Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay.
The current paper describes 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 is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-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 affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-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.
Van Driel
I R
IR
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Brown
M S
MS
Goldstein
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D011487:Protein Conformation; D011869:Radioligand Assay; D011973:Receptors, LDL / Q000032:analysis / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011973:Receptors, LDL