Waveguide interrogated optical immunosensor (WIOS) for detection of sulfonamide antibiotics in milk.

An immunosensor was developed for the detection of sulfonamide antibiotics in milk. Detection relied on a competitive immunoassay format, using immunoreagents previously developed for the generic detection of sulfonamide antibiotics and evaluated by enzyme-linked immunosorbent assay. The immunoassay was implemented onto a microsystem platform, the wavelength interrogated optical sensing device, which uses the evanescent field to probe changes at the interface of a waveguiding layer, and thus allows sensitive detection of biomolecule adsorption. The immunoreagents were immobilized onto the surface of the waveguide chip, and a fluidic cell allowed flowing analyte and detection solutions above the surface. Sulfapyridine was used as reference sulfonamide and detected with the immunosensor in buffer and in milk with a limit of detection (IC(90)) of 0.2+/-0.1 microg L(-1) and 0.5+/-0.1 microg L(-1), respectively. The analysis time was below 30 min, including regeneration of the sensing surface, with minimum sample preparation required. The reproducibility of the detection was better than 10%. A blind assay allowed identifying milk samples that were contaminated with different sulfonamide antibiotics at or above the maximum residue limits established by the European Union for these compounds (100 microg L(-1)). Thus, the developed immunosensor presents great potential as a generic sensing device for the fast and early detection of food contaminants on the field by non-skilled users.

Lab-on-a-chip for multiplexed biosensing of residual antibiotics in milk.

A multiplexed immunoassay-based antibiotic sensing device integrated in a lab-on-a-chip format is described. The approach is multidisciplinary and involves the convergent development of a multi-antibiotic competitive immunoassay based on sensitive wavelength interrogated optical sensor (WIOS) technology and a polymer-based self-contained microfluidic cartridge. Immunoassay solutions are pressure-driven through external and concerted actuation of a single syringe pump and multiposition valve. Moreover, the use of a novel photosensitive material in a 'one step' fabrication process allowed the rapid fabrication of microfluidic components and interconnection port simultaneously. Pre-filled microfluidic cartridges were used as binary response rapid tests for the simultaneous detection of three antibiotic families - sulfonamides, fluoroquinolones and tetracyclines - in raw milk. For test interpretation, any signal lower than the threshold value obtained for the corresponding Maximum Residue Limit (MRL) concentration (100 microg L(-1)) was considered negative for a given antibiotic. The reliability of the multiplexed detection system was assessed by way of a validation test carried out on a series of six blind milk samples. A test accuracy of 95% was calculated from this experiment. The whole immunoassay procedure is fast (less than 10 minutes) and easy to handle (automated actuation).

Generation of broad specificity antibodies for sulfonamide antibiotics and development of an enzyme-linked immunosorbent assay (ELISA) for the analysis of milk samples.

Immunoreagents appropriately produced to detect a wide range of sulfonamide antibiotic congeners have been used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA). The selectivity has been achieved by combining antibodies raised against 5-[6-(4-aminobenzenesulfonylamino)pyridin-3-yl]-2-methylpentanoic acid (SA1), covalently coupled to horseshoe crab hemocyanin (HCH), and 5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2), coupled to ovalbumin (OVA), on an indirect ELISA format. The immunizing hapten has been designed to address selectivity against the common aminobenzenesulfonylamino moieties, using theoretical calculations and molecular modeling tools. Hapten SA1 has been synthesized in four steps from methyl 5-(4-amino-3-pyridinyl)-2-methyl-4-pentenoate through a Heck reaction, under Jeffery conditions, to avoid introduction of additional epitopes in the linker. The microplate immunoassay developed is able to reach the necessary detectability for the determination of the sulfonamide antibiotics most frequently used in the veterinary field, in compliance with the EC Regulation 2377/90. As an example, the IC(50) and LOD values accomplished for sulfapyridine are 2.86 +/- 0.24 and 0.13 +/- 0.03 microg L(-1), respectively. Studies performed with different types of milk samples demonstrate that direct and accurate measurements can be performed in this type of matrix without any previous sample cleanup method.

A multianalyte ELISA for immunochemical screening of sulfonamide, fluoroquinolone and ss-lactam antibiotics in milk samples using class-selective bioreceptors.

A multianalyte ELISA has been developed for the simultaneous determination of the most frequently used antibiotic families in the veterinary field following the typical planar microarray configuration, where the identity of the target analyte is encoded by its location in the detection platform (Master et al. in Drug Discovery Today 11:1007-1011, 2006). To accomplish this aim, two individual enzyme-linked immunosorbent assays for sulfonamide and fluoroquinolone antibiotics and an enzyme-linked receptor assay for ss-lactam antibiotics have been combined. The strategy uses microplates coated with the corresponding haptenized proteins in specific sections of the microplate. The samples are mixed with a cocktail containing the bioreagents, and distributed in the wells of the microplate. Identification of the antibiotic present in a particular sample is consequently accomplished by detecting a positive response on the corresponding microplate section. Since the bioreceptors used show a wide recognition of the congeners of each antibiotic family, the multianalyte method is able to detect more than 25 different antibiotics from the three most important antibiotic families. The detectability reached in full-fat milk samples is below the European maximum residue limits. The accuracy and reliability of this multiplexed bioanalytical method have been demonstrated by analyzing blind spiked samples.

Validation and comparison of the Copan Milk Test and Delvotest SP-NT for the detection of antimicrobials in milk.

The Delvotest SP-NT and Copan Milk Test, two microbiological tests designed for screening antimicrobial substances in milk were compared and validated. The performance criteria described by the European Decision 2002/657/EC were used for the study. Both tests were evaluated with visual and automated reading (scanner) and the validation was performed on 10 different antibiotics (penicillin-G, cloxacillin, sulfamethazine, sulfadiazine, oxytetracycline, gentamicin, cephalexin, cefquinome, dihydrostreptomycin and trimethoprim). Both tests were found to detect penicillin, cloxacillin, sulfamethazine, sulfadiazine, cephalexin and gentamicin at or below the EU maximum residue limits (MRLs). Some other antibiotics such as oxytetracycline, dihydrostreptomycin, trimethoprim and cefquinome were not detected or only with a low sensitivity. Both tests were found easy to use, robust and fulfilled EU requirements.