INSTRUCTIONS FOR USE
For Veterinary Use Only. For In Vitro Use Only.
PrioCHECK™ Calf Enteritis Ag Feces Kit
Immunoenzymatic test for simultaneously detecting the antigens Rotavirus (Group A),
Coronavirus, E. coli K99, and Cryptosporidium parvum in ruminant feces
Catalog Number LSIDTT1
Doc. Part No. 100020263 Pub. No. MAN0008594 Rev. B.0
Technology
Species
Sample matrix
Test type
Protocol
Double-well Antigen-capture
ELISA Strip plates
Ruminants Feces Individual Short incubation
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves.
Safety Data Sheets (SDSs) are available from thermofisher.com/support.
WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this
product’s page at thermofisher.com. Wear appropriate protective eyewear, clothing, and gloves.
General information
Diarrhea constitutes one of the leading causes of death in calves and all young ruminants. Bovine neonatal
gastroenteritis is a multifactor disease. It may be induced by a virus (Rotavirus and Coronavirus), bacteria
(Salmonella, Enterotoxigenic Colibacillus) or protozoans (Cryptosporidium parvum). Laboratory tests are
obligatory for determining causes of diarrhea since identifying the causative agent based on clinical
symptoms is impossible.
Procedure overview
The test is based on the principle of an Antigen-capture ELISA.
1. The samples and controls are deposited in the plate coated with anti-pathogenic antibodies (AbP) and
control antibodies (AbC). The antigens, including specific antigens of the potentially present
pathogens being investigated, bind to the AbP while only the non-specific Ag bind to the AbC.
2. After washing, specific monoclonal conjugates labeled with peroxidase are added, binding to the
antigens previously attached to the microwells.
3. The unbound conjugates are eliminated by washing before the addition of a chromogenic substrate. A
blue color results from substrate oxidation by the peroxidase in the conjugate.
4. After stopping the reaction, the color turns yellow. The results are read by an ELISA plate reader.
The appearance of a yellow coloration indicates the presence of antigens. A non-specific reaction
results in an equivalent coloration between the AbP and AbC wells. A specific reaction results in a
more significant coloration in the AbP well compared to the AbC well. The difference between these
two colorations indicates the level of positivity in the sample.
2 PrioCHECK™ Calf Enteritis Ag Feces Kit Instructions for Use
Kit contents and storage
Component Description
Quantity
(12 tests)
Storage
1 - Coated microplate DTT
DTT-coated microplate, 12 strips of 8 wells:
• Line A: specific anti-
Rotavirus
group A antibody
• Line B: control antibody
• Line C: specific anti-
Coronavirus
antibody
• Line D: control antibody
• Line E: specific anti-
E. coli
K99 antibody
• Line F: control antibody
• Line G: specific anti-
Cryptosporidium
antibody
•
Line H: control antibody
1 unit 5±3°C(1)
3a - Positive C.
Rotavirus
Rotavirus
positive control (red)
1.4 mL
5±3°C
3b - Positive C.
Coronavirus
Coronavirus
positive control (yellow)
1.4 mL
3c - Positive C.
E. coli
K99
E. coli
K99 positive control (blue)
1.4 mL
3d - Positive C.
Cryptosporidium
Cryptosporidium
positive control (green)
1.4 mL
4a - Conjugate
Rotavirus
Rotavirus
HRP conjugate (red)
3.5 mL
4b - Conjugate
Coronavirus
Coronavirus
HRP conjugate (yellow)
3.5 mL
4c - Conjugate
E. coli
K99
E. coli
K99 HRP conjugate (blue)
3.5 mL
4d - Conjugate
Cryptosporidium
Cryptosporidium
HRP conjugate (green)
3.5 mL
A - Wash (10x)
Wash solution, 10X concentrate
60 mL
B - Sample DB (5x) DTT
Sample dilution buffer (5X) DTT, 5X concentrate
25 mL
C - Substrate
Substrate solution
12 mL
D - Stop
Stop solution
12 mL
Adhesive plate covers
2
RT(2)
Plastic caps
6 × 8
(1) Unused strips can be stored in the sealed pouch with desiccant (supplied with the kit) at 5±3°C until the kit’s expiry date.
