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1 材料和方法 1.1 材料 人清蛋白放免试剂盒由北方原子能研究所提供。人清蛋白放射免疫检测,采用抗原竞争法。原试剂盒灵敏度为0.25μg/mL,不能满足实验需要,经反复摸索,调整抗体、~(125)I-标记抗原浓度及实验条件,最终得出如下结果:~(125)I-人清蛋白放射性比活度为25μCi/μg,每一样品测定用量2.5μCi,抗体1:8稀释,并将温育方式改为4℃ 24h,可提高灵敏度至3ng/mL左右。 1.2 方法 1.2.1 清蛋白标准曲线、线性关系及灵敏度 取洁净试管分别标上记号,非特异性结合管(NSB),标准;管S0～S7(n=5),然后用微量加样器加样,缓冲液:NSB管加300μL,S0管200μL;标准品S1～S7管加200μL;~(125)I-人清蛋白:各管均加100μL;抗体:S0管、S1～S7管各100/μL。4℃ 24h温育后分别加免疫分离试剂1000μL,混匀后室温放置15min,任取5管测放射性计数(cpm)取平均值为总T.3000r/min离心15min,测
1 Materials and Methods 1.1 Materials Human albumin RIA kit provided by the North Institute of Atomic Energy. Human albumin radioimmunoassay, the use of antigen competition law. The original kit sensitivity of 0.25μg / mL, can not meet the experimental needs, after repeated exploration, adjust the antibody, 125I-labeled antigen concentration and experimental conditions, the following results: ~ (125) I- The specific activity of radioactivity was 25μCi / μg, and the dosage of 2.5μCi for each sample was measured. The dilution of antibody 1: 8 and the incubation method to 4 ° C for 24 hours increased the sensitivity to about 3ng / mL. 1.2 Methods 1.2.1 albumin standard curve, linear relationship and sensitivity Take clean tubes were marked with non-specific binding tube (NSB), standard; tube S0 ~ S7 (n = 5), and then with a micro sampler , Buffer: NSB tube plus 300μL, S0 tube 200μL; Standard S1 ~ S7 tube plus 200μL; ~ (125) I-human albumin: each tube plus 100μL; antibody: S0 tube, S1 ~ μL. After incubation at 4 ° C for 24 h, immunosuppressants (1000 μL) were added separately. After mixing, the cells were allowed to stand at room temperature for 15 min, and 5 tubes were used for radioactivity counting (cpm). The average value was T.3000 r / min for 15 min.