In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoylecgonine and the low affinity (Kd > 30 μM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

在这项工作中，我们确定与重组人源化抗可卡因 mAb (h2E2) 的 Fab 片段结合的可卡因可以使用简单且廉价的吸收和荧光测量直接且容易地定量，使用通常用于差示扫描荧光法、DASPMI 和SYPRO 橙色。对于 Fab 片段的浓缩样品，使用这些染料的吸收光谱显示存在的可卡因位点的数量，使用 DASPMI（通过测量染料吸光度的增加）或 SYPRO Orange（通过测量染料最大吸光度波长的变化）。有趣的是，我们观察到与 Fab 片段结合的可卡因对 SYPRO Orange 染料吸光度的影响与之前报道的完整 h2E2 mAb 的影响大不相同，与之前使用完整 h2E2 mAb 的结果相比，导致 Fab 片段的总染料吸光度大幅下降。对于 Fab 片段的稀释样品，开发了一种染料荧光发射光谱测定法，以通过配体诱导的这两种外在染料的荧光发射减少来量化可卡因（和其他高亲和力可卡因代谢物）结合位点的数量。使用该测定法，高亲和力（Kd < 30 nM）配体、可卡因、可卡乙烯和苯甲酰芽子碱和低亲和力（Kd > 30 μM）配体、去甲可卡因、芽子碱甲酯和芽子碱的可卡因滴定差异很明显。这些简单、直接且廉价的技术应证明可用于评估其他小分子抗原结合 Fab 片段，