Search

Phage display has become a powerful tool for antibody discovery in a wide variety of fields. This technology allows specific binders for a given antigen to be selected from combinatorial libraries. A key step in the process is characterizing and evaluating antibody clones thus selected to reliably identify the best antigen binders. Novel characterization methods can provide essential insight into the binding mechanism and supplement the information obtained with conventional techniques. In this work, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to determine the kinetic and thermodynamic binding parameters for phage-displayed VH antibody fragments. Phytohemagglutinin (PHA), a legume lectin of analytical interest, was used as a complex model antigen to select specific VH fragments from a phage-displayed library. Eight VH fragments with a unique amino acid sequence were identified as PHA binders by using the well-established enzyme-linked immunosorbent assay (ELISA). QCM-D measurements, structural analysis and principal component analysis (PCA) were used to evaluate the antibody fragments and identify clone clusters with similar binding characteristics and molecular interaction mechanisms. This unprecedented study has enabled the identification of high-affinity phage-displayed VH antibody fragments for PHA, which could be useful for PHA analysis (apparent association constant ranged from 10e8 to 10e10 1/M). In fact, the proposed methodology provides a useful tool for evaluating and characterizing antibody fragments with capabilities beyond those of conventional techniques., European Regional Development Fund (ERDF), Depto. de Química Analítica, Fac. de Ciencias Químicas, TRUE, pub

Phage display has become a powerful tool for antibody discovery in a wide variety of fields. This technology allows specific binders for a given antigen to be selected from combinatorial libraries. A key step in the process is characterizing and evaluating antibody clones thus selected to reliably identify the best antigen binders. Novel characterization methods can provide essential insight into the binding mechanism and supplement the information obtained with conventional techniques. In this work, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to determine the kinetic and thermodynamic binding parameters for phage-displayed VH antibody fragments. Phytohemagglutinin (PHA), a legume lectin of analytical interest, was used as a complex model antigen to select specific VH fragments from a phage-displayed library. Eight VH fragments with a unique amino acid sequence were identified as PHA binders by using the well-established enzyme-linked immunosorbent assay (ELISA). QCM-D measurements, structural analysis and principal component analysis (PCA) were used to evaluate the antibody fragments and identify clone clusters with similar binding characteristics and molecular interaction mechanisms. This unprecedented study has enabled the identification of high-affinity phage-displayed VH antibody fragments for PHA, which could be useful for PHA analysis (apparent association constant ranged from 108 to 1010 M−1). In fact, the proposed methodology provides a useful tool for evaluating and characterizing antibody fragments with capabilities beyond those of conventional techniques.

[EN] Microanalytical systems have huge potential for prognosis and diagnosis of diseases where the quantification of biomarkers is the critical factor. Immunoglobulin E (IgE)-associated allergy is the most common immune disorder and reliable multiplex quantification of specific IgE antibodies is of key importance for in vitro allergy diagnosis. Here, we report an unprecedented highly sensitive multiplex colorimetric microanalytical system for reliable quantification of ß-lactam-specific IgE. The biosensor microsystem includes a regular digital versatile surface where nuclear antigens are immobilized in microarray format to determine simultaneously specific IgEs to amoxicillin, penicillin G, piperacillin and aztreonam, using a hacked disc drive as the optoelectrical sensor. The analytical microsystem was successfully tested in a cohort of 101 human serum samples, showing a limit of detection of 0.06 IU/mL (144¿pg IgE/mL), a clinical sensitivity of 42.3 % for amoxicillin, and an excellent specificity (100 %). The analytical performances were good as compared and validated with both in vivo and in vitro reference clinical immunofluorescence assay, settling a good correlation between the techniques. The compact microanalytical system can be easily implemented in primary care laboratory settings without the need for any additional instrumentation, facilitating massive drug allergy screening and delabeling campaigns to better define drug sensitization profiles.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.0c03020, [EN] beta-lactams (BLCs) are the most widely used antibiotics and consequently the most common cause of drug allergy in the world. The diagnosis of drug allergy is complex and represents a serious challenge that includes a wide variety of methods. In vitro tests are based on the immunological determination of allergen-specific IgE, but the tests in the market lack the required sensitivity and specificity. In addition, the large sample volume, long incubation times, and single-plex configuration have brought their use into question to complement the clinical information. Here, we report a chemiluminescence immunoassay (CLIA) for multiparametric quantification of specific IgE to penicillin G, penicillin V, amoxicillin, and piperacillin, using histone H1 as a carrier. The developed CLIA allowed the determination of BLC-specific IgE below 0.1 IU/mL, thus allowing identification of allergic patients with better sensitivity, using only 25 mu L of a sample (serum). The immunoassay was successfully applied in a cohort of 140 human serum samples, showing good sensitivity (64.6%) as well as specificity (100%), which significantly improve the predictive character of existing BLC-allergy in vitro tests.

[EN] Food allergy is reported as the commonest adverse reaction to food components, whose prevalence has increased in recent years. As food avoidance is mainly in practice the only way to prevent hypersensitive consumers from ingesting allergenic substances, it is imperative to provide complete and accurate in-formation on food ingredients. In this scenario, there is a need for precise, fast and cost-effective methods for the high-throughput screening of specific allergen content in food products. This work reviews recent approaches, existing kits for food-borne allergen detection and cutting-edge applications by focusing on the sensitivity, selectivity and applicability of current methods in food samples. In addition, the advantages, benefits and limitations of each approach are discussed to establish the most suitable methods and which challenges are to be addressed in forthcoming years from an analytical viewpoint. (C) 2020 Elsevier B.V. All rights reserved.

[EN] The synthesis of structurally new haptens and the development of suitable antigens are essential for boosting the sensitivity of drug allergy diagnostic testing. Unprecedented structural antigens for benzylpenicillin and amoxicillin are characterised and evaluated in a cohort of 70 subjects with a turnkey solution based on consumer electronics.

[EN] New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for beta-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty beta-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-beta-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-beta-lactam IgE antibodies with high sensitivity (<0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of beta-lactam antibiotics renders improved molecular recognition events when the 4-member beta-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.

[EN] People respond differently to food components, which can sometimes cause dramatic and unpredictable effects, such as intolerance, toxicity and allergy, and are a major global problem today. Of all the different reactions induced by food allergens, the most frequent are hypersensitivity mediated by specific immunoglobulins E (IgE). The in vitro biosensing of specific IgE levels is an alternative approach to invasive risky in vivo tests for diagnosing food allergy in humans. This work reviews the current analytical approaches in food-specific IgE biosensing, including immunochemical and lateral flow assays, electrochemical biosensors, array technologies, nanomaterial-based techniques, label-free sensors and microfluidic systems. The aim is to draw a closer more stimulating scenario for innovation in the food allergies topic. Perspectives on the future of biosensor technology applied to in vitro IgE determinations are also offered from an analytical viewpoint.