睾酮固相放射免疫分析方法的建立

摘要

目的 建立一种睾酮固相放射免疫分析方法,实现睾酮液相放射免疫分析药盒的固相包被管分离.方法 利用本公司专利技术纯化出亲和纯驴抗兔抗体,将其包被于聚苯乙烯试管上,兔抗-睾酮抗体能与驴抗兔抗体结合而被间接包被上,从而制备出睾酮抗体包被管,再采用标准抗原与标记抗原竞争包被在聚苯乙烯试管上定量抗体的技术思路,建立睾酮固相放射免疫分析方法.结果 睾酮抗体包被管中亲和纯驴抗兔抗体包被浓度为20μg/mL,兔抗-睾酮抗体包被滴度为1∶5万;本方法的反应条件为37℃温育1h,测量范围为0.1～20 ng/mL,灵敏度为0.02 ng/mL,批内变异为4.37％～6.84％,批间变异为6.31％ ～9.03％,回收率为93.6％～104.1％,稀释实验测定值与理论值呈线性相关,相关系数为r=0.999;与合格睾酮液相放射免疫分析药盒的相关性方程为Y=1.05X+0.01,相关系数r=0.982;睾酮抗体包被管、125I-睾酮标记物以及睾酮标准品在37℃存放1周稳定性良好.结论 本方法学鉴定结果符合免疫分析的基本要求.%Objective To develop a kind of solid-phase RIA for testosterone to change the separation method of testosterone liquid-phase radioimmunoassay kit by solid-phase coated tube.Methods Affinity pure donkey anti-rabbit antibody was made by latest patented technique of our company,which was used to be coated on polystyrene tube,and rabbit anti-testosterone antibody could be coated on the tube indirectly by combining with donkey anti-rabbit antibody.In this way testosterone solid-phase coated tube was successfully made.Because of the technical principle that standard antigen would compete with labeled antigen for limited antibody coated on the tube,testosterone solid-phase radioimmunoassay could be established easily.Results The concentration of affinity pure donkey anti-rabbit antibody was 20 μg/mL and the dilution of rabbit antitestosterone antibody was 1 ∶ 50000.The reaction condition was incubated in 37℃ for an hour.The standard range of the method was 0.1 ～ 20 ng/mL.The assay sensitivity was 0.02 ng/mL.The intra-assay and interassay coefficients of variance were 4.37％ ～6.84％ and 6.31％ ～9.03％ respectively.Analytical recovery was 93.6％ ～ 104.1％.The correlation coefficients between measured and expected values were 0.999 after serial diluted.The correlative equation between qualified testosterone liquid-phase radioimmunoassay kit and solid-phase radioimmunoassay method was Y =1.05X + 0.01,and correlation coefficient was 0.982.Testosterone antibody coated tubes,labeled Testosterone antigen,and testosterone standard antigen could keep good stability at 37 ℃ for a week.Conclusion This method met the basic requirements of immunoassay.