Electrochemical nanoprobe-based immunosensor for deoxynivalenol mycotoxin residues analysis in wheat samples.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by several species of Fusarium fungi, which can be predominantly found in agricultural crops such as wheat. In livestock, deoxynivalenol-contaminated grain can produce vomiting, feed refusal, weight loss, and diarrhea. This paper reports an electrochemical immunosensor for the detection of residual DON mycotoxin in food samples. The device uses electrochemical nanoprobes (CdSNP-AbDON) and antigen biofunctionalized magnetic µ-particles (DON-BSAMP) to detect the mycotoxin. CdSNP-AbDON are prepared by labelling the DON-specific antibodies with CdS nanoparticles (CdSNPs). Nanoparticle size and CdSNP-AbDON conjugation ratio are characterized using TEM images. The metal ions released by the CdSNP are reduced at the working electrode and read by anodic stripping voltammetry. DON can be detected in PBST buffer with an IC50 of 6.74 ± 0.19 µg L-1. The high detectability of the immunosensor developed allows detection of DON residues in 50-fold diluted wheat extracts. The limit of detection (LOD, IC90) accomplished in wheat is of 342.4 µg kg-1, which is below the maximum residue limit (MRL, 1750 µg kg-1 for unprocessed durum wheat, 750 µg kg-1 for cereals intended for direct human consumption) established by the EU for this mycotoxin. The working range is in the interval between 610 and 6210 µg kg-1. The performance of the immunosensor was compared with the ELISA assay. DON naturally contaminated wheat samples were analyzed with the immunosensor, showing acceptable recoveries. Graphical abstract.

Immunochemical Determination of Pyocyanin and 1-Hydroxyphenazine as Potential Biomarkers of Pseudomonas aeruginosa Infections.

A novel immunochemical approach to diagnose Pseudomonas aeruginosa infections is reported, which is based on the quantification of relevant and specific virulence factors secreted by this microorganism. Specific antibodies have been raised using hapten PC1 (a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids), designed to recognize 1-hydroxyphenazine (1-OHphz), which is the main metabolite of pyocyanin (PYO). PYO is one of the most important virulence factors produced by nearly all P. aeruginosa strains, and other species do not produce this factor. With these antibodies, an immunochemical analytical procedure able to quantify both 1-OHphz and PYO in complex clinical samples has been developed. 1-OHphz can be directly measured in solubilized sputum samples diluted 20 times with the assay buffer. Quantification of PYO is accomplished after conversion to 1-OHphz in just 20 min under basic conditions. A LOD of 0.60 ± 0.01 nM (4.80 ± 0.08 nmol kg(-1) sputum) is reached for both biomarker targets under the conditions established, a value that is much below the reported concentrations on sputum samples obtained from infected patients (up to 100 µM). The assay is robust, reproducible, accurate, can be run in about 2 h, and many samples can be measured simultaneously. The present reported assay could represent a significant improvement in the diagnosis of infectious diseases caused by this pathogen.

Electrochemical detection of fluoroquinolone antibiotics in milk using a magneto immunosensor.

An amperometric magneto-immunosensor (AMIS) for the detection of residues of fluoroquinolone antibiotics in milk samples is described for the first time. The immunosensor presented combines magnetic beads biomodified with an antibody with a broad recognition profile of fluoroquinolones, a haptenized enzyme and a magnetic graphite-epoxy composite (m-GEC) electrode. After the immunochemical reaction with specific enzyme tracer, the antibody biomodified magnetic beads are easily captured by an electrode made of graphite-epoxy composite containing a magnet, which also acts as transducer for the electrochemical detection. In spite of the complexity of milk, the use of magnetic beads allows elimination of potential interferences caused by the matrix components; hence the AMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. The immunosensor is able to detect up to seven different fluoroquinolones far below the MRLs defined by the UE for milk; for example ciprofloxacin is detected directly in milk with an IC50 of 0.74 µg/L and a LOD of 0.009 µg/L. This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis fluoroquinolones in complex samples.

Immunochemical determination of fluoroquinolone antibiotics in cattle hair: a strategy to ensure food safety.

