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BDI ANTIBODY
  • OVERVIEW
  • TECHNICAL CAPABILITIES
  • ADVANTAGES
  • APPLICATIONS
  • CASE STUDIES

Antibody Research & Commercialisation

A core competence of the BDI is its vast expertise, capability and resources in antibody research, engineering, screening and characterisation. Led by Professor Richard O'Kennedy, the 30-strong team of researchers has a track record of research commercialisation and industry engagement, having worked with many national and international companies on a wide range of projects. Applications include human and animal health, food safety, environmental monitoring and drug detection.

BDI Antibody is a commercially-focused entity established to exploit this expertise for the benefit of Irish-based industry and the Irish economy. BDI Antibody will work with SME's, start-ups and multinationals to help them exploit new technologies and develop next generation higher performing competitive products. It will also work with foreign-based multinationals seeking to establish a presence in Ireland.

If you wish to learn more about BDI Antibody, please contact:
Mr. Kieran Flynn / E: k.flynn@dcu.ie / P: 01 700 7663

Antibody Characterisation Capability

BDI Antibody offers one of the most comprehensive and advanced antibody characterisation facilities in Europe. Having supported the development of the surface plasmon resonance (SPR) technology at the heart of GE Healthcare's new Biacore 4000 system, BDI Antibody now operates Biacore 4000, Biacore 3000 and Biacore 1000 systems in its characterisation facility. The high throughput capacity of these systems enables BDI Antibody to offer an extensive characterisation service to determine binding affinity and specificity of antibody-antigen interactions.

Characterisation Facilities

  • BiacoreTM 4000 SPR system, BiacoreTM 3000 SPR system, BiacoreTM 1000 SPR system, Mbio diagnostics SnapEsi®-beta detection platform
  • High-throughput custom designed TECAN based robotic antibody screening system
  • BioMek 2000 for rapid sample processing/rapid analysis: antibody testing
  • HPLC and FPLC systems
  • Image analyser, Cell culture suite, PCR/Cycler, Bacterial shaker incubators

Click here to learn more about SPR and Biacore system.

Custom Antibody Generation

BDI Antibody specialises in the development and production of custom-designed polyclonal, monoclonal and recombinant antibodies. Polyclonal antibodies are produced using a variety of different hosts including rabbits, chickens and mice.

A particular strength is the development of recombinant antibodies, so-called antibody fragments, which can be engineered to have higher affinity and higher specificity than their monoclonal or polyclonal counterparts. BDI Antibody has the option to use either phage display or its own proprietary technology in the development of recombinant antibodies.

BDI Antibody has successfully produced antibodies for the following target applications:

  • Cardiac disease Biomarkers
    Cardiac troponin I (cTnI), C-reactive protein (CRP), Cardiac biomarker heart-type fatty acid binding protein hFABP, Myeloperoxidase (MPO)

  • Cancer Biomarkers
    Prostate Specific Antigen (PSA), Fetuin A/AHSG (r2-HSglycoprotein)- hepatocellular carcinoma

  • Animal Health Biomarkers - Mastitis
    Phospholipase D (PLD), N-acetyl-β-D-glucosaminidase (NAGase)

  • Food Safety
    Internalin A (InlA) , Internalin B (InlB) - Listeria monocytogenes, , Benzimidazole carbamate (BZT), Halofuginone, Aflatoxin B1 (AFB1)

  • Marine toxins
    Microcystin

  • Drugs of abuse
    Morphine-3-glucuronide (M3G)

Whilst BDI Antibody produces polyclonal, monoclonal and recombinant antibodies (rAb) it has particular expertise in rAb development. Recombinant antibodies can be used in all applications in which traditional mAb are used and have inherent advantages over their animal-derived counterparts.

Recombinant Antibody Engineering

In our lab we use phage display in combination with animal-based immune cDNA libraries to successfully produce large and diverse panels of recombinant antibodies. The power of phage display lies in the precise control over selection conditions which can be tailored to retrieve antibody fragments that have high-affinity and specificity. In addition, the genetic coding material of the antibody fragment resides within the phage particle and therefore, during selection a direct link between the antibody's genotype and phenotype is maintained. Thus, the genes encoding the antibody fragment can be readily retrieved from the phage DNA. We have successfully applied phage display to a wide variety of target molecules. In addition, through the use of antibody engineering techniques we have successfully generated antibodies with enhanced sensitivity and specificity.

