Development of a sandwich ELIME assay exploiting different antibody combinations as sensing strategy for an early detection of Campylobacter

Abstract

Campylobacter is an important cause of acute bacterial disease in human worldwide. Within the genus Campylobacter, C. jejuni and C. coli are the two species responsible for over 95% of human infections. The purpose of this work is the development of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay for a simple and rapid detection of both Campylobacter species. Tosyl-activated magnetic beads (MBs), supporting a sandwich immunological complex, were coupled to a strip of 8-magnetized screen-printed electrodes, as electrochemical transducing element. With the aim to simultaneously detect C. jejuni and C. coli, four anti-campylobacter antibodies were screened in ELISA paying the attention to the selection of both culture medium and cell treatment (whole campylobacter cells inactivated with NaN3 and heat killed cells) for a suitable antigen-antibody interaction. Six combinations of the selected anti-campylobacter antibodies, unconjugated and conjugated with Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), were exploited as sensing strategy to set-up the ELIME assay. The antibody pair, consisting of a goat polyclonal antibody anti-campylobacter (coated on the surface of MBs) and the same antibody conjugated with AP (to label the captured cells), was selected given the best response for both Campylobacter species and the lowest detection limit (9 × 103 CFU/mL for C. jejuni and 2 × 104 CFU/mL for C. coli). The selectivity of the ELIME assay was proved by analyzing other C. jejuni strains and non target microorganisms attaining promising results.