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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,R2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 establish a sandwich ELISA method for the detection of porcine anti-human T lymphocyte porcine immunoglobulin (ALG) in human serum. Methods Rabbit anti-porcine Ig G coated ELISA plate, HRP-labeled mouse anti-porcine Ig G as secondary antibody, the establishment of human serum ALG content of double antibody sandwich ELISA detection method and the method of coating concentration and secondary antibody Concentration was optimized, at the same time to verify the repeatability, sensitivity, accuracy, specificity and stability of the method. Fourteen patients with severe aplastic anemia (SAA) underwent optimized drug therapy (TDM) with ALG. The results showed that the optimal concentration of coating was 1: 2 000 and the dilution of the best secondary antibody was 1: 8000. The four-parameter Logistic curve fitting of the standard curve with the reference concentration of 4.9-5000μg / ml is better, the R2 value is 0.991 1, the linearity is good in the range of 4.9-156μg / ml, the R2 value is 0.991 5, the intra-assay CV values were all <10%; the detection limit was 0.39μg / ml; the recoveries were 91.2% -107.0% at the concentrations of 500, 50 and 5μg / ml reference substance; The ALG levels in the serum of ALG patients were measured. The ELISA plate and secondary antibody coated with ALG were incubated for 1 to 3 weeks at 4 and 37 ℃, respectively, and the results were stable after 1 to 3 days. According to the DAS 3.1.4 software, the area under the curve (AUC) of serum ALG in 14 ALA-treated patients was (7.3 ± 5.1) mg / (L · d) (95% CI) and half-life ) Was (20.65 ± 38.67) d (95% CI). Conclusion The method has good repeatability, accuracy and stability and can be used for TDM of ALG.