Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of anti-listeriolysin O (LLO) IgG in sheep serum and plasma to diagnose listerial infection and to screen flocks/herds for Listeria monocytogenes exposure
Sample Type
Serum, Plasma
Analytical Method
Quantitative
Components
- 12 x 8-well strips : coated with LLO recombinant protein, blocked with BSA 2% and protected with gelatine. - 1 x Buffer A: 50ml ready to use, with preservative. - 1 x Buffer B (Wash Buffer 10X concentrate) : 100ml, with preservative - 1 x HRP-conjugated secondary antibody : 10l - Chromogen Solution: 1 x 30ml Buffer C, 1 x 30l Buffer C1, 1 x 2 ABTS tablets.
Material not included
Microplate reader capable of measuring absorbance at 415 nm Thermomixer, shaking water bath or rocking platform at 37 °C Precision pipettes and pipette tips Glass or plastic pipettes Deionized or distilled water Multi-channel pipette, semi-automated or automated microplate washer 1000 mL graduated cylinder for preparation of 1X Wash Buffer Vortex mixer Glass tubes
Listeria monocytogenes is a facultative intracellular Listeria monocytogenes is a facultative intracellular Gram-positive food-borne bacterium, increasingly recognized as responsible for severe infections in both animals and humans (1). Ingestion of contaminated food causes an infection, named listeriosis, which affects especially immunocompromised patients, new-borns and pregnant women and is characterized by a variety of severe syndromes, such as encephalitis, meningoencephalitis, septicemia and abortion (2). Since dairy products are involved in several outbreaks of listeriosis, the identification of infected animals could be very important for human health. Listeriolysin O (LLO), the major virulence factor produced by all pathogenic strains of L.monocytogenes, has been identified as a candidate antigen for a serological assay (3, 4). Antibodies against LLO have been already detected in the serum of goats (5), sheep (6, 7), lambs (4), cows (8) and humans (9) by Western blot, dot blot or ELISA analysis. L. monocytogenes releases in the culture medium low levels of LLO, therefore the isolation of this toxin requires high culture volumes, is time-consuming and provides a very low yield (10, 11). For this reason LLO protein, used in the assay as test antigen, was expressed in Escherichia coli and purified as described i