Immunoglobulin M Capture Assay for Serologic Confirmation of Early Lyme Disease: Analysis of Immune Complexes with Biotinylated Borrelia burgdorferi Sonicate Enhanced with Flagellin Peptide Epitope

Abstract

ABSTRACT We previously reported on the efficacy of the enzyme-linked immunoglobulin M capture immune complex (IC) biotinylated antigen assay (EMIBA) for the seroconfirmation of early Lyme disease and active infection with Borrelia burgdorferi . In earlier work we identified non-cross-reacting epitopes of a number of B. burgdorferi proteins, including flagellin. We now report on an improvement in the performance of EMIBA with the addition of a biotinylated form of a synthetic non-cross-reacting immunodominant flagellin peptide to the biotinylated B. burgdorferi B31 sonicate antigen source with the avidin-biotinylated peroxidase complex detection system used in our recently developed indirect IgM-capture immune complex-based assay (EMIBA). As in our previous studies, the enzyme-linked immunosorbent assay (ELISA) reactivities of antibodies liberated from circulating ICs (by EMIBA) were compared with those of antibodies in unprocessed serum (antibodies found free in the serum, thus as an IgM-capture ELISA, but not EMIBA, because the antibodies were not liberated from ICs), the sample usually used in standard ELISAs and Western blot assays. The addition of the flagellin epitope enhanced the ELISA signal obtained with untreated sera from many Lyme disease patients but not from healthy controls. In tests with both free antibodies and ICs, with or without the addition of the flagellin epitope to the sonicate, we found the most advantageous combination was IC as the source of antibodies and sonicate plus the flagellin epitope as the antigen. In a blinded study of sera obtained from patients with early and later-phase Lyme disease, EMIBA with the enhanced antigenic preparation compared favorably with other serologic assays, especially for the confirmation of early disease.