Instructions for Use
Version: 6.0.0
Revision date: 19 Nov 2024
Contagious Caprine Pleuropneumonia (CCPP) ELISA Kit
Catalog No: abx055277
Size: 96T
Range: Qualitative
Sensitivity: Qualitative
Storage: Store at 2-8°C.
Application: The qualitative detection of CCPP in Goat tissue homogenates, cell lysates and other biological fluids.
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Principle of the Assay: This kit is based on sandwich enzyme-linked immuno-sorbent assay technology. An antibody is pre-coated onto a
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96-well 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 CCPP will produce a blue coloured product, which then changes to yellow after adding the acidic stop solution. The intensity of the
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yellow colour is proportional to the CCPP amount bound on the plate. The Optical Density (OD) is measured spectrophotometrically at 450
nm in a microplate reader, from which the presence of CCPP 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 • Ice
• Stop Solution: 6 ml • Microplate reader (wavelength: 450 nm)
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• Plate Sealer: 3 • ELISA Shaker
• Hermetic Bag: 1
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v10.1.5 Abbexa LTD, Cambridge, UK · Phone: +44 (0) 1223 755950 · Fax: +44 (0) 1223 755951 1 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: 6.0.0
Revision date: 19 Nov 2024
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. Keep samples on ice during preparation. Equivalate samples to room temperature prior to analysis.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type – this is just an example.
Rinse tissues with ice-cold PBS to remove the excess of blood. Weigh before homogenization. Finely mince tissues and
homogenize with a tissue homogenizer on ice in PBS and sonicate the cell suspension. Centrifuge the homogenates at 10000 × g
for 5 mins and collect the supernatant.
Cell Lysates: Detach adherent cells with trypsin and collect by centrifugation and remove the supernatant. Wash the cells three
times in ice-cold PBS and re-suspend cells in PBS. Lyse the cells by ultra-sonification 4 times, or freeze at -20°C and thaw to room
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temperature 3 times. Centrifuge at 1500 × g for 10 mins at 2-8°C to remove cellular debris. Collect the supernatant.
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Other Biological Fluids: Centrifuge at approximately 1000 × g for 20 mins to remove precipitate. Analyse immediately or aliquot
and store at -20°C or -80°C.
Notes:
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Analyse samples at a 5-fold (1/5) dilution (i.e. add 10 µl of sample to 40 µl of Sample Diluent Buffer).
Store frozen samples undiluted. Once ready to analyse, thaw samples and dilute.
erroneous results.
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Fresh samples, or recently obtained samples, are recommended to prevent protein degradation and denaturation that may lead to
NaN3 cannot be used as a test sample preservative, since it inhibits HRP.
If possible, prepare solid samples using sonication and/or homogenization, as lysis buffers may (on occasion) interfere with the kit's
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performance.
If a sample is not indicated in the manuals applications, a preliminary experiment to determine the suitability of the kit will be
required.
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B. Reagent Preparation
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
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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 samples in duplicate.
1. Set 2 positive and 2 negative control, test sample and control (zero) wells on the pre-coated plate respectively, and record their
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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.
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
v10.1.5 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: 6.0.0
Revision date: 19 Nov 2024
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.
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 ≥ 1.00.
Mean OD of the Negative Control should be ≤ 0.20.
CUT OFF value = Negative Control + 0.15
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If the Positive Control value is ≥ 1.00, and the Negative Control value is ≤ 0.20, 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.
If OD of Samples ≥ CUT OFF, the test samples are considered positive.
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Precautions:
Before using the kit, centrifuge the tubes to bring down the contents trapped in the lid.
exceed 10 mins.
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Do not leave the wells uncovered for extended periods between incubations. The addition of reagents for each step should not
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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Do not reuse pipette tips and tubes.
Do not use expired components, or components from a different kit.
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.
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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
protein.
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Technical Support
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For troubleshooting, frequently asked questions, and assistance, please visit:
https://www.abbexa.com/scientific-support/troubleshooting-and-faqs/elisa-kit-scientific-support or email us at support@abbexa.com.
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v10.1.5 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
