Isolation of antibodies to hypochlorite-modified low-density lipoproteins from human serum and study of their specificity.

BACKGROUND: Modified low-density lipoproteins (LDL) have immunogenic properties and induce the production of antibodies. In this case, HOCl promotes the formation of subsequent active halogen-containing compounds interacting with proteins and lipid parts of LDL, which leads to their modification and the production of antibodies to them. The aim of this work is to isolate antibodies to hypochlorite-LDL from human blood sera and study their specificity.

MATERIALS AND METHODS: Malondialdehyde (MDA-LDL), acetic anhydride (acet-LDL) and sodium hypochlorite (hypochlorite-LDL) were used to obtain modified LDL. IgG antibodies to hypochlorite-LDL were isolated by affinity chromatography. The total IgG fraction antibodies was previously isolated from human blood sera using MabSelect Xtra. The specific antibodies to hypochlorite-LDL were isolated from this IgG pool by affinity chromatography. CNBr-Sepharose 4B conjugated with human serum albumin modified with NaOCl was used as a sorbent. The specificity of antibodies against hypochlorite-LDL was tested using a competitive enzyme-linked immunosorbent assay (ELISA). The competitors were hypochlorite-LDL, acet-LDL and MDA-LDL in concentrations (1-250 μg/ml).

RESULTS: IgG antibodies against hypochlorite-modified proteins that interact with hypochlorite-LDL were detected in human blood. According to ELISA date the binding of the isolated antibodies to hypochlorite-LDL was almost completely inhibited only by appropriately modified LDL, that is, hypochlorite-LDL, but not native LDL or acet-LDL. MDA-LDL also showed some competitive activity, but much weaker than hypochlorite-LDL. Hypochlorite-LDL itself and, to a lesser extent, MDA-LDL competed for binding with antibodies of human serum to hypochlorite-LDL. Acet-LDL and native LDL did not reduce the efficiency of antibody binding to their antigen.

CONCLUSIONS: Hypochlorite-LDL forms epitopes independent of other LDL modifications studied. These epitopes are responsible for the formation of specific antibodies.