Virus Strain Discrimination Using Recombinant Antibodies

Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological facilities. Single chain Fv antibody fragments (scFv) have been selected from a synthetic phage-antibody library by affinity selection with purifiedPotato virus Y, ordinary strain (PVYO). The scFv selected was specific for PVY and detected 7 out of 9 isolates of PVYOwhilst it did not detect 15 isolates from the closely related necrotic strains PVYNand PVYNTN. In ELISA the scFv could be used to detect virus at concentrations of 50 ng/ml in plant sap and was shown to have similar limits of detection as commercially available PVY monoclonal antibodies. These results highlight the potential of the technology for the selection of strain specific antibodies with an affinity and assay sensitivity similar to traditional monoclonal antibodies and their use in viral diagnostics.

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Selection of Human Single Chain Fv Antibody Fragments Binding and Inhibiting Helicobacter pylori Urease

Tumor Biology ◽

10.1159/000030153 ◽

2001 ◽

Vol 22 (1) ◽

pp. 36-44 ◽

Cited By ~ 17

Author(s):

Mehdi Houimel ◽

Irène Corthesy-Theulaz ◽

Igor Fisch ◽

Cindy Wong ◽

Blaise Corthesy ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibody Fragments ◽

Single Chain ◽

Single Chain Fv ◽

Fv Antibody ◽

Single Chain Fv Antibody ◽

Selection Of

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Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3343-3349.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3343-3349 ◽

Cited By ~ 40

Author(s):

William J. J. Finlay ◽

Iain Shaw ◽

Joanna P. Reilly ◽

Marian Kane

Keyword(s):

Domoic Acid ◽

Recombinant Antibody ◽

Variable Region ◽

Antibody Fragments ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

High Affinity ◽

Single Chain Fv ◽

Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

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