Exploiting recombinant antibodies in point-of-care (POC) diagnostics: The combinatorial advantage

Abstract

Antibodies are ubiquitously deployed on in vitro diagnostic (IVD) platforms for detecting a panoply of analytes indicative of environmental and food contamination, residue adulteration and both veterinary and medical diagnostics. In the clinical realm, rapid and accurate determination of disease status is paramount. The significance of immunodiagnostic performance cannot be overemphasized and in many cases reliable diagnosis informs medical intervention which can mean the difference between patient recovery and demise. Cardiovascular disease (CVD) is the single biggest cause of adult mortality in the western world and principal burden on the healthcare services. Although the troponin (Tn) family, in particular troponin I (TnI), are regarded as the gold standard for diagnosis of myocardial damage, over the last decade much research has focused on the identification of alternative cardiac biomarker molecules that can either supplant or complement TnI metrics to add value to cardiac risk stratification criteria. In particular, markers that appear earlier than TnI in the pathophyisiology of cardiac disease are highly sought after. The subject of this addendum represents part of a broader challenge to deliver novel rapid point-of-care (POC) diagnostics to provide a chip-based multi-plexed platform for more comprehensive profiling of cardiac status with additive diagnostic and prognostic value. Specifically, it outlines proof-of-concept direct myeloperoxidase (MPO) detection, demonstrates the benefits of using recombinant antibodies in POC diagnostics and describes optimized strategies for generation of superior candidate antibody panels.

Reconciling the Structural Attributes of Avian Antibodies

“…The antibodies were generated from immunized repertoires by phage display and interacted with their cognate antigens with high affinity (scFv B8, 1 nM; scFv 180, 20 pM). These antibodies exemplify the power of display technologies, in particular phage display, to not only recapitulate (scFv B8) but also exceed (scFv 180) the diversity of host immune system to generate antibodies that surmount the theoretical affinity ceiling (8,49). Combinatorial libraries facilitate combinations of heavy and light chains that are truly randomized, not represented or accessible in the natural B-cell repertoire, and routinely permit isolation of antibodies with sub-single digit nanomolar affinities (8).…”

“…The natural immune repertoire is dynamic, with the capacity to generate a repertoire of 10 8 by affinity maturation in vivo, which is substantiated by continual exposure or sensitization to antigens from the environment or by immunization (8). Artificial manipulation of antibody genes has facilitated in vitro selection of truly unique antibodies from extremely large combinatorial libraries, which can be constructed from virtually any species, isolated from B-cells derived from naïve, immunized, or infected subjects or are partially or wholly synthesized in vitro (8).…”

“…Artificial manipulation of antibody genes has facilitated in vitro selection of truly unique antibodies from extremely large combinatorial libraries, which can be constructed from virtually any species, isolated from B-cells derived from naïve, immunized, or infected subjects or are partially or wholly synthesized in vitro (8). Display technologies such as phage, yeast, and ribosome display, when combined with high-throughput approaches for judicious library screening, have enabled the development of antibodies with highly tailored affinities, specificities, and biophysical properties (9,10).…”

“…Display technologies such as phage, yeast, and ribosome display, when combined with high-throughput approaches for judicious library screening, have enabled the development of antibodies with highly tailored affinities, specificities, and biophysical properties (9,10). The merits of in vitro-based antibody approaches, although largely under-appreciated by the research community, allow one to isolate antibodies with properties extremely or if not impossible to attain using the immune system alone (6,8,11). These powerful avenues of antibody development are of considerable importance for development of novel therapeutic entities and next generation diagnostic reagents and address the consternation among researchers arising from a preponderance of subpar research antibodies (12)(13)(14).…”

Point-of-Care Compatibility of Ultra-Sensitive Detection Techniques for the Cardiac Biomarker Troponin I—Challenges and Potential Value

“…Recombinant antibodies are produced by exploiting genetic techniques to generate a variety of antibody constructs, such as Fab (fragment antigen binding) Abs and scFv (single-chain fragment variable) Abs [44]. The production method used here offers considerable advantages, such as the Ab size being smaller, enabling a greater density of Abs to be immobilised, and the capacity to add tags to enhance immobilisation and ensure correct orientation [45,46]. A study comparing the use of Ab fragments has shown that their incorporation into an assay may reduce the interference from matrix biomolecules, in addition to reducing reagent consumption and increasing the assay sensitivity [47].…”