Fv antibodies to aflatoxin B1 derived from a pre-immunized antibody phage display library system

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible

The production of a dextran binding antibody by phage display library and its applications to sugar processing

A phage collection (M1114), which is a mixture of two collections, M710-3R and Silica 426 each derived from a human synthetic phage antibody library (Fab 2lox), was used further enriched for dextran binding using enzyme-linked immunosorbent assays (ELISA) screening. The effects of dextran concentration on phage binding affinity were tested using indirect sandwich ELISA on phage collections, M1114-m74 and AE-M1114-m74-2R. Most of the phage bound to dextran (T2000) coated on a sandwich ELISA. The combination of ELISA screening and a sephadex columns enriched dextran binding 7 fold over enrichment by a single ELISA screen. Phage collection (M1114-m74) produced by combination screening showed the greatest binding on ELISA. The color intensity produced by phage collection (AE-M1114-m74-2R) obtained after the 2nd round of selection was 3.5 fold higher than that of phage collections, AE-M1114-m74-1R after the 1st round. Dextran binding by phage collection (AE-M1114-m74-2R) was illustrated using image analysis of transmission electron micrographs. Sephadex bead agarose electrophoresis (SBAE) screening produced phage collections (AE-M1114-m74-1R and 2R) which were used in a paper-dip assay. A dip stick assay using a protein blocked paper with adsorbed high molecular size dextran (T10,000, 107) produced the most color (59 ±5) using anti-dextran phage enzyme linked assays. Low molecular size dextran (T40, 4x104) produced significantly lower color (15 ±1). Phage collection (AE-M1114-m74-2R) was tested for specificity against dextran (T2000), corn starch, sucrose, dextrose, and chitin. Dextran produced up to 18 fold the normalized intensity of the other carbohydrates. The presence of Fab inserts in the phage collections was confirmed using PCR, and the presence of the same insert in the host E.coli was checked using a â-galactosidase linked assay. DNA sequencing of phage collection (AE-M1114-m74-2R) confirmed that human origin antibody was present. The PCR products of ë, ê light chains and heavy chain from phage collection (AE-M1114-m74-2R) were approximately 420 bp, 550 bp and 600 bp. This research used various selection methods to isolate anti-dextran phages from a library. These were used to develop a paper-dip stick method for dextran detection used for routine screening of sugar juices

Antibody fragments as probe in biosensor development

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed

The production of polyclonal, monoclonal and genetically-derived scFv antibody fragments for the detection of the ß-lactam antibiotic, cephalexin, in milk

This thesis describes the production of antibodies for the detection of cephalexin and their application in ELISA and Biacore ‘real-time’ surface plasmon resonance (SPR)-based biosensor assays. The use of antibiotics in food-producing animals has many beneficial effects (e.g. treatment of mastitis in dairy cattle). However, their use and misuse has also been implicated in the increased incidence of resistant microbial strains with the associated public health risks and economic losses for food processors. Antibiotic residues can adversely affect bacterial starter cultures used in the production of milk-based products such as cheese and yoghurts. The P-lactam antibiotic cephalexin was chosen as the target for the production of antibodies due to its generic cephem-based structure, solubility, and, in particular its use of this antibiotic in treatment of udder infections (e.g. mastitis). Cephalexin-protein conjugates were produced and used for the generation of polyclonal and monoclonal antibodies that were then purified and characterised by SDS-PAGE, Western blotting and ELISA. The specificity of these antibodies was determined and they were used in displacement immunoassays for the detection of the relevant antibiotic in ‘spiked’ samples of PBS and whole milk. Recombinant single chain Fv (scFv) antibody fragments were produced using phage-display technology. Naive and immune phage-display libraries were panned using cephalexin conjugates. In addition, a monoclonal antibody-secreting hybridoma cell line was used in the production of a phage-displayed recombinant scFv. The recombinant scFv (wild type) derived from the parental hybridoma cell was purified and characterised by SDS-PAGE, Western blotting and ELISA. Wild type scFv was subsequently used as a template for the production of a mutant scFv phage-displayed library using random mutagenesis and error-prone PCR.Mutant scFv antibodies were isolated, expressed and then purified using immobilised metal affinity chromatography (IMAC). Two mutant clones expressing scFv antibodies selected showed improved functional expression and assay performance when compared to wild type scFv. Comparative gene and amino-acid sequence analysis and protein modelling was carried out on wild type and selected mutant recombinant scFv antibodies and confirmed the presence of mutations in the framework and variable heavy chain complemetarity determining region amino acid sequences. All antibodies were evaluated in ELISA and Biacore assay formats for the detection of cephalexin in milk samples. The polyclonal, monoclonal and recombinant scFv antibodybased assays demonstrated varying sensitivity and reproducibility but achieved cephalexin detection below the legislated European Union maximum residue limit (EUMRL) in milk ( 10 0 Mgflig)

Generation and application of recombinant antibody fragments for prostate cancer detection.

