DEVELOPMENT OF AN ENZYME‐LINKED IMMUNOSORBENT ASSAY (ELISA) FOR ANALYSIS OF LISTERIOLYSIN O PRODUCED BY LISTERIA MONOCYTOGENES

KIM, KEE‐TAE; MURANO, ELSA A.; OLSON, DENNIS G.

Listeriolysin O (LLO) is a heat‐labile hemolysin produced by Listeria mono‐cytogenes. Its hemolytic activity has been evaluated qualitatively by sodium dodecyl sulfate (SDS) electrophoresis and immunoblotting. In this experiment, an enzyme‐linked immunosorbent assay (ELISA) was developed for quantitative analysis of LLO by using Streptolysin O (SLO) and antistreptolysin O (ASO) as the reagents. The selected coating and blocking buffers were 0.05 M Tris buffer (pH 8.5) and 0.25% casein solution with phosphate‐buffered saline solution + 0.05% Tween 20 (PBS‐T), respectively. A relationship between ASO and antibody was achieved with 5 mg/ml ASO and a 1:1,000 dilution of conjugate. The heat stability of LLO at 48, 62, 72, and 80C was examined by using this method and compared with a traditional hemolysis assay. Although the LLO is inactivated easily at those temperatures, the protein structure was not affected at temperatures lower than 80C for 3 min, pointing to a need for both hemolysis and ELISA to be conducted in determining both the activity and presence of LLO in foods.