Validation of a novel ELISA for measurement of electronegative low-density lipoprotein.
BACKGROUND
Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(-) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes.


METHODS
We developed and validated a sandwich ELISA for LDL(-) in human plasma using two monoclonal antibodies against LDL(-) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed.


RESULTS
The calibration range of the assay is 0.625-20.0 mU/L at 1:2000 sample dilution. ELISA validation showed intra- and inter-assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(-), respectively. The intra- and inter-assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(-) ranged from 92.8% to 105.1%.


CONCLUSIONS
This ELISA represents a very practical tool for measuring LDL(-) in human blood for widespread research and clinical sample use.
Santo Faulin
Tanize do Espírito
Tdo E
Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
de Sena
Karine Cavalcanti Maurício
KC
Rodrigues Telles
Andréia Elisa
AE
de Mattos Grosso
Daniela
D
Bernardi Faulin
Edson José
EJ
Parra Abdalla
Dulcineia Saes
DS
D002852:Chromatography, Ion Exchange; D004563:Electrochemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood*; D015203:Reproducibility of Results
D008077:Lipoproteins, LDL