Prostate cancer (PCa) is the most prevalent adenocarcinoma and the second highest cause of cancer-related deaths in men. Early diagnosis is required to identify the development of PCa to reduce the risk of the disease metastasising to different regions of the body. Multiple biomarkers in serum have been identified for the diagnosis of PCa. However, prostate-specific antigen (PSA) and its isoforms remain the gold standard test for PCa detection. Many antibodies have been generated for the detection of PSA with good sensitivities and limits of detection. To ensure reliable assay performance a number of biomarkers for PCa are required to increase specificity and sensitivity thus reducing the number of false positive results upon initial examination. The overall aims of the work presented in this thesis were to generate, select and characterise antibodies to PSA, an important biomarker in PCa. The generation and selection of murine and avian recombinant antibody fragments to free PSA (fPSA) was performed. Animals were immunised, RNA extracted and cDNA synthesised. The antibody variable genes were amplified by PCR, gel-purified and cloned into a phagemid vector. Electrocompetent cells were transformed and library sizes of 2.02 x 106 cfu/mL and 3.10 x 108 cfu/mL were obtained for the murine and avian clones, respectively. Specific clones were isolated by ‗biopanning‘ which involved a selection process on fPSA-coated immunotubes, enrichment and affinity maturation. Five and three rounds of selection were performed on the murine and avian anti-fPSA single chain variable fragments (scFv) recombinant antibody libraries, respectively. Preliminary analysis by ELISA indicated a large panel of positive binding clones for both screened libraries with approximately 60% of the analysed clones binding to fPSA. The murine recombinant antibody fragments were reformatted and expressed as single chain antibodies (scAb). This construct greatly enhanced soluble protein expression and stability while also facilitating the rapid kinetic analysis of candidate antibodies by Biacore. A differential expression profile of an anti-fPSA scFv and reformatted scAb was performed by ELISA. Individual antibodies isolated from the ‗biopanning‘ process were then characterised. The murine anti-fPSA antibodies were further analysed using the Biacore 3000 in a capture assay format. This facilitates correct affinity ranking of the selected antibodies where the antibody fragment was captured using the appropriate secondary tag antibody (anti-human constant kappa Ab) and a fixed concentration of fPSA (1 g/mL) passed over the captured antibody fragment. Ninety four antibody fragments were evaluated and ranked on their percentage complex stability values after a 5 minute dissociation period. Eleven of the ninety four were subjected to further analysis and their affinities ranged from 3.99 x 10-9 M to 3.62 x 10-10 M. They were then expressed in host Escherichia coli (E. coli) cells on a large scale and purified by immobilised metal affinity chromatography (IMAC). The cell lysates were characterised by ELISA, SDS-PAGE and Western blotting. The antibodies were sequenced and analysed for variability and similarities between the coding regions of the variable domains. Inhibition ELISA-based assays with inter and intra-day analysis were performed on the purified antibodies and a reproducible and accurate assay format was demonstrated with a limit of detection of 2.6 ng/mL. The avian clones were analysed, ranked and affinities evaluated by Dr. Paul Leonard using the Biacore A100 employing the same assay format used for analysis of the murine antibody fragments. Imperative to assay design is the selection of optimal antibody candidates for improved performance. For assay development antibody pairs which bind different regions of the fPSA molecule were determined using the capture assay format on the Biacore 3000. One murine scAb, two avian Fabs and one avian scFv were shown to bind different epitopes of the fPSA molecule with no non-specific binding evident. Following the successful purification of the murine and avian antibody fragments immunohistochemistry studies were performed on PCa tissue samples. These antibodies were shown to bind to different zones of the prostate gland (epithelial and stromal regions) confirming that the antibodies were binding to different epitopes of the fPSA molecule. The purified murine anti-fPSA scAb was biotinylated and this was employed in a sandwich ELISA assay whereby the murine anti-fPSA biotinylated scAb was captured on a neutravidin-coated ELISA plate and fPSA sandwiched using the avian antibody fragments as the detection antibodies. The assay sensitivity was improved using this format with a limit of detection of 1.8 ng/mL in PBST solution. Female serum was spiked with fPSA and inter-day studies performed by sandwich ELISA. Patient serum samples with diagnosed PCa were evaluated by sandwich ELISA to determine levels of fPSA in the sample and data were compared to documented tPSA levels which were comparable to the levels obtained from a commercially available sandwich assay

Production of antibodies for use in a biosensor-based assay for Listeria monocytogenes

