Smartphone-based magneto-immunosensor on carbon black modified screen-printed electrodes for point-of-need detection of aflatoxin B1 in cereals.

Considering the complexities and speed of modern food chains, there is an increasing demand for point-of-need detection of food contaminants, particularly highly regulated chemicals and carcinogens such as aflatoxin B1. We report a user-friendly smartphone-based magneto-immunosensor on carbon black modified electrodes for point-of-need detection of aflatoxin B1 in cereals. For buffered analyte solutions and a corn extract sample, the assay demonstrated a low limit of detection of 13 and 24 pg/mL, respectively. The assay was also highly reproducible, exhibiting mean relative standard deviations of 3.7% and 4.0% for the buffered analyte and corn extract samples. The applicability of the assay was validated on the basis of EU guidelines and the detection capability was lower than or equal to 2 µg/kg, which is the EU maximum residue limit for aflatoxin B1 in cereals. False-positive and false-negative rates were less than 5%. Additionally, an open-source android application, AflaESense, was designed to provide a simple interface that displays the result in a traffic-light-type format, thus minimizing user training and time for data analysis. AflaESense was used for smartphone-based screening of spiked corn samples containing aflatoxin B1 (0.1, 2, and 10 ng/mL), and naturally contaminated corn containing 0.15 ng aflatoxin B1/mL. The measured values were in close agreement with spiked concentrations (r2 = 0.99), with recovery values ranging between 80 and 120%. Finally, contaminated samples correctly triggered a red alert while the non-contaminated samples led to the display of a green color of AflaESense. To the best of our knowledge, this is the first smartphone-based electrochemical system effective for screening samples for contamination with aflatoxin B1.

A high-specificity immunoassay for the therapeutic drug monitoring of cyclophosphamide.

Personalized medicine is pushing forward new diagnostic techniques to aid in controlling drug therapeutic levels and their toxic effects. This study aims to develop a high-throughput screening method for therapeutic drug monitoring (TDM) and occupational exposure of cyclophosphamide (CP), an alkylating agent used as a chemotherapeutic and immunosuppressive drug. In order to achieve this goal, an immunizing hapten that exposes the cyclophosphamide moiety has been designed for the first time. Antibodies produced against this hapten have been used to develop an indirect competitive ELISA for the quantification of CP with high specificity and low cross-reactivity with some metabolites and other anticancer drugs. The assay obtained showed a LOD of 22 ± 6 nM in serum samples, with concentrations much below the blood CP levels of patients treated with the drug. A new tool for the detection and quantification of CP is provided which could be relevant for future pharmacokinetic studies and for therapeutic index improvement.

Immediate hypersensitivity to penicillins. Identification of a new antigenic determinant.

The study of adverse drug reactions (ADRs) constitutes a challenge in the area of Medicine. Drugs generate a large number of the total registered hypersensitivity reactions, where penicillins are responsible for more than half of them. In vitro tests in the market are not efficient enough since they lack in sensitivity and specificity. This is the reason why in vivo tests are carried out, with the subsequent danger to the patient's life. It is essential to discover new ß-lactam antigenic determinants to develop more effective detection systems and thus, obtain better explanations of the allergic mechanisms related to these drugs. We propose a strategy based on the use of "peptide probes", small labeled and chemical active peptides which have been structurally modified for reacting with the ß-lactam moiety at different conditions. The probes also contain a biotin group for application in an immunoassay format. Three different amoxicillin adducts have been obtained, purified and characterized by HPLC-MS and NMR techniques. These results have helped us to elucidate and propose a new antigenic determinant for ß-lactams, named the "penamidyl" epitope. All the adducts have been validated and evaluated with sera from different penicillin allergic patients by means of a Magneto-ELISA, immunochemical technique that has allowed us to detect specific IgEs in a very high percentage of the serum samples. An immunoassay has been developed, validated and applied as a diagnostic tool for the detection of specific IgEs in the sera of penicillin allergic patients using a new antigenic determinant.

Biobarcode assay for the oral anticoagulant acenocoumarol.

A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target.

Quantification of interacting cognate odorants with olfactory receptors in nanovesicles.

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.

Sandwich NP-based biobarcode assay for quantification C-reactive protein in plasma samples.

