Sujatha N, Suryakala S, Rao B S
Osmania University, University College of Science, Department of Biochemistry, Hyderabad, India.
J AOAC Int. 2001 Sep-Oct;84(5):1465-74.
A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-alpha-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-alpha-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thin-layer chromatography showed a single spot (Rf = 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of the AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 +/- 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50,000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 +/- 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 microg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 +/- 4.37 microg/mg albumin.
开发了一种简单的程序，用于使用黄曲霉毒素B1、N-α-乙酰赖氨酸和间氯过苯甲酸（MCPBA）体外合成和表征黄曲霉毒素B1-赖氨酸加合物。在1:16（黄曲霉毒素B1:N-α-十六烷基赖氨酸）的摩尔比下，加合物的回收率为62%。通过薄层色谱分析加合物显示为一个斑点（比移值Rf = 0）。加合物的吸收光谱在275和335 nm处显示出2个峰。黄曲霉毒素B1-赖氨酸加合物的液相色谱（LC）分析显示相对保留时间为2.1分钟。使用相同的环氧化程序，分别合成了牛血清白蛋白-黄曲霉毒素B1加合物和卵清蛋白-黄曲霉毒素B1加合物，用于制备抗体和作为包被抗原。向对照大鼠血清中加入黄曲霉毒素B1-赖氨酸加合物并进行LC分析，显示保留时间为2.1分钟，这与合成的黄曲霉毒素B1-赖氨酸参考标准品的保留时间相似。此外，向经酶水解的对照大鼠血清中加入牛血清白蛋白-黄曲霉毒素B1加合物，显示出2个峰，保留时间分别为2.1和2.7分钟。基于LC分析，以黄曲霉毒素B1-赖氨酸加合物计，牛血清白蛋白-黄曲霉毒素B1的回收率为67±5%。主要峰（2.1分钟）占加合物的72%；第二个小峰（2.7分钟）占形成的总黄曲霉毒素B1-赖氨酸加合物的28%。对合成的黄曲霉毒素B1-赖氨酸加合物进行的稳定性研究表明，它在1个月内是稳定的。当使用卵清蛋白-黄曲霉毒素B1作为包被抗原时，抗体捕获试验在1:50,000的稀释度下显示吸光度为0.9至1.0。间接竞争ELISA显示，在黄曲霉毒素B1-赖氨酸浓度为13 ng时，抗体的50%被置换（半数抑制浓度IC50），而黄曲霉毒素B1的IC50为7 ng。向对照大鼠血清中加入黄曲霉毒素B1-赖氨酸加合物，然后进行酶水解和免疫分析（间接ELISA），回收率为93±6%。酶免疫测定通过啮齿动物模型进行了验证，在该模型中，动物暴露于黄曲霉毒素B1（20μg黄曲霉毒素B1/千克体重/天）。大鼠血清中黄曲霉毒素B1-赖氨酸加合物的水平为27.3±4.37μg/毫克白蛋白。
