E. Coli Verotoxin 1+2 Ag ELISA Kit

Sample

Stool specimens, broth cultures, from individual colonies or colony sweeps of agar plates.

Detection Method

iELISA

Intended Use

The EHEC test is a rapid in vitro microwell ELISA for the detection of Shiga Toxins I and II (Verotoxins) in stool specimens, broth cultures, from individual colonies or colony sweeps of agar plates. The EHEC test is intended for use as an aid in the research of enterohemorrhagic E. coli (EHEC) infections.

Contents of Kit

The maximum number of tests obtained from this kit is listed on the outer box.
1. EHEC Antibody Coated Microwells – Breakaway plastic wells coated with monoclonal antibodies specific for E. coli Shiga toxin I and II.
2. EHEC Positive Control – Inactivated Shiga toxin in a buffered protein solution with preservative.
3. EHEC Negative Control – Buffered solution with a preservative.
4. EHEC Sample Diluent – Buffered Protein solution with a preservative.
5. 20× Wash Buffer II – Concentrated wash buffer with a preservative.
6. EHEC Detection Antibody – Rabbit antibodies specific for Shiga toxins in buffered protein solution containing preservative.
7. EHEC Enzyme Conjugate – Goat anti-rabbit antibody conjugated to horseradish peroxidase in buffered protein solution containing preservative.
8. Substrate II – Buffered solution containing urea peroxide.
9. Stop Solution II – 2N Sulfuric Acid. CAUTION: Avoid contact with skin. Flush with water if contact occurs.
10. Transfer pipettes - Each pipette is marked to indicate 50 μL, 100 μL, 200 μL and 300 μL volumes.
11. Microwell strip holder
12. Microwell strip sealers

Storage

The expiration date is indicated on the kit label. Store the kit at 2-8°C and return the kit promptly to the refrigerator after each use.

Performance Characteristics

The EHEC test was evaluated at three major medical centers and one state health laboratory in the United States. Each study site tested diarrheal stools submitted for routine culture of enteric pathogens. Each stool was tested directly and by the broth method in the EHEC assay. A specific ST cytotoxin neutralization assay was performed at CD on these specimens. The results obtained for the direct stool assay and the broth assay are summarized in the following table.

Relative Sensitivity = 78.9% (56.6-91.5%)* 100% (85.1-100%)
Relative Specificity = 95.8% (93.4-97.4%) 97.9% (95.2-99.1%)
Relative Agreement = 95.1% (92.6-96.8%) 98.1% (95.6-99.2%)
ST producing EHEC were isolated from two of the 17 EHEC positive/cytotoxin negative direct stool specimens: one O157:H7 and one O6:H untypeable. In addition, PCR** confirmed the presence of the ST gene in two EHEC positive/cytotoxin negative and one positive/cytotoxin indeterminate stool specimens. The 15 EHEC positive/cytotoxin positive direct stool specimens were comprised of: nine – O157:H7, two – O6:H untypeable, one – O untypeable: H12, 41w or 51, and two – O111:NM EHEC strains. An isolate could not be obtained from the remaining stool specimen.
ST producing EHEC were isolated from two of five EHEC positive/cytotoxin negative broths: one O157:H7 and one O111:NM. The 22 EHEC positive/cytotoxin positive samples were comprised of 13 O157:H7, three O6:H untypeable, one O untypeable H:12, 41w or 51, and one O111:NM ST producing strains of EHEC. No isolates could be found in the remaining four broth specimens.
*95% confidence intervals calculated by the normal method as indicated in parenthesis.
**PCR is a research tool and not intended for in vitro diagnostic use.
Various E. coli ST producing organisms were tested in the ELISA by both the Plate and Broth Methods (Specimen Collection And Preparation). Each strain is a clinical isolate and each was tested by a cytotoxin assay and by a polymerase chain reaction (PCR) to confirm the presence of the ST gene. All organisms generated positive results when tested in this manner. The number in parenthesis represents the number of different strains tested in that group type. Also, the type of toxin produced by each strain is indicated.

Detection Limit

The limit of detection for ST-I and ST-II are approximately 7 and 15 pg/well respectively.

General Description

Enterohemorrhagic E. coli (EHEC) have been isolated from patients who have hemorrhagic colitis and hemolytic-uremic syndrome (HUS). To date, O157:H7 is the most frequently encountered EHEC strain in stools from these patients. This is probably due to the fact that conventional diagnostic strategies such as O157 latex agglutination assays rely on the unique sorbitol negative fermentation property of this strain. The major weakness in this approach is that at least 50 other EHEC serotypes have been reported to be associated with the development of hemorrhagic colitis and HUS. One virulence trait of all EHEC strains is the ability to produce cytotoxin(s) called Shiga toxin (ST) or verotoxin (VT) ST-I and ST-II are the two most common toxins and individual EHEC strains have the ability to produce both or either, in varying quantities. Therefore, ST production and not individual (O157:H7) serotype identification is a better diagnostic strategy for the determination of EHEC associated disease. Cytotoxin can be identified by a specific cytotoxin assay described by Karmali. The cytotoxin assay, however, is labor intensive, requires tissue culture facilities, has not been standardized and may take up to 72 hours to confirm the presence of cytotoxin. Exploiting the EHEC attribute to produce these toxins, EHEC was developed for the direct detection of ST producing strains from stool specimens or culture systems.