E. coli O157 ELISA Assay Kit
E. coli O157 ELISA Developed and Manufactured in the USA
Size: 1×96 wells
Sensitivity: Cut-Off Control
Incubation Time: 2 hours
Sample Type: Stool
Sample Size: 250 mg
Alternative Names: Enterohemorrhagic Escherichia coli O157:H7, E. Coli O157:H7
For Research Use Only
Specificity: The E. coli O157 ELISA Assay Kit does not cross react to the following: Toxin A, Toxin B, Helicobacter pylori, glutamate dehydrogenase 1 (GDH), Cryptosporidium parvum, Giardia lamblia, rotavirus and adenovirus.
Precision: The precision of this assay is validated by measuring two samples both in a single assay of 16-replicate determinations (intra-assay)
Sample 1: Mean 1.687, Std Dev 0.049, %CV 2.9%
Sample 2: Mean 1.975, Std Dev 0.058, %CV 2.9%
Most E. coli strains harmlessly colonize the gastrointestinal tract of humans and animals as a normal flora. However, there are some strains that have evolved into pathogenic E. coli by acquiring virulence factors through plasmids, transposons, bacteriophages, and/or pathogenicity islands.
Enterohemorrhagic Escherichia coli O157:H7, also known as EHEC, is a major foodborne pathogen causing severe disease in humans worldwide. Healthy cattle are a reservoir of E. coli O157:H7, and bovine food products and fresh produce contaminated with bovine waste are the most common sources for disease outbreaks in the United States. E. coli O157:H7 also survives well in the environment.
E. Coli O157:H7 is the most predominant strain of Shiga Toxin producing E. Coli in the United States, Japan, and the United Kingdom. The Centers for Disease Control and Prevention (CDC) has estimated that E. coli O157:H7 infections cause 73,000 illnesses, 2,200 hospitalizations, and 60 deaths annually in the United States. Healthy adults usually recover from infection with E. coli O157:H7 within a week, but young children and older adults have a greater risk of developing a life-threatening form of kidney failure called hemolytic uremic syndrome.
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The Eagle Biosciences Fecal E. coli 0157 ELISA Assay Kit is a “sandwich” ELISA that is designed, developed and produced for the qualitative measurement of E.coli 0157 in stool specimen. The assay utilizes the microplate-based enzyme immunoassay technique by coating highly purified antibody onto the wall of microtiter wells. Controls and extracted fecal specimen are added to microtiter wells of microplate that was coated with a purified monoclonal anti-E. coli 0157 on its wall. During the assay, the E. coli Antibody will be bound to the antibody coated plate after an incubation period. The unbound material is washed away and another HRP-conjugated monoclonal antibody which specifically recognizes the protein of E. coli 0157 is added for further immunoreactions. After an incubation period, the immunocomplex of “Anti-E. coli 0157 Capture Antibody – E.coli – HRP-conjugated Anti-E.coli Tracer Antibody” is formed if E. coli 0157 is present in the test sample. The unbound tracer antibody and other proteins in buffer matrix are removed in the subsequent washing step. HRP conjugated tracer antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the tracer antibody bound to E. coli 0157 proteins captured on the wall of each microtiter well is directly proportional to the amount of E. coli 0157 level in each test specimen.
- Place a sufficient number of E. coli 0157 monoclonal antibody-coated microwell strips in a frame.
- Add 100 µL of controls and extracted patient stool samples into the designated microwell. Mix by gently tapping the plate. Cover the plate with one plate sealer. Cover with foil or other material to protect from light.
- Incubate plate at room temperature, static, for 1 hour.
- Remove the aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well, then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
- Add 100 µL of E. coli 0157 Tracer Antibody to each well. Mix by gently tapping the plate.
- Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
- Incubate plate at room temperature, static, for 30 minutes.
- Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
- Add 100 µL of ELISA HRP Substrate into each of the wells.
- Cover the plate with a new plate sealer and also with aluminum foil to avoid exposure to light.
- Incubate plate at room temperature for 20 minutes.
- Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
- Read the absorbance at 450 nm.
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