A NP-based biobarcode for C-reactive protein (CRP) quantification in plasma samples is reported for the first time. The assay uses capture antibody functionalized magnetic beads (pAbCRP2-MP), multifunctional oligonucleotide encoded probes modified with a detection antibody (pAbCRP1-ePSP), and a fluorescent DNA microarray. Thus, magnetic beads are added to the sample to form immunocomplexes that will be isolated, to then add the codified particles to form a sandwich complex with both particles and the target protein, subsequently the complexes are treated to release the oligonucleotide codes, which are finally hybridized in a fluorescent DNA microarray. The assay has been implemented to the analysis of plasma samples being able to quantify this biomarker within 900 ng mL-1 to 12500 ng mL-1 with an excellent accuracy (mean of recovery of 99.5 ± 4.2%, N = 3). The CRP biobarcode has been used on a small pilot clinical study in which plasma samples from patients suffering different pathologies, most of them related to cardiovascular diseases (CVDs). The samples have been analyzed and the results compared to a reference method demonstrating that the assay can be useful for monitoring this biomarker on patients being suspicious to be under risk of suffering CVDs or other diseases involving inflammatory processes.

A high throughput immunoassay for the therapeutic drug monitoring of tegafur.

Cancer is a group of diseases in which abnormal cells grow and divide without control, with the potential to invade other parts of the body. Chemotherapy is a type of treatment that uses chemical agents to treat cancer. These drugs are toxic and produce undesirable adverse drug reactions due to their narrow therapeutic window and highly variable pharmacokinetics, thus, they need to be monitored to establish personalized treatment to achieve maximal efficiency and reduce drug toxicity. Nowadays, therapeutic drug monitoring (TDM) is not routinely used for chemotherapy agents, however, TDM has the potential to improve the clinical benefit of chemotherapy drugs. Tegafur, a prodrug of 5-fluorouracil (5FU), is one of the main anti-cancer drugs used worldwide. Herein, a reproducible and sensitive indirect competitive ELISA has been developed and validated in plasma samples. The assay reports an IC50 of 35.6 nM, reaching a limit of detection of 2.7 nM. It is highly reproducible and does not show cross-reactivity with any related compound. In summary, this assay provides a sensitive, accurate and high throughput analytical method for tegafur quantification in plasma, which fits TDM requirements.

Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.

Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

Current bioanalytical methods for detection of penicillins.

With the worldwide use of penicillin antibiotics comes the need for tighter controls. Bacterial resistance is a genuine problem and governmental and international bodies, for example the European Medicines Agency (EMA) and the World Health Organization (WHO), have designed strategies to overcome this unfortunate consequence of antibiotic use. Foodstuffs are monitored to ensure they contain very low quantities of antibiotics, so they are not prejudicial to health and the environment. Detection is based on chromatographic methods. However, screening can be performed by use of simpler, rapid methods of detection, e.g. microbial inhibition test, lateral flow assays, immunoassays, and use of biosensors, to reduce the final number of samples to be analyzed by chromatography. In this review, we have gathered information regarding all such screening methods for the penicillins and have critically assessed their capability and specificity for detection of penicillins.

Nonlinear immunofluorescent assay for androgenic hormones based on resonant structures.

We report for the first time the use of two photon fluorescence as detection method of affinity binding reactions. We use a resonant grating waveguide structure as platform enhancement for detecting the interaction between fluorescent labeled Boldenone, a non-natural androgenic hormone, and a specific anti-anabolic antibody. We were able to detect a surface coverage of approximately 0.7 ng/mm(2).

Immunochemical assays for direct sulfonamide antibiotic detection in milk and hair samples using antibody derivatized magnetic nanoparticles.

Two direct enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of sulfonamide antibiotic residues in milk samples. One of them is using magnetic nanoparticles (MNP) for target capture/enrichment (Ab-MNP-ELISA), and the second is performed using microtiter plates. Selective polyclonal antibodies, raised against 5-[6-(4-amino-benzenesulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid (SA1), used in combination with an enzyme tracer prepared with the same hapten, has allowed us to reach a limit of detection (LOD) lower than 0.5 microg L(-1) for both ELISA formats. Sulfapyridine, sulfamethoxypyridazine, sulfathiazole, and sulfachloropyridazine are detected below the maximum residue limits established by the European Union for these antibiotics in milk (100 microg L(-1)). Matrix effects and accuracy studies performed with full-cream milk and hair extracts indicated a lack of interference from these sample matrices and very good recovery values, especially when using the Ab-MNP format. Milk samples and hair extracts can be measured without any previous treatment. The results demonstrate the high potential of these methods as screening tools for food safety and inspection controls.

