Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1.

H Sun,Y.H Yue,Y.Y Chen,G.L Zhang,Y Tian,G.M Wu

doi:10.1590/1414-431x20143276

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.

Highlights

Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin supergene family and is a cell surface ligand for lymphocyte function-associated antigen-1 (LFA-1)
To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19610–8 M) against human ICAM-1
The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods

Summary

Introduction

Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin supergene family and is a cell surface ligand for lymphocyte function-associated antigen-1 (LFA-1). Many diseases are associated with overexpressed ICAM-1, such as acute pancreatitis [4], inflammatory bowel disease and colonic neoplasms [5], inflammation associated with organ transplantation [6,7,8], angiocardiopathy [9,10], ischemiareperfusion injury [11], and cancer [12,13,14] These reports suggested that the anti-ICAM-1 strategy has a potential application in the treatment of ICAM-1-mediated immunological and inflammatory diseases. ScFv has a wide range of applications in diagnosis and therapy It is possible for anti-ICAM-1 scFv to block the biological activity of ICAM-1, and it may be effective in preventing the progression of the above-mentioned diseases. This study paves the way for preparing large quantities of anti-ICAM-1 scFv for application against diseases associated with inflammation

Material and Methods

Results

Discussion