RESUMEN
A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.
Asunto(s)
Anticuerpos Monoclonales/química , Residuos de Medicamentos/análisis , Oxitetraciclina/química , Penaeidae/química , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Oxitetraciclina/análisis , Reproducibilidad de los Resultados , Rolitetraciclina/química , Sensibilidad y Especificidad , Albúmina Sérica BovinaRESUMEN
An understanding of the detailed energetics and mechanism of the binding of drugs with target proteins is essential for devising guidelines to synthesize new drugs. Binding of the antibiotic drugs tetracycline and rolitetracycline with serum albumin has been studied by a combination of isothermal titration calorimetry, differential scanning calorimetry, steady-state and time-resolved fluorescence, and circular dichroism spectroscopies. Both tetracycline and rolitetracycline bind to bovine serum albumin in a sequential manner with first binding being the major binding event with an association constant of the order of 10(4) for tetracycline and 10(3) for rolitetracycline, respectively. Ionic strength dependence and binding in the presence of tetrabutylammonium bromide and sucrose indicate involvement of a mix of hydrophobic, ionic, and hydrogen bonding interactions. The isothermal titration calorimetry results for the binding of these drugs to bovine serum albumin in the presence of warfarin and in the presence of each other indicate that both these drugs share binding site 2 on bovine serum albumin. The differential scanning calorimetry results provide quantitative information on the effect of drugs on the stability of bovine serum albumin. A comparison of isothermal titration calorimetry and fluorescence results demonstrates that the former technique has been able to explain the sequential binding events that can be missed by the fluorescence measurements.
Asunto(s)
Antibacterianos/metabolismo , Rolitetraciclina/metabolismo , Albúmina Sérica Bovina/metabolismo , Tetraciclina/metabolismo , Animales , Antibacterianos/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Bovinos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Rolitetraciclina/química , Albúmina Sérica Bovina/química , Espectrometría de Fluorescencia , Tetraciclina/química , TermodinámicaRESUMEN
One of the major pathological features of Alzheimer's disease is the deposition of beta-amyloid peptide (Abeta). Cellular toxicity has been shown to be associated with fibrillar forms of Abeta; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Abeta fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Abeta fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Abeta and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.