(2) Room temperature
Materials required but not provided
Unless otherwise indicated, all materials are available through thermofisher.com.
Disposable pipette tips
Single- and multi-channel micropipettes
Distilled or deionized water
Disposable containers
Hemolysis tubes
ELISA reader equipped with a 450 nm filter or 450 and 620 nm filters
Important procedural guidelines
• Do not mix reagents from different kit batches.
• Avoid contaminating the reagents by using single-use sampling equipment.
• Do not pipette reagents by mouth.
Information on samples
Fresh feces, refrigerated feces (8 days at 5±3°C) or frozen feces (1 year at < −16°C) can be used.
Dilute the feces to 1:2 for the test.
Prepare reagents
• The reagents 1 - Coated microplate DTT, 3a - Positive C. Rotavirus, 3b - Positive C. Coronavirus,
3c - Positive C. E. coli K99, 3d - Positive C. Cryptosporidium, 4a - Conjugate Rotavirus,
4b - Conjugate Coronavirus, 4c - Conjugate E. coli K99, 4d - Conjugate Cryptosporidium,
C - Substrate, and D - Stop are ready to use.
• Dilute the reagent B - Sample DB (5x) DTT 1:5 in distilled/deionized water.
Example: dilute 10 mL of the reagent B - Sample DB (5x) DTT in 40 mL of water.
Mix after diluting. The diluted solution Sample DB DTT may be stored at 5±3°C for use within
8 days.
PrioCHECK™ Calf Enteritis Ag Feces Kit Instructions for Use 3
• A - Wash (10x) solution should be diluted 1:10 in distilled/deionized water.
Example: for one strip: 2 mL of A - Wash (10x) solution in 18 mL of water; for one plate: 25 mL of
A - Wash (10x) solution in 225 mL of water.
Mix after diluting. The diluted Wash solution can be stored for 1 month at 5±3°C.
NOTE: Due to the high salt concentration, crystals may form in the A - Wash (10x) solution. The
crystals can be dissolved by shaking. The solution should be mixed thoroughly prior to dilution.
Perform the ELISA test
NOTE: Bring the reagents to room temperature (21±4°C) before performing the test. The tolerance range
for incubation times is ±10%. The use of disposable containers is recommended for distribution of
components.
If one or more samples are not tested using all of the pathogens, cover all wells containing unused
pathogens with plastic caps.
1. Preparation of samples
Dilute the fecal matter 1:2 in the diluted solution Sample DB DTT (see “Prepare reagents”). We
recommend diluting 500 µL of feces in 500 µL of the diluted solution Sample DB DTT using a hemolysis
tube. If the sample consistency renders volume measurement or mixing difficult, add a volume of the
diluted solution Sample DB DTT equal to the sample volume into a hemolysis tube and vigorously shake
together in the vortex. Numerous agitation cycles may be required. Do not centrifuge.
2. Distribution of samples and controls
Test the positive controls without additives or dilution, adding 100 µL of:
• 3a - Positive C. Rotavirus: wells A1 and B1
• 3b - Positive C. Coronavirus: wells C1 and D1
• 3c - Positive C. E. coli K99: wells E1 and F1
• 3d - Positive C. Cryptosporidium: wells G1 and H1
NOTE: The positive control 3d - Positive C. Cryptosporidium may contain aggregates, thus making it
harder to pipette. In this case, we recommend that you vortex the tube well and cut the cone tip to sample
the control.
Add 100 µL of the diluted samples to each well according to the following layout: sample 1 in the
column 2 wells, sample 2 in the column 3 wells, etc.
Gently shake the plate, then cover it with an adhesive plate cover. Incubate the plate for 1 hour at 21±4°C.
