Premier EHEC Benutzerhandbuch

EIA for the detection of the Toxins Produced by Enterohemorrhagic E. coli in Stool
Specimens or Culture Systems
608096
INTENDED USE
The Premier EHEC test is a rapid in vitro microwell EIA 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 Premier EHEC test
is intended for use as an aid in the diagnosis of enterohemorrhagic E. coli (EHEC) infections.
SUMMARY AND EXPLANATION OF THE TEST
Enterohemorrhagic E. coli (EHEC) have been isolated from patients who have hemorrhagic colitis and hemolytic-
5, 8, 14
uremic syndrome (HUS). To date, O157:H7 is the most frequently encountered EHEC strain in stools from
9, 13
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.
weakness in this approach is that at least 50 other EHEC serotypes have been reported to be associated with the
7-11, 16
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
9, 14
(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.
by a specific cytotoxin assay described by Karmali.
tissue culture facilities, has not been standardized and may take up to 72 hours to confirm the presence of
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cytotoxin.
Exploiting the EHEC attribute to produce these toxins, Premier EHEC was developed for the direct detection of ST
producing strains from stool specimens or culture systems.
BIOLOGICAL PRINCIPLES
The Premier EHEC test utilizes monoclonal anti-Shiga toxin capture antibodies absorbed to microwells.
samples are added to the wells and incubated at room temperature. A wash is performed to remove unbound
material. Polyclonal anti-Shiga-like toxin antibodies are added for detection and incubated at room temperature.
Another wash is used to remove unbound antibody. Enzyme conjugated anti-IgG polyclonal antibody is added
and incubated at room temperature. If toxin is present, a reactive antibody-enzyme complex is formed. After
washing to remove unbound conjugate, substrate is added and incubated for 10 minutes at room temperature.
Color develops in the presence of bound enzyme. Stop Solution is added and the results are interpreted visually
or spectrophotometrically.
REAGENTS/MATERIALS PROVIDED
The maximum number of tests obtained from this kit is listed on the outer box.
1. Premier EHEC Antibody Coated Microwells – Breakaway plastic wells coated with monoclonal
antibodies specific for E. coli Shiga toxin I and II.
2. Premier EHEC Positive Control – Inactivated Shiga toxin in a buffered protein solution with preservative.
3. Premier EHEC Negative Control – Buffered solution with a preservative.
4. Premier EHEC Sample Diluent – Buffered Protein solution with a preservative.
5. Premier 20X Wash Buffer II – Concentrated wash buffer with a preservative.
6. Premier EHEC Detection Antibody – Rabbit antibodies specific for Shiga toxins in buffered protein
solution containing preservative.
7. Premier EHEC Enzyme Conjugate – Goat anti-rabbit antibody conjugated to horseradish peroxidase in
buffered protein solution containing preservative.
8. Premier Substrate II – Buffered solution containing urea peroxide.
9. Premier 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
MATERIALS NOT PROVIDED
1. Wooden applicator sticks
2. Test tubes (12 x 75 mm) for dilution of sample
3. Distilled or deionized water
4. Squirt bottle
5. Graduated cylinder for making 1X Wash Buffer
6. EIA plate reader capable of reading absorbance of 450 nm or 450/630 nm*
7. STAT Fax™ 2200 plate shaker or equivalent (Optional – Broth Method Only)*
* NOTE: It is the operator's responsibility to validate readers and plate shakers prior to their use with this
product.
PRECAUTIONS
1. All reagents are for in vitro diagnostic use only.
2. Patient specimens may contain infectious agents and should be handled at Biosafety Level 2 as
recommended in the CDC/NIH manual, "Biosafety in Microbiology and Biomedical Laboratories".
3. All reagents should be mixed gently before use.
4. Do not interchange Microwells, Detection Antibody, Enzyme Conjugate or Positive Control Reagent
between different kits. 20X Wash Buffer II, Sample Diluent, Substrate II and Stop Solution II can be
interchanged provided they are within their expiration dates at the time of testing.
5. Allow reagents to warm to room temperature before use.
6. Hold reagent vials vertically at suitable distance above the well to insure proper drop size and delivery.
7. Do not use kit components beyond labeled expiration date.
8. Replace colored caps on correct vials.
9. Dispose of used wash buffer and all test materials in appropriate container. Treat as potential biohazard.
10. The Positive Control regent contains inactivated Shiga toxin.
potentially hazardous material.
