Ciprofloxacin and Clinafloxacin Antibodies for an Immunoassay of Quinolones: Quantitative Structure⁻Activity Analysis of Cross-Reactivities.

A common problem in the immunodetection of structurally close compounds is understanding the regularities of immune recognition, and elucidating the basic structural elements that provide it. Correct identification of these elements would allow for select immunogens to obtain antibodies with either wide specificity to different representatives of a given chemical class (for class-specific immunoassays), or narrow specificity to a unique compound (mono-specific immunoassays). Fluoroquinolones (FQs; antibiotic contaminants of animal-derived foods) are of particular interest for such research. We studied the structural basis of immune recognition of FQs by antibodies against ciprofloxacin (CIP) and clinafloxacin (CLI) as the immunizing hapten. CIP and CLI possess the same cyclopropyl substituents at the N1 position, while their substituents at C7 and C8 are different. Anti-CIP antibodies were specific to 22 of 24 FQs, while anti-CLI antibodies were specific to 11 of 26 FQs. The molecular size was critical for the binding between the FQs and the anti-CIP antibody. The presence of the cyclopropyl ring at the N1 position was important for the recognition between fluoroquinolones and the anti-CLI antibody. The anti-CIP quantitative structure⁻activity relationship (QSAR) model was well-equipped to predict the test set (pred_R² = 0.944). The statistical parameters of the anti-CLI model were also high (R² = 0.885, q² = 0.864). Thus, the obtained QSAR models yielded sufficient correlation coefficients, internal stability, and predictive ability. This work broadens our knowledge of the molecular mechanisms of FQs' interaction with antibodies, and it will contribute to the further development of antibiotic immunoassays.

Heterologous strategy enhancing the sensitivity of the fluorescence polarization immunoassay of clinafloxacin in goat milk.

BACKGROUND: Clinafloxacin is used for the treatment of disease in food-producing animals, e.g. Brucella melitensis, which often occurs in goats; however, the clinafloxacin residue in goat milk may harm human health and result in the development of drug-resistant bacterial strains or allergies. Despite this, there is not a rapid, sensitive and accurate analytical method in goat milk for rapid screening or monitoring purposes. RESULTS: One homologous and five heterologous tracers were designed and compared for fluorescence polarization immunoassay (FPIA) optimization. Based on the combination of a heterologous tracer (PAZ-FITC, synthesized with pazufloxacin and FITC) and the antibody against clinafloxacin, a highly sensitive FPIA was established for the detection of clinafloxacin residue in goat milk for the first time. The IC50 value was 29.3 µg L(-1) for clinafloxacin in the heterologous format - six times lower than that of the combination of the homologous tracers and the antibody. The recoveries ranged from 86.8% to 104.5%, with the relative standard deviation ranging from 4.1% to 7.2%. Validation by high-performance liquid chromatography (HPLC) confirmed that the results obtained from the proposed FPIA were in agreement with those of HPLC. CONCLUSION: This proposed heterologous strategy for enhanced FPIA is sensitive and rapid enough for the high-throughput detection of clinafloxacin residue in goat milk.

Rapid immunochromatographic assay for ofloxacin in animal original foodstuffs using native antisera labeled by colloidal gold.

An immunochromatographic assay was developed to detect fluoroquinolone antibiotic ofloxacin based on the competitive binding of ofloxacin and the membrane-immobilized ofloxacin-protein conjugate to colloidal gold-labeled antibodies in the course of the labeled antibodies, and to test sample flow through the membrane. The specific feature of labeling by colloidal gold is that native antiserum is used instead of purified immunoglobulins or specific antibodies. This makes the synthetic procedure easier, with no sacrifice in the detection limit. The proposed test makes it possible to detect down to 30 ng mL(-1) of ofloxacin, which corresponds to the demands of food safety assessment. The assay time is 10 min. The assay provides reliable information on the ofloxacin content in milk without the sample preparation and in chicken and pork meat with the minimum sample preparation (the separation of the insoluble fraction of the homogenate by centrifugation). The high degree of detection of ofloxacin in foodstuffs by the proposed assay (70-112%) was shown by a comparison with the data obtained with the use of a commercial immunoenzymatic kit.