三硝基甲苯血红蛋白加合物的竞争抑制性酶联免疫吸附试验测定

建立了竞争抑制性酶联免疲吸附试验方法(CI—ELISA)以定量测定三硝基甲苯(TNT)血红蛋白加合物,该法的线性范围0.5 ng·ml~(-1)～1μg·ml~(-1),每个样品最小可测值为0.05 ng;批内变异系数8.0％左右,批间变异系数约为15.0％;正常大鼠血浆和血红蛋白溶液中外加2-氨基-4,6-二硝基甲笨(2A)或4-氨基-2,6-二硝基甲苯(4A)的回收率为84～101％,用此法首次检测TNT染毒大鼠中血红蛋白加合物,证实TNT血红蛋白加舍物水平与染毒剂量之间有明显的依赖关系,并可存留较长时间;反复染毒时,血红蛋白中2A,4A量有明显蓄积现象。

A competitive inhibition enzyme-linked immunosorbent assay for trinitrotoluene-hemoglobin adducts

Trinitrotoluene (TNT) is one of the most commonly used explosives. Acute or prolonged exposure to TNT has been reported to result in serious toxic effects, such as liver injury and cataract. There is a great need to develop a sensitive method for bio-monitoring. Our previous studies showed that TNT hemoglobin adducts appeared to be a good monitoring parameter. So a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) method for quantitative measuring TNT hemoglobin adducts was developed and described in this paper.Artificial antigens were prepared by coupling 2A or 4A, which are major contents released from the adducts, to bovine serum albumin, diphtheria toxoid and hemocyanin from Limulus polyphenus, resulting in conjugates containing 8.9-27.9 mol of 2A or 4A per 100 kDa. Antibodies to two of these artificial antigens were raised in rabbits. The rabbit antisera are strongly reactive and the liter varies from 1:8 ×103 to 1:64 ×103. The linear range of this method is between 0.5-1000 ng ·ml-1 and 0.05 ng of 2A or 4A can be detected in each sample. The coefficients of the variationare approximately 8.0% and 15.0% for intra- and inter-assay respectively. The recoveries of 2A or 4A in the plasma and hemoglobin of the control rats are 84-101%.By using CI-ELISA, TNT hemoglobin adducts were measured in the TNT treated rats for the first time. The dose-response and time-response relationships were shown in all the experiments, including administration of different dosages of TNT (10, 25, 50 and 75 mg·kg-1 ip) or different time points (1, 4, 24, 72, 120 and 168 h) after dosing. After a single administration of TNT, the adducts could be detected for a period of time. The amount of 2A and 4A in hemoglobin accumulated obviously upon repeated exposure.The advantages of this method including sensitivity, reliability and practicality showed that CI-ELISA method might be used for quantitating TNT hemoglobin adducts as a dose monitor in a large scale investigation.