3. Washing steps
Empty the plate and perform 4 washes with the diluted Wash solution (see “Prepare reagents”) using
300 µL per well. Empty and tap the plate on absorbent paper to eliminate any traces of liquid. Washes can
be performed either manually or using an automatic washer. Do not allow the plate to dry out.
4. Distribution of conjugate
Add 100 µL of the conjugates to each well, according to the following layout:
• 4a - Conjugate Rotavirus: lines A and B
• 4b - Conjugate Coronavirus: lines C and D
• 4c - Conjugate E. coli K99: lines E and F
• 4d - Conjugate Cryptosporidium: lines G and H
Gently shake the plate, and cover the plate using a new adhesive plate cover. Incubate the plate for
1 hour at 21±4°C.
5. Washing steps
Repeat the step outlined above in “3. Washing steps.”
6. Test development
Add 100 µL of solution C - Substrate to each well. Gently shake the plate for 2 seconds. Incubate for
10 minutes at room temperature (21±4°C) and in darkness. Do not cover the plate.
Add 100 µL of solution D - Stop to each well and in the same order as solution C - Substrate. Gently
shake the plate for 2 seconds.
7. Reading
Dry the bottom of the plates with a soft tissue to remove any dust. Read the plate within 30 minutes of
stopping the reaction, at 450 nm (monochromatic) or at dual wavelengths of 450 and 620 nm.
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13 October 2016
Calculation
Calculate the corrected Optical Density (ODc) for
each sample and pathogen by deducting the OD
for the corresponding control well from the OD
obtained on each specific well.
ODc = OD AbP – OD AbC
Carry out the same operation for the Antigen
Positive Controls (PC).
NOTE: For negative samples, ODc may be < 0.
Validation
The test is validated if each positive control
reaches: ODc ≥ 0.400.
Interpretation of results
The interpretation of results for the 4 pathogens is
as follows:
Results
Interpretation
ODc ≥ 0.150
Positive
OD
c
< 0.150
Negative
Example (For 2 samples using 3 strips):
OD
ODc
Interpretation
Pathogens
PC
Samp1
Samp2
PC
Samp1
Samp2
PC
Samp1
Samp2
A
2.269
2.237
0.184
2.139
2.055
0.032 Positive
Validated
Pos
Neg
Rotavirus
B
0.130
0.182
0.152
C
1.865
0.090
1.567
1.798 0.008
1.469
Positive
Validated
Neg
Pos
Coronavirus
D
0.067
0.082
0.098
E
2.352
0.260
0.067
2.250
0.170
−0.005
Positive
Validated
Pos
Neg
E. Coli
K99
F
0.102
0.090
0.072
G
1.955
0.212
0.320
1.874 0.012 0.060 Positive
Validated
Neg Neg
Cryptosporidium
H
0.081
0.200
0.260
Technical Support can provide you with an Excel file for automatically carrying out calculations and
interpretations of results.
Documentation and support
Customer and technical support
Technical support: visit
thermofisher.com/askaquestion
Visit thermofisher.com/support for the latest in
services and support, including:
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• Safety Data Sheets (SDSs; also known as
MSDSs)
NOTE: For SDSs for reagents and chemicals
from other manufacturers, contact the
manufacturer.
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affiliate(s) warrant their products as set forth in the
Life Technologies' General Terms and Conditions
of Sale found on Life Technologies' website at
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questions, please contact Life Technologies at
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8211 AR Lelystad | The Netherlands
Translated from the French Pub. No. MAN0008595 Rev. B.0.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE
TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE
FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR
CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING
FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0008594 (English)
Rev.
Date
Description
B.0 13 October 2016
Updated to the current document
template, with associated updates to
the warranty, trademarks, and logos.
A.0
22 January 2014
Baseline for revision history
Limited Use Label License No. 473: Veterinary Diagnostic Assay.
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only to perform veterinary diagnostic services including reporting
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