Maria J. Moreno

Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection. An analytical comparison.

Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60-90% range by ELISA and in the 50-90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation.

Development of Monoclonal Immunoassays for the Determination of Triazole Fungicides in Fruit Juices

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution.

A monoclonal immunoassay for carbofuran and its application to the analysis of fruit juices

Abstract Monoclonal antibodies (MAbs) for the carbamate pesticide carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were obtained by immunizing mice with the hapten 3-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]amino] propanoic acid (BFNP) conjugated to bovine serum albumin (BSA). Based on one of these MAbs in combination with an heterologous hapten, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of carbofuran. For standards, the detection limit of the ELISA was 0.2 ng ml−1, with a I50 value of 1.8 ng ml−1. Cross-reactivity studies showed that the immunoassay was quite specific for carbofuran since, of the six N-methylcarbamates assayed, only bendiocarb was significantly recognized (17.4%). The ELISA was applied to the determination of carbofuran in apple, grape, and pineapple juices. Recovery and precision of the method were evaluated by spiking juices with carbofuran at 25, 50, and 100 ng ml−1. Coefficients of variation were below 15% in most cases, and mean recoveries were 112.6, 107.4, and 115.4% for apple, grape, and pineapple juices, respectively. Therefore, the developed ELISA was able to determine carbofuran at levels below the maximum residue limits simply by diluting the sample, without the need for cleanup or sample concentration.

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well–known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I50 value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

Validation of an immunoassay for fast screening of bisphenol A in canned vegetables

The effects of BPA exposure on human health are an issue of concern and controversy. In the present work, the validation for the first time of a monoclonal antibody-based enzyme-linked immunoassay (ELISA) for BPA determination in canned vegetables is described, using HPLC as the reference method. From a collection of monoclonal antibodies, a high-sensitivity immunoassay was selected on the basis of its tolerance to organic solvents and the influence of matrix effects. This ELISA displayed a limit of detection of 3 μg kg−1 of BPA in the whole product of canned vegetables and 15 μg L−1 of BPA in the liquid portion. For assay validation, processed vegetables were fortified with BPA at 10, 50, and 200 μg kg−1. Sample treatment rendered crude and purified extracts. Purified extracts were analyzed by HPLC and ELISA, while crude extracts could be analyzed only by ELISA. Depending on the crop and the fortification level, good recoveries were obtained for both methods: 70.6–105% for HPLC and 61.4–115% (purified extracts) or 82–120% (crude extracts) for ELISA. HPLC was more precise than ELISA. Finally, crude extracts of canned peas were analyzed by ELISA. Results (33–62 μg kg−1) also compared well with those obtained by HPLC on purified extracts (23–44 μg kg−1). In all samples, BPA concentration was significantly lower than the specific migration level of 600 μg kg−1 established by the European Commission. Therefore, the ELISA herein validated constitutes a sensitive, fast, and high-throughput technique for BPA screening in canned vegetables.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection

BACKGROUND Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. METHOD & MATERIAL Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. RESULTS & DISCUSSION For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.

Advances in the development of a piezoelectric immunosensor for the detection of a tuberculosis biomarker

The progress in the development of a piezoelectric immunosensor for the detection of a secretion protein (38 kDa) from Mycobacterium tuberculosis are described. A high affinity and specificity monoclonal antibody was obtained from a recombinant form of the target protein. This immunoreagent was employed for the development of a competitive ELISA, as a previous diagnostic test and reference assay for the characterization of the future biosensor. The ELISA detection limit was 14 ng/mL of the 38kDa antigen. The first experimental approach to the piezoelectric immunosensor was undertaken using bovine serum albumin (BSA) as a model antigen and 10 MH piezoelectric crystals as the biosensor transducer element. The obtained results show that high frequency crystals (≥ 100 MHz) should be used in order to reach the high sensitivity required for diagnostic purposes. These crystals would allow obtaining high and stable assay signals using very low protein concentrations for crystal functionalization, thus enhancing immunosensor sensitivity. Preliminary experiments have proved the successful and efficient immobilization of the 38 kDa antigen on high fundamental frequency (HFF) crystals (100 MHz). Moreover, the functionalized surface seems to be able to selectively bind the monoclonal antibody specific to the target antigen.