11. Avoid skin contact with Stop Solution II (2N Sulfuric Acid). Flush with water immediately if contact occurs.
12. Do not reuse microwells.
13. Unused microwells must be placed back inside resealable pouch. It is important to protect strips from
moisture.
14. Transfer pipettes provided must be used for specimen preparation and transfer. Use one per specimen.
15. Avoid splashing when dispensing diluted samples into microwell by placing transfer pipette tip about
halfway down the well and dispensing slowly down the side of well.
16. Microwell washing is to be performed precisely as directed in assay procedure. Inadequate washing will
cause elevated background and false positive results.
17. All reagents except the 20X Wash Buffer II are provided already diluted to the proper concentration.
18. Any deviation below or above set incubation times may affect sensitivity and specificity and should be
avoided.
19. Do not use microwells with pouches that have been damaged (ie, show holes or punctures).
HAZARD and PRECAUTIONARY STATEMENTS
Refer to the SDS, available at www.meridianbioscience.com
SHELF LIFE AND 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.
In Vitro Diagnostic Medical Device
2, 9, 13, 14
7, 8, 16
Cytotoxin can be identified
8
The cytotoxin assay, however, is labor intensive, requires
It should be handled, however, as a
for Hazards and Precautionary Statements.
PROCEDURAL NOTES
The Premier EHEC transfer pipette is diagrammed below:
REAGENT PREPARATION
1. Bring the entire kit, including microwell pouch, to room temperature before use.
2. Prepare 1X Wash Buffer as needed.
For example: 4.0 mL of 20X Wash Buffer II + 76.0 mL of distilled or deionized water is sufficient to wash one strip.
Place in a clean squirt bottle. The 1X Wash Buffer can be stored at room temperature for up to three months.
SPECIMEN COLLECTION AND PREPARATION
1. CDC Recommendations for Stool Specimen Storage, Isolation and Identification of EHEC Organisms:

Raw stool specimens should be examined as soon as they are received in the laboratory. If not
processed immediately, they should be placed at 2-8 C or frozen at ≤ -70 C.
 Refrigerated raw stool specimens should be examined within 1-2 hours. If stools cannot be examined
within this time, they should be placed in a Cary-Blair-based transport medium. All rectal swabs should
be placed immediately in a Cary-Blair-based transport medium.

If specimens in transport medium will be examined in 2-3 days, they should be refrigerated.
The major
specimens in transport medium are not examined in 2-3 days, they should be frozen immediately at ≤ -
70 C. Specimens in transport medium should not be refrigerated for days, then frozen or left for any
time at room temperature.
2. Stool and Broth Storage Prior to Premier EHEC Testing: Stool specimens and broths may be held up to
seven days at 2-8 C before testing in the EIA. If testing is not performed within this time period, the stool
and/or broth should be frozen at (≤ -70 C). Repeated freeze-thaws should be avoided.
A. Direct Stool:
1. Add 200 µL of Sample Diluent to a clean 12 x 75 mm test tube. (NOTE: Third marking from tip
on transfer pipette represents 200 µL.)
2. Mix stool as thoroughly as possible prior to pipetting.
a. Liquid, semi-solid or stools in transport medium: Using a transfer pipette, draw stool to the
50 µL calibration point (first mark from the tip of the pipette). Dispense the stool into the
Sample Diluent. Using the same pipette, gently withdraw and expel the stool suspension
several times, then vortex 15 seconds. Leave transfer pipette in tube for later use.
b. Non-pipetable stools: Using a wooden applicator stick, transfer a small "BB" sized portion
(3-4 mm diameter) of thoroughly mixed stool into Sample Diluent. Emulsify stool using the
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Diluted
wooden applicator stick, then vortex 15 seconds. Place a transfer pipette in the tube.
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 or colony sweeps can be removed with a loop and placed in 200 µL of
Sample Diluent for EIA testing.
C. Broth Method:
1. Add 10-50 µL of stool to 5 mL of MacConkey's Broth, or GN Broth. Vortex 10-15 seconds.
2. Incubate 16-24 hours at 37 C.
3. Add 50 µL of growth to 200 µL Sample Diluent for EIA testing.
TEST PROCEDURE
NOTE: With large numbers of specimens, repetitive or multichannel pipettes may be used for dispensing the
reagents.
