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ABSTRACT: Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species and the binding event is visualized in real-time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns both concentration and affinity data but when used correctly can also resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.
Methods in molecular biology (Clifton, N.J.) 01/2012; 907:411-42.
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ABSTRACT: Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.
Analytical Biochemistry 08/2011; 415(2):158-67. · 3.00 Impact Factor
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ABSTRACT: Antibody-based separation methods, such as immunoaffinity chromatography (IAC), are powerful purification and isolation techniques. Antibodies isolated using these techniques have proven highly efficient in applications ranging from clinical diagnostics to environmental monitoring. IAC is an efficient antibody separation method which exploits the binding efficiency of a ligand to an antibody. Essential to the successful design of any IAC platform is the optimisation of critical experimental parameters such as: (a) the biological affinity pair, (b) the matrix support, (c) the immobilisation coupling chemistry, and (d) the effective elution conditions. These elements and the practicalities of their use are discussed in detail in this review. At the core of all IAC platforms is the high-affinity interactions between antibodies and their related ligands; hence, this review entails a brief introduction to the generation of antibodies for use in IAC and also provides specific examples of their potential applications.
Methods in molecular biology (Clifton, N.J.) 01/2011; 681:35-59.
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ABSTRACT: Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilised to purify proteins, carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently proteins G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification of antibodies which may be implemented for a selection of different applications such as immunodiagnostics. This chapter is designed to inform the researcher about the basic techniques involved in the affinity chromatography-based purification of monoclonal, polyclonal, and recombinant antibodies. Examples are provided for the use of proteins A and G. In addition, tables are provided that allow the reader to select the most appropriate protein for use in the isolation of their antibody.
Methods in molecular biology (Clifton, N.J.) 01/2011; 681:369-82.
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ABSTRACT: The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1,179 papers that involved the application of biosensor data were identified for the year 2007 alone (Rich and Myszka, J Mol Recognit 21:355-400, 2008), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay set-up while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterisation of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilised surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.
Methods in molecular biology (Clifton, N.J.) 01/2011; 681:403-18.
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[show abstract]
[hide abstract]
ABSTRACT: Antibody-based separation methods, such as immunoaffinity chromatography (IAC), are powerful purification and isolation techniques.
Antibodies isolated using these techniques have proven highly efficient in applications ranging from clinical diagnostics
to environmental monitoring. IAC is an efficient antibody separation method which exploits the binding efficiency of a ligand
to an antibody. Essential to the successful design of any IAC platform is the optimisation of critical experimental parameters
such as: (a) the biological affinity pair, (b) the matrix support, (c) the immobilisation coupling chemistry, and (d) the
effective elution conditions. These elements and the practicalities of their use are discussed in detail in this review. At
the core of all IAC platforms is the high-affinity interactions between antibodies and their related ligands; hence, this
review entails a brief introduction to the generation of antibodies for use in IAC and also provides specific examples of
their potential applications.
Key wordsImmunoaffinity chromatography–Antibody–Purification
12/2010: pages 35-59;
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[show abstract]
[hide abstract]
ABSTRACT: The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1,179
papers that involved the application of biosensor data were identified for the year 2007 alone (Rich and Myszka, J Mol Recognit
21:355–400, 2008), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate
and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently
execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance,
and assay set-up while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based
screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this
protocol describes the kinetic characterisation of single chain Fv (scFv) antibody fragments from crude bacterial lysates
using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments
to an anti-HA tag monoclonal antibody-immobilised surface prior to kinetic analysis, the same methodologies are universally
applicable and can be used for practically any affinity pair and most Biacore systems.
Key wordsAntibody–Surface plasmon resonance–Biacore–Kinetics–Biosensor analysis–Protein–protein interactions.
12/2010: pages 403-418;
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[show abstract]
[hide abstract]
ABSTRACT: Affinity chromatography permits the isolation of a target analyte from a complex mixture and can be utilised to purify proteins,
carbohydrates, drugs, haptens, or any analyte of interest once an affinity pair is available. It involves the exploitation
of specific interactions between a binding affinity pair, such as those between an antibody and its associated antigen, or
between any ligand and its associated binding receptor/protein. With the discovery of protein A in 1970, and, subsequently
proteins G and L, immuno-affinity chromatography has grown in popularity and is now the standard methodology for the purification
of antibodies which may be implemented for a selection of different applications such as immunodiagnostics. This chapter is
designed to inform the researcher about the basic techniques involved in the affinity chromatography-based purification of
monoclonal, polyclonal, and recombinant antibodies. Examples are provided for the use of proteins A and G. In addition, tables
are provided that allow the reader to select the most appropriate protein for use in the isolation of their antibody.
Key wordsProtein A–Protein G–Protein L–Affinity chromatography–Monoclonal–Polyclonal–Recombinant antibodies
12/2010: pages 369-382;
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ABSTRACT: Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody's assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.
Analytical Biochemistry 11/2010; 410(1):27-33. · 3.00 Impact Factor
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ABSTRACT: Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from naïve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.
Journal of immunological methods 09/2010; 364(1-2):14-20. · 2.35 Impact Factor
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ABSTRACT: Antibody phage display is a powerful biomolecular selection technology now routinely used for refining antibody diversity towards analytes of both therapeutic and diagnostic interest. Post selection, automated robotic systems can be utilised to pick, express and analyse large numbers of putative analyte-specific clones allowing the parallel screening of thousands of antibodies in less time. Most screening techniques involve a spatial addressing process whereby the selected antibodies are extracted from the cells and analysed to verify specificity. Using a simple 'on-plate' growth and screening approach, we show that antibody-expressing clones can be simultaneously cultured and analysed rapidly in antigen-coated ELISA plate wells yielding high binding signals and saving valuable selection time, while also eliminating the necessity for antibody extraction. The utilisation of the 'on-plate' technique for the screening of Fab and scFv antibodies, and a comparative analysis with commonly used antibody extraction processes, are described.
