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-Aß10, VEGF-GRP, VEGF-CETPC, and VEGF-ßhCGCTP37 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.
Subject(s)
Antibodies/analysis , Enzyme-Linked Immunosorbent Assay/instrumentation , Epitopes/immunology , Gonadotropin-Releasing Hormone/immunology , Vascular Endothelial Growth Factor A/immunology , Animals , Antibodies/immunology , Antigens/genetics , Antigens/immunology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/genetics , Gonadotropin-Releasing Hormone/genetics , Immunization , Male , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Vascular Endothelial Growth Factor A/geneticsABSTRACT
To define the molecular basis of ethanol dependence, changes in the phosphorylation of cAMP response element binding protein (CREB) in the nucleus accumbens of rats after acute and chronic ethanol administration were detected using immunohistochemistry. The results demonstrate that the expression of phospho-CREB (p-CREB) protein in the rat nucleus accumbens significantly increased after 15 min of acute ethanol exposure, reaching a peak at 30 min after ethanol administration. The increment remained after 1 or 6 h of ethanol exposure compared to the control rats. In contrast, chronic intake of ethanol solution obviously decreased the expression of p-CREB protein compared to the control rats. The decrement remained 24 h or 72 h after ethanol withdrawal, and returned to the control levels after 7 d of ethanol withdrawal. The results suggest that an acute ethanol administration led to an increase in the phosphorylation of CREB in the nucleus accumbens, but chronic ethanol administration produced a decrement, which is possibly one of the molecular mechanisms of alcohol dependence.
