Catalog # 608096 – 96 tests

Description

Microplate Enzyme-Immunoassay for the qualitative detection of Shiga-toxins produced by Enterohemorrhagic E. coli in human stools or culture systems.

Features

-          Direct detection of Shiga-toxins I and II

-          Break-away microtiter strips

-          Color coded ready-to-use reagents

-          Visual or spectrophotometer reading

-          1 positive + 1 negative controls per run

Specimen handling

The stool specimen, collected in an empty container, should either be frozen (-20°C to -80°C) or placed at 2-8°C immediately after collection. The refrigerated specimens should be cultured within 2 hours. If culturing cannot be performed within 2 hours, the specimen should be placed in a Cary-Blair based transport media. Samples in transport media should be stored at 2-8°C if they can be examined within 3 days. If culturing cannot be performed within this time, the specimen should be frozen (-20°C to -80°C) immediately upon receipt.

Kit Components

Antibody coated microwells, Positive control, Negative control, Sample diluent, Wash buffer, Detection antibody, Enzyme conjugate, Substrate, Stop solution, Transfer pipets, Strip holder, Strip sealer.

Specimen preparation:

A. Direct stool :

1. Add 200 µl of sample diluent to a test tube.

2. Mix stool and tranfer 50 µl of liquid stool, or a small portion of solid stool (3-4 mm diameter) to the tube. Emulsify and vortex 15 seconds.

B. Plate method :

1. Add 20 µl of stool or specimen to MacConkey or Sorbitol/MacConkey plate and spread with an inoculating loop.

2. Incubate 16-24 hours at 37°C.

3. Individual colonies can be removed with a loop and placed in 200 µl of sample diluent for testing.

C. Broth method :

1. Add 10-50 µl of stool to 5 ml of MacConkey's broth, GN broth or E-Z-Coli broth. Vortex 10-15 seconds.

2. Incubate 16-24 hours at 37°C.

3. Add 50 µl of growth to 200 µl of sample diluent for EIA testing.

Assay Protocol:

1.         Add 100 µl of diluted specimen and 2 drops of positive and negative controls to the appropriate wells.

2.         Mix well, seal and incubate the plate for 1 hour at room temperature.

3.         Wash 5 times with the wash buffer.

4.         Add 2 drops of detection antibody to each well, and swirl the plate for 30 seconds.

5.         Seal and incubate the plate for 30 minutes at room temperature.

6.         Wash 5 times with the wash buffer.

7.         Add 2 drops of enzyme conjugate to each well, and swirl the plate for 30 seconds.

8.         Seal and incubate the plate for 30 minutes at room temperature.

9.         Wash 5 times with the wash buffer.

10.     Add 2 drops of substrate to each well, and swirl the plate for 30 seconds.

11.     Seal and incubate the plate for 10 minutes at room temperature.

12.     Add 2 drops of stop solution to each well, and swirl the plate for 30 seconds.

13.     Read at 450 nm or visually.

Storage

The kit must be stored at 2-8°C.

Additional Information

Click here for more information (complete procedure, safety data sheet, literature).