Disulfide symmetric dimers as stable pre-hapten forms for bioconjugation: a strategy to prepare immunoreagents for the detection of sulfophenyl carboxylate residues in environmental samples.

A convenient, generic synthesis of bioconjugates from haptens with a thiol group has been established. The corresponding haptens are synthesized as stable symmetric dimmers through a disulfide bond that is reduced immediately before conjugation with the aid of a di(n-butyl)phenylphosphine polystyrene (DBPP) resin. This strategy was used to prepare haptenized biomolecules and to raise antibodies against short-alkyl-chain sulfophenyl carboxylates (X-C(z)-SPCs; X is the position of the benzylic group and z is the alkyl-chain length) formed after degradation of the widely used domestic and industrial linear alkylbenzene sulfonates (LASs) surfactants. Because of the complexity of the LASs technical mixture, homologous and pseudo-heterologous immunization strategies have been studied with the aim of broadening antibody recognition of the SPC family. With this purpose, two types of immunizing haptens have been synthesized and used to prepare bioconjugates and raise antibodies. Type-A bioconjugates (SPC(A)-protein) were prepared by synthesizing type-A haptens as stable symmetric dimers, generically 2,2'-dithiobis[5-{4-(N-ethylsulfamoyl)}phenylalkanoic acids] (X-C(z)-S-SPC). On the other hand, type-B bioconjugates (SPC(B)-protein) were prepared by treating the carboxylic groups of the corresponding 4-sulfophenylalkanoic acids (X-C(z)-SPC) with the amino groups of the lysine residues by using classical carbodiimide procedures. Type-A haptens produced antibodies with a much higher avidity for the target analyte. Under competitive immunochemical configurations (As112/2-C(5)-ovalbumin), these antibodies can reach a limit of detection (LOD) of 40 ng L(-1) with an IC(50) value of 200 ng L(-1) for 3-C(6)-SPC, which opens up the possibility of trace contamination of edible waters by surfactants with 3-C(6)-SPC as a marker of LAS pollution. A comparative study of the properties of the three families of polyclonal antibodies produced revealed that antibodies raised through pseudo-heterologous immunization strategies produced antibodies with a broader specificity versus the SPC family. These results indicate that this approach could be useful in avoiding synthetic difficulties associated with preparing haptens that preserve all the most important chemical functionalities of the molecule.

Evaluation of a newly developed enzyme-linked immunosorbent assay for determination of linear alkyl benzenesulfonates in wastewater treatment plants.

A recently developed enzyme-linked immunosorbent assay (ELISA) for the determination of linear alkyl benzenesulfonates (LAS) and long chain sulfophenyl carboxylates (SPCs) has been evaluated for its application in wastewater control analysis. This ELISA based on the use of polyclonal antibodies in an indirect format shows an IC50 of 28.1 +/- 3.2 microg L(-1) and a limit of detection (LOD) of 1.8 +/- 0.6 microg L(-1) in buffer. The assay uses antibodies raised through a pseudoheterologous immunization strategy using an equimolar mixture of two immunogens, N-(4-alkylphenyl)sulfonyl-3-aminopropanoic acid covalently coupled to keyhole limped hemocyanin (SFA-KLH) and sulfophenyl carboxylate 13C13 coupled to KLH (13C13-SPC-KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the LAS molecule. To assess the performance of this immunoassay in complex real samples, a cross reactivity study was carried out, and the possible interference of other surfactants commonly detected in wastewater, including nonylphenol ethoxylates (NPEOs), nonylphenol (NP), octylphenol (OP), and coconut fatty acid diethanol amides (CDEA), have been evaluated. Additionally, a study of the matrix effects of different types of wastewater was achieved. This ELISA has been evaluated and validated by measuring the LAS content of 22 samples collected from the influents and the effluents of six wastewater treatment plants (WWTP) located in Catalonia, Spain. A solid-phase extraction followed by liquid chromatography coupled to mass spectrometry detection (SPE-LC-MS) has been used as a validation method of the new ELISA test.

