Roger Galve

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.

Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine

A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2 microg L(-1). The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods.

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).

Chapter 2.8 Application of bioassays/biosensors for the analysis of pharmaceuticals in environmental samples

Abstract With the ever-increasing use of pharmaceuticals and the subsequent release into the environment comes the need for reliable detection methods. While the emphasis continues to be on chromatographic methods, every time, there are more and more biologically based assays being developed for the detection of pharmaceuticals in the environment. This chapter affords an overview of the currently available bioassays, biochemical assays, and biosensors focusing on the most commonly used pharmaceuticals—antibiotics, hormones, and analgesics. Bioassays rely on biological indicators, whether it be cells, tissues, or whole organisms, to evaluate an analyte according to its biological activity, whereas biochemical assays and biosensors incorporate biorecognition elements such as enzymes, proteins, or antibodies but the signal is enhanced by physical or chemical means. There has been more emphasis given on biosensors, especially immunologic sensors, for as the reader will appreciate, they show greater promise as alternatives to chromatography as they are fast, robust, specific, and quantifiable and may be performed on-site.

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, ~100u2009nm 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.

High-sensitive nonlinear detection of steroids by resonant double grating waveguide structures-based immunosensors

We report the non linear fluorescence real-time detection of methylboldenone, an androgenic anabolic steroid used illegally as growth promoter based on a resonant sensing chip: a double grating waveguide structure. The limit of detection of this synthetic steroid is two orders of magnitude lower than the Minimum Required Performance Limit required by the World Anti-Doping Agency. The immunoreagents have been have been immobilized onto the surface of the resonant sensor after being activated with phosphonohexanoic acid spacers. The developed immunosensor presents great potential as a robust sensing device for fast and early detection of illegal dopants and food contaminants.

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.

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.

Immediate hypersensitivity to penicillins. Identification of a new antigenic determinant

Graphical abstract Figure. No Caption available. HighlightsPeptide probes, small labeled and chemically active peptides, have been used for identification of new epitopes.A new antigenic determinant of penicillins has been isolated and identified using these peptides probes.A magneto‐ELISA has been developed for the detection of specific IgEs from sera of penicillin allergic patients. ABSTRACT 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 &bgr;‐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 &bgr;‐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 &bgr;‐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.

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 900xa0ngxa0mL-1 to 12500xa0ngxa0mL-1 with an excellent accuracy (mean of recovery of 99.5xa0±xa04.2%, Nxa0=xa03). 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.