Journal of immunological methods 07/2010; 359(1-2):61-4. · 2.35 Impact Factor
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ABSTRACT: Recent trends in the development of analytical procedures are directed toward platforms and devices that are speedy, small, sensitive, and specific. However, these aims may require new reagents and approaches that are tailor-made for the application envisaged, or re-tooling of existing systems. Whichever approach is taken, the problems and demands remain similar. They include non-specific binding and its elimination, oriented immobilization of ligands and other molecules, matrix effects, the need for sample “clean-up,” single or multiple use, miniaturization, microfluidic needs, ease-of-use, user, legislator and consumer appreciation, acceptance and requirements, and cost considerations. These and other issues will be critically analyzed in the light of recent achievements and the potential to overcome current obstacles will be discussed.
Analytical Letters 07/2010; 43(10-11):1630-1648. · 1.02 Impact Factor
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Nature Nanotechnology 01/2010; 5(1):9-10. · 27.27 Impact Factor
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ABSTRACT: Currently, the reliable detection and quantification of a multitude of different analytes is crucial in many applications and settings. Biosensors have revolutionised diagnostics for use in point-of-care testing (POC), the detection of food and environmental contaminants, biological warfare agents, illicit drugs and human/animal disease markers. Antibodies continue to play a pivotal role in many sensor devices due to their exquisite specificity for their cognate antigens. In this review current biosensor platforms employing antibodies for molecular recognition are briefly described. The use of molecular biological techniques for the generation and improvement of antibodies is critically examined. Such recombinant antibodies possess improved attributes for use in biosensor development in terms of design, stability, affinity and specificity.
Seminars in Cell and Developmental Biology 03/2009; 20(1):10-26. · 6.65 Impact Factor
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ABSTRACT: Cardiovascular disease (CVD) is the single greatest cause of adult mortality in the western world and, consequently, places a massive burden on healthcare services and the economy. Lifestyles, lack of clearly defined risk assessment criteria, consistently high incidences of misdiagnosis and inappropriate referrals, all contribute significantly to this problem. It also correlates directly with inefficient or non-accessible early detection systems. Over the last decade much research has focused on the identification of cardiac biomarkers that can be used for the detection of cardiac distress and that add value to current risk stratification criteria. An exposition of some of the most consistently cited biomarkers is provided and their current status and potential value as early CVD risk predictors, more accurate diagnostic markers of acute myocardial damage and as reliable prognostic indicators, is evaluated. The particular importance of early prediction and the integral role that point-of-care (POC) testing is expected to play in the future of cardiac care is critically discussed.
Clinical biochemistry 03/2009; 42(7-8):549-61. · 2.02 Impact Factor
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ABSTRACT: Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.
Journal of Immunological Methods 07/2007; 323(2):172-9. · 2.20 Impact Factor
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ABSTRACT: This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.
Biosensors and Bioelectronics 06/2007; 22(11):2724-9. · 5.60 Impact Factor
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ABSTRACT: Prostate-specific antigen (PSA) is the best serum marker currently available for the detection of prostate cancer and is the forensic marker of choice for determining the presence of azoospermic semen in some sexual assault cases. Most current assays for PSA detection are processed on large analyzers at dedicated testing sites, which require that samples be sent away for testing. This leads to delays in patient management and increased administration costs. The recent emphasis placed on the need for point-of-care patient management has led to the development of novel biosensor detection strategies that are suitable for the miniaturization of assays for various targets including PSA. This review highlights the current and novel analytical technologies used for PSA detection, which will benefit clinicians, patients and forensic workers in the future.
Trends in Biotechnology 04/2007; 25(3):125-31. · 9.15 Impact Factor
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ABSTRACT: Listeria monocytogenes is an important food-borne pathogen with an extremely high mortality rate (approximately 30%). Therefore, a highly sensitive, reproducible and rapid assay for its detection is vital. L. monocytogenes cells employ two surface bound proteins, Internalin A (InlA) and Internalin B (InlB) to promote invasion into host cells. Recombinant forms of both proteins were previously cloned and expressed in Escherichia coli. In this paper we describe how the InlB protein was sub-divided into three shorter overlapping peptide fragments yielding truncated functional protein of M(R) 23, 35 and 45 kDa, respectively. Purification of the InlB fragments by immobilised metal affinity chromatography (IMAC) was optimised and confirmed by electrophoresis and Western blotting. Identification of the antibody binding regions was achieved by probing the expressed polypeptide domains with a panel of antibodies and antibody fragments. The cloned peptide fragments were also used to develop novel fluorescence-based immunoassays incorporating quantum dots. The application of quantum dot-labelled anti-InlA monoclonal antibodies for immunostaining L. monocytogenes was also demonstrated.
International Journal of Biological Macromolecules 09/2006; 39(1-3):127-34. · 2.45 Impact Factor
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ABSTRACT: A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) 'mAb2B3' was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non-Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1x10(7) cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody 'mAb2B3', described herein.
Journal of Microbiological Methods 09/2006; 66(2):294-312. · 2.09 Impact Factor
