Instructions for Use

Version: 5.0.0
Revision date: 22 Jul 2022

Duck Virus Enteritis Antibody (DVE-Ab) ELISA Kit

Catalog No: abx054975

Size: 96T

Range: Qualitative

Sensitivity: Qualitative

Storage: Store at 2-8°C for 6 months.

Application: The qualitative detection of DVE-Ab in Duck serum, plasma and other biological fluids.

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Principle of the Assay: This kit is based on enzyme-linked immuno-sorbent assay technology. An antigen is pre-coated onto a 96-well

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plate. Controls, test samples and HRP-conjugated reagent are added to the wells and incubated. Unbound conjugates are removed using
wash buffer. TMB substrate is used to quantify the HRP enzymatic reaction. After TMB substrate is added, only wells that contain sufficient
DVE-Ab will produce a blue coloured product, which then changes to yellow after adding the acidic stop solution. The intensity of the yellow

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colour is proportional to the DVE-Ab amount bound on the plate. The Optical Density (OD) is measured spectrophotometrically at 450 nm in
a microplate reader, from which the presence of DVE-Ab can be determined.

Kit Components Materials Required But Not Provided

• Pre-coated 96-Well Microplate: 12 x 8
• Wash Buffer: (30X) 20 ml
• Positive Control: 0.5 ml
• 37°C incubator ce
• Multi and single channel pipettes and sterile pipette tips
• Squirt bottle or automated microplate washer
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• Negative Control: 0.5 ml • 1.5 ml tubes
• Sample Diluent Buffer: 6 ml • Distilled water
• Detection Reagent: 6 ml • Absorbent filter papers
• TMB Substrate A: 6 ml • 100 ml and 1 liter graduated cylinders
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• TMB Substrate B: 6 ml • Microplate reader (wavelength: 450 nm)

• Stop Solution: 6 ml • ELISA Shaker
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• Plate Sealer: 3
• Hermetic Bag: 1
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Abbexa LLC, Houston, TX USA · Phone: +1 832 327 7413
Abbexa BV, Leiden, NL
Website: www.abbexa.com · Email: info@abbexa.com
Instructions for Use
Version: 5.0.0
Revision date: 22 Jul 2022

Protocol
A. Sample Preparation

Analyse immediately or store samples at 2-8°C (within 24 hrs). For long term storage, aliquot and store at -20°C or -80°C. Avoid multiple
freeze-thaw cycles.
Serum: Samples should be collected into a serum separator tube. Coagulate the serum by leaving the tube undisturbed in a
vertical position overnight at 4°C or at room temperature for up to 1 hr. Centrifuge at approximately 1000 × g for 20 mins. If
precipitate appears, centrifuge again. Assay immediately or aliquot and store at -20°C or -80°C.
Plasma: Collect plasma using EDTA or citrate as an anticoagulant. Centrifuge for 15 mins at 1000 x g, within 30 mins of collection.
If precipitate appears, centrifuge again. Avoid hemolytic samples.
Other Biological Fluids: Centrifuge at approximately 1000 × g for 20 mins to remove precipitate. Analyse immediately or aliquot

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and store at -20°C or -80°C.

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Notes:

Analyse samples at a 5-fold (1/5) dilution (i.e. add 10 µl of sample to 40 µl of Sample Diluent Buffer).

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Store frozen samples undiluted. Once ready to analyse, thaw samples and dilute.
Fresh samples, or recently obtained samples, are recommended to prevent protein degradation and denaturation that may lead to
erroneous results.
NaN3 cannot be used as a test sample preservative, since it inhibits HRP.
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If possible, prepare solid samples using sonication and/or homogenization, as lysis buffers may (on occassion) interfere with the
kit's performance.
If a sample is not indicated in the manuals applications, a preliminary experiment to determine the suitability of the kit will be
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required.

