Validation of a novel ELISA for measurement of MDA-LDL in human plasma.
The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 micro g/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F(2)-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F(2)-isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 +/- 8.8 micro g/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk.
Bevan
Ruth J
RJ
Department of Clinical Biochemistry, University of Leicester, Leicester, UK
Durand
Mireille F
MF
Hickenbotham
Peter T
PT
Kitas
George D
GD
Patel
Parul R
PR
Podmore
Ian D
ID
Griffiths
Helen R
HR
Waller
Helen L
HL
Lunec
Joseph
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001055:Apolipoproteins B / Q000097:blood / Q000276:immunology; D016022:Case-Control Studies; D003300:Copper / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D028441:F2-Isoprostanes / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology; D015227:Lipid Peroxidation / Q000502:physiology; D008077:Lipoproteins, LDL / Q000097:blood*; D008297:Male; D008315:Malondialdehyde / Q000097:blood*; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D017202:Myocardial Ischemia / Q000097:blood* / Q000473:pathology; D010084:Oxidation-Reduction; D011817:Rabbits; D012307:Risk Factors; D013858:Thiobarbiturates / Q000378:metabolism
D001055:Apolipoproteins B; D028441:F2-Isoprostanes; D007136:Immunoglobulins; D008077:Lipoproteins, LDL; D013858:Thiobarbiturates; D008315:Malondialdehyde; D003300:Copper; C029684:thiobarbituric acid