Development of a chemiluminescent immunosensor for chloramphenicol

A direct competitive chemiluminescent immunosensor system that exploits the competition between chloramphenicol (CAP) as an analyte and CAP-horseradish peroxidase conjugate as a tracer for binding to an anti-CAP antibody on a solid support was devised by installing a flow-through cell which was connected to an injector and a peristaltic pump inside a dark box, followed by positioning a photomultiplier tube as light detector in front of it. The anti-CAP antibody was immobilized onto positively charged Biodyne B membrane pieces by a dipping procedure. The operating conditions for the immunosensor were selected with respect to substrate composition (0.25, 13.3 and 0.66 mM for luminol, H2O2 and p-iodophenol, respectively), injection volume of the substrate solution (200 microL) and the concentrations of antibody for immobilization (0.10 mg mL(-1)) and tracer (0.030 mg mL(-1)). At these conditions, sensor response according to analyte concentration was well fitted to a linear equation when plotted in semi-logarithmic scale, with the limit of detection for CAP of 10(-8) M. By using the immunosensor, CAP measurement in the model samples prepared from five food materials was conducted.