B. Reagent Preparation
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Wash Buffer: Dilute the concentrated Wash Buffer 30-fold (1/30) with distilled water (i.e. add 20 ml of concentrated wash buffer into 580 ml
of distilled water). If crystals have formed in the concentrated Wash Buffer, warm to room temperature and mix gently until the crystals have
completely dissolved.
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C. Assay Protocol

Equilibrate the kit components and samples to room temperature prior to use. It is recommended to measure in duplicate.
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1. Set 2 positive and 2 negative control, test sample and control (zero) wells on the pre-coated plate respectively, and record their
positions. Add the solution to the bottom of each well without touching the side walls. Pipette the standards and samples up and
down to mix before adding to the wells. Avoid foaming or bubbles.
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2. Aliquot 50 μl of negative and 50 μl positive control into the set wells. Aliquot 50 µl of sample diluent buffer in the control (zero) blank
well.
3. Aliquot 50 μl of appropriately diluted sample (diluted 5-fold) into the test sample wells. Gently tap the plate to mix, or use a
microplate shaker.
4. Cover the plate with a plate sealer and incubate for 30 mins at 37°C.
5. Remove the cover and discard the liquid. Wash the plate 5 times with 1X Wash Buffer. Fill each well completely with Wash buffer
(300 µl) using a multi-channel Pipette or autowasher (1-2 mins soaking period is recommended). Complete removal of liquid at
each step is essential for good performance. After the final wash, remove any remaining Wash Buffer by aspirating or decanting.
Invert the plate and blot it against clean absorbent paper towels.
6. Aliquot 50 µl of Detection Reagent to each well (except the blank well). Cover the plate with a plate sealer and incubate for 30 mins
at 37°C.
7. Remove the cover, discard the liquid, and repeat the wash process as described above, 5 times.
8. Aliquot 50 μl of TMB Substrate A and 50 μl of TMB Substrate B into each well. Cover the plate with the plate sealer and gently tap
the plate to mix thoroughly. Incubate at 37°C for 10-15 mins. The incubation time is for reference only, the optimal time should be
determined by end user. Avoid exposure to light.

v4.2.0 Abbexa LTD, Cambridge, UK · Phone: +44 (0) 1223 755950 · Fax: +44 (0) 1223 755951 2 of 3
Abbexa LLC, Houston, TX USA · Phone: +1 832 327 7413
Abbexa BV, Leiden, NL
Website: www.abbexa.com · Email: info@abbexa.com
Instructions for Use
Version: 5.0.0
Revision date: 22 Jul 2022

9. Aliquot 50 µl of Stop Solution into each well. It is important that the Stop Solution is mixed quickly and uniformly throughout the
microplate to inactivate the enzyme completely.
10. Ensure that there are no fingerprints or water on the bottom of the plate, and that the fluid in the wells is free of bubbles. Measure
the OD at 450 nm immediately.

Data Analysis:

Mean OD of the Positive Control should be ≥ 1.0.

Mean OD of the Negative Control should be ≤ 0.2.
CUT OFF value = Negative Control + 0.15

If the Postive Control value is ≥ 1.0, and the Negative Control value is ≤ 0.2, the test is valid, otherwise, the test is invalid.

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If OD of Samples < CUT OFF, the test samples are considered negative.

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If OD of Samples ≥ CUT OFF, the test samples are considered positive.

Precautions:

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Before using the kit, centrifuge the tubes to bring down the contents trapped in the lid.
Do not leave the wells uncovered for extended periods between incubations. The addition of reagents for each step should not
exceed 10 mins.

Do not reuse pipette tips and tubes.

Do not use expired components, or components from a different kit.
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Ensure that the plate is properly sealed or covered during the incubation steps, and that the time and temperature are controlled.
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The TMB substrate should be used under sterile conditions, and light exposure should be minimised. Unused substrate should be
colorless, or a very light yellow in appearance. Do not discard any residual solution back into the vial.
Please note that this kit is optimised for detection of native samples, rather than recombinant proteins or synthetic chemicals. We
are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native
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protein.
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v4.2.0 Abbexa LTD, Cambridge, UK · Phone: +44 (0) 1223 755950 · Fax: +44 (0) 1223 755951 3 of 3
Abbexa LLC, Houston, TX USA · Phone: +1 832 327 7413
Abbexa BV, Leiden, NL
Website: www.abbexa.com · Email: info@abbexa.com