Enrofloxacin (ERFX) is a synthetic antibiotic of the fluoroquinolone (FQ) family, which is commonly administered in veterinary medicine. ERFX and its metabolite, ciprofloxacin (CPFX), have been reported to accumulate in hair of treated animals. Therefore, hair analysis is an attractive non-invasive alternative to control misuse of such antibiotic and to ensure food safety by preventing such food derived products arrive to the consumer. In this context, an immunochemical analytical protocol has been established to detect ERFX and CPFX residues in cattle hair samples. Unpigmented and pigmented hair were collected from ERFX-treated and non-treated calves, and the aqueous NH4OH extracts were directly analyzed by ELISA, being possible to achieve limits of detection in the range of 10-30 µg kg(-1). A good concordance between HPLC and ELISA measurements was observed. The results demonstrate the potential of the immunochemical procedure reported here to rapidly screen and quantitate FQ residues in hair samples.

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples.

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite-epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18 ± 0.09 µg L(-1). Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90% of the maximum signal) of 2.18 ± 2.08 µg kg(-1), far below the maximum residue level (20 µg kg(-1)) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.

Development of a Coulombimetric immunosensor based on specific antibodies labeled with CdS nanoparticles for sulfonamide antibiotic residues analysis and its application to honey samples.

A new electrochemical immunosensor has been developed to detect sulfonamide antibiotic residues in food samples. The immunosensor presented uses immunoreagents specifically developed for the broad recognition of the sulfonamide antibiotic family, a graphite composite electrode (GEC), biofunctionalized magnetic µ-particles and electrochemical nanoprobes prepared by labeling the specific antibodies with CdS nanoparticles (CdSNP). After the immunochemical reaction, the CdSNP are dissolved and the metal ions released are reduced at the electrode and read as in the form of current or charge signal, by the well-known anodic stripping technique. Due to the amplification effect on the amperometric/coulombimetric signal produced by the CdSNP, a high detectability can be reached. Thus, sulfapyridine (SPY), one of the most widely used sulfonamide congeners, can be detected in buffer with an IC50current of 0.20±0.25µgL(-1). The immunosensor has been applied to the analysis of residues of this antibiotic in honey samples. Due to the reported formation of sulfonamide-sugar conjugates in this type of matrix, honey samples are first hydrolyzed in acidic media. The use of magnetic particles minimizes the matrix effect allowing to reach a detectability (LOD, limit of detection) of 0.11µgkg(-1) (current measurements), far below the limits established in some countries for these types of residues in honey samples. Due to the use of magnetic racks, multiple samples can be run simultaneously. The whole analysis process can be performed in around 22min.

Molecular modeling assisted hapten design to produce broad selectivity antibodies for fluoroquinolone antibiotics.

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.

Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples.

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 µg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 µg L(-1).

Nanogold probe enhanced Surface Plasmon Resonance immunosensor for improved detection of antibiotic residues.

An exhaustive study is reported on the effect that antibody nanogold probes produce on the performance of a Surface Plasmon Resonance (SPR) immunosensor. The paper studies the improvement that different nanogold probes prepared at different antibody:gold nanoparticle (IgG:AuNP) ratios and AuNP sizes produce on the maximum signal and detectability of a simple SPR immunosensor developed to analyze fluoroquinolone (FQ) antibiotic residues (SPReeta system). The investigation compares the features of sensor enhanced formats using both, secondary and primary nanogold probes (anti-IgG and IgG coupled to AuNP, on double and single-antibody immunochemical assay steps, respectively), in respect to the unenhanced format. For this purpose, a reproducible bioconjugation procedure for preparing gold biohybrid nanoparticles has been established, involving the formation of a mixed self-assembled monolayer (m-SAM) with PEGylated cross-linkers around the AuNP followed by the covalent attachment of the antibodies. The procedure allows controlling the IgG:AuNP ratio of the nanogold probes on a reproducible manner and the functionalized NPs have been found to be stable during assay and storage. Both formats, using secondary and primary nanogold probes, are excellent strategies to improve immunosensor detectability. Thus, using anti-IgG-AuNP, the detectability could be improved by a factor of 14 (LOD 0.07±0.01 µg L(-1) vs. 0.98±0.38 µg L(-1)) reducing at the same time the amount of primary antibody used (30,000 vs. 1000 dilution factor). Likewise, the format using IgG-AuNP also allows improving detectability (LOD 0.11±0.01 µg L(-1)), but reducing the number of needed steps.

Portable surface plasmon resonance immunosensor for the detection of fluoroquinolone antibiotic residues in milk.

