Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1
Braz. j. med. biol. res
;
47(7): 540-547, 07/2014. tab, graf
Article
in English
| LILACS
| ID: lil-712968
ABSTRACT
Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Immunoglobulin Fragments
/
Gene Expression
/
Intercellular Adhesion Molecule-1
/
Protein Renaturation
/
Single-Chain Antibodies
/
Protein Refolding
Limits:
Animals
/
Female
/
Humans
/
Male
Language:
English
Journal:
Braz. j. med. biol. res
Journal subject:
Biology
/
Medicine
Year:
2014
Type:
Article
/
Project document
Affiliation country:
China
Institution/Affiliation country:
Academy of Military Medical Sciences/CN
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