Specimen Collection and Handling: Specimens should be clear, non-hemolyzed and non-lipemic. Blood samples should be collected using approved venipuncture techniques. Allow samples to clot and separate serum by centrifugation. Alternatively, use a serum separator tube (BD Vacutainer® Cat.No. 366430) and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at < -20°C. Avoid repeated freeze-thaw cycles.
Additional Materials Required
1. Distilled or deionized water
2. Graduated cylinder, one liter
3. Wash bottle or automated washer
4. Precision pipettes
5. Tubes or microtiter plate to prepare standard dilutions
6. Plate sealers or parafilm
7. Laboratory timer
8. Graph paper or automated data reduction software
9. Microplate reader capable of measuring absorbance at 450 nm
Recommended Assay Procedure
1. Prepare Wash Buffer by dissolving 1 packet of powdered Tris in 1L of distilled water.
2. Prepare the CRP standard by diluting the provided 10x stock standard 1:10. (Example of the dilution scheme is in the figure above). Thereafter, perform 1:3 serial dilutions three times. For example, to prepare standard for one well:
Standard #1: Dilute the stock standard 1:10 (i.e. 18 µL of 10x stock standard mixed with 162 µL of Wash Buffer)
Standard #2: Dilute Standard #1 1:3 (i.e. 60 µL of Standard #1 mixed with 120 µL of Wash Buffer)
Standard #3: Dilute Standard #2 1:3 (i.e. 60 µL of Standard #2 mixed with 120 µL of Wash Buffer)
Standard #4: Dilute Standard #3 1:3 (i.e. 60 µL of Standard #3 mixed with 120 µL of Wash Buffer)
3. Serum samples will likely need to be diluted before testing and a titration of the sample is strongly recommended. The suggested dilution for canine serum is 1:500 (i.e. 2 µL of serum into 1 mL Wash Buffer).
4. Add 100 µl to each well and incubate at room temperature for 30 minutes.
5. Wash the plate 3-4 times with a gentle stream of wash buffer from a wash bottle or a plate washer. Tap plates on a stack of absorbent paper towels to remove residual buffer.
6. Prepare a 1x working concentration of the detection antibody/enzyme conjugate by diluting the provided 100x detection antibody/enzyme conjugate 1:100 with Wash Buffer (e.g. add 50 µL of 100x detection antibody/enzyme conjugate to 5 mL of Wash Buffer).
7. To each microwell being testing, add 100 µl of the 1x detection antibody/enzyme conjugate.
8. Cover the plate and incubate for 30 minutes at room temperature.
9. Wash the plate as described in step 5.
10. Add 100 µL of TMB substrate solution and incubate 5-10 minutes at room temperature.
11. Stop reaction by adding 100 µL of Stop solution.
12. Read absorbance at 450 nm within 30 minutes of stopping the reaction. If wavelength correction is available, absorbance at 630 nm may be subtracted from absorbance at 450 nm.
13. The provided 10x stock standard represents pre-diluted serum. To determine sample CRP concentrations, multiply measured values by dilution factor used (eg. by 500, if a 1:500 dilution was performed) to account for the dilution during sample preparation.
Limitations of the Procedure
1. The BD™ ELISA canine CRP kit is intended for use as an integral unit. Do not mix reagents from different kit batches.
2. Samples that generate absorbance values higher than the standard curve should be diluted and retested. Occasionally, antigen excess may be encountered in serum with high CRP values. In this situation, all the available CRP may not have reacted with the detection antibody and testing the serum at higher dilutions (e.g. 1:1000, 1:2000, or 1:4000) may be necessary.
3. Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.
4. Do not use components past the expiration date.
Handle all serum and plasma specimens in accordance with NCCLS guidelines for preventing transmission of blood-borne infections. The control serum has not been screened for infectious agents. Since no testing can assure the complete absence of infectious agents, serum specimens and equipment coming in contact with these specimens should be handled with good laboratory practices.
Warning: PBS-T Wash Buffer Powder (component 51-915X001) contains 75.8% sodium chloride (w/w) and 1.9% (w/w) potassium chloride (w/w).
May be harmful if swallowed.
Call a POSION CENTER/doctor if you feel unwell.
Danger: Stop Solution (component 51-946P001) contains 15.23% phosphoric acid (w/w).
Causes severe skin burns and eye damage.
Wear protective gloves / eye protection.
Wear protective clothing.
IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
Dispose of contents/container in accordance with local/regional/national/international regulations.