Development of a novel and broadly applicable sandwich ELISA assay based on rabbit single-chain variable fragments and a modified Ig-binding domain of protein L fused to a polystyrene-binding peptide

Abstract

Most of currently available sandwich-type enzyme-linked immunosorbent assays (ELISA) require the use of full-length animal-derived antibodies which poses welfare criticisms and are often expensive to produce. There is therefore a strong demand for the development of more affordable and animal-free methods to produce antibodies for sandwich ELISA assay. To address these issues, we propose here the development of a new technology based on two complementary rabbit single-chain variable fragments (scFvs) and an Ig-binding domain of protein L (PpL1) fused to a polystyrene-binding peptide (PS-tag) that can be recombinantly produced in bacteria. Toward this goal, we developed a rabbit scFv capable to bind the antigen via its variable regions while engaging protein L through its constant framework domain. To enhance the density of captured scFv and enable a better solvent exposure, we generated multiple PpL1 variants bearing polystyrene-binding peptides (PS) tags fused to its ends. The tandem trimer of PpL1 variant bearing PS-tags located at the N-terminus (PpL1′-T-PSN) revealed increased antigen-binding signal when immobilized on hydrophilic polystyrene (phi-PS) plates. By CDR-grafting different antigen-binding specificities into our engineered protein L-binding scFv we validated our technology against a different antigen. Finally, to further enhance the sensitivity of our assay, we implemented a protein L-based pretreatment to remove potential inhibitory immunoglobulin often present in the blood samples. The ability to rapidly and cost-effectively generate animal-free recombinant antibody fragments that can be adsorbed and specifically oriented on plates while retaining their antigen-binding properties could lead to the development of innovative and widely applicable sandwich ELISA systems for the efficient, versatile and sensitive detection of different types of antigens.