A solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands aisd ded "'I-LDL or an 12'I-labeled monoclonaal ntibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for "'I-LDL and for '2'I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinitisy the same at 4 "C and 37 "C. The binding of "'I-LDL and "'IIgG- HL1 occurs with 1:l molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of lZ6I-LDL and '261-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.
For additional information: http://www.jbc.org/content/264/16/9533.long