The inclusion of L. monocytogenes in the list of organisms subject to HACCP has recently driven the search for detection methods suitable for on-line monitoring. The aim of the work presented in this thesis was the development of a biosensor-based immunoassay for the detection of Listeria monocytogenes using SPR. Two polyclonal antibodies were generated from Listeria monocytogenes MB extract and heat-treated Listeria monocytogenes cells. Both antibodies were purified and characterised by ELISA, SDS-PAGE and Western blotting. An inhibition ELISA-based immunoassay was developed with each antibody for the detection of Listeria monocytogenes. Intra- and interday studies were performed to evaluate the accuracy and intermediate precision of the assays. The feasibility of detecting Listeria monocytogenes cells in chocolate milk, a food matrix which has been reported to have been the cause of a well known Listeria monocytogenes-&ssoc\atQ<\ food poisoning outbreak, was also examined. To determine the potential cross reactivity of each antibody, inhibition ELISAs were performed with a number of bacterial strains. It was concluded that both antibodies can be used as valuable tools for the genus-specific detection of Listeria cells, but were severely limited for the species-specific detection of Listeria monocytogenes cells. Expressing Listeria monocytogenes invasion-associated proteins in E. coli allows the safe and efficient production of high quantities of pure protein for use in the generation of Listeria monocytogenes specific antibodies and immunoassay development. Two Listeria monocytogenes invasion-associated proteins, Intemalin B (InlB) and p60 (also known as iap), were cloned and expressed in E. coli XL-10 Gold cells. Expressed protein was purified by immobilised affinity chromatography (IMAC) and used for the selection of specific antibodies (Chapter 5) and for the development a biosensor-based immunoassay for the detection of Listeria monocytogenes (Chapter 6). The emergence of recombinant antibody phage display technology has transformed the way we generate antibodies for the specific detection of a chosen analyte. Chapter 5 describes the development of two combinatorial antibody libraries from mice immunised with Listeria monocytogenes cells and InlB protein extract. Phage scFv antibodies were selected from both murine antibody libraries and from a large naive human antibody library against Listeria monocytogenes cells and invasion associated protein. A number of the selected phage-scFv antibodies could not be expressed as soluble scFv and also showed tendencies to cross react with various bacterial strains tested. A soluble scFv antibody was selcctcd that recognised the Listeria monocytogenes invasion-associated protein, Internalin B, but did not recognise Listeria monocytogenes cells. An inhibition ELISA was developed with this antibody to indircctly detect Listeria monocytogenes. The two polyclonal antibodies and the selected anti-InlB scFv antibody were used in development of three biosensor-based immunoassays using a BIAcore 3000 instrument (chapter 6 ). Various assay formats and sensor chip surfaces were evaluated. Intra- and interday assay variability studies performed to determine the precision and reproducibility of each assay. The assay developed with the polyclonal anti-InlB polyclonal proved to be most sensitive and cost effective while the assay developed with the anti-InlB scFv antibody fragment proved most specific for the detection of Listeria monocytogenes

Genetic engineering of antibody fragments for the detection of illicit drugs

The monitoring of illicit drugs is now of major importance. Key areas of analysis include the detection of illicit use by drivers, workplace testing, forensic investigations and general monitoring to detect or reduce abuse. Thus, the development of a highly sensitive and reliable array-based assay for saliva, an assay-friendly matrix for detection of illicit drugs, would be of major benefit. This thesis describes the production and affinity maturation of antibodies for the detection of illicit drugs and their application in ELISA, Biacore surface plasmon resonance -based biosensor assays and, finally, in a multidrug-based assay. The first phase of this work focused on the development of a repertoire of antibody fragments against various illicit drugs including morphine-3-glucuronide (M3G), amphetamine and tetrahydrocannabinol (THC). Antibody libraries were generated from murine, avian and leporine immune systems. A number of antibody fragments were produced, purified and characterised by SDS-PAGE, Western blotting and ELISA. A pre-characterised, murine anti-M3G, whole IgG structure was used for conversion to scFv and Fab antibody fragments. This permitted SPR-based comparative studies regarding the validity of using either scFv or Fab fragments in current high throughput screening strategies. The Fab fragment revealed an apparent off rate 40-fold higher than that of the scFv, that did not translate to higher affinity for the target in question. A murine anti-amphetamine Fab fragment demonstrated a 3-fold higher affinity than that of the parent IgG clone. Diverse leporine immune libraries were utilised in the development of novel recombinant Fab and scFv fragments against amphetamine and THC. Subsequently the antibodies were solubly expressed in Escherichia coli, and fully characterised with respect to their binding capabilities on ELISA and Biacore-based analytical platforms. The next phase of the work involved the in-vitro affinity maturation of the anti-M3G Fab and anti-THC scFv fragments. The wild-type antibodies were used as templates for 22 construction of mutant recombinant antibody libraries. Initial mutagenesis strategies exploited error prone-PCR, DNA shuffling, light-chain shuffling and various methods of site-directed mutagenesis to generate improved affinity antibody fragments. Kunklestyle, site-directed mutagenesis, proved to be the most successful method for the generation of an anti-M3G Fab mutated library. The highest affinity anti-THC clones, characterised by ELISA, were carried forward for light-chain shuffling and subjected to specifically-tailored affinity selection conditions. Comparative gene and amino-acid sequence analyses were carried out on the wild-type and selected mutants and confirmed the presence of mutations in the selected CDR’s. The final phase of this research involved the incorporation of the antibodies onto multianalyte assay formats. Optimisations were carried out with respect to immobilisation strategies, surface chemistries, recombinant antibody fragment selection, use of drugprotein conjugates and detection strategies

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development

Acknowledgments: The authors would like to acknowledge the funding support for this work from Scottish Enterprise [VNAR_001(2012)] and the Biotechnology and Biological Sciences Research Council (BB/K010905/1).Peer reviewedPublisher PD

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided