酶联免疫吸附法测定红细胞内多聚谷氨酸化甲氨蝶呤浓度的方法建立及其初步应用

A novel method to establish and its preliminary application for determination of concentrations of methotrexate polyglutamates in erythrocytes by enzyme-linked immunosorbent assay

目的建立检测红细胞内多聚谷氨酸化甲氨蝶呤（methotrexate polyglutamates,MTXPG）的酶联免疫吸附测定法（ELISA）方法。方法将含有MTXPG的红细胞采用冻融法、化学裂解法、酶法、超声破碎法等方法裂解,加入血清与裂解液在抗坏血酸、巯基乙醇保护液作用下将MTXPG转化为甲氨蝶呤（methotrexate,MTX）,转化液采用高氯酸、三氯乙酸及饱和硫酸铵溶液沉淀蛋白,最后将溶液p H值调整为3.0～9.0,采用ELISA检测MTX浓度。经方法学考核后,检测临床标本对方法准确性进行评价。结果发现冻融法与冻融法裂解红细胞显著优于酶法和化学裂解法（P〈0.05）,抗坏血酸配制的保护液明显优于巯基乙醇（P〈0.05）,转化液37℃孵育3 h时MTXPG转化完全,高氯酸、三氯乙酸、饱和硫酸铵溶液3种蛋白沉淀剂无明显差异（P〉0.05）,最适p H为6～8,最佳p H值为6.8。浓度在0.2～10.0 ng/m L范围内线性关系良好（r=0.9967）,回收率为96.6%～103.3%,RSD为8.0%～14.3%,日内差异为1.7%～11.7%,日间差异为7.4%～13.5%,RSD均小于15%。检测患者红细胞内MTXPG浓度为（64.75±28.45）ng/m L。结论建立的红细胞内MTXPG的ELISA法有良好的准确性和特异性,方法操作简单易行,可用于临床红细胞内MTXPG浓度的监测。

Objective To establish a novel method that determines concentrations of methotrexate polyglutamates（MTXPG） in erythrocytes by enzyme-linked immunosorbent assay（ELISA）. Methods MTXPG-containing erythrocytes were lysed with a series of different methods, including freeze-thawing, chemical lysis, enzymatic lysis, ultrasonication.MTXPG was transformed into methotrexate（MTX） in the treatment of ascorbic acid or mercaptoethanol after serum and cell lysis buffer were added. Perchloric acid, trichloroacetic acid and saturated ammonium sulfate solution were adopted to precipitate proteins of the transforming solution. ELISA was adopted to measure concentrations of MTX following adjustment of p H value from 3.0 to 9.0. The accuracy of the method were evaluated by detecting clinical samples after methodology validation. Results Freeze-thawing and ultrasonication for lysis of erythrocytes were significantly better than enzymatic lysis and chemical lysis（P 〈0.05）. In the process of MTXPG transforming, scorbic acid was significantly better than mercaptoethanol. MTXPG was completely transformed in the condition of incubation of transforming solutions incubation at 37℃ for 3 h. Protein precipitation was showed no statistical difference in the Perchloric acid,trichloroacetic acid and saturated ammonium sulfate solution（P 〉0.05）. The scope of p H was 6-8 and the optimal p H value was 6.8. The results showed a good linear relation of concentration in the range of 0.2 to 10.0 ng/m L（r=0.9967,recovery rate was 96.6%-103.3%, RSD=8.0%-14.3%, within-day and between-day accuracy were 1.7%-11.7%, 7.4%-13.5%. Concentrations of MTXPG in erythrocytes of patients was（64.75 ±28.45） ng/m L. Conclusion Besides simpleand easy operation, the established method shows excellent accuracy and specificity, which can provide a novel pathway for clinical detection for concentrations of MTXPG in erythrocytes.