间接竞争ELISA方法检测HM-3血液药物浓度的研究

Research of McAb-Based Competitive ELISA for Detecting HM-3 Residues in Serum

为检测血液中小分子多肽药物HM-3含量,以HM-3-BSA为包被抗原、HM-3标准品为竞争抗原,利用HM-3的单克隆抗体,初步建立了的间接竞争ELISA(ic-ELISA)方法。经优化,理想的抗原包被浓度为1∶6 400稀释、单抗工作浓度为1∶128 000稀释,酶标二抗工作浓度为1∶2 000稀释,得到线性回归方程(y=-40.234x+159.54,R2=0.9804)和标准曲线,最小检测量为40 ng/mL,该方法对恩度无交叉反应。采用高温水浴法处理血清,回收率高。通过ELISA方法测得HM-3大鼠体内消除半衰期(t1/2β)为(9.375±4.777)min,与液相色谱/质谱联用法(LC-MS)测定结果接近。该间接竞争ELISA方法的成功建立将对HM-3血药浓度的临床监测提供参考。

An indirect competitive ELISA（ic-ELISA） for detecting HM-3 in serum was established by using man-made coating antigen of HM-3-BSA,competitive antigen of HM-3 and monoclonal antibody of HM-3.The results of the experiment demonstrated that the optimal concentration of the coating antigen,mcAb against HM-3 and goat anti-mouse IgG were 1∶6 400,1∶128 000 and 1∶2 000 respectively.A standard curve was obtained,and the regression equation was y=-40.234x＋159.54,R2=0.980 4.The detection limit was 40 ng/mL.There was no cross reactivity to endostatin which was closely related with HM-3.The recoveries of HM-3 spiked in serum were good by high temperature water bath treatment.The elimination half life（t1/2β） of HM-3 in rats in vivo by ELISA assay was（9.375±4.777） min,closely to the results detected by liquid chromatography/mass spectrometry（LC-MS）.A competitive ELISA has been established,which can be used to monitor HM-3 concentration in serum clinically.