Uses of Recombinant Antibody

As with traditional animal-generated antibodies, rAbs can be used in blotting techniques (Western, Northern, Southern, etc.), immunostaining, flow cytometry, and enzyme linked immunosorbent assays (ELISAs). rAbs are also used in the diagnosis of infectious diseases by detecting pathogens and can be used in protein arrays that analyze relative protein expression associated with disease or other biological change. Regulatory agencies have also realized the utility of rAbs in vaccine potency testing and in therapeutics.

Benefits of Recombinant Antibody

  1. Decreased time to produce rAbs: an antigen-specific antibody suitable for research purposes can be produced in as little as 8 weeks –significantly less time than the months required for hybridoma-based methods.
  2. Exceptional avidity: in vitro selection for high affinity binding is high-throughput and robust, and can result in antibodies with affinities unobtainable in vivo
  3. Reproducibility: batch-to-batch variation is avoided due to tightly controlled production processes which are suitable for large scale production.
  4. Customizable: Can be engineered to facilitate specific immobilisation on customised surfaces e.g. for sensors; for isolation; for size, and for enhanced affinity.
  5. Specific Binding: rAbs can be engineered for recognition of a broad range of similar compounds or for highly specific unique antigens.
  6. Conditional Binding: a high degree of control is possible, making selection of rAbs that bind in a particular pH, salinity, or in other specific buffer conditions precise and practical.
  7. Unlinked to Immune Response: recombinant antibodies can be isolated from existing antibody libraries without the need for immunization.
  8. Isotype Switching Possible: once a desirable antibody fragment is found it can be easily converted into any antibody isotype (e.g. IgA, IgM IgG etc.) from any species by adding the appropriate constant domain, making these methods highly adaptable and very practical.
  9. rAbs to highly toxic/non-immunogenic proteins possible: Antibodies to highly toxic or non-immunogenic antigens can be created using library methods.
  10. Removal or minimisation of animal use: eliminating the use of animals throughout the process through the use of prepared libraries also minimises/eliminates animal welfare concerns Advances in molecular biology have led to the ability to synthesize antibodies de novo in vitro – completely without the use of animals.
  11. Can be tagged with specific markers: e.g. for detection.

References: John Hopkins Bloomberg School of Public Health (2013) http://altweb.jhsph.edu/mabs/rabsaptamer.html

The improved ability to engineer and characterise antibodies has resulted in their significant impact and extensive use in a wide range of in vivo and in vitro applications. BDI Antibody offers the ideal biomolecular recognition element for numerous analytical platforms including:

  • Immunodiagnostics
  • Western blotting (immunoblotting)
  • Immunohistochemistry
  • Immunocytochemistry
  • Immunoprecipitation
  • Enzyme-linked immunosorbant assay (ELISA)
  • Antibody microarrays
  • Antibody-imaging/immunoscintigraphy
  • Radioimmunoassays
  • Flow cytometric analysis
  • Immunosensors
  • Immuno-polymerase chain reactions (IPCRs)

Antibodies are also useful in immunoaffinity chromatography for separation of antigens from complex mixtures of proteins, drug targeting, immunotherapy and immunoprophylaxis 1

Immunoassay Development

Advances in Immunoassay and Biomedical device development are reliant on the integration of research and development across a wide range of multidisciplinary fields The BDI provides the ideal research environment for the incorporation of all facets of assay development including antibody engineering, transduction science, signal amplification, microfluidic platforms and surface sciences. It is this practical and multidisciplinary experience that gives BDI Antibody researchers the expertise to engineer and develop specific and robust antibodies that underpin advanced immunoassay and multi-array development both within the BDI and by our industry partners. Our researchers can work with you to understand your complete immunoassay needs and thereby develop the best antibody for your application.