An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 µg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 µg L(-1) and a LOD of 2.0 ± 0.2 µg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestlé Co.

Quantum dot-based array for sensitive detection of Escherichia coli.

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL(-1) without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (-) bacteria was negligible at low concentrations of bacteria.

A label-free and portable multichannel surface plasmon resonance immunosensor for on site analysis of antibiotics in milk samples.

Techniques for immunosensing like surface plasmon resonance (SPR) may respond to the need for rapid screening methods to improve food safety. This paper describes the development of a novel portable six channel SPR biosensor based on the plasmon of gold diffraction grating surface for simultaneous multianalyte antibiotic detection in milk samples. Representative congeners from three important antibiotic families (FQs: fluoroquinolones, SAs: sulfonamides and CAP: phenicols) were chosen for this study. The chips are covalently biofunctionalized with haptenized proteins by means of a previously formed mixed self assembled monolayer (m-SAM) prepared using two types of mercapto alkyl reagents containing polyethyleneglycol (PEG) units. The samples or standards are mixed with specific polyclonal antibodies and injected into the sensor device. The detectability accomplished is very good (i.e. in buffer, enrofloxacin, 0.30 µg L(-1); sulfapyridine, 0.29 µg L(-1); and chloramphenicol, 0.26 µg L(-1)) and whole milk samples can be analyzed directly without clean-up steps, by just diluting the sample five times with water to remove non-specific interferences caused by the matrix. Although the detectability of CAP regarding the MRPL (minimum required performance limit) is slightly compromised by the dilution, the detectability accomplished by FQs and SAs was far below the maximum residue levels (MRLs) established by the European Union.

Evaluation of immunoassays as an alternative for the rapid determination of pesticides in wine and grape samples.

The main objective of this paper is to address the performance of immunochemical assays for the detection of the residues of three pesticides [atrazine, bromopropylate, and 2,4,6-trichlorophenol (TCP)] in real winery samples, such as wine, grapes, and grape juice. Different approaches have been evaluated to minimize interferences from the matrixes, and suitable working protocols have been established in order to achieve the necessary LODs, accuracy, and precision for real samples. A simple dilution of the sample proved to be sufficient for the determination of atrazine and bromopropylate in red and white wine and grape juice at the required levels of concentration. However, for TCP, an SPE procedure has been optimized using amino cartridges. The recoveries were above 85% in all cases, and the LOD values were below the parts per billion level, except for bromopropylate, which ranged between 2 and 50 microg/L, depending on the matrix. The grape matrix effect could be resolved by a simple extraction with methanol. Complete recoveries were obtained, and the final measurement procedures were able to determine selected pesticides below their maximum residue levels. The newly developed methods have been compared with standard chromatographic methods.

Competitive multi-immunosensing of pesticides based on the particle manipulation with negative dielectrophoresis.

In this work, we have applied particle manipulation based on negative dielectrophoresis (n-DEP) to develop rapid and separation-free immunosensing systems. Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to demonstrate competitive immunosensing based on the rapid manipulation of microparticles. A suspension of the fluorescence microparticles modified with a specific antibody was injected into the n-DEP device consisting of the interdigitated microarray (IDA) electrode and indium-tin-oxide (ITO) substrate immobilized by protein conjugation with antigen. The application of 2 MHz AC voltage (16 V peak-to-peak) to the IDA forced most of the particles to form a line pattern on the upper ITO over the gaps of IDA within 60s. In the absence of analytes, patterned microparticles were irreversibly captured on the ITO by the construction of immuno-complexes. When the microparticles bearing anti-atrazine IgG antibody were suspended in an analyte (atrazine) solution, irreversible capturing of microparticles on the ITO was inhibited because of the occupation of the binding sites of the antibodies with free-atrazine. As a result, the analyte molecules were re-dispersed from the ITO to disintegrate the line formation after turning off the voltage. We could discriminatively detect the fluorescence intensity of the captured microparticles at the designated areas from that of the uncaptured microparticles suspended in the solution. Thus, the separation steps usually required for conventional immunoassay are eliminated in the present procedure. A pre-incubation of microparticles for 3 min in an orange juice solution containing analyte allowed for the determination of the atrazine and bromopropylate concentrations with a limit of detection of 4 and 1.5 microg L(-1), respectively, providing sufficient detectability to achieve international regulations regarding pesticide residues in food samples. The assay was significantly accelerated by the rapid particle manipulation with n-DEP and totally accomplished within 5 min. We also demonstrated the possibility of the simultaneous determination of two pesticide residues by using the DEP devices with two channels modified with specific competitors for atrazine and bromopropylate.

