C-Reactive Protein Assay
Intended Use:
C-Reactive Protein is used as an in vitro diagnostic marker to aid in the evaluation of the amount of injury to human body tissue. Produced mainly in the liver, C-Reactive Protein is a plasma protein whose concentration increases dramatically during the acute phase inflammatory response. Inflammation and other pathological processes such as coronary artery disease, stroke, heart attack, and others may cause CRP levels to be elevated as much as 2000 fold. CRP is also useful as a prognostic indicator for the risk of cardiovascular disease in general. CRP levels are very helpful in determining, not only the degree of damage from a cardiac episode as related to the post-CRP concentration, but to aid in assessing the risk of future serious cardiac conditions associated with inflammation and other pathology.
Principle of
Procedure:
Solid phase
capture sandwich ELISA assay using a
microwell format.
Shelf Life:
The expiration date for the
package and each
component is stated on the label(s).
Store all
components at 2°- 8°
degrees C except for the standard,
which should be stored at -20oC.
Patient and Standard Dilutions:
Standards are provided pre-diluted at 1:10,000. In order to construct a reasonable curve within the diagnostic range of interest standards should be diluted at least 1:10 with the diluent CRP Specimen diluent provided to form a final dilution of 1:10,000. Prepare two-fold serial dilutions of the diluted standard, i.e. Neat (N), 1:2, 1:4,1:8 1:16 using the CRP Specimen Diluent provided. The diluent without standard serves as the zero or blank control. Patient specimens should be diluted at least 1:1,000 in PBS and subsequently 1:10 or greater with the CRP Specimen Diluent provided. Greater dilutions may be necessary in order to get them on the standard curve used. A mathematical correction should be applied to correct for the difference between the dilution used for standard verses specimen. For example if both are diluted the same there is no correction factor. But if specimens were diluted 1:20,000 and the standard curve used and an initial dilution of 1: 10,000 then interpolated answers would be multiplied by a factor of two.
Materials Supplied:
Anti-Human CRP 12x8 microwell coated strip plate - (1)
Anti-Human CRP