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] Discovering nanoscale phenomena to sense biorecognition events introduces new perspectives to exploit nano science and nanotechnology for bioanalytical purposes. Here we present Bio Bragg Gratings (BBGs), a novel biosensing approach that consists of diffractive structures of protein bioreceptors patterned on the surface of optical waveguides, and tailored to transduce the magnitude of biorecognition assays into the intensity of single peaks in the reflection spectrum. This work addresses the design, fabrication, and optimization of this system by both theoretical and experimental studies to explore the fundamental physicochemical parameters involved. Functional biomolecular gratings are fabricated by microcontact printing on the surface of tapered optical microfibers, and their structural features were characterized. The transduction principle is experimentally demonstrated, and its quantitative bioanalytical prospects are assessed in a representative immunoassay, based on patterned protein probes and selective IgG targets, in label-free conditions. This biosensing system involves appealing perspectives to avoid unwanted signal contributions from non-specific binding, herein investigated in human serum samples. The work also proves how the optical response of the system can be easily tuned, and it provides insights into the relevance of this feature to conceive multiplexed BBG systems capable to perform multiple label-free biorecognition assays in a single device.

The final publication is available at link.springer.com., [EN] The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (-df/dD and Delta h/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure.

[EN] Protein-protein interactions are key in virtually all biological processes. The study of these interactions and the interfaces that mediate them play a key role in the understanding of biological function. In particular, the observation of protein¿protein interactions in their dynamic environment is technically difficult. Here two surface analysis techniques, dual polarization interferometry and quartz crystal microbalance with dissipation monitoring, were paired for real-time mapping of the conformational dynamics of protein¿ protein interactions. Our approach monitors this dynamics in real time and in situ, which is a great advancement within technological platforms for drug discovery. Results agree with the experimental observations of the interaction between the TRIM21a protein and circulating autoantibodies via a bridging bipolar mechanism. This work provides a new chip-based method to monitor conformational dynamics of protein¿protein interactions, which is amenable to miniaturized high-throughput determination.

[EN] Microcontact printing (mu CP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect mu CP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard mu CP. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect mu CP on different materials, reports the limitations of standard mu CP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect mu CP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect mu CP in an immunoassay to detect human immunoglobulin E.

[EN] An autoantigen piezoelectric sensor to quantify specific circulating autoantibodies in human serum is developed. The sensor consisted on a quartz crystal microbalance with dissipation monitoring (QCM-D) where TRIM21 and TROVE2 autoantigens were covalently immobilized, allowing the selective determination of autoantibodies for diagnosis and prognosis of Systemic Lupus Erythematosus (SLE). The sensitivity of the biosensor, measured as IC50 value, was 1.51 U/mL and 0.32 U/mL, for anti-TRIM21 and anti-TROVE2 circulating autoantibodies, respectively. The sensor is also able to establish a structural interaction fingerprint pattern or profile of circulating autoantibodies, what allows scoring accurately SLE patients. Furthermore, a statistical association of global disease activity with TRIM21-TROVE2 interaction was found (n=130 lupic patient samples, p-value=0.0413). The performances of the biosensor were compared with standard ELISA and multiplex DVD-array high-throughput screening assays, corroborating the viability of piezoelectric biosensor as a cost-effective in vitro assay for the early detection, monitoring or treatment of rare diseases.