Thakur M L, DeFulvio J D
Thomas Jefferson University.
Biotechniques. 1990 May;8(5):512-6.
Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which generates a cysteine-ninhydrin complex (520 nm), has a molar extinction coefficient of 30 250 and can determine 0.04 micrograms/ml cysteine units with an absorbance of 0.01. The method has been applied to determine the quantity of cysteine groups produced by the reduction of an immunoglobin G antibody with five different reducing agents in normal to five times the previously determined optimal molar ratios. The quantities of cysteine units produced from the controlled reduction from 240 micrograms immunoglobin G ranged from 0.073 +/- 0.01 to 1.07 +/- 0.04 micrograms, which were merely 0.54 +/- 0.08% to 7.9 +/- 0.28% of the total available disulfide groups in the protein.
将二硫键还原为巯基以直接对抗体进行放射性标记用于免疫闪烁成像和治疗应用一直备受关注。已对灵敏的分光光度法进行了评估，这将使研究人员能够确定产生的半胱氨酸单位的亚微克量，以确保还原过程得到控制。一种方法可生成半胱氨酸 - 茚三酮复合物（520纳米），其摩尔消光系数为30250，在吸光度为0.01时可测定0.04微克/毫升的半胱氨酸单位。该方法已用于测定用五种不同还原剂还原免疫球蛋白G抗体时产生的半胱氨酸基团的量，还原剂的摩尔比为正常比例到先前确定的最佳摩尔比的五倍。从240微克免疫球蛋白G进行受控还原产生的半胱氨酸单位量在0.073±0.01至1.07±0.04微克之间，仅占蛋白质中总可用二硫键基团的0.54±0.08%至7.9±0.28%。