A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples.

A high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to enzyme-linked immunosorbent assay (ELISA) has been established for the analysis of stanozolol (St) and its main metabolite in cattle, 16beta-hydroxy-stanozolol (16betaOH-St), in cow urine samples. The chemical structure of the immunizing hapten 2'H-androst-2-eno[3,2-c]-pyrazol-17-hemiglutarate 5 (hapten A) has been designed to accomplish simultaneous detection of St and 16betaOH-St. The antibodies obtained have been used to establish a microplate ELISA method able to detect these metabolites with IC(50) values of 0.57microgL(-1) and 1.46microgL(-1), respectively in PBST. Immunosorbents prepared by covalently attaching the antibodies to Sepharose, efficiently removed the matrix interferences caused by the cattle urine samples. Moreover, St and 16betaOH-St were efficiently extracted from urine samples as demonstrated by LC-MS/MS analysis. The immunosorbents are filled on small mini-columns arranges on a 96-SPE-setup compatible with the microplate based ELISA methods. Samples and standards can be run in parallel which increment considerably the speed of the screening method. The recovery values of the whole HTS-IS-SPE-ELISA procedure has found to be 112+/-10% and St can be detected in hydrolyzed urine samples with LOD of 1.26+/-0.46microgL(-1) using just 1mL of sample. As proof-of-concept the urinary excretion profile of St treated animals has been investigated by analyzing individual sampling points. Results from pooled urine samples have also been compared with the results obtained by GC-MS analysis demonstrating the StIR equiv. measured with the HTS-IS-SPE-ELISA protocol are in accordance with the St and 16betaOH-St levels found with the chromatographic method. The analytical procedure is rapid, effective and the detectability achieved is below the MPRL (minimum performance required levels) recommended by CRL (Community Reference Laboratory) to the European Community.

Impedimetric immunosensor based on a polypyrrole-antibiotic model film for the label-free picomolar detection of ciprofloxacin.

This paper describes the construction of an impedimetric immunosensor for the label-free detection of ciprofloxacin, an antibiotic belonging to synthetic fluoroquinolones. A poly(pyrrole-N-hydroxysuccinimide) film was electrogenerated onto electrodes and then used for the reagentless covalent binding of a fluoroquinolone model bearing an amino group. The resulting electrodes were utilized to immobilize a layer of anticiprofloxacin antibody onto the polymer surface by immunoreaction. In presence of ciprofloxacin, the antibody was displaced in solution inducing marked changes in the impedance of the sensor electrodes. These phenomena were detected and characterized by electrochemical impedance spectroscopy allowing the selective detection of extremely low ciprofloxacin concentration, namely, 1 x 10(-12) g mL(-1) or 3 pmol L(-1). Sensors exposed to ciprofloxacin showed a decrease in the sum of the interfacial resistances with the increase in ciprofloxacin concentration from 1 x 10(-12) to 1 x 10(-6) g mL(-1).

Traceability of sulfonamide antibiotic treatment by immunochemical analysis of farm animal hair samples.

The use of hair to trace use of unauthorized substances, therapeutic agents, or their misuse is becoming very attractive since residues can be detected for a long time after treatment. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) has been evaluated for its capability to trace sulfonamide antibiotic treatment by analyzing cattle and pig hair samples. Pigmented and nonpigmented hair samples from control and sulfamethazine (SMZ)-treated pigs and calves were collected, extracted under different alkaline conditions, and analyzed by ELISA after just diluting the extracts with the assay buffer. Data analysis following the European recommendations for screening methods demonstrates that the ELISA can detect SMZ in hair samples with a limit of detection (90% of the zero dose (IC(90))) between 30 and 75 ng g(-1). The same samples have been analyzed by HPLC after a dual solid-phase extraction. The ELISA results matched very well those obtained by the chromatographic method, demonstrating that the immunochemical method can be used as a screening tool to trace animal treatments. Between the benefits of this method are the possibility to directly analyze hair extracts with sufficient detectability and its high-throughput capability. Preliminary validation data are reported using an experimental approach inspired on the Commission Decision 2002/657/EC criteria for screening methods.

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.

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.