Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins

Abstract

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

Integrating scFv into xMAP Assays for the Detection of Marine Toxins

“…Like monoclonal and polyclonal antibodies, scFvs have been incorporated into ELISAs, SPR, and LFIs [38]. Although the ELISA format affords amplification through the action of an enzyme, it is not easily multiplexed to examine multiple toxins in one well.…”

“…A viable alternative is recombinant antibodies including single-chain variable fragment (scFv), and single domain antibodies (sdAb) [36]. Recombinant constructs (scFv) that consist of linked heavy and light variable domains that make up the binding domains of conventional antibodies have been produced for the sensitive detection of DA, as well as a number of other biotoxins [37,38]. Recombinantly produced binding elements serve to “preserve” mAbs as they can be easily recreated from their sequence information, thereby reducing variability and eliminating issues with loss of cell lines.…”

“…Recombinantly produced binding elements serve to “preserve” mAbs as they can be easily recreated from their sequence information, thereby reducing variability and eliminating issues with loss of cell lines. An additional advantage of scFv over mAbs is the ability to produce reagent recombinantly in E. coli and the potential to produce fusion constructs with enhanced utility that can potentially be tailored to particular sensor systems [38,39,40,41]. While not universal, improvements in stability, affinity, and diversity have been observed in scFvs, for example, improvement in both stability and affinity was demonstrated by McConell et al [42].…”

Establishment of an ultrasensitive indirect competitive time-resolved fluoroimmunoassay for vancomycin determination

“…Indirect competitive immunoassay, containing IC-ELISA and IC-TRFIA which were the most common method for the small molecule hazards (such as small molecule pesticides, biotoxins and antibiotics, etc.) determination (Kavanagh, Elliott, & Campbell, 2015;Ni et al, 2019;Watanabe, Miyake, & Yogo, 2013). Because of its superiority, the key evaluation index of antibody to antigen, containing IC 10 (LOD), IC 50 and IC 20 -IC 80 (linear working range), which could be calculated out directly.…”

Identification and characterisation of the proteins bound by specific phage‐displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts

“…Traditionally, those specific antibodies have been raised in animals . However, there is a tendency towards the replacement of antibodies raised in animals with antibodies synthesised in vitro , products of synthetic libraries . Filamentous bacteriophage libraries that display antibody fragments fused to one of the phage coat proteins have been successfully used for this purpose …”