Development of recombinant antibody fragments capable of detecting azaspiracid

1
Development of recombinant antibody fragments
capable of detecting azaspiracid
Beaufort Marine Research Initiative

• Edwina Stack, Richard O Kennedy, Linda Steward
  and Chris Elliott
• School of Biotechnology and National Centre for
  Sensor Research, Dublin City University, Dublin
  9, Ireland
• and
• Institute of Agri-Food and Land Use, Queen's
  University, Belfast, UK

2
Azaspiracid Toxin

• Discovery 1995 in The Netherlands following the
  consumption of mussels
• grown in Killary Harbour
• Structure Nitrogen-containing polyether toxin
  with a cyclic amine (aza group) a unique spiral
  ring assembly and a carboxylic acid group
• MW 841.5 Da
• Structure elucidation Yasumoto et.
• al. 1998, revised by Nicolaou et al., 2004
• Suspected causative species Protoperidinium
• Symptoms (Severe Acute)
• Nausea
• Vomiting
• Diarrhoea
• Stomach cramps

Yasumoto, et al., J. Am. Chem. Soc. 1998
120(38) 9967 9968 Nicolaou, et al., Angew Chem
Int Ed Engl. 2004 Aug 2043(33)4312-18 Nicolaou
et al., Angew Chem Int Ed Engl. 2004 Aug
2043(33)4318-24.
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Introduction

• Azaspiracids
• Phycotoxin known to accumulate in shellfish
  causing severe gastrointestinal human
  intoxication
• Currently
• NO means for rapid and reliable detection of the
  azaspiracid (AZA) biotoxin in algal blooms or in
  shellfish meat exists
• Aims
• Production of genetically engineered, highly
  stable and specific biorecognition ligands for
  detection of AZA
• Development of a rapid, sensitive and specific
  immunosensor
• Development of matrices for immunoaffinity
  purification and clean-up prior to HPLC analysis

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Exploitation of Azaspiracid toxin Outbreak in
Blue Mussel

• Collaboration Institute of Agri-Food and Land
  Use, Queens University Belfast

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Antibody Library Production

• First Round PCR Amplification

V? long linker
V? long linker
VH
M 1kB DNA ladder (Invitrogen) N Negative
Control VH and VL Annotation for heavy
chain/light chain primer combinations
M 1 2 3 M
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Antibody Library Production

• Splice by extension Overlap (SOE PCR)
  Amplification
• scFv insert incorporated into pComb3x vector and
  transformed into E. coli electrocompetent cells

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Ongoing and Future Work
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BIAcore A100Rapid Kinetic Characterisation
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Antibody Engineering

• Random Mutagenesis for higher affinities
• Mutator strains - XLI-Red deficient in DNA repair
  pathways (mutS, mutD amd mutT)
• Error Prone PCR
• Rolling circle error-prone PCR
• DNA shuffling
• Insertion mutagenesis

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Conjugate Issues

• Issues with conjugate preparation
• Lack of toxin
• High Price
• Approx 9,000 for 50µg AZA1
• Have obtained 200µg AZA-2 from Luis Botana,
  Universidade de Santiago de Compostela Fac.
  Veterinaria, Lugo, Spain
• Purchasing 200µg AZA-2 in conjunction with Chris
  Elliott

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Conjugate Production
NHS, DCC
Protein-NH2

• Conjugations prepared by NHS ester method
• Two protein carriers selected
• Bovine Thyroglobulin (BTG) MW 670 kDa
• Keyhole Limpet Hemocyanin (KLH) MW
  8,000-9,000 kDa
• Characterisation needs to be carried out by Mass
  Spectrometry

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Cytotoxicity Experiments

• Analysis of effects of azaspiracid toxin on
  immune system
• Using J774 cell line derived from murine
  macrophage cell
• Lactate Dehydrogenase (LDH) Assay for analysis of
  cell death and cell lysis
• WST-1 Assay for measurement of cell proliferation
  and viability

LDH Assay on J774 at 24 hour time point
WST-1 Assay on J774 at 24 hour time point
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Summary

• Rabbits immunised with AZA protein conjugates
• Recombinant scFv Antibody Library prepared
• Research hindered by lack of purified toxin,
  conjugates and characterisation methods
• New AZA protein conjugates have been prepared
• Next stage to isolate high affinity antibody
  clone from scFv library
• Potential for development of immunoaffinity
  purification columns for purification and
  enrichment
• Positive results from toxicological studies on
  murine macrophage cell line
• Presented at the 4th safefood Biotoxin Research
  Network Meeting and Workshop
• New Technologies for the Detection of Biotoxins
  in Foods