Detection of antibodies against customized epitope: use of a coating antigen employing VEGF as fusion partner

Abstract

Diagnosis of many infectious, autoimmune diseases and cancers depends on the detection of specific antibodies against peptide epitope by enzyme-linked immunosorbent assay (ELISA). However, small peptides are difficult to be coated on the plate surfaces. In this study, we selected GnRH as a model hapten to evaluate whether VEGF121 would be suitable as an irrelevant hapten-carrier to develop a universal platform for specific antibodies detection. Firstly, GnRH was fused to the C terminus of VEGF121 and the resultant fusion protein VEGF-GnRH expressed effectively as inclusion bodies in Escherichia coli. Thereafter, VEGF-GnRH was easily purified to near homogeneity with a yield of about 235 mg from 2.1 L induced culture. At last, VEGF-GnRH was used to perform ELISA and western blot, and our results suggested that VEGF-GnRH was capable of detecting anti-GnRH antibodies in sera both qualitatively and quantitatively. Indeed, previous studies of our laboratory had demonstrated that other fusion proteins such as VEGF-Aβ10, VEGF-GRP, VEGF-CETPC, and VEGF-βhCGCTP37 were able to detect their corresponding antibodies specifically. Therefore, VEGF121 may be a suitable irrelevant fusion partner of important diagnostic peptide markers. Our works would shed some light on the development of a universal platform for detection of specific antibodies.