检测血清ATG浓度ELISA方法的建立

An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration

目的建立检测人血清中兔抗人胸腺免疫球蛋白(ATG)的检查方法并进行初步临床应用。方法用鼠抗兔IgG单克隆抗体做固相,辣根过氧化物酶(HRP)标记的羊抗兔IgG多克隆抗体做二抗,建立双抗体夹心ELISA方法。并检测使用ATG的异基因造血干细胞移植患者血中ATG的浓度。结果ATG浓度最低检测值为31.25ng/ml,在40～1000ng/ml范围内呈线性关系,组内和组间的变异系数分别为7.91%和5.22%。用该法测定7例干细胞移植预处理方案中使用ATG患者的浓度,ATG随时间在体内浓度逐渐下降,但预处理后90天仍能测得ATG,表明ATG在体内作用时间比较久,可能会影响移植后免疫功能重建。结论双抗体夹心法具有灵敏性高、特异性强的特点,可用于人血清中ATG水平的定量测定。干细胞移植预处理方案中使用ATG后体内浓度进行性降低,ATG将会比较长时间在体内起作用。

Objective To establish an enzyme-linked immunosorbent assay（ELISA） for determining anti-themocyte globulin（ATG） levels in serum samples.Methods The microplate was coated with mouse anti-rabbit IgG monoclonal antibody,and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation.Result The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 μg/ml,1：40 and 1：2500,respectively.The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection.A linear relationship was established within the concentration range from 40 to 1000 ng/ml,with the coefficients of variation of 7.91 within assay and 5.22 between assays,respectively.Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG,and after 90 days ATG could still be detected.Conclusion The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting.In patients undergoing stem cell transplantation with ATG pretreatment,the ATG concentration gradually decreases but remains detectable 90 days after the administration.