Iznaga Escobar N, Morales A, Núñez G
Center of Molecular Immunology, Havana, Cuba.
Nucl Med Biol. 1996 Jul;23(5):641-4. doi: 10.1016/0969-8051(96)00028-5.
A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (microgram/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 micrograms, the lowest detection limit for cysteine quantification was 0.03 microgram. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.
本文描述了一种简单、快速且可重复的微量方法，用于定量用2-巯基乙醇（2-ME）还原单克隆抗体（MAb）二硫键后产生的巯基（SH）基团。使用Ellman试剂显色，根据半胱氨酸标准曲线计算2-ME和其他还原剂中每分子抗体的SH基团数量。结果以405 nm处的吸光度对半胱氨酸浓度（微克/毫升）作图。扣除Ellman试剂产生的背景后，得到一条通过原点的直线关系。对黄色产物的吸收光谱进行了控制，发现在412 nm和405 nm处的光密度之间没有显著差异。使用约37微克的少量抗体，半胱氨酸定量的最低检测限为0.03微克。半胱氨酸浓度与吸光度之间发现了极好的线性相关性（r = 0.999），样品中半胱氨酸定量的相对误差平均值为2.8%。统计学上的Student t检验表明半胱氨酸标准品与样品之间具有极好的线性和平行性。
