RESUMO
The study of antibody-antigen interactions, through epitope mapping, enhances our understanding of antibody neutralization and antigenic determinant recognition. Epitope mapping, employing monoclonal antibodies and mass spectrometry, has emerged as a rapid and precise method to investigate viral antigenic determinants. In this report, we propose an approach to improve the accuracy of epitopic peptide interaction rate recognition. To achieve this, we investigated the interaction between the nucleocapsid protein of fig mosaic virus (FMV-NP) and single-chain variable fragment antibodies (scFv-Ab). These scFv-Ab maintain high specificity similar to whole monoclonal antibodies, but they are smaller in size. We coupled this with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The experimental design involved using two different enzymes to digest FMV-NP separately. The resulting peptides were then incubated separately with the desired scFv-Ab at different incubation times and antibody concentrations. This allowed us to monitor the relative rate of epitopic peptide interaction with the antibody. The results demonstrated that, at a 1:1 ratio and after 2 h of interaction, the residues 122-136, 148-157, and 265-276 exhibited high-rate epitopic peptide binding, with reductions in peak intensity of 78%, 21%, and 22%, respectively. Conversely, the residues 250-264 showed low-rate binding, with a 15% reduction in peak intensity. This epitope mapping approach, utilizing scFv-Ab, two different enzymes, and various incubation times, offers a precise and dependable analysis for monitoring and recognizing the binding kinetics of antigenic determinants. Furthermore, this method can be applied to study any kind of antigens.
RESUMO
Protein A, a major cell wall component of Staphylococcus aureus, is one of the first immunoglobulin-binding proteins that is discovered about 80 years ago. However, a great deal of development in both purification methods and application of antibodies in treatment have been done. There are many publications based on the untargeted (size exclusion, ion exchange and hydrophobic interactions) and targeted (affinity) methods by scientists in academic/industry groups. In this review, we have focused on the study of both native and engineered Protein A to understand its mechanism in the purification of antibodies. What domain of Protein A dose interact with antibody? Where are contact regions? What is the non-covalent interaction mechanism of Protein A and antibody? Does alkaline condition, in the washing step, influence on antibody structure and activity? On the other hand, the immobilization of Protein A on various sorbents such as agarose, silica, polysaccharide, polymers, and magnetic nanoparticles have investigated. Also, the application of Protein A as biosensor for detection of the antibody is discussed. We have tried to find interesting and stimulating answers to all these questions.