Shiga-like Toxin (Stx) ELISA Kit

Catalog No. ABIN997080

Product Details

Target: Shiga-like Toxin (Stx)

Reactivity: E. coli

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: E. coli Verotoxin assay is an enzyme-linked immunosorbent assay (ELISA) that may be used to screen food products for the presence of Verotoxin.

Analytical Method: Qualitative

Application Details

Comment:

Quality Control:
The use of a positive and negative control allows easy validation of kit stability. For a valid test, the positive control must be over 0.5 OD units and the negative control must be under 0.15 OD units. Should the values fall outside these ranges, the kit should not be used. TROUBLESHOOTING Problem: Negative control has substantial color development. Correction: Washings were insufficient. Repeat test with more vigorous washings.

Plate: Pre-coated

Protocol: During the first incubation, Verotoxin antigen in the sample is captured by antibodies attached to the wells. The second incubation adds an additional anti-Verotoxin antibody cocktail that "sandwiches" the antigen. The next incubation attaches horseradish peroxidase to the sandwich. After washings to remove unbound enzyme, a chromogen is added which develops a blue color in the presence of the enzyme complex and peroxide. The stop solution ends the reaction and turns the blue color to yellow.

Reagent Preparation:

WASH BUFFER
Remove cap and add contents of one bottle to 475 mL DI water. Transfer contents of diluted wash buffer into a squeeze bottle (small tip bottle).
MEDIUM PREPARATION
Modified EC broth with Novobiocin (mEC+n)
1. Combine the following components with 1 liter of distilled water (if prepared media is not used): Tryptone 20.0 g Lactose 5.0 g K2HPO4 4.0 g KH2PO4 1.5 g NaCl 5.0 g Bile Salt #3 1.5 g pH adjusted to 6.9 ± 0.1
2. Autoclave at 121 °C for 15 minutes.
3. Allow to cool to room temperature and add 1 mL of a filter sterilized aqueous solution of 20 mg/mL Novobiocin. (for 225 mL add 0.225 mL of Novobiocin solution.) Final concentration should be 20 pg/mL. SAMPLE PREPARATION 1. Add 225 mL of mEC+n to 25 g food product in a sterile stomacher bag or blender jar.
2. Stomach or blend sample and broth for 2 minutes.
3. Transfer stomacher bag to shaker at 37 °C or alternatively transfer contents of blender jar to a sterile flask and attach to shaker.
4. Incubate stomacher bags or flasks at 37 °C with shaking (120 rpm) for 18 hours.
5. Remove a 1 mL aliquot from each sample and place in a separate clean screw top test tube. This is the sample that will be used in the assay.

Assay Procedure:

1. Break off number of wells needed (number of samples plus 2 for controls) and place in strip holder.
   2. Add 100 pL of the negative control to well #1 and 100 pL of positive control to well #2 (use both as undiluted).
   3. Add 100 pL of the test supernatant to the appropriate test well.
   4. Incubate at room temperature for 30 minutes, then wash.
   5. Add 2 drops of Reagent 1 (blue solution) to each well.
   6. Incubate at room temperature for 30 minutes, then wash.
   7. Add 2 drops of Reagent 2 (red solution) to each well.
   8. Incubate at room temperature for 30 minutes, then wash. Slap out excess fluid against an absorbent towel.
   9. Add 2 drops of Chromogen to each well.
   10. Incubate at room temperature for 10 minutes.
   11. Add 2 drops of Stop Solution to each well. Mix wells by tapping strip holder.
   12. Read results visually or on a spectrophotometer using a bichromatic reading, with the filters set at 450 nm and 620-650 nm. Zero the reader on air. * Each washing consists of dumping the contents of the wells into an appropriate container with disinfecting solution (e.g. 3 % bleach in water) and using the diluted wash buffer to fill to overflowing in each well, shaking out the contents and refilling the wells for a total of 3 times. Samples with sticky particulate matter may require more thorough washing than other samples. The potential exists for false positive results if the sample is not thoroughly washed from the well before addition of subsequent reagents.

Calculation of Results:

VISUAL Reactive: Any sample well that has distinct and substantial yellow color. Negative: Any sample well that does not have distinct yellow color.
NOTE: The negative control, as well as some samples, may show some slight color.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store at 2-8 °C

Target Details

Target: Shiga-like Toxin (Stx)

Background: The Verotoxin (VT), or shiga-like toxin family is a group of closely related toxins produced by certain strains of Escherichia coli. These strains are a significant cause of human hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). In addition, they are both water borne and food borne and may also be transmitted from person-to-person by the oral-fecal route. In adults, illness caused by Verotoxin may last several days, while in children and the elderly, the illness can be fatal. Infections occur as sporadic illnesses or outbreaks, and are distributed throughout North and South America, Europe, and Asia. The peak incidence of infection occurs during summer months. In the past, lengthy and cumbersome diagnostic procedures have been utilized for the detection of verotoxin. Currently, simple to perform enzyme-linked immunosorbent assays (ELISA) are being used as an alternative to these longer tedious methods. These antigen-detection assays provide a cost effective tool for the management of diarrheal disease with an accurate and rapid test.