Anterior gradient-3 protein-antibody interaction at charged interfaces. Label-free chronopotentiometric sensing Veronika Ostatn  a a, * , Stanislav Haso  n a , Veronika Kasalov  a a , Michal  Durech b , Roman Hrstka b a Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kr alovopolsk a 135, 612 65 Brno, Czech Republic b Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology,  Zlutý kopec 7, 656 53 Brno, Czech Republic article info Article history: Received 16 September 2018 Received in revised form 7 December 2018 Accepted 8 December 2018 Available online 10 December 2018 Keywords: Constant current chronopotentiometric stripping Anterior gradient-3 protein Antigen-antibody interactions Mercury electrodes Electrochemical analysis abstract We developed a fast, simple, label-free method useful for the study of interactions between an antibody (Ab) and antigen based on chronopotentiometric stripping analysis and the catalytic hydrogen evolution reaction. The specific interaction of the Ab, adsorbed at a mercury electrode, with AGR3 protein induced a significant increase in chronopotentiometric peak H in comparison to both the CPS response of the Ab alone and that after incubation with nonspecifically binding proteins. Qualitatively similar results were obtained with another polyclonal antibody specific for AGR2 protein. The demonstrated results, along with previous findings indicate that the proposed technique shows promise as a new option for studying the dynamics, not only of surface-attached antibody-antigen complexes, but also other protein-protein interactions. © 2018 Elsevier Ltd. All rights reserved. 1. Introduction Antibodies (or immunoglobulins) represent a large family of glycoproteins, recognizing antigens with high specificity. They are widely used for immunoassays in combination with various other methods for many applications [1e3]. There are two antibody (Ab) types, depending on the number of epitopes (region of an antigen that interacts with an antibody). Polyclonal Ab can bind multiple epitopes on an antigen and can indicate a heterogeneous immu- nological response. Monoclonal Ab is more specific, binding at a single epitope, which diminishes the chance of cross-reactivity. Abs are quite large proteins composed of one or more copies of a ‘Y’ shape unit. Each ‘Y’ unit contains four polypeptide chains e two identical heavy chains (molecular weight ~ 50 kDa each) and two identical light chains (molecular weight ~ 25 kDa each) [4,5]. Despite the huge development of OMICS methods, antibodies are still an indispensable tool in various fields of human endeavor including cancer diagnostics. The use of Abs has expanded from simple diagnostics to the detection of the fine structure of molecules, elucidation of gene function, localization of gene prod- ucts, rapid screening of biological effectors for drug discovery and testing, development of therapeutic Abs and utilization of anti- bodies as carriers of drugs or prodrugs to directed sites. We can analyze any protein using electrochemical approaches [6e9] based on the ability of the amino acid residues with exchangeable protons (cysteine, histidine, arginine and lysine [10e12]) to catalyze hydrogen evolution [6]. Using constant current chronopotentiometric stripping (CPS) in combination with mercury electrodes, proteins produce a high electron yield catalytic response, so-called peak H [13], which is sensitive to structural changes in the proteins [6, 14]. We tested by CPS analysis not only free proteins but also those in complex with other molecules, such as nucleic acid, peptides, etc. [6, 15, 16]. We extended these efforts to study lectin-glycoprotein interactions, where we used two ap- proaches. The first approach dealt with the interactions between lectin Concanavalin A and the glycoprotein ovalbumin [17]; their complex was prepared in a test-tube followed by adsorption at the electrode. In the second work [15], we analyzed the interaction of the surface-attached glycoprotein and biomarker, prostate specific antigen (PSA), with a lectin in solution. This arrangement was more convenient for the detection of differences in PSA glycosylation [15]. Here, we tested an additional interaction couple, namely * Corresponding author. Institute of Biophysics CAS, v.v.i., Kr alovopolsk a 135, 612 65 Brno, Czech Republic. E-mail address: ostatna@ibp.cz (V. Ostatn a). Contents lists available at ScienceDirect Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta https://doi.org/10.1016/j.electacta.2018.12.049 0013-4686/© 2018 Elsevier Ltd. All rights reserved. Electrochimica Acta 297 (2019) 974e979