ELISA case study:

BDI researchers have developed an improved and optimised approach to produce rapid and highly-sensitive ELISAs. This method involves covalently immobilising antibodies on chemically modified polymeric surfaces and yields a 16-fold increase in sensitivity compared with established methods of testing. This ELISA format could be used as a quantitative analytical approach in clinical situations or as a faster approach to screen phage-displayed antibodies during development. Combining research and insights from multidisciplinary fields, this protocol vastly improves ELISA sensitivities and highlights the advantage of the diverse expertise readily available to BDI Antibody. 2

Publications

1 Affinity chromatography as a tool for antibody purification. Vijayalakshmi Ayyar et al.

2 Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays. Chandra Kumar Dixit, Sandeep Kumar Vashist, Brian D MacCraith, & Richard O'Kennedy. Nature Protocols 6, 439–445 (2011).

The following exemplar case studies provide examples of the applications addressed by BDI Antibody. In all of these cases the work was stimulated by industry or clinical need. We will work with you to develop the optimum solutions for your business.

Human Health - Cardiac Troponin I (cTn I)

Cardiac troponin I (cTnI) is a highly specific biomarker and the gold standard for CVD diagnosis. BDI Antibody has produced anti-cTnI scFvs against this target. Panels of monoclonal antibodies have also been generated against cTnI. 1 2

Food Safety - Aflatoxin B1 (AFB1)

Aflatoxins are naturally occurring mycotoxins produced by the fungal species, Aspergillus flavus and Aspergillus parasiticus. There are four main isoforms; aflatoxin B1, B2, G1 and G2 which act as harmful food contaminants. Aflatoxin M1, the metabolic product makes up the fifth member of the aflatoxin family. Aflatoxins are known to contaminate a variety of foods including cereals (corn, wheat, maize, rice, etc.), spices, nuts, seeds and dried fruit and exposure is extremely harmful to humans. AFB1, the predominant isoform, is a potent hepatocarcinogen, and there is a positive association between aflatoxin exposure and development of hepatocellular carcinoma (HCC). As a result, all 5 aflatoxins B1, B2, G1, G2 and M1 have been listed by the International Agency for Research on Cancer as Group 1 human carcinogens.

An aflatoxin B1-specific single chain variable fragment (scFv) has been developed and converted to a chimeric Fab format by the addition of human constant regions. A mutant Fab fragment has also been generated. These antibodies permit improved sensitivity for aflatoxin detection. 3 4 5 6 7

Marine Biotoxins - Microcystin-LR

Polyclonal antibodies and recombinant antibody fragments against microcystin-LR have been developed. In particular the scFv recombinant antibody fragment offers increased specificity, sensitivity and stability relative to existing products. The BDI Antibodies were immomilised onto the Mbio SnapEsi-betaThe BDI Antibodies were immomilised onto the Mbio SnapEsi-beta novel planar wave guide cartridge system. This system enables the detection of microcystin below the World Health Organisation regulatory limit. 8

Drug Detection - Morphine-3-Glucuronide (M3G)

Morphine-3-glucuronide (M3G) is the main metabolite of heroin and morphine. A polyclonal antibody raised against M3G was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor for drug detection. Furthermore, a model anti-M3G recombinant antibody in both scFv and chimeric Fab formats was developed to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore. We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was also produced with bovine serum albumin (M3G-BSA) conjugate. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. 9

Publications

1 Cardiac biomarkers and the case for point-of-care testing. Barry McDonnell et al.

2 Cardiac troponin I: a case study in rational antibody design for human diagnostics. P.J. Conroy et al.

3 Aflatoxins B1, B2 and G1 modulate cytokine secretion and cell surface marker expression in J774A.1 murine macrophages. Johanna C. Bruneau et al.

4 Development of surface plasmon resonance-based immunoassay for aflatoxin B(1). Daly S.J. et al.

5 Advances in biosensors for detection of pathogens in food and water. Paul Leonard et al.

6 Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins. Barry Byrne et al.

7 The application of biosensors for the sensitive detection of agricultural contaminants, pathogens and food-borne toxins. Barry Byrne et al.

8 Sensing technologies for monitoring the marine environment. Lorna Fitzsimons et al.

9 Production of a recombinant anti-morphine-3-glucuronide single-chain variable fragment (scFv) antibody for the development of a "real-time" biosensor-based immunoassay. Dillon Paul P et al.