Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods

Abstract

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A-and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-l and <100 CFU of S. aureus ml-'. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 103 g-1) with samples of Cheddar cheese. MATERIALS AND METHODS Reagents. Human immunoglobulin G, protein A (from S. aureus Cowan strain), rabbit anti-protein A antibody, antirabbit immunoglobulin G (whole molecule) alkaline phosphatase (EC 3.1.3.1) conjugate, ,3-NAD 2'3'-cyclic mono-3278

BIOTIN‐AVIDIN ENZYME‐LINKED IMMUNOSORBENT ASSAY FOR BACILLUS SPORES IN BUFFER AND FOOD1

“…We have demonstrated the feasibility of using an immunological procedure for semiquantitative detection of bacterial spores (Foegeding and Chang 1993). Typically, enzyme-based assays require lo5-lo6 organisms/ml for a detectable signal (Anderson and Hartman 1985;Brooks et al 1990;Van Poucke 1990). The detection of spores required about 106-107 spores/ml (Foegeding and Chang 1993).…”

“…There have been several approaches to increasing the sensitivity of an ELISA. Typically, these have involved utilization of a biotin-avidin amplification system, an enzyme amplification step using NAD-specific redox cycling, chemiluminescent enzyme immunoassay, or a fibrinogen amplification system (Brooks et al 1990;Catty and Raykundalia, 1989;Kendall et al 1983). The biotin-avidin complex can increase assay sensitivity or may function as one type of conjugation system in an ELISA.…”

Immunoelectrochemical Assays for Bacteria: Use of Epifluorescence Microscopy and Rapid-Scan Electrochemical Techniques in Development of an Assay for Salmonella

“…Most applications of IEC assays have been in clinical pharmaceutical analysis, with relatively few studies aimed at microbial analysis. Kroll's group − utilized electrochemical detection in conjunction with conventional ELISA methods. Bacteria were captured by antibody adsorbed to the walls of polystyrene microwell plates, labeled with antibody−enzyme conjugate, and incubated with substrate.…”

Development of Biosensors for the Detection of Biological Warfare Agents: Its Issues and Challenges

“…Mirhabibollahi et al 26,27 detected Staphylococcus aureus and Salmonella in pure cultures and in foods with the help of an enzyme-linked amperometric immunosensor. This immunosensor could detect 10 4 to 10 5 CFU mL -1 of Staphylococcus aureus.…”