title: Development of capture assays for different modifications of human low-density lipoprotein.
abstract: Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), Nepsilon(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r=0.706, P<0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.
authors.affiliation: Medical University of South Carolina, Department of Microbiology and Immunology, 173 Ashley Ave., PO Box 250504, Charleston, SC 29425, USA. virellag@musc.edu
authors.forename: Gabriel
authors.initials: G
authors.lastname: Virella
authors.forename: M Brooks
authors.initials: MB
authors.lastname: Derrick
authors.forename: Virginia
authors.initials: V
authors.lastname: Pate
authors.forename: Charlyne
authors.initials: C
authors.lastname: Chassereau
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Thorpe
authors.forename: Maria F
authors.initials: MF
authors.lastname: Lopes-Virella
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000276:immunology; D003300:Copper / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008077:Lipoproteins, LDL / Q000032:analysis* / Q000276:immunology*; D008239:Lysine / Q000031:analogs & derivatives* / Q000276:immunology; D008315:Malondialdehyde / Q000276:immunology; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000936:Antigen-Antibody Complex; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D008315:Malondialdehyde; C048496:N(6)-carboxymethyllysine; D003300:Copper; D008239:Lysine