Abstract
The 24 kDa protein from the gag of the bovine leukaemia virus was cloned and expressed as a fusion protein GST-p24. This recombinant protein was then used to immunize a Leghorn chicken. The partially purified chicken anti-GST IgY was used to develop a solid-phase assay by binding the IgY to an ELISA plate. When the fusion protein contacts the antibody, it binds it by its N-terminal, leaving the C-terminal, which carries the sequence that acts as a capture antigen in solution maximally exposed, reducing the risk of epitope masking. The conditions of the fusion protein on the solid phase maximize the presentation of the antigens' epitopes in solution. For the first time, a system has been developed with a non-mammalian coating antibody. Besides optimizing the recognition of low-molecular-weight antigens synthesized as fusion proteins, it avoids cross-reactions with commonly used secondary antibodies, mostly raised in mammalian hosts.
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Abbreviations
- BLV:
-
bovine leukaemia virus
- GST:
-
glutathione S-transferase
- PCR:
-
polymerase chain reaction
- FLK:
-
fetal lamb kidney
- GS4B:
-
glutathione agarose 4B
- IPTG:
-
isopropyl-β -d-thiogalactopyranoside
- PMSF:
-
phenylmethylsulphonyl fluoride
- FCA:
-
Freund’s complete adyuvant
- BCI/TNBT:
-
5-bromo-4-chloro-3-indolyl phosphate/tetranitroblue tetrazolium
- SDS-PAGE:
-
sodium dodecyl sulphate–polyacrylamide gel electrophoresis
- TMB:
-
3,3′,5,5′-tetramethylbenzidine
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Juliarena, M., Gutierrez, S. & Ceriani, C. Chicken Antibodies: A Useful Tool for Antigen Capture ELISA to Detect Bovine Leukaemia Virus without Cross-reaction with Other Mammalian Antibodies. Vet Res Commun 31, 43–51 (2007). https://doi.org/10.1007/s11259-006-3422-1
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DOI: https://doi.org/10.1007/s11259-006-3422-1