BIOO ANIMAL HEALTH PRODUCTS
NovareDxTM- EHDV Antibody Test Kit Manual
Catalog #: 7006-01 (1 X 96 wells); 7006-02 (2 X 96 wells)
©BIOO Scientific Corp. • 2014
TABLE OF CONTENTS
GENERAL INFORMATION .......................................................................................... 1
Product Description ........................................................................................................................ 1
Procedure Overview ........................................................................................................................ 1
Kit Contents, Storage and Shelf Life ............................................................................................... 2
Specificity (Cross-Reactivity) .......................................................................................................... 2
Required Materials Not Provided With the Kit ............................................................................... 2
Warnings and Precautions .............................................................................................................. 2
SAMPLE PREPARATION .............................................................................................. 3
Serum/Plasma ................................................................................................................................. 3
Cell culture Supernatant ................................................................................................................. 3
EHDV ANTIBODY ELISA TEST KIT PROTOCOL ................................................... 4
Reagent Preparation ....................................................................................................................... 4
ELISA Protocol ............................................................................................................................... 4
RESULTS: ....................................................................................................................................... 5
Interpretation of Controls ............................................................................................................... 5
Interpretation of Samples ................................................................................................................ 5
TROUBLESHOOTING ................................................................................................... 6
No Color Development or No Signals with Standards .................................................................... 6
Low Optical Density (OD) Readings in Positive Control ............................................................... 6
High Optical Density (OD) Readings in Negative Control ............................................................. 6
EXAMPLES OF THE KIT TEST FOR DEER SERUM .............................................. 7
The NovareDxTM EHDV Antibody ELISA Test Kit is intended for laboratory use only, unless otherwise indicated. This
product is NOT for clinical diagnostic use.
NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
The NovareDxTM EHDV Antibody Test Kit is designed for the detection of antibodies specific to Epizootic
Hemorrhagic Disease (EHD) virus in cervids. EHDV is an orbivirus that infects most wild and domestic
ruminants. EHD is endemic in many countries, particularly affecting white-tailed deer in North America,
with a morbidity and mortality rate as high as 90%. Deer, elk, and moose can seroconvert. Edema,
hemorrhage, and ulcerations are seen in the mouth and lips in the acute form of the disease. In the
chronic form of the disease, affected animals exhibit sloughing of the hoof wall, erosions, and scars in
the rumen. Spontaneous abortions and stillbirths have also been reported in some epidemics.
The detection of antibodies to EHDV is done using blood samples (serum or plasma) from infected
hosts. The NovareDxTM EHDV Antibody Test Kit provides a simple, rapid, sensitive and cost-effective
enzyme-based immunoassay (ELISA) screening method. This enables white-tailed deer breeders,
ranchers, veterinarians, and government agencies to detect EHDV serogroup-specific antibodies.
Bluetongue virus (BTV), a member of the genus Orbivirus, produces similar symptoms, and differential
diagnosis is important to prevent widespread outbreak and economic loss. The ELISA method
accurately detects antibodies specific to EHDV, without cross-reactivity between the serogroups of BTV
and EHDV.
Procedure Overview
The method is based on a colorimetric ELISA. Purified EHDV antigens have been coated in the plate
wells. During analysis, the serum or plasma sample is added. If antibodies to EHDV are present in the
sample, it will bind to the coated antigen. The secondary antibody, tagged with a peroxidase enzyme,
targets anti-EHDV antibodies, and the resulting color intensity, after the addition of substrate, is related
to the amount of anti-EHDV antibodies in the sample.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
Kit Contents, Storage and Shelf Life
The NovareDxTM EHDV Antibody ELISA Test Kit has the capacity for testing of 46 samples (Cat #
7006-01) or 92 samples (Cat # 7006-02) in duplicate.
Return any unused microwells to the foil bag and reseal them with the desiccant provided in the original
package.
The shelf life is 6 months when the kit is properly stored.
Kit Contents
EHDV antigen-coated Plate
EHDV Positive Control
EHDV Negative Control
50X HRP-Conjugated Antibody
Solution
Secondary Antibody Diluent
Solution
Serum/Plasma Diluent
20X Wash Solution
TMB Substrate
Stop Solution
Cat # 7006-01
1 x 96-well plate
0.5 mL
0.5 mL
Cat # 7006-02
2 x 96-well plate
1.0 mL
1.0 mL
Storage
4°C
-20°C
-20°C
200 L
400 mL
10 mL
15 mL
20 mL
10 mL
10 mL
20 mL
30 mL
40 mL
20 mL
20 mL
-20°C
4°C
4°C
4°C
4°C
4°C
Specificity (Cross-Reactivity)
This kit can broadly detect EHDV serogroup-specific antibodies.