Proatherogenic effects of the cholesterol ozonolysis products, atheronal-A and atheronal-B.

The proatherogenic properties of the cholesterol 5,6-secosterols (atheronal-A and atheronal-B), recently discovered in atherosclerotic arteries, have been investigated in terms of their effects on monocyte/macrophage function. A fluorescent analogue of atheronal-B (1) (50 microM), when cultured in either aqueous buffer (PBS) or in media containing fetal calf serum (10%), is rapidly taken-up into cultured macrophage (J774.1 or RAW 264.7) cells and accumulates at perinuclear sites (within 1 h). Co-incubation of macrophage cells (J774.1) with atheronal-A (25 microM) and atheronal-B (25 microM) when complexed with low-density lipoprotein (LDL) (100 microg/mL) leads to a significant upregulation of scavenger receptor class A (approximately 3-fold increase relative to LDL alone, p < 0.05) but not CD36, showing that cultured macrophages respond to LDL-complexed atheronals in a manner highly analogous to acetylated LDL rather than oxidized LDL. Both atheronal-A and atheronal-B in solution exhibit a dose-dependent (0-25 microM) induction of chemotaxis of cultured macrophages (p < 0.001). When complexed with LDL (100 microg/mL), atheronal-A (but not atheronal-B) induces a dose-dependent (0-25 microM, p < 0.05) upregulation of the cell-surface adhesion molecule endothelial (E)-selectin on vascular endothelial cells (HUVECs). LDL (100 microg/mL) complexed atheronal-B (25 microM) but not atheronal-A induces cultured human monocytes (THP-1) to differentiate into macrophage cell lineage. When these in vitro data are taken together with the already known effects of cholesterol 5,6-secosterols on foam cell formation and macrophage cytotoxicity, the atheronals possess biological effects that if translated to an in vivo setting could lead to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, with the major leukocyte player in inflammatory artery disease. As such, the atheronal molecules may be a new association, in the already complex inter-relationship, between inflammation, cholesterol oxidation, the tissue macrophage, and atherosclerosis.

Development of an enzyme-linked immunosorbent assay for the determination of the linear alkylbenzene sulfonates and long-chain sulfophenyl carboxylates using antibodies generated by pseudoheterologous immunization.

ELISA methods have been developed for screening contamination of water resources by linear alkyl benzene sulfonates (LAS) or the most immediate degradation products, the long chain sulfophenyl carboxylates, SPCs. The assay uses antibodies raised through pseudoheterologous immunization strategies using an equimolar mixture of two immunogens (SFA-KLH and 13C(13)-SPC-KLH) prepared by coupling N-(4-alkylphenyl)sulfonyl-3-aminopropanoic acid (SFA) and p-(1-carboxy-13-tridecyl)phenylsulfonic acid (13C(13)-SPC) to keyhole limpet hemocyanin (KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the molecule. The SFA hapten maximizes recognition of the alkyl moiety while preserving the complexity of the different alkyl chains present in the LAS technical mixture. The 13C(13)-SPC hapten addresses recognition of the common and highly antigenic phenylsulfonic group. The antisera raised using this strategy have been shown to be superior to those obtained through homologous immunization procedures using a single substance. By using an indirect ELISA format, LAS and long-chain SPCs can be detected down to 1.8 and 0.2 microg L(-1), respectively. Coefficients of variation of 6 and 12% within and between assays, respectively, demonstrate immunoassay reproducibility. The assay can be used in media with a wide range of pH and ionic strength values. Preliminary experiments performed to assess matrix effects have demonstrated the potential applicability of the method as a screening tool to assess contamination by these types of surfactants in natural water samples.

Direct competitive enzyme-linked immunosorbent assay for the determination of the highly polar short-chain sulfophenyl carboxylates.

