A linear-polymer-based lactoferrin-selective recognition element for an ELISA mimic: A proof of concept

Abstract

The synthesis of polymers with tailored properties for the recognition of macromolecules such as proteins is challenging. In this work, the synthesis of a new polymer format, a linear polymer (LP), as the selective recognition element for the globular protein lactoferrin (LF) is proposed as a proof-of-concept study. For the synthesis, a solid-phase strategy using the reversible deactivation radical polymerisation (RDRP) mechanism is proposed. This approach, which is usually used in molecular imprinting, involves the immobilisation of LF on the surface of a solid support, but, unlike classical imprinting, a cross-linker in the polymerisation mixture is not required. Consequently, the copolymer is soluble and flexible, thus overcoming the drawbacks associated with traditional synthetic polymers for macromolecule imprinting. This new polymer format has great potential for replacing natural antibodies in bioassays such as enzyme-linked immunosorbent assays (ELISA), dot blot, western blot, or pull-down. In our case, the linear polymer was used as a recognition element to replace natural antibodies in a LF-selective ELISA. The responses of the linear polymer between LF concentrations of 0.1 nM and 0.25 μM were studied, and a significant difference was observed between the non-specific signals and the signals measured in the presence of the polymeric material. Further, the response versus log concentration curves were fitted to a logistic equation, allowing estimation of the EC₅₀ value: 11.8 ± 1.4 nM. We also confirmed the selective detection of LF using the competitive inhibition of the selective LF-biotin conjugate (LF-Bi) binding to the plastic receptor (LP) for closely related proteins (e.g. those having similar molecular weights or isoelectric points) such as human lysozyme, trypsin, and albumin, which are present in human body fluids. The system presents a cross-reactivity value or selectivity of 1.95% for lysozyme, 0.028% for trypsin, and 0.016% for albumin. The applicability of this method for the determination of urine LF levels in inflammatory and infectious diseases of the human urinary tract is also demonstrated.

Keywords: ELISA mimic; Lactoferrin; Linear polymer; Plastic antibody; Reversible deactivation radical polymerisation; Urine sample.