人血清脂联素竞争性酶免疫分析方法的建立及初步应用

Establishment and preliminary application of a competitive enzyme immunoassay for human adiponectin in serum

目的建立测定人血清脂联素(APN)水平的酶免疫分析法(EIA),以评价APN水平检测的临床意义。方法采用重组人APN蛋白免疫小鼠,获得高效价的抗人APN多克隆抗体,再以APN包被塑料孔条作固相抗原,与样本中的液相抗原竞争结合限量的APN抗体,从标准曲线判读待测样本的浓度,建立人APN竞争性EIA。结果方法的灵敏度为10μg/L,可测范围为10～800μg/L,批内和批间变异系数均<10%。用本方法测得101名健康正常人的APN水平为男11.7±7.4mg/L、女16.2±6.8mg/L,女高于男;64例2型糖尿病(T2DM)患者为男4.3±2.0mg/L、女5.9±2.5mg/L;T2DM患者低于同性别的正常人(P均<0.01)。结论新建立的APN-EIA法具有简便准确等优点,可广泛应用于APN临床意义的研究。

Objective To develop an enzyme immunoassay for measurement of the circulating levels of human adiponectin （hAPN） and evaluate its clinical significance. Methods Mouse anti-hAPN polyclonal antibodies （PcAb） with high titre were prepared by immunizing BALB/C mouse with recombinant hAPN. The hAPN protein was also absorbed to the microtitre strip wells as solid antigen, and competed with the liquid antigen in unknown sample to bind to the limited PcAb. A simple competitive ELA method was developed. Results The detectable low limit of the assay was 10 ug/L, and the working range was 10ug/L～800ug/L. Both of the inter-assay and intra-assay coefficient variation （CV） were less than 10%. Mean serum APN levels detected by us in-house EIA in health male subjects（11.7 ± 7.4mg/L） were lower than the levels in female（ 16.2 ± 6.8mg/L ）. Furthermore, as compared with health subjects of the same sex, APN levels in type 2 diabetes mellitus （4.3±2.0 mg/L in male, 5.9 ±2.5mg/L in female） were much lower （P〈0.01）. The results agreed well between our EIA and the commercially available Phoenix Pharmaceuticals ELISA （r = 0. 96, n = 114 ）. Conclusion With high quality the EIA assay provides us a new tool to study the clinical significance for measuring adiponectin concentraions in human serum.