Department of Chemical and Biomolecular Nanotechnology, IQAC-CSIC, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain.
Anal Chem. 2012 May 15;84(10):4527-34. doi: 10.1021/ac300263m. Epub 2012 Apr 30.
Antibodies with a wide recognition profile of fluoroquinolone antibiotics have been produced based on chemical criteria, theoretical studies, and molecular modeling assisted hapten design. The immunizing hapten preserves the most important and characteristic epitopes of this antibiotic family. The studies have taken into consideration the zwitterionic character of most of the fluoroquinolones and the relative concentration of the different species in equilibrium at physiologic pH. The hapten is prepared in the form of a stable prehapten through a 5 step synthetic pathway. Immediately before conjugation, the immunizing hapten is obtained by removing the diphenylmethane protecting group. The specificity of the antibodies obtained is directed toward the common area defined by the fluorine atom at position 6 and the β-ketoacid moiety. The ELISA developed is able to recognize with very good detectability important fluoroquinolones used in the veterinary field such as ciprofloxacin (CPFX, IC(50), 0.35 μg L(-1)), enrofloxacin (ERFX, IC(50), 0.65 μg L(-1)), danofloxacin (DNFX, IC(50), 7.31 μg L(-1)), difloxacin (DFX, IC(50), 0.91 μg L(-1)), sarafloxacin (SRFX, IC(50), 0.96 μg L(-1)), norfloxacin (NRFX, IC(50), 0.78 μg L(-1)), ofloxacin (OFX, IC(50), 1.84 μg L(-1)), flumequine (Flume, IC(50), 3.91 μ gL(-1)), marbofloxacin (MBFX, IC(50), 4.30 μ gL(-1)), and oxolinic acid (OXO, IC(50), 23.53 μg L(-1)). The results presented here demonstrate that the antibody affinity is strongly affected by the presence of divalent cations, owing to their complexation with the fluoroquinolone molecules. Moreover, the outcome from the effect of the pH on the immunochemical assays suggests that the selectivity could be modulated with the pH due to the zwitterionic character of the fluoroquinolones and as a function of their different pK(a) values.
基于化学标准、理论研究和分子建模辅助半抗原设计，已经生产出了对氟喹诺酮类抗生素具有广泛识别特性的抗体。免疫半抗原保留了该抗生素家族最重要和最具特征性的表位。这些研究考虑到了大多数氟喹诺酮类药物的两性离子特性以及在生理 pH 值下处于平衡状态的不同物种的相对浓度。半抗原通过五步合成途径制备成稳定的前半抗原形式。在进行缀合之前，通过去除二苯甲烷保护基获得免疫半抗原。所获得的抗体的特异性针对由 6 位氟原子和β-酮酸部分定义的共同区域。开发的 ELISA 能够非常好地检测到在兽医领域使用的重要氟喹诺酮类药物，如环丙沙星（CPFX，IC（50），0.35μg L（-1））、恩诺沙星（ERFX，IC（50），0.65μg L（-1））、达氟沙星（DNFX，IC（50），7.31μg L（-1））、二氟沙星（DFX，IC（50），0.91μg L（-1））、沙拉沙星（SRFX，IC（50），0.96μg L（-1））、诺氟沙星（NRFX，IC（50），0.78μg L（-1））、氧氟沙星（OFX，IC（50），1.84μg L（-1））、氟甲喹（Flume，IC（50），3.91μgL（-1））、马波沙星（MBFX，IC（50），4.30μgL（-1））和恶喹酸（OXO，IC（50），23.53μg L（-1））。这里呈现的结果表明，抗体亲和力受二价阳离子的存在强烈影响，这是由于它们与氟喹诺酮类分子的络合作用。此外，免疫化学测定中 pH 值的影响结果表明，由于氟喹诺酮类的两性离子特性以及它们不同的 pK（a）值，选择性可以通过 pH 值进行调节。