1. After the pouch has reached temperature, break off the required number of microwells (1 well for each
specimen plus 1 positive and 1 negative control well per batch). Place the microwells in the microwell strip
holder and record the location of all wells. Unused microwells must be resealed in the pouch immediately.
2. Add 100 µL of diluted specimen (second mark from the tip of the pipette) to the appropriate well (place
pipette tip halfway into well and let sample slowly run down side of well).
3. Add 2 free-falling drops of Positive and Negative Control to the appropriate wells. Mix wells by firmly
shaking/swirling the plate for 30 seconds.
4. Cut plate sealer to size and press firmly onto top of microwells to seal. Incubate the plate for 1 hour at
room temperature (22-27 C). Broth Method Only: Alternatively, laboratories equipped with a heated plate
shaker (STAT Fax™-2200) can incubate and rotate the plate for 30 minutes at 25 C at 1000 rpm (setting
5).
5. Carefully, remove the plate sealer and wash wells:
a. Dump plate contents firmly into a biohazard receptacle.
b. Bang the inverted plate on a clean stack of paper towels.
c. Fill all wells with 1X Wash Buffer. Use of a squirt bottle is recommended.
d. Repeat washing cycle (dump, bang on fresh towels, fill) 4 additional times. After the last fill,
dump and bang plates on fresh towels hard enough to remove as much excess Wash Buffer as
possible but do not allow wells to completely dry at any time.
6. Add 2 free-falling drops of Detection Antibody to each well. Firmly shake/swirl the plate for 30 seconds.
7. Press plate sealer firmly onto top of microwells to seal.
temperature (22-27 C). Broth Method Only: Alternatively, incubate and rotate the plate for 15 minutes on
the STAT Fax™ at 25 C and at 1000 rpm (setting 5).
8. Repeat wash procedure (Step #5)
9. Add 2 free-falling drops of Enzyme Conjugate to each well. Firmly shake/swirl the plate for 30 seconds.
10. Press plate sealer firmly onto top of microwells to seal.
temperature (22-27 C). Broth Method Only: Alternatively, incubate and rotate the plate for 15 minutes on
the STAT Fax™ at 25 C and at 1000 rpm (setting 5).
11. Repeat wash procedure (Step #5)
12. Clean the underside of all wells with a lint free tissue.
13. Add 2 free-falling drops of Substrate Solution II to each well. Firmly shake/swirl the plate for 30 seconds.
Incubate for 10 minutes at room temperature.
14. Add 2 free-falling drops of Stop Solution II to each well. Firmly shake/swirl the plate for 30 seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of Stop Solution II.
15. Observe Reactions:
a. Visual Determination – Read within 15 minutes after adding Stop Solution II.
b. Spectrophotometric Determination – Zero EIA reader on air. Wipe underside of wells with a lint free
tissue. Read absorbance at 450 nm or 450/630 nm within 30 minutes of adding Stop Solution II.
INTERPRETATION OF RESULTS
1.
Visual Reading
Negative – colorless
Positive = definite yellow color
A faint yellow color must be evaluated spectrophotometrically
NOTE: In view of the epidemiological importance of obtaining bacterial isolates it is recommended that all toxin
positive samples be submitted for isolation of Shiga toxin positive organisms.
microbiology laboratories coordinate bacterial isolation with their local state health laboratories.
2.
Spectrophotometric Single Wavelength (450 nm)
Negative = OD < 0.180
450
Positive = OD ≥ 0.180
450
3.
Spectrophotometric Dual Wavelength (450/630 nm)
Negative = OD
450/630
Positive = OD ≥ 0.150
450/630
NOTE: A positive result indicates the presence of Shiga toxins. A negative result indicates the absence of Shiga
toxins, or that the level of toxin is below that which can be detected by the test.
NOTE: See Quality Control section (Item #5) regarding low positive results. Extremely strong positive reactions
may yield a purple precipitate within a few minutes of stopping the reaction.
1
Incubate the plate for 30 minutes at room
Incubate the plate for 30 minutes at room
< 0.150
If
We suggest that individual