Abraham R, Moller D, Gabel D, Senter P, Hellström I, Hellström K E
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121.
J Immunol Methods. 1991 Nov 5;144(1):77-86. doi: 10.1016/0022-1759(91)90233-6.
Eight monoclonal antibodies of different classes and isotypes and rabbit IgG were oxidized under a variety of conditions with 5-50 mM periodate. The number of aldehyde groups generated per immunoglobulin were measured by reduction with tritiated sodium borohydride or coupling of fluoresceinthiosemicarbazide. There were up to 25.5 aldehyde groups detected on the periodate-oxidized antibody 96.5, measured by borohydride reduction whereas the same conditions led to only 9.6 aldehydes per IgG on the antibody L6 of the same IgG2A isotype. Fluoresceinthiosemicarbazide bound to oxidized antibodies but not to the same extent as tritium. On mildly oxidized IgMs it was possible to generate more than 200 aldehyde groups per antibody molecule. Depending on the conditions and the antibody used periodate oxidation could lead to antibody crosslinking. The avidities of the modified antibodies were determined by Scatchard analyses and inhibition assays. A new mathematical method to evaluate the immunoreactivities of modified antibodies relative to the unlabeled native antibody from inhibition binding data was established. Periodate concentrations higher than 50 mM decreased the avidities and immunoreactivities of all IgGs tested. This effect is more pronounced if the oxidation is performed at pH 5.6 and 25 degrees C instead of pH 4.6 and 0 degree C. The BR96 antibody is inactivated even under mild oxidation conditions.
八种不同类别和亚型的单克隆抗体以及兔免疫球蛋白G（IgG）在多种条件下用5 - 50 mM高碘酸盐进行氧化。通过用氚化硼氢化钠还原或与荧光硫代半卡巴腙偶联来测定每个免疫球蛋白产生的醛基数量。通过硼氢化钠还原测定，在高碘酸盐氧化的抗体96.5上检测到多达25.5个醛基，而在相同IgG2A亚型的抗体L6上，相同条件下每个IgG仅产生9.6个醛基。荧光硫代半卡巴腙与氧化抗体结合，但程度不如氚。在轻度氧化的IgM上，每个抗体分子可能产生超过200个醛基。根据条件和所用抗体的不同，高碘酸盐氧化可能导致抗体交联。通过Scatchard分析和抑制试验测定修饰抗体的亲和力。建立了一种新的数学方法，用于根据抑制结合数据评估修饰抗体相对于未标记天然抗体的免疫反应性。高于50 mM的高碘酸盐浓度会降低所有测试IgG的亲和力和免疫反应性。如果在pH 5.6和25℃而不是pH 4.6和0℃下进行氧化，这种效应会更明显。即使在温和的氧化条件下，BR96抗体也会失活。
