P-选择素时间分辨荧光免疫分析法的建立及临床应用

Time-resolved fluroimmunoassy of P-selectin and it's clinical application

目的 建立时间分辨荧光免疫分析法 (TRFIA)用于检测血栓形成相关疾病患者的血浆P 选择素的含量 ,并探讨其临床意义。方法 用抗人血小板单抗SZ 5 1 IgG包被 ,将Eu3+ N (对 异硫氰酸苄基 ) 二乙烯四乙酸与另一个血小板单抗S12连接以制备Eu3+ S12示踪剂 ,β 二酮为发光增强剂 ,采用平衡饱和法建立时间分辨荧光免疫分析法 (TRFIA)用于分析糖尿病、高血脂、脑梗塞患者及正常人血浆中P 选择素含量的变化 ,并与流式细胞术 (FCM)和酶标记免疫吸附分析法 (ELISA)作比较。结果 方法灵敏度为 0 98ng/ml,批内和批间CV分别为 4 2 3 %和 6 6 7% ,平均回收率为 97 2 4% ,可测范围为 4 1～ 80ng/ml。应用TRFIA测定 2 2例高血脂、18例脑梗塞患者和 2 0例正常人血浆中P 选择素含量 ,分别为 8 0 8± 1 4ng/ml,12 5 1± 3 6ng/ml和19 0 4± 7 9ng/ml,显著高于正常人 (3 0 7± 1 6 4ng/ml) (P <0 0 1)。同时与ELISA和FCM方法检测血浆内P 选择素和血小板膜表面表达情况作比较 ,结果一致。结论 TRFIA检测血浆P 选择素的方法可靠、灵敏、方便 。

Objective To establish a two-site time-resolved fluoroimmunoassay(TRFIA)of P selectin and evaluate it on clinical.Methods The solided MoAb(SZ-51)which against at a specific antigenic site on P selectin was prepared,and europium-labelled another MoAb(S12) by with helpful of the chelate of N (P isothiocyanatobenzyl) diethylenetriamine N,N,N,N tetraacetic acide.The luminescent enhancement system was the solution which contained mainly 2 naphthoyltrifluoroacetone.The TRFIA of P selection was developed based on the direct sandwhich technique,on the equilibrium method.Results The intra and inter assay CV of the TRFIA were 4.23% and 6.67%,respectively.The recovery rate was 97.24% and the sensitivity was 0.98 ng/ml.The MoAbs provided a linear response from 4.1 80 ng/ml.The concentration of P selection on plasma by this method was 8.08±1.4 ng/ml for 32 patients with diabetes,12.51±3.6 ng/ml for 22 patients with hyperlipemia, 19.04±7.9 ng/ml for 18 patients with apoplexia.They are much higher than the contral group or normal human(3.7±1.64 ng/ml , P < 0.01). Compared with ELISA and FCS,the better correlative results were obtained in three methods.Conclusion This method to analysis the concentration of P selectin on plasma is simple,senstiive and suitable,and it is favorable to apply to clinical.