Development of immunoassays for anti-electronegative LDL autoantibodies and immune complexes

Abstract

Background: Electronegative low-density lipoprotein (LDL-) promotes atherosclerosis through inflammatory and immunologic mechanisms that lead to the production of anti-LDL(-) autoantibodies and to the subsequent formation of immune complexes (IC) and macrophage foam cells. We described the development and validation of an ELISA for the quantification of free anti-LDL(-) autoantibodies and an ELISA for the quantification of IC consisting of LDL(-)-bound IgG in human plasma.

Methods: LDL(-) purified from human plasma, and anti-LDL(-) monoclonal antibody Fab fragments were adsorbed onto ELISA plates to capture anti-LDL(-) autoantibodies and IC-LDL(-), respectively. The performance characteristics of both ELISAs, including the limits of detection and quantification, accuracy and inter- and intra-assay precision were evaluated. Linearity, interference and stability tests were also performed.

Results: The calibration range of the anti-LDL(-) assay was 0.004-0.125 mU/l and plasma demonstrated a dilutional linearity when diluted 1:100, 1:200, 1:400 and 1:800. The calibration range of the IC-LDL(-) assay was 0.06-4 U/l, and plasma demonstrated a dilutional linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. Both ELISAs showed intra- and inter-assay precision and recovery within the required limits for immunoassays.

Conclusion: These ELISAs can be used in clinical studies and for the biochemical investigation of atherosclerosis. In addition, they will enable the comprehensive evaluation of the importance of bound or free autoantibodies against LDL(-) in this disease.