The TECO® Canine CRP kit is a sensitive sandwich enzyme linked immunosorbent assay for the quantitative determination of CRP in canine serum, plasma and cell culture.

  • Range: 5 – 50mg/l (50 – 500ng/ml before dilution)
  • Sensitivity: 2.9mg/l
  • Incubation time: 2hrs
  • Sample volume: 20μl (dilute 1:100)
  • Sample type: Serum and cell culture.
  • Reference values: Serum of healthy canine < 5mg/l

 

Symbol Description Format
 1  96-well plate coated with canine CRP specific antibody with plate cover
12 break apart strips of 8 wells (12 x 8 in total), in a frame. Ready to use.		 1 plate
 A  Standard Stock Solution
500ng in 100μl		 1 x 0.4ml
 L  Control 1
Range as indicated on data sheet		 1 x 0.05ml
 H  Control 2
Range as indicated on data sheet		 1 x 0.05ml
 2  Wash Buffer 50x
50 times concentrated		 1 x 30ml
 3  Sample Diluent
Ready to use		 1 x 100ml
 4  Assay Buffer
Ready to use		 1 x 12ml
 6  HRP Antibody Conjugate
Ready to use		 1 x 12ml
 7  TMB Substrate
Ready to use		 1 x 12ml
 8  Stop Solution – 1M HCI
1M hydrochloric acid, ready to use		 1 x 12ml
 I  Kit Instructions 1 x

 

Materials Required and not Supplied

  • Pipettes capable of accurately dispensing 20μl, and 100 to 1000μl
  • Multichannel pipette for 100μl
  • Graduated cylinders for reconstituting or diluting reagents
  • Automatic washer or equivalent plate washing system
  • Distilled or deionized water
  • Vortex mixer
  • ELISA plate shaker (orbital shaker, 500rpm)
  • ELISA plate reader suitable for 96 well formats and capable of measuring at 450nm (Reference Filter: 590–650nm)
  • ELISA plate reader software for data generation and analysis

ASSAY PRINCIPLE

The TECO® Canine CRP EIA Kit is a 96 well immuno-capture ELISA product. Samples are incubated with a specific polyclonal antibody coated on the plate. After incubation, the unbound material is washed away. Then a HRP linked polyclonal antibody that specifically recognizes canine CRP is added to the wells. After a further incubation and washing step, a TMB substrate is added which reacts with the HRP and resulting in a concentration-depended color level. The reaction is stopped with HCl and the plate is read using a plate reader at 450nm.

Color development is proportional to the amount of canine CRP in the sample.

ASSAY PROCEDURE

It is recommended that all determinations (Standards, Controls and samples) are assayed in duplicate. When performing the assay, Standards, Controls and samples should be pipetted as fast as possible (< 15 minutes).

To avoid distortions due to differences in incubation times, Substrate Solution and Stop Solution should be added to the plate in the same order and with the same time interval.

Before use, allow all reagents to stand at room temperature (20–25°C) for at least 30 minutes. During all incubation steps, plates should be sealed with the adhesive foil or a plastic cover. For light protection, incubate in a dark chamber or cover plate with aluminum foil.

  1. Allocate the wells of the Microwell Plate  1  for Standards, Controls and samples.
  2. Add 100μl Assay Buffer  4  to each well (multichannel pipette).
  3. Pipette 20μl of each Standard (  A  till  F  ), diluted Controls (  L  and  H  ) and diluted samples into the corresponding wells.
    Note: Controls have to be diluted like samples, e. g. 1:100 (see Note page 8)
  4. Incubate the plate for 1h at room temperature (20–25°C) on a shaker (500rpm).
  5. After incubation, aspirate the content of the wells and wash 3 times with 350μl diluted Wash Buffer  2  The use of an automatic plate washer is recommended.
  6. Add 100μl of HRP Antibody Conjugate  6  (multichannel pipette).
  7. Incubate the plate for 30min in the dark at room temperature (20–25°C) on a shaker (500rpm).
  8. After incubation, aspirate the content of the wells and wash 5 times with 350μl diluted Wash Buffer  2  The use of an automatic plate washer is recommended.
  9. Pipette 100μl of the TMB Substrate Solution  7  in each well (multichannel pipette).
  10. Incubate the plate for 15-20min in the dark at room temperature (20–25°C) on a shaker (500rpm).
  11. Stop the reaction by adding 100μl of Stop Solution  8  (multichannel pipette).
  12. Measure the color reaction within 10 minutes at 450nm (reference filter between 590–650nm).

Example only. Not for use in calculation of actual results

Standards Concentration (ng/ml) Absorption at 450nm
Stock Standard A 500 2.632
Standard B 278 1.413
Standard C 154 0.645
Standard D 86 0.254
Standard E 48 0.121
Standard F 0 0.014

 

 

OBSERVED VALUES

 

 

 

 

The measurement of CRP is used for research of inflammatory diseases. Although CRP is not disease-specific, the detection of an increased CRP level provides a valuable indication of the presence of inflammatory processes. CRP levels in serum/blood rise within hours after the onset of inflammation and reach a peak during the acute phase. As soon as the inflammation subsides, the CRP levels begin to decrease. As the erythrocyte sedimentation rate may be influenced by other physiological processes, the determination of CRP is a more reliable and more sensitive indicator of inflammation for:

  • Rapid and easy detection of inflammatory processes
  • Research of immune-mediated inflammatory processes
  • Research before and follow-up after surgery
  • Research of infectious diseases
  • Research of health status, e. g. before travelling or vaccination
  • Health research (especially with older dogs)
  • Pregnancy research
  • Cancer research

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