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

Abstract

BACKGROUND: Modified low-density lipoproteins 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 low-density lipoproteins, which leads to their modification and the production of antibodies to them.

AIM: The aim of this work is to isolate antibodies to hypochlorite modified low-density lipoproteins from human blood sera and study their specificity.

MATERIALS AND METHODS: Malondialdehyde, acetic anhydride and sodium hypochlorite were used to obtain modified low-density lipoproteins. IgG antibodies to hypochlorite modified low-density lipoproteins were isolated by affinity chromatography. The total IgG fraction antibodies was previously isolated from human blood serum using MabSelect Xtra. The specific antibodies to hypochlorite modified low-density lipoproteins 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 modified low-density lipoproteins was tested using a competitive enzyme-linked immunosorbent assay. The competitors were hypochlorite modified low-density lipoproteins, acetic anhydride modified low-density lipoproteins and malondialdehyde modified low-density lipoproteins in concentrations (1–250 μg/ml).

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

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

Keywords

antibodies / affinity chromatography / low-density lipoproteins / hypochlorite-low-density lipoproteins / enzyme-linked immunosorbent assay

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