RESUMO
BACKGROUND: Antibiotics have been implicated in the reactivation of exanthema and systemic involvement in drug reaction with eosinophilia and systemic symptoms (DRESS); however, it is not clear whether these patients become sensitized to the antibiotic. OBJECTIVE: To evaluate if, after DRESS, patients become sensitized to antibiotics. METHODS: We retrospectively reviewed the patch test (PT) data and clinical files of DRESS patients who were administered antibiotics during DRESS from other culprits. RESULTS: Nine patients out of 17 (53%) were positive to antibiotics in PT: six to the penicillin group and three to cephalosporins (including one patient with additional positivity to vancomycin). Considering the eight patients who were negative to antibiotics in PT, seven were exposed to a fluoroquinolone. Four cases were patch tested again and three remained positive to antibiotics 2 to 5 years thereafter. Two patients with positive PT results had an accidental re-exposure to antibiotics and developed a maculopapular exanthema without systemic symptoms. CONCLUSION: Exposure to antibiotics during DRESS or its prodromal phase could enhance sensitization to antibiotics, as confirmed by a positive PT. Reproducibility of positive PTs to antibiotics after several years and reactivation after re-exposure support that T-cell-mediated hypersensitivity to antibiotics in the setting of DRESS is a specific reaction.
Assuntos
Antibacterianos/efeitos adversos , Síndrome de Hipersensibilidade a Medicamentos/etiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/imunologia , Cefalosporinas/efeitos adversos , Cefalosporinas/imunologia , Criança , Síndrome de Hipersensibilidade a Medicamentos/imunologia , Feminino , Fluoroquinolonas/efeitos adversos , Fluoroquinolonas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Penicilinas/efeitos adversos , Penicilinas/imunologia , Estudos RetrospectivosRESUMO
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 structureâ»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.
Assuntos
Anticorpos/metabolismo , Ciprofloxacina/química , Fluoroquinolonas/análise , Animais , Anticorpos/química , Especificidade de Anticorpos , Ciprofloxacina/imunologia , Fluoroquinolonas/química , Fluoroquinolonas/imunologia , Imunização , Imunoensaio , Masculino , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , CoelhosRESUMO
On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens.
Assuntos
Anticorpos/imunologia , Fluoroquinolonas/análise , Fluoroquinolonas/imunologia , Haptenos/química , Haptenos/imunologia , Técnicas Imunoenzimáticas/métodos , Luminescência , Modelos Moleculares , Oxazinas/análise , Oxazinas/imunologia , Animais , Fluoroquinolonas/síntese química , Leite/química , Estrutura Molecular , Oxazinas/síntese químicaRESUMO
Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.
Assuntos
Anticorpos Monoclonais/imunologia , Fluoroquinolonas/imunologia , Hibridomas/imunologia , Imunização/métodos , Animais , Anticorpos Monoclonais/biossíntese , Reações Antígeno-Anticorpo , Bovinos , Fluoroquinolonas/química , Hibridomas/metabolismo , Imunoensaio/métodos , Coelhos , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologiaRESUMO
The prescription ot fluoroquinolones has been constantly increasing over the past decade. consequently, an increasing number of hyper-sensitivity reactions and adverse events have been reported. The aim of the review is to discuss the incidence of hypersensitivity reactions either IgE (immediate) or T cells mediated (delayed). We will make an overview ofthe diagnostic tools available to detect such hypersensitivity reactions. Finally, the specific adverse events associated with fluoroquinolones, including tendinopathy, chondrotoxicity, peripheral neuropathy or retinal detachment will be discussed.
