Peptide-based radioimmunoassay specific for GLUT1 glucose transporter.

ABSTRACT

A radioimmunoassay for the GLUT1 glucose transporter was developed with a synthesized peptide based on the sequence of the cDNA for GLUT1. A peptide corresponding to the COOH-terminal domain of the GLUT1 glucose transporter (Thr-Pro-Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val) was synthesized and conjugated to keyhole limpet hemocyanin through the NH2-terminal of the peptide. An antibody was raised against this complex and affinity purified with the immobilized peptide. A second peptide, with tyrosine residue added to the NH2-terminal of the above peptide, was synthesized and used as a standard and iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the COOH-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose-transporter isoforms GLUT2 and GLUT4. Furthermore, the results obtained with this radioimmunoassay on the number of glucose transporters in human erythrocytes were in good agreement with previous studies based on cytochalasin B binding, suggesting that this radioimmunoassay is able to quantify the number of glucose transporters. The assay is completed within 4 h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.