Purpose: This study was designed with the objective to check the ability of Wheat Germ Agglutinin (WGA) lectin to specifically bind with Listeria monocytogenes based on fluorescence based assay.
Methods: The fluorescence activity of WGA, mixture of WGA and L. monocytogenes and WGA with different sugars was evaluated using Tecan multimode plate reader by an optimized protocol. Association constant (Ka) of WGA with different sugars and L. monocytogenes was evaluated using fluorescence-based detection followed by evaluation of assay sensitivity at different cell levels.
Results: WGA showed an excitation/emission spectra of 290/350 nm and the native fluorescence of WGA was greater at increased concentration of WGA while relative fluorescence change was maximum at 125ug/ml of WGA concentration. In the presence of L. monocytogenes, the maximum binding response was observed at the same concentration of WGA and least fluorescence was observed at lower concentration of WGA. Higher Binding constant (Ka) of WGA lectin was observed at 8.0 logs CFU/ml of L. monocytogenes and a decreased binding constant was observed at 5.5 log cfu/ml of L. monocytogenes. The Ka of WGA was also higher in the presence of NAcGlu which confirms the presence of NAcGlu on the cell surface of L. monocytogenes cell wall which helps in specific binding of WGA on L. monocytogenes cell surface. The detection sensitivity of the fluorescence-based detection of L. monocytogenes was found to be 5.5 log CFU/ 100ul in the buffer and 4.9 log cfu/100ul in spiked milk. However, at lower counts of cells (< 3.5 log cfu/100ul) no specific binding was observed even after increase in incubation time for upto 2 hrs.
Conclusions: The fluorescence-based assay using WGA lectin showed specific binding of L.monocytogenes that would be a promising protein for the further development of sensor for the detection of L. monocytogenes in milk.
Keywords: Detection, Fluorescence, Listeria monocytogenes, milk, WGA