Assuntos
Antibacterianos/efeitos adversos , Hipersensibilidade a Drogas/etiologia , Fluoroquinolonas/efeitos adversos , Antibacterianos/imunologia , Fluoroquinolonas/imunologia , Humanos , Hipersensibilidade Tardia/etiologia , Hipersensibilidade Tardia/imunologia , Imunoglobulina E/imunologia , Incidência , Linfócitos T/imunologiaAssuntos
Antibacterianos/imunologia , Meios de Contraste/efeitos adversos , Síndrome de Hipersensibilidade a Medicamentos/diagnóstico , Testes Intradérmicos/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Reações Cruzadas , Síndrome de Hipersensibilidade a Medicamentos/imunologia , Feminino , Fluoroquinolonas/efeitos adversos , Fluoroquinolonas/imunologia , Glicopeptídeos/efeitos adversos , Glicopeptídeos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , beta-Lactamas/efeitos adversos , beta-Lactamas/imunologiaRESUMO
A recombinant bispecific single-chain diabody (scDb), recognizing fluoroquinolones (FQs) and sulfonamides (SAs), was successfully constructed with two single-chain variable fragment antibodies (scFvs). The scDb gene was cloned into the expression vector pJB33, and 6×His-tagged scDb was expressed as soluble bodies in Escherichia coli RV308 host, then purified by one step affinity chromatography of immobilized metal ion affinity chromatography (IMAC). SDS-PAGE and Western blotting analysis of the purified scDb indicated that the prepared scDb was successfully expressed as a â¼60 kDa and the final purity of the scDb protein was up to 95% with yields of approximately 6 mg/L of bacterial culture. The scDb was further characterized by indirect competitive enzyme linked immunosorbent assay (icELISA), showing that the affinity and specificity of scDb were fully retained from the two parental scFvs, capable of simultaneously binding FQs and SAs. The 50% inhibition concentration (IC50) values of the optimized immunoassay were 0.45 ng mL(-1) for FQs and 0.75 ng mL(-1) for SAs, respectively. The scDb exhibited high affinity to 20 FQs and 14 SAs. Taken together, these findings suggested that the prepared scDb could be used to develop future novel immunoassay for simultaneous determination of 20 FQs and 14 SAs.
Assuntos
Antibacterianos/imunologia , Anticorpos Biespecíficos/imunologia , Escherichia coli/genética , Fluoroquinolonas/imunologia , Anticorpos de Cadeia Única/imunologia , Sulfonamidas/imunologia , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/isolamento & purificação , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Due to the widespread occurrence in the environment and potential risk toward organisms of fluoroquinolones (FQs), it is of importance to develop high efficient methods for assessing their occurrence and environmental risk. A monoclonal antibody (Mab) with broad cross-reactivity to FQs was produced by immunizing BALB/c mice with a synthesized immunogen prepared by conjugating ciprofloxacin with bovine serum albumin. This developed Mab (C2F3C2) showed broad and high cross-reactivity (40.3-116%) to 12 out of the 13 studied FQs. Using this Mab and norfloxacin conjugated with carrier protein ovalbumin as coating antigen, a time-resolved fluoroimmunoassay (TRFIA) method was developed for determining the total concentration of at least 12 FQs in environmental waters. The respective detection limit (LOD) and IC(50) calculated from the standard curve were 0.053 µg/L and 1.83 µg/L for enrofloxacin (ENR). The LODs of the other FQs, estimated based on the corresponding cross-reactivity and the LOD of ENR, were in the range of 0.051-0.10 µg/L. The developed TRFIA method showed good tolerance to various interfering substances present in environmental matrix at relevant levels, such as humic acids (0-10 mg/L DOC), water hardness (0-2% Ca(2+) and Mg(2+), w/v), and heavy metals (0-1 mg/L). The spiked recoveries estimated by spiking 0.5, 1, and 2 µg/L of five representative FQs into various water samples including paddy water, tap water, pond water, and river water were in the range of 63-120%. The measured total FQ concentration by TRFIA agreed well with that of liquid chromatography-tandem mass spectrometry and was applied to directly evaluate the occurrence and environmental risk of FQs in the surface water of a case area. TRFIA showed high efficiency and great potential in environmental risk assessment as it measures directly the total concentration of a class of pollutants.