Required Materials Not Provided With the Kit



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Microplate reader (450 nm)
Incubator (at 37°C)
Vortex mixer
10, 20, 100 and 1000 L pipettes
Multi-channel pipette: 50-300 L (Optional)
Warnings and Precautions
Bioo strongly recommends that you read the following warnings and precautions to ensure your full
awareness of ELISA techniques and other details you should pay close attention to when running the
assay. More information can be found in Troubleshooting section. Periodically, optimizations and
revisions are made to the kit and manual. Therefore, it is important to follow the protocol that comes with
the kit. If you need further assistance, you may contact your local distributor or Bioo Scientific at
[email protected].
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


Handle the positive and negative controls with care.
Do not use the kit past the expiration date.
Do not mix reagents from different kits or lots. Antibodies and plates are lot and kit specific.
Make sure that the HRP conjugated antibody and diluent are mixed in correct volumes.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
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Maintain a laboratory temperature of 20°– 25°C (68°– 77°F). Avoid running assays under or near
air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays
in direct sunlight, as this may cause excessive heat and evaporation.
Water quality is very important; make sure you are using only distilled or deionized water.
When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the
lower corner of the well, making contact with the plastic.
Incubations of assay plates should be timed as precisely as possible.
Add your controls first, and then your samples.
Always refrigerate plates in sealed bags with desiccant to maintain the plate(s) stability.
Prevent condensation from forming on plates by allowing them equilibrate to room temperature
(20 – 25C / 68 – 77F) while in the packaging.
SAMPLE PREPARATION
SAMPLE PREPARATION
Serum/Plasma
Dilute serum or plasma samples using the serum/plasma diluent provided in the kit (e.g. 50 L of
serum/plasma is added to 500 L of diluent to obtain a 1:10 dilution). Perform dilution at room
temperature and use immediately. Store any other serum samples at -20°C.
Cell culture Supernatant
Cell culture supernatants from hybridoma or engineered cell lines can be directly used in the assay.
Frozen supernatants should be thawed at room temperature before use in the assay.
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its
products are made are of standard quality. There is no warranty of merchantability of this product, or of the
fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from the use of this product.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
EHDV
ANTIBODY
ELISA
KIT PROTOCOL
EHDV
ANTIBODY
ELISA
TESTTEST
KIT PROTOCOL
Reagent Preparation
IMPORTANT: All frozen reagents should be brought up to room temperature before use (1 – 2
hours at 20 – 25C / 68 – 77F); Make sure you read the “Warnings and Precautions” section on
page 2-3. Solutions should be prepared just prior to performing the ELISA. All reagents should be
mixed by inversion prior to use. Do not return used pipette tips or any reagents to the original stock
tubes/bottles. Use disposable reservoirs when handling reagents to minimize the risk of contamination.
1. Preparation of 1X Wash Solution
Mix 1 volume of 20X Wash Solution with 19 volumes of distilled water.
2. Preparation of 1X HRP-Conjugated Secondary Antibody
Based on the needed amount, add 1 volume of 50X HRP-Conjugated Secondary Antibody solution
to 49 volumes of the Secondary Antibody Diluent Solution and mix well by inverting. The Secondary
Antibody solution is then ready to use. The diluted Secondary Antibody is stable for 2 weeks at
-20C.
ELISA Protocol
Amount of reagent required for one reaction is shown in the table below.
Component
Volume per well
Sample/Positive Control/Negative Control
100 L
1X HRP-Conjugated Antibody
100 L
1X Wash Solution
3 mL
TMB Substrate
100 L
Stop Solution
100 L
Note: All samples and controls should be run in duplicate
1.
2.
3.
Bring the EHDV antigen-coated plate and all reagent components to room temperature
(20°– 25°C/ 68°– 77°F) for at least an hour.
Aliquot EHDV Positive Control (100 L/well) to two wells of the antigen coated plate.
Aliquot EHDV Negative Control (100 L/well) to two wells of the antigen coated plate.
Aliquot the Positive and Negative Controls separate from each other, for e.g., the upper left-hand and
bottom right-hand corner wells of the plate
4. Add 100 L of previously diluted (1:10) serum/plasma sample (or neat cell culture supernatant) per
well in duplicate.
5. Cover plate with foil and incubate for 60 minutes at 37C.
Avoid direct sunlight and air vents during incubation.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
6. Discard the solution in the wells.
7. Wash the plate by adding 300 L of 1X Wash solution to each well of the plate. Discard the wash
solution and tap dry on paper towels. Repeat four additional times for a total of five washes.
Perform the next step immediately. Do not allow the plate to dry.
8. Add 100 L of 1X HRP-Conjugated Secondary Antibody to each well of the plate. Cover plate with
foil and incubate for 60 minutes at 37C.
9. Wash plate with 1X Wash solution as described in Step 7.
10. Add 100 L of TMB Substrate to each well of the plate. Cover plate with foil and incubate for 5
minutes at room temperature (20 – 25C / 68 – 77F).
