCLUSION: A successful establishment of chemiluminescent immunoassay provides a way for clinical determination of AFP.
　　【Keywords】  alphafetoproteins; chemiluminescent  measurements; quanti tative analysis
　　引言
　　甲胎蛋白（AFP）为最常用的肿瘤标志物之一. 是目前最好的可实际用于早期诊断原发性肝癌的指标，可在症状出现前6~12 mo作出诊断. 其他一些转移性肝癌及肝外肿瘤如睾丸肿瘤， 卵巢肿瘤，胃癌，胰腺癌，结肠癌，支气管癌，肾癌，乳腺癌和白血病等血液中的AFP也可增高. AFP属糖蛋白，分子量70000，含糖40 g/L，正常人血清AFP含量在2~8 μg/L之间，AFP正常值一般低于25 μg/L;本文根据化学发光免疫分析法的原理建立并研制了甲胎蛋白化学发光定量测定试剂盒，现将结果报道如下.
　　1材料和方法
　　1.1材料
<P>　　抗AFP单抗腹水购自OEM公司；AFP多克隆抗体为自制；AFP纯品抗原购自Fitzgeral公司；碱性磷酸酶（ALP）由华美生物工程公司提供；化学发光底物CSPD及增强剂购自PE公司；过碘酸钠（NaIO4）、硼氢化钠（NaBH4）、乙二醇、2，4二硝基氟苯（DNFB）均购自Sigma；Sephadex G200凝胶柱层析购自LKB公司，DE52离子交换柱购自Sigma，聚苯乙烯塑料白色不透明微孔板购自深圳金灿华；luminescent化学发光仪（芬兰雷勃）；洗板机（BioRad）；AFP国家标准品购自中国药品生物制品检定所.  标本均为血清，清亮无溶血，-20℃冻存. 分别为健康体检门诊的成年男性200份，成年女性（非妊娠女性）200份，青少年200份，老年人标本200份，其中男女比例为1∶1；