Assuntos
Anticorpos Monoclonais/imunologia , Fluoroquinolonas/análise , Haptenos/imunologia , Poluentes Químicos da Água/análise , Animais , Monitoramento Ambiental , Feminino , Fluorimunoensaio , Fluoroquinolonas/imunologia , Haptenos/química , Camundongos , Camundongos Endogâmicos BALB C , Medição de Risco , Soroalbumina Bovina/química , Soroalbumina Bovina/imunologia , Poluentes Químicos da Água/imunologiaRESUMO
A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously, where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model.
Assuntos
Afinidade de Anticorpos , Antineoplásicos/análise , Epitopos/imunologia , Fluoroquinolonas/análise , Imunoensaio/métodos , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Enrofloxacina , Epitopos/química , Fluoroquinolonas/imunologia , Limite de Detecção , Modelos MolecularesRESUMO
BACKGROUND: The residues of fluoroquinolone drugs in foods of animal origin are dangerous to the consumers. The objective of this study was to produce a generic monoclonal antibody for determination of fluoroquinolone residues in meat. RESULTS: Two novel haptens of ciprofloxacin containing a free amidogen group on the piperazinyl ring were synthesised that were used to produce the monoclonal antibodies. The antibodies obtained simultaneously recognised 12 fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, sarafloxacin, diflocaxin, danofloxacin, ofloxacin, marbofloxacin, pefloxacin, lomefloxacin, amifloxacin and enofloxacin). After evaluation of different coating antigen-antibody combinations, a heterologous competitive indirect ELISA was used to determine the 12 drugs. The cross-reactivities were in the range of 23-120% and the limits of detection were in the range of 1.0-4.5 ng mL(-1). Eight fluoroquinolone drugs licensed as veterinary drugs in China were fortified into blank chicken for analysis. The recoveries were in the range of 61.5-82.5% with coefficients of variation in the range of 7.5-15.2%. CONCLUSION: This method could be used as a rapid screening tool for routine monitoring the residues of these fluoroquinolone drugs in animal-derived foods.
Assuntos
Anticorpos Monoclonais/biossíntese , Ciprofloxacina/imunologia , Monitoramento Ambiental/métodos , Fluoroquinolonas/análise , Contaminação de Alimentos/análise , Haptenos/imunologia , Carne/análise , Animais , Galinhas , China , Reações Cruzadas , Dieta , Fluoroquinolonas/imunologia , Humanos , Limite de Detecção , Drogas Veterinárias/análise , Drogas Veterinárias/imunologiaRESUMO
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 ß-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.
Assuntos
Antibacterianos/imunologia , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Fluoroquinolonas/imunologia , Haptenos/imunologia , Modelos Moleculares , Antibacterianos/química , Cátions/química , Fluoroquinolonas/química , Haptenos/química , Concentração de Íons de HidrogênioRESUMO
Fluoroquinolones (FQs) are a group of synthetic, broad-spectrum antibacterial agents. Due to its extensive use in animal industry and aquaculture, residues of these antibiotics and the emergence of bacteria resistant to FQs have become a major public health issue. To prepare a generic antibody capable of recognizing nearly all FQs, a single-chain variable fragment (scFv) was generated from the murine hybridoma cells C49H1 producing a FQ-specific monoclonal antibody. This scFv was characterized by indirect competitive enzyme-linked immunosorbent assay (ciELISA), and it showed identical binding properties to parental monoclonal antibody: it was capable of recognizing 17 of 20 targeted FQs below maximum residue limits, except for sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO) which are highly concerned members in the FQs family. In order to broaden the specificity of this scFv to SAR and its analogues (DIF and TRO), protein homology modeling and antibody-ligands docking analysis were employed to identify the potential key amino acid residues involved in hapten antibody. A mutagenesis phage display library was generated by site directed mutagenesis randomizing five aminoacid residues in the third heavy-chain complementarity determining region. After one round of panning against biotinylated norfloxacin (NOR) and four rounds of panning against biotinylated SAR, scFv variants we screened showed up to 10-fold improved IC(50) against SAR, DIF, and TRO in ciELISA while the specificity against other FQs was fully retained.