11. Add 100 L of Stop Solution to each well. Read the plate as soon as possible on a plate reader at
450 nm wavelength.
RESULTS:
Interpretation of Controls
Positive Control:
Absorbance readings at 450 nm for positive control wells should be greater than O.D of 1.0.
Negative Control:
Absorbance readings at 450 nm for negative control wells should be less than O.D of 0.4.
Controls must be within the limits above for proper interpretation of samples.
Interpretation of Samples
O.D ≥ 1:
Sample is interpreted as positive for antibodies to EHDV if the absorbance reading at 450 nm is close
to greater than O.D of 1.0.
O.D ≤ 0.4:
Sample is interpreted as negative for antibodies to EHDV if the absorbance reading at 450 nm is close
to or less than 0.4.
O.D values between 0.4 and 1.0 can be interpreted as weak positive for antibodies to EHDV.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
TROUBLESHOOTING
TROUBLESHOOTING
No Color Development or No Signals with Standards
Possible Causes
Recommended Action
Reagents were used in the wrong
order or a step was skipped.
Wrong antibodies were used, or
secondary antibody was prepared
incorrectly or has deteriorated.
TMB substrate has deteriorated.
Follow the protocol carefully and repeat the assay.
Make sure that the antibodies used are the ones that came with the kit. All
antibodies are kit- and lot-specific. Make sure that the secondary antibody and
diluent are mixed in correct volumes.
Use a new set of BIOO TMB substrate.
Low Optical Density (OD) Readings in Positive Control
Possible Causes
Recommended Action
Reagents were expired or mixed
with a different lot number.
Wash solution was prepared
incorrectly.
Too many wash cycles were used.
Incubation times were too short.
Lab temperature was too low.
Reagents and plates were too cold.
Reader was at wrong wavelength,
or reader was malfunctioning.
Excessive kit stress has occurred.
Assay plates were compromised
Verify the expiration dates and lot numbers.
Use the wash solution in the kit and make sure that it is prepared correctly.
Make sure to use the number of washes per the protocol instruction.
Time each plate separately to ensure accurate incubation times, follow protocol.
Maintain the lab room temperature between 20°– 25°C (68°– 77°F). Do not run
assays under air conditioning vents or near cold windows.
Make sure plates and reagents are brought up to room temperature. Keep the
kit components out of the kit box for at least 1 hour before starting the assay.
Make sure the wavelength is 450 nm for the assay and read the plate again.
Verify reader calibration and lamp alignment.
Check records to see how many times the kit has cycled from the refrigerator.
Check to see if the kit was left at extreme temperatures for too long.
Always refrigerate plates in sealed bags with a desiccant to maintain stability.
Prevent condensation from forming on plates by allowing them to equilibrate to
room temperature (20 – 25C / 68 – 77F) while in the packaging.
High Optical Density (OD) Readings in Negative Control
Possible Causes
Recommended Action
Poor quality water was used in
wash solution.
Substrate solution has deteriorated.
If water quality is questionable, try substituting an alternate distilled water
source to prepare the wash solution.
Make sure the substrate is colorless prior to addition to the plate.
Use the number of washes per the protocol instruction. Make sure that at least
250 L of wash solution is dispensed per well per wash. Verify the performance
of the washer system; have the system repaired if any ports drip, dispense or
aspirate poorly.
There was insufficient washing or
poor washer performance.
Reader was malfunctioning or not
blanked properly. This is a high
possibility if the OD readings were
high and the color was light.
Verify the reader’s performance using a calibration plate and check the lamp
alignment. Verify the blanking procedure, if applicable, and reblank.
Lab temperature was too high
Maintain the room temperature between 20°– 25°C (68°– 77°F). Avoid running
assays near heat sources or in direct sunlight.
Reagents were intermixed,
contaminated or prepared
incorrectly.
Ensure that the correct reagents were used, that working solutions were
prepared correctly and that contamination has not occurred.
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NovareDxTM- EHDV Antibody Test Kit Manual – 7006-01; 7006-02
EXAMPLES
OF THE
FOR DEER
SERUMSERUM
EXAMPLES
OFKIT
THETEST
KIT TEST
FOR DEER
Deer (n=292) exhibiting clinical signs for EHDV (edema, hemorrhage, ulcers) were tested for virus-specific
antibody by NovareDxTM-EHDV Antibody Test Kit. Absorbance (at 450 nm) is plotted for each sample.
Absorbance (450 nm)
3
2.5
2
1.5
1
0.5
0
1
41
81
121
161
201
241
Individual deer serum samples
Independent field trial, January 2014
Bioo Scientific Corporation
7050 Burleson Rd
Austin, TX 78744 USA
Tel: 1.888.208.2246
Fax: (512) 707-8122
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O.D Negative control
Made in USA
BIOO Animal Health Products
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