Adsorption of low-density lipoprotein, its oxidation, and subsequent binding of specific recombinant antibodies: An in situ ellipsometric study.
BACKGROUND
Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system.


METHODS
In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl₂-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl₂ while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data.


RESULTS
Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies.


CONCLUSIONS
Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state.
Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden. Maria.Stollenwerk@mah.se
Maria M
MM
Stollenwerk
Olof
O
Svensson
Alexandru
A
Schiopu
Bo
B
Jansson
Thomas
T
Arnebrant
Gunilla Nordin
GN
Fredrikson
D000327:Adsorption; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053299:Apolipoprotein B-100 / Q000276:immunology; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D008315:Malondialdehyde / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012031:Refractometry / Q000379:methods; D012822:Silicon Dioxide / Q000737:chemistry* / Q000378:metabolism; D013499:Surface Properties
D000906:Antibodies; D053299:Apolipoprotein B-100; D000939:Epitopes; D007074:Immunoglobulin G; D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; C099252:oxidized low density lipoprotein; D008315:Malondialdehyde; D012822:Silicon Dioxide; D003300:Copper; C029892:cupric chloride