Orientation and capturing of antibody affinity ligands: Applications to surface plasmon resonance biochips

Abstract

A surface plasmon resonance (SPR) sensor chip with immobilized protein G was used for simultaneously capturing, purifying and orienting antibody ligands. The ligands were further stabilized by chemical cross-linking. This procedure of designing the sensor chip improved efficient use of the ligands and could prolong the analytical use.The procedure was evaluated on standard dextran-coated sensor chips onto which commercial semi-purified antibodies toward human serum albumin and human troponin where captured and used for analysing their antigens.The procedure demonstrates a general design approach for presenting the biorecognition element on a biosensor surface which enhances sensitivity, stability and selectivity at the same time as an impure ligand is purified.

Comparative Assessment of Affinity-Based Techniques for Oriented Antibody Immobilization towards Immunosensor Performance Optimization

“…The main challenge posed in the successful application of this immobilization technique is surface instability due to affinity interactions, as antibodies coupled to the surface might get displaced by naturally occurring plasma or serum proteins [43]. One way to address this is the use of various bifunctional crosslinking reagents, most commonly dimethyl pimelimidate (DMP) [43,44]. Despite the fact that DMP has been recently replaced by BS3 (bis(sulfosuccinimidyl)suberate) in the majority of protocols for antibody crosslinking to Fc-binding ligands, we opted for DMP to crosslink the secondary antibodies to the immobilized protein A/G since it has been shown that it disrupts less the antigen-binding sites of the Fab region of the antibodies [45].…”

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

“…[162–165] However, crosslinkers like DMP may also modify the amine groups on the antigen biding sites, leading to a decrease in antigen binding efficiency. [166] Konrad et. al.…”

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

“…These proteins specifically bind to the Fc region of immunoglobulin G (IgG) 1 of many mammalian species, leaving antigen-binding sites of IgG fully available to antigens. For instance, Protein A or Protein G was immobilized on the surface of sensor chips, and antibodies were site-specifically immobilized on the layers of those Fc-binding proteins [6][7][8]. It is also known that the oriented immobilization of Fc-binding proteins on planar surfaces enhances the antigen-binding efficiency of antibodies in comparison to immobilization in a random manner [9,10].…”