Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies.
Two methods are described for the assay of low-density-lipoprotein (LDL) receptor protein based on the binding of receptor to microtitre plate wells coated with a specific monoclonal antibody or with LDL, followed by detection with radioactive antibody that recognizes a different part of the molecule. The two-antibody procedure detected approx. 2 ng of pure bovine receptor at twice background, and there was a linear relationship on a double-logarithm plot between radioactive antibody bound and the amount of receptor added, up to at least 500 ng of receptor protein per well. The procedure using immobilized LDL was less sensitive and the binding of receptor was inhibited by low concentrations of NaCl, which restricted its usefulness for routine assay of tissue extracts. LDL receptor protein could be readily assayed using the two-antibody procedure in normal human skin fibroblast extracts prepared by bulk-elution from small columns of DEAE-cellulose followed by rapid desalting. No radioactive antibody bound with extracts of cells from a receptor-negative familial hypercholesterolaemic subject. The LDL receptor content of normal fibroblasts preincubated with lipoprotein-deficient serum was estimated, using bovine receptor as standard, to be approx. 60 ng of receptor protein/mg of cell protein.
D000302:Adrenal Cortex / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008077:Lipoproteins, LDL; D011973:Receptors, LDL / Q000032:analysis* / Q000187:drug effects / Q000276:immunology
D000911:Antibodies, Monoclonal; D003902:Detergents; D008077:Lipoproteins, LDL; D011973:Receptors, LDL