人血清中猪抗人淋巴免疫球蛋白双抗体夹心ELISA检测法的建立及验证

Development and verification of a double antibody sandwich ELISA for anti-human lymphocyte porcine immunoglobulin in human serum

目的建立人血清中猪抗人淋巴免疫球蛋白（anti-human T lymphocyte porcine immunoglobulin,ALG）双抗体夹心ELISA检测法,并进行验证。方法以兔抗猪Ig G包被酶标板,HRP酶标记的鼠抗猪Ig G为二抗,建立人血清中ALG含量的双抗体夹心ELISA检测法,并对方法中包被浓度及二抗浓度进行优化,同时验证该方法的重复性、敏感性、准确性、特异性及稳定性。采用优化后的方法对14位严重型再生障碍性贫血（severe aplastic anemia,SAA）患者进行ALG的血药浓度监测（therapeutic drug monitoring,TDM）。结果最佳包被浓度为1∶2 000,最佳二抗稀释度为1∶8 000。参考品浓度在4.9-5 000μg/ml范围内标准曲线的四参数Logistic曲线拟合较好,R2值为0.991 1,在4.9-156μg/ml范围内的线性拟合也较好,R^2值为0.991 5,批内重复性CV值均〈10%;其检测底限为0.39μg/ml;浓度为500、50及5μg/ml参考品的回收率为91.2%-107.0%;该方法可特异性检测出注射ALG患者血清中ALG含量;包被好的ELISA板及二抗、底物于4及37℃条件下分别保存1-3周及1-3 d后检测结果均较稳定。经DAS 3.1.4软件分析,14名经ALG治疗的SAA患者血清中ALG的曲线下面积（AOC）为（7.3±5.1）mg/（L·d）（95%CI）,半衰期（t1/2）为（20.65±38.67）d（95%CI）。结论该方法具有良好的重复性、准确度及稳定性,可用于ALG的TDM。

Objective To develop and verify a double antibody sandwich ELISA for anti-human lymphocyte porcine immunoglobulin（ALG）in human serum. Methods A double antibody sandwich ELISA was developed using rabbit antiporcine Ig G as coating antibody, and HRP-labeled mouse anti-porcine Ig G as the secondary antibody. The concentrations of coating antibody and the secondary antibody were optimized, and the developed method was verified for repro-ducibility,sensitivity, accuracy, specificity and stability and used for the therapeutic drug monitoring（TDM） of patients with severe aplastic anemia（SAA） after intravenous infusion with ALG. Results The optimal antibody concentration for coating was1 ∶ 2 000, while the optimal dilution of the secondary antibody was 1 ∶ 8 000. The calibration curve was at reference concentrations of 4. 9 - 5 000 μg / ml（R^2 = 0. 991 1） fitted with four parameter logistic curve with well goodness and4. 9 - 156 μg / ml（R2 = 0. 991 5）fitted with linear. The CVs in intra-assays were less than 10%. The detection limit of was concluded as 0. 39 μg / ml. The recovery rates of references at concentrations of 500, 50 and 5 μg / ml ranged from91. 2% - 107. 0%. The ALG content in sera of patients was detected by the developed method specifically. The detection results were stable after the microtiter plate, the secondary antibody and substrate were stored at 4 ℃ for 1 - 3 weeks and at 37 ℃ for 1 - 3 d. Analysis by DAS 3. 1. 4 software showed that the area under curve（AOC）of ALG in sera of patients with SAA was（7. 3 ± 5. 1 mg / L·d）（95% CI）, while the half life period（t1 / 2）was（20. 65 ± 38. 67）d（95% CI）.Conclusion The developed double antibody sandwich ELISA showed high reproducibility, accuracy and stability, which might be used for TDM of ALG.