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Canine C-Reactive Protein [CRP] ELISA Kit

Canine C-Reactive Protein [CRP] ELISA Kit

(RUO)
商品详情
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BD™
ELISA (Tested During Development)
RUO


描述

Materials Provided

Capture antibody coated microplate

One plate of 96 breakable wells coated with a goat anti-canine CRP antibody

Detection antibody/Enzyme Conjugate (100x)

130 µL of concentrated horseradish peroxidase (HRP) conjugated to a goat anti-canine CRP antibody containing stabilizers and preservative. Protect from light.

Standard (10x)

250 µL of canine serum with elevated levels of CRP.

Wash Buffer

One packet of powdered Tris with 0.05% Tween-20. Reconstitute with 1L distilled water.

TMB Substrate

A 12 mL solution containing 3,3',5,5'-tetramethylbenzidine (TMB) supplied in a protective opaque bottle. Protect from light.

Stop Solution

12 mL of diluted phosphoric acid.

The Canine C-Reactive Protein (CRP) ELISA Kit is designed for the detection and quantitation of canine CRP in canine serum.  CRP is an acute-phase protein produced by the liver during conditions of inflammation, bacterial infection, or tissue trauma. Quantitation of CRP can be useful for the determination of inflammatory conditions that would be otherwise difficult to detect and monitor.  This ELISA for canine CRP is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes an antibody specific for canine CRP coated on a 96-well plate.  Standards or samples are added to the wells, and any canine CRP present binds to the immobilized antibody. After the appropriate incubation, wells are washed and a horseradish peroxidase conjugated anti-canine CRP is added to produce an antibody-antigen-antibody "sandwich".  Following another incubation period, the wells are washed again and a substrate solution is added, which produces a blue color in direct proportion to the amount of CRP present in the initial sample (development of a blue color indicates a positive reaction while negative reactions appear colorless).  The reaction is interrupted with the Stop Solution, which changes the color from blue to yellow (negative reactions remain colorless or faintly yellow).  The color is then measured at a wavelength of 450 nm (absorbance) on a spectrophotometer or on an ELISA reader.

准备和存储

Store undiluted at 4°C.

Kit components should be brought to room temperature (20-25°C) before opening bottles and plate pouches. Allow at least 30 minutes for this process. Do not use the kit after the expiration date.

推荐的实验流程

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).

Hazard statements

May be harmful if swallowed.

Precautionary statements

Call a POSION CENTER/doctor if you feel unwell.

Danger:  Stop Solution (component 51-946P001) contains 15.23% phosphoric acid (w/w).

Hazard statements

Causes severe skin burns and eye damage.

Precautionary statements

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.

557826 Rev. 8
报价单和参考
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研发参考 (10)

  1. Burton SA, Honor DJ, Mackenzie AL, Eckersall PD, Markham RJ, Horney BS. C-reactive protein concentration in dogs with inflammatory leukograms. Am J Vet Res. 1994; 55(5):613-618. (Biology). 查看参考
  2. Casals C, Varela A, Ruano ML, et al. Increase of C-reactive protein and decrease of surfactant protein A in surfactant after lung transplantation.. Am J Respir Crit Care Med. 1998; 157(1):43-9. (Biology). 查看参考
  3. Conner JG, Eckersall PD, Ferguson J, Douglas TA. Acute phase response in the dog following surgical trauma.. Res Vet Sci. 1988; 45(1):107-10. (Biology). 查看参考
  4. Eckersall PD, Conner JG, Harvie J. An immunoturbidimetric assay for canine C-reactive protein.. Vet Res Commun. 1991; 15(1):17-24. (Biology). 查看参考
  5. Lindbäck S, Hellgren U, Julander I, Hansson LO. The value of C-reactive protein as a marker of bacterial infection in patients with septicaemia/endocarditis and influenza.. Scand J Infect Dis. 1989; 21(5):543-9. (Biology). 查看参考
  6. Ndung'u JM, Eckersall PD, Jennings FW. Elevation of the concentration of acute phase proteins in dogs infected with Trypanosoma brucei.. Acta Trop. 1991; 49(2):77-86. (Biology). 查看参考
  7. Otabe K, Sugimoto T, Jinbo T, et al. Physiological levels of C-reactive protein in normal canine sera. Vet Res Commun. 1998; 22(2):77-85. (Biology). 查看参考
  8. Rikihisa Y, Yamamoto S, Kwak I, et al. C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis.. J Clin Microbiol. 1994; 32(4):912-7. (Biology). 查看参考
  9. Yamamoto S, Shida T, Okimura T, et al. Determination of C-reactive protein in serum and plasma from healthy dogs and dogs with pneumonia by ELISA and slide reversed passive latex agglutination test.. Vet Q. 1994; 16(2):74-7. (Biology). 查看参考
  10. Yamashita K, Fujinaga T, Miyamoto T, Hagio M, Izumisawa Y, Kotani T. Canine acute phase response: relationship between serum cytokine activity and acute phase protein in dogs.. J Vet Med Sci. 1994; 56(3):487-92. (Biology). 查看参考
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557826 Rev. 8

Please refer to Support Documents for Quality Certificates

Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.