A direct enzyme-linked immunosorbent assay for the detection of the short-chain sulfophenylcarboxylic acids (SPCs), the main metabolites of the linear alkylbenzenesulfonates, is reported. Six SPCs (2C3, 2C4, 3C4, 2C5, 3C5, 3C6), differing in the length of the alkyl chain (between C3 and C6) and in the position of the phenylsulfonic group versus the carboxylic group, have been synthesized. Antibodies have been raised against a mixture of the corresponding horseshoe crab hemocyanin conjugates prepared by coupling the carboxylic acid to the lysine amino acid residues. The immunoassay As115/3C4-HRP achieves an IC50 value of 23 nM (6.67 microg L(-1)) and a detection limit of 0.85 nM (0.24 microg L(-1)), using as standard analyte an equimolar mixture of the six SPCs. The immunoassay has found to work better in media with low or moderate ionic strength (4-30 mS cm(-1)). The decrease in the detectability produced by the potential formation of SPC salts with divalent cations such as Ca2+ can be prevented by lowering the pH of the assay medium below the pKa value of the SPC carboxylic group and using a buffer chelating with properties such as citrate buffer. The assay can be considered specific for short-chain SPCs since congeners with longer alkyl chains and other pollutants containing sulfonic groups in their structure do not interfere significantly in the assay. Preliminary experiments addressed to evaluate the potential application of this assay to environmental water samples demonstrate the usefulness of the assay.

Evidence for ozone formation in human atherosclerotic arteries.

Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.

Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay.

Biological monitoring of 2,4,5-trichlorophenol (II): evaluation of an enzyme-linked immunosorbent assay for the analysis of water, urine, and serum samples.

Chlorophenols are frequently found in the urine of the population as consequence of the widespread use of chlorophenols and other organochlorinated compounds. An immunoassay for 2,4,5-trichlorophenol (2,4,5-TCP) has been evaluated as a tool to assess risk exposure of the population to these substances. The immunoassay is stable in media with pH values ranging from 6.6 to 10.5 units and ionic strength values varying within 20 and 80 mS cm(-)(1). Considering these parameters, the optimized immunoassay shows a limit of detection of 0.05 microg L(-)(1) and the dynamic range is placed between 0.09 and 0.72 microg L(-)(1). It shows a good accuracy and the coefficients of variation within and between assays are around 12% or lower. However, matrix effects can diminish the efficiency and detectability of the immunochemical methods. In this paper, the effect of water and complex biological sample matrices, such as serum and urine, on the immunoassay for 2,4,5-TCP has been evaluated. Simple sample treatment procedures have been developed for the analysis of these matrices. The final analytical protocols allow straightforward immunochemical determination of 2,4,5-TCP in natural waters, urine, and serum with detection limits of 0.07, 0.26, and 0.8 microg L(-)(1), respectively.

Development and evaluation of an immunoassay for biological monitoring chlorophenols in urine as potential indicators of occupational exposure.

Trichlorophenols (TCP) eliminated by the urine can be considered as potential biomarkers of exposure of many chemicals (chlorophenols, chlorophenoxy acid herbicides, prochloraz, lindane, hexachlorobenzene, etc). High-throughput screening methods are necessary to carry out efficient monitoring programs that may help to prevent certain occupational health diseases. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) for 2,4,6-trichlorophenol detection has been developed using polyclonal antisera raised against 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid (hapten 5) covalently coupled by the mixed anhydride (MA) method to keyhole limpet hemocyanin (KLH). The indirect ELISA uses a heterologous coating antigen prepared by conjugation of 3-(2-hydroxy-3,6-dichlorophenyl)propanoic acid (hapten 4) to bovine serum albumin (BSA) using the active ester (AE) method. The optimum hapten density for the coating antigen was found to be 3 mol of hapten/mol of protein. The assay shows a limit of detection of 0.245 +/- 0.116 microg L(-1), and it is performed on 96-well microtiter plates in about 1.5 h. The ELISA reported recognizes on a much less extent other chlorinated phenols, such as 2,3,4,6-tetrachlorophenol (2,3,4,6-TtCP, 21%), 2,4,5-TCP (12%) and 2,3,5-TCP (15%); however, brominated phenols (BP) are even more recognized than the corresponding chlorinated analogues (ex. 2,4,6-TBP, 710%; 2,4-DBP, 119%). With the aim of finding an explanation for this behavior, theoretical calculations have been performed for those and other halogenated phenols (2,4,6-triiodophenol and 2,4,6-trifluorophenol) to clarify which physicochemical parameter can explain better the recognition pattern observed. Finally, the assay has been adapted to the analysis of urine samples. The studies have shown that a limit of detection of 1 microg L(-1) can be accomplished on this biological matrix by combining the ELISA procedure with a C18 solid-phase extraction method.