The E. coli Verotoxin 1+2 Ag ELISA is an in vitro diagnostic device for direct detection of verotoxin 1 and 2 (shiga-toxin 1 and 2) in faecal specimens and stool culture supernatants.
The E. coli Verotoxin 1+2 Ag ELISA is an indirect two-site-immunoassay for the qualitative determination of verotoxin 1 and 2 based on polyclonal and monoclonal antibodies. Verotoxin 1 and/or 2 of specimens and the positive control react with polyclonal anti-verotoxin 1 and 2 antibodies coated on the solid phase of the microplate. After incubation for 60 minutes at room temperature (RT) non-bound material is removed by a washing step. Subsequently bound toxins specifically react with biotinylated monoclonal anti-verotoxin 1
and anti-verotoxin 2 antibodies during a second incubation period of 30 min at RT. Non-bound material is separated from the solid-phase immune complexes by a subsequent washing step. During the next incubation period of 30 min at RT horseradish peroxidase (HRP) conjugated streptavidin reacts with the bound biotinylated antibodies. Unbound
conjugate is removed by a washing step. HRP converts the subsequently added colorless substrate solution of 3,3Õ,5,5Õ-tetramethylbenzidine (TMB) into a blue product. The enzyme reaction is terminated by sulphuric acid dispensed into the wells after 15min incubation at RT turning the solution from blue to yellow.
The optical density (OD) of the solution read at 450/³ 620 nm is directly proportional to the specifically bound amount of verotoxin 1 and/or verotoxin 2. Considering the cut-off value results are interpreted as positive or negative.