Site‐specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced ebola detection

Abstract

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

“…33–38 These methods utilize the nucleotide-binding site (NBS) of antibody as a binding pocket for conjugation of small indole molecules such as indole 3-butyric acid (IBA) and tryptamine. Although the NBS has no known function, it is a highly conserved region located in the variable domain of the Fab fragment.…”

“…34,39 The UV-NBS method utilizes UV energy to initiate a reactive radical driven reaction between the indole molecule and the NBS, 34 which provides an opportunity to produce functionalized antibodies and fab fragments with various functional groups such as affinity tags, imaging molecules, chemotherapeutics and peptides. 33–38 In multiple publications, we established that the UV-NBS method can be used to functionalize antibodies without unfavorably impacting their antigen binding activity. Furthermore, we also demonstrated many utilities that the NBS can provide in a variety of applications beyond covalent linking such as oriented immobilization, 35,36 enhanced antigen detection, 35,37,38 as well as inhibition of allergic reactions 39–41 .…”

“…33–38 In multiple publications, we established that the UV-NBS method can be used to functionalize antibodies without unfavorably impacting their antigen binding activity. Furthermore, we also demonstrated many utilities that the NBS can provide in a variety of applications beyond covalent linking such as oriented immobilization, 35,36 enhanced antigen detection, 35,37,38 as well as inhibition of allergic reactions 39–41 .…”

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

“…They also used the same photo-cross-linking strategy to prepare biotin labeled antibodies and coated such conjugates onto a streptavidin plate for enhanced antigen detection. [96,97] Although NBS is present in the variable region of an antibody but it is still distant from the antigen binding site ( Figure 6 ),[93,94] and doesn't affect antigen binding capacity of the antibodies. In addition to NBS, other unconventional binding sites on antibodies, which are specific for small molecules, have been identified and may offer alternative conjugation/immobilization strategies.…”

Polymeric Nanoparticles Decorated with Monoclonal Antibodies: A New Immobilization Strategy for Increasing Lipase Activity

“…In this case, a protein of interest is fused to an enzyme that reacts selectively with an immobilized substrate analogue or inhibitor to form a covalent bond. Following this approach, enzyme-mediated site-directed covalent immobilization techniques have been developed for immunosensing applications [11][12][13][14] As is well known, the use of monoclonal antibodies (MoAbs) acquired relevance in basic immunological research in the past, finding various applications ranging from industrial technologies in the biotechnological field to diagnostics and therapy [15,16]. In the industrial field, MoAbs are used as a purification tool for proteins of biotechnological interest (i.e., cytokines, growth factors).…”