Assuntos
Antibacterianos/análise , Antibacterianos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fluoroquinolonas/análise , Fluoroquinolonas/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Clonagem Molecular , Hibridomas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Sensibilidade e Especificidade , Anticorpos de Cadeia Única/genéticaRESUMO
We developed fluorescent biosensor systems that are either general or selective to fluoroquinolone antibiotics by using a single-chain variable-fragment (scFv) as a recognition element. The selectivity of these biosensors to fluoroquinolone antibiotics was rationally tuned through the structural modification on the pharmacophore of fluoroquinolone antibiotics and the subsequent selection of scFv receptor modules against these antibiotics-based antigens using phage display. The resulting A2 and F9 scFv's bound to their representative antigen with a moderate affinity (K(D) in micromolar range as determined by surface plasmon resonance). A2 is a specific binder for enrofloxacin and did not cross-react with other fluoroquinolone antibiotics including structurally similar ciprofloxacin, while F9 is a general fluoroquinolone binder that likely bound to the antigen at the common pyridone-carboxylic acid pharmacophore. These scFv-based receptors were successfully applied to the development of one-step fluorescent biosensor which can detect fluoroquinolone antibiotics at concentrations below the level suggested in animal drug application guidelines. The strategy described in this report can be applied to developing convenient field biosensors that can qualitatively detect overused/misused antibiotics in the livestock drinking water.
Assuntos
Antibacterianos/análise , Especificidade de Anticorpos , Anticorpos de Cadeia Única/imunologia , Detecção do Abuso de Substâncias/métodos , Ressonância de Plasmônio de Superfície/métodos , Antibacterianos/imunologia , Anticorpos Imobilizados/imunologia , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Fluoroquinolonas/análise , Fluoroquinolonas/imunologia , Biblioteca de Peptídeos , Espectrometria de FluorescênciaRESUMO
Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider specificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody.
Assuntos
Antibacterianos/análise , Anticorpos Monoclonais/imunologia , Fluoroquinolonas/análise , Imunoensaio/métodos , Engenharia de Proteínas , Antibacterianos/síntese química , Antibacterianos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Biotinilação , Fluoroquinolonas/síntese química , Fluoroquinolonas/imunologia , Mutagênese , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
The study of immunomodulating, antioxidant and hepatoprotective activity of some fluoroquinolones (norfloxacin, ofloxacin, levofloxacin) immobilized into erythrocyte and leukocyte carriers was made on Wistar rats with body mass about 150-180 g. It is shown that toxic action on the kidneys of mercury dichloride, especially a combined action of mercury dichloride and staphylococcal infection, raised the levels of urea and creatinine, caused immunosuppression, activated hepatotoxic, cytolytic and oxidative processes, decreased antioxidant and energetic potentials of erythrocytes. Unbound fluoroquinolones intensified the above processes. Introduction of fluoroquinolones immobilized into erythrocyte and leukocyte carriers, respectively, decreased and normalized intensity of lipid peroxidation, cholestasis, cytolysis, improved and normalized immune system functions, antioxidant and energetic potentials of erythrocytes.
Assuntos
Antioxidantes/farmacologia , Eritrócitos/imunologia , Fluoroquinolonas/farmacologia , Nefropatias/imunologia , Leucócitos/imunologia , Infecções Estafilocócicas/imunologia , Animais , Antioxidantes/metabolismo , Desinfetantes/efeitos adversos , Desinfetantes/farmacologia , Fluoroquinolonas/imunologia , Fluoroquinolonas/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/terapia , Cloreto de Mercúrio/efeitos adversos , Cloreto de Mercúrio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Ratos , Ratos Wistar , Infecções Estafilocócicas/induzido quimicamente , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/terapiaRESUMO
A polyclonal anti-gatifloxacin antibody has been prepared, and an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed on the basis of the antibody prepared for the first time. The antibody shows high sensitivity with an IC50 value of 2.6 ppb and excellent specificity with only a minor cross-reaction with lomefloxacin (3.0%) among common (fluoro)quinolones evaluated in this study. The high specificity of the antibody was explained by the molecular structures of related drugs by comparison with published research. The cELISA test kit developed has a detection limit of 0.05 ppb and could be used as a screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to the detection of milk samples spiked by gatifloxacin. The recovery rates were in the range of 86-106%, whereas the intra- and interassay coefficients of variation were <14.3 and <19.6%, respectively.
Assuntos
Anti-Infecciosos/análise , Anticorpos/imunologia , Resíduos de Drogas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Fluoroquinolonas/análise , Fluoroquinolonas/imunologia , Leite/química , Animais , Especificidade de Anticorpos , Contaminação de Alimentos/análise , GatifloxacinaRESUMO
A polyclonal antibody against the quinolone drug pazufloxacin (PAZ) but with surprisingly broad specificity was raised to simultaneously detect 24 quinolones (QNs). The developed competitive indirect enzyme-linked immunosorbent assay (ciELISA) exhibited limits of detection (LODs) for the 24 QNs ranging from 0.45 to 15.16 ng/mL, below the maximum residue levels (MRLs). To better understand the obtained broad specificity, a genetic algorithm with linear assignment of hypermolecular alignment of data sets (GALAHAD) was used to generate the desired pharmacophore model and superimpose the QNs, and then advanced comparative molecular field analysis (CoMFA) and advanced comparative molecular similarity indices analysis (CoMSIA) models were employed to study the three-dimensional quantitative structure-activity relationship (3D QSAR) between QNs and the antibody. It was found that the QNs could interact with the antibody with different binding poses, and cross-reactivity was mainly positively correlated with the bulky substructure containing electronegative atom at the 7-position, while it was negatively associated with the large bulky substructure at the 1-position of QNs.
Assuntos
Fluoroquinolonas/farmacologia , Oxazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Quinolonas/farmacologia , Algoritmos , Ensaio de Imunoadsorção Enzimática , Fluoroquinolonas/imunologia , Oxazinas/imunologia , Sensibilidade e EspecificidadeRESUMO
In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 µg L(-1) to as low as 0.07 µg L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fluoroquinolonas/análise , Haptenos/química , Animais , Enrofloxacina , Fluoroquinolonas/imunologia , Haptenos/imunologia , Soros Imunes/imunologiaRESUMO
Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC(50)) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.
Assuntos
Anticorpos Monoclonais/imunologia , Técnicas de Química Analítica , Fluoroquinolonas/análise , Fluoroquinolonas/imunologia , Análise de Alimentos/métodos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Bovinos , Galinhas , Clonagem Molecular , Reações Cruzadas , DNA Complementar/genética , Enrofloxacina , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Concentração Inibidora 50 , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
A unique analytical method using nanoparticles and laser-induced fluorescence microscopy (LIFM) was developed to determine enrofloxacin in this work. For sample pretreatment, two different kinds of particles, i.e., synthesized dye-doped core-shell silica nanoparticles and magnetic micro-particles (MPs), were used for fluorescent tagging and concentrating the enrofloxacin, respectively. The antibody of enrofloxacin was immobilized on the synthesized FITC-doped core-shell nanoparticles, and the enrofloxacin target was extracted by the MPs. At this moment, the average number of antibodies on each core-shell silica nanoparticle was ~0.9, which was determined by the fluorescence ratiometric method. The described method was demonstrated for a meat sample to determine enrofloxacin using LIFM, and the result was compared with enzyme-linked immunosorbent assay (ELISA). The developed technique allowed the simplified analytical procedure, improved the detection limit about 54-fold compared to ELISA.