老年男性肾脏雄激素调节蛋白检测方法的建立及参考范围  

Establishment of methodology and reference range for testing kidney androgen-regulated protein in elderly males

作　　者：巴怡平[1] 余卫[2] 靳雅丽[1] 刘惠萍[1] 吴毅琴[1] 伍绍国[3] 

机构地区：[1]广州市第十二人民医院心血管内科,510620 [2]广州市第十二人民医院血液科,510620 [3]广州市第十二人民医院检验科,510620

出　　处：《中华生物医学工程杂志》2014年第4期298-301,共4页Chinese Journal of Biomedical Engineering

基　　金：广州市科技计划项目（2013J4100121）;广东省医学科研基金（A2013548）

摘　　要：目的 探索建立检测人尿和血清中肾脏雄激素调节蛋白（KAP）含量的ELISA试剂盒,并初步提出在老年男性中的参考值范围.方法 采用间接竞争ELISA法,筛选酶标亲和素及生物素化二抗的最适滴度.以KAP标准品对数值作为横坐标,加入抗体后测得的A450值作为纵坐标,绘制标准曲线.利用此试剂盒检测100例正常老年男性的尿和血清中KAP的含量,每组做3个复孔,间隔6个月后再重复1次.结果 包被原为0.5μg,生物素化抗体为1∶3 200,酶标亲和素的稀释度为1∶2000时所获的A450值最佳.间接竞争ELISA法标准曲线的绘制：以0.008～0.500 mg/L、0.250 mg/L、0.125 mg/L、0.063 mg/L、0.031 mg/L、0.016 mg/L、0.008 mg/L的对数值为横坐标,其对应的A450值为纵坐标绘制标准曲线,其线性关系良好（r=0.980）.本试剂盒的灵敏度为0.008 ～ 0.5 mg/L.100例样本中尿KAP的含量为（0.042±0.031） mg/L,血清KAP的含量为（0.014±0.011）mg/L.老年男性尿及血清KAP参考值范围分别为（0.000～ 0.103） mg/L及（0.000～ 0.041） mg/L.组内差异为1.7％,组间差异为8％,每次检测耗时小于2h.结论 本试剂盒具有较高的灵敏度及特异性,检测速度较快.同时,我们提供了部分正常老年男性尿及血清中KAP的含量,为今后对KAP与疾病关系的研究提供了依据.Objective To establish the methodology and the reference range of testing kidney androgen-regulated protein （KAP） in elderly males.Methods The optimal titer of the enzyme-labeled avidin and biotinylated antibody was determined by using indirect competitive inhibition enzyme-linked immunosorbent assay.The standard curve was prepared by utilizing the logarithms of KAP standard as the horizontal axis,and A450 values as the vertical axis.Urine and plasma samples from 100 normal elderly males were assayed using the above-mentioned standard curve,and the wells were measured in triplicates.The samples were stored for 6 months at 4 ℃ for repeated measurement.Results The optimal A450 corresponded to the titer of coating antigen of 0.5 μg,enzyme labeled avidin of 1 ∶ 3 200 and biotinylated antibody of 1 ∶ 2 000,respectively.The linear correlation of standard curves was ideal （r=0.980） by using logarithms of 0.008～0.500 mg/L,0.250 mg/L,0.125 mg/L,0.063 mg/L,0.031 mg/L,0.016 mg/L and 0.008 mg/L.The detection range of the assay kit was 0.008～0.500 mg/L.Of the 100 samples,the KAP of urine and plasma was 0.042±0.031） mg/L and （0.014±0.011） mg/L,respectively.The reference ranges of urine and plasma in elderly males were （0-0.103） mg/L and （0-0.041） mg/L,respectively.The intra-and inter-group differences were 1.7％ and 8％,respectively.Less than 2 hours were required for the measurement.Conclusion The newly established assay kit is characterized by high assay sensitivity and specificity and rapid operating procedures.Meanwhile,the normative range of urine and serum KAP in normal elderly males has provided good eridence for further studies.

关 键 词：肾脏 雄激素 抗体 

分 类 号：R692[医药卫生—泌尿科学]