Swinecheck PEDV
Immunoenzymatic assay for the detection of antibodies to porcine epidemic diarrhea virus (PEDV) in porcine serum samples.
Porcine epidemic diarrhea (PED) is a swine disease characterized by acute diarrhea and vomiting. The disease is very contagious and affects pigs of all ages. The mortality may reach 100% in young piglets but is negligible in mature pigs. PED is caused by a coronavirus distinct from the other porcine coronaviruses more especially the transmissible gastroenteritis virus (TGEV). PED was first reported in Europe in the late ’60 and is now prevalent in China, the Asian South East and more recently in the USA. PED diagnosis relies on the demonstration of PEDV nucleic acids or antigens in various specimens (e.g., intestines, feces or oral fluids) or of PEDV antibodies in serum. Several serological tests have been developed to detect PEDV antibodies (e.g., IFA, ELISA). Enzyme-Linked Immuno Assays (ELISA) are easy to perform and to be automatized.
Controls and diluted porcine serum samples are incubated in wells coated with PEDV antigens (Ag). The antibodies (Ab) specific to PEDV present in positive samples bind to the Ag in the wells. After several washes to eliminate unbound substances, biotinylated polyclonal Ab targeted at porcine immunoglobulins G (IgG) (conjugate A) are added. After incubation, the unbound material is eliminated by several washes and streptavidin conjugated to horseradish peroxidase (HRPO) (conjugate B) is added. After incubation, the excess of conjugate is eliminated by several washes and a chronogeneous substrate (TMB) is added. During the incubation of the substrate, HRPO, if present, reacts with the substrate and a blue color develops. The reaction is then stopped (the color changes from blue to yellow) and the optical densities (OD) are read. The intensity of the color allows the determination of the status of the sample tested. A negative sample will show a weak reaction (pale yellow) whereas a strong positive will show a strong reaction (dark yellow). All shades of yellow between dark and pale represent various degrees of positivity.
Materials Provided:
- 2 x 6 strips of 16 wells coated with PEDV antigen
- 1 x 2.5 ml Ready-to-use positive control
- 1 x 2.5 ml Ready-to-use negative control
- 1 x 100-300 µl Concentrated conjugate A
- 1 x 500-1000 µl Concentrated conjugate B
- 3 x 100 ml Ready-to-use dilution buffer
- 2 x 100 ml Concentrated wash solution (10X)
- 1 x 25 ml Ready-to-use substrate
- 1 x 25 ml Ready-to-use stop solution
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Materials Required but not Provided:
- Purified water
- Adjustable single- and multi-channel micropipettes
- Single-use micropipette tips
- Test tubes for sample dilution
- Containers for dilution of other solutions
- ELISA microplate washer (optional)
- ELISA 96-well microplate reader equipped with 450 nm filter
