Development of a confirmatory method for detecting recombinant bovine somatotropin in plasma by immunomagnetic precipitation followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

Recombinant bovine somatotropin (rbST), a synthetic growth hormone, is used to stimulate growth and enhance milk production in dairy cows. Both its use and the sale of dairy products from treated animals are prohibited in the European Union, as well as in Australia, Canada, Japan, and New Zealand, but authorised in several countries (e.g. Brazil, USA). Screening methods involve detecting anti-rbST antibodies (biomarkers) in treated cows. Confirmatory methods are required to prove rbST abuse. The major challenges in determining rbST are its potentially low levels, its high similarity to native bST, and matrix interferences. To overcome these obstacles, we have developed a method involving immunomagnetic precipitation followed by UHPLC-MS/MS for rbST detection. Briefly, protein G magnetic beads pre-coated with an in-house produced monoclonal antibody were added to plasma. Incubation at room temperature allowed rbST present in the sample to bind to the magnetic beads. After that, magnetic beads were isolated by centrifugation and thoroughly washed (PBS, PBS + 0.2% Tween 20). Finally, rbST was released by alkalinisation and the samples were trypsin digested prior to UHPLC-MS/MS analysis in the MRM mode. Validation was done in accordance with European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used. The decision limit (CCα) reached with this approach was 0.11 µg l-1.

Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron.

Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator forchlorfenuron (CPPU) is described. The competitive lateral flow immunoassay (LFIA) was based on the immobilization onto a nitrocellulose membrane of an ovalbumin-CPPU conjugate (test line) and on the use of an immunodetection ligand consisting of carbon nanoparticles labeled with an anti-CPPU monoclonal antibody through interaction with a secondary antibody. The presence of CPPU in horticultural samples was visually interpreted by the decrease in the black signal intensity of the test line, according to the competitive character of the format. The quantitative determination of the analyte was easily performed by a two-step procedure consisting of flatbed scanning of the strips followed by computer-based image analysis of the pixel gray volumes of the test lines. Under optimized conditions, the immunochromatographic test afforded a limit of quantification in buffer of 89 ng/L. The accuracy of the strip test was assessed by the analysis of fruit samples with incurred residues, and the obtained results were compared with those derived from two reference methods, ELISA and HPLC. The LOQ of the CPPU-specific LFIA in kiwifruits and grapes was established at 33.4 µg/kg. The excellent analytical performance of the developed strip test demonstrates the potential of immunochromatographic assays for the quantitative monitoring of small organic molecules in complex matrices.

Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron.

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L(-1) in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 µg kg(-1). The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC-UV determinations showed an excellent correlation (r(2)=0.998), whereas analysis of the slope (0.99±0.01) and of the intercept (-1±3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels.

Forchlorfenuron-mimicking haptens: from immunogen design to antibody characterization by hierarchical clustering analysis.

To obtain highly-specific and selective forchlorfenuron binders, a collection of functionalized derivatives with different spacer arm locations and lengths was prepared. By immunization with target-mimicking haptens, a large battery of monoclonal and polyclonal antibodies against this synthetic cell regulator was produced and exhaustively characterized in two immunoassay formats using homologous and heterologous conjugates. Antibodies with IC(50) values lower than 0.3 nM were successfully raised from the prepared immunogens, thus evidencing the efficacy of the explored strategies. In order to identify significant epitopes in the antibody-antigen interaction, a series of new chemical forchlorfenuron analogues, with slight modifications at both rings of the target molecule, were synthesized and evaluated in competitive assays. As a novel approach in hapten recognition studies, data processing was performed by computational classification methods based on hierarchical clustering. This strategy was shown to be highly valuable for a straightforward profiling of antibodies according to analogue recognition patterns. A relationship could be established between the antigen binding properties of antibodies and the structure of the immunogen. Whereas antibodies with equivalent affinities had been obtained from all of the derivatives, their specificity was found to be largely influenced by the differential exposition of the molecule to the immune system.

Hapten synthesis and polyclonal antibody-based immunoassay development for the analysis of forchlorfenuron in kiwifruit.

High-affinity polyclonal antibodies directed against the synthetic cytokinin forchlorfenuron (CPPU) were produced from three immunizing haptens with equivalent spacer arms located at different positions. A competitive immunoassay was developed with a limit of detection in buffer of 12.42 +/- 3.06 ng/L. In addition, the ability of the produced antibodies to recognize a set of synthetic CPPU analogues was studied. It was evidenced that the linker position had a strong impact on the specificity of the generated polyclonals, which were more sensitive to changes at moieties of the target analyte located furthest from the derivatization site of the immunogen. Finally, matrix effects of gold and green kiwifruit over assay parameters were evaluated. Excellent recoveries and low coefficients of variation were found with just a 100-fold dilution of the sample in buffer, hence indicating the effectiveness of the developed immunoassay as an analytical tool for monitoring this agrochemical in kiwifruit samples.

Production and characterization of monoclonal and polyclonal antibodies to forchlorfenuron.

The development of immunoassays for the detection of the plant growth regulator forchlorfenuron (CPPU) is described. To achieve that purpose, a set of CPPU derivatives has been obtained by the previous synthesis of the adequate p-aminophenyl alkanoic acid. Protein conjugates of these compounds have been used as immunogens to produce rabbit polyclonal antibodies and a collection of mouse monoclonal antibodies. Additionally, a battery of structural analogues of the target analyte has been synthesized and used for the characterization of antibody binding. This strategy has demonstrated that most antibodies followed Landsteiner's principle, although some monoclonal antibodies showing important deviations from this behavior have also been found. Finally, different assay formats have been developed with a variety of antibodies and conjugates, and a rapid procedure has been optimized for the indirect ELISA format using monoclonal and polyclonal antibodies. In the indirect competitive ELISA, assay IC50 values for CPPU below 0.5 nM were found with LODs as low as 0.013 nM.

Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin.

Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.

Hapten synthesis and monoclonal antibody-based immunoassay development for detection of the fungicide trifloxystrobin.

High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).

Hapten synthesis and monoclonal antibody-based immunoassay development for the detection of the fungicide kresoxim-methyl.

Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 microg/kg) and lower than the detection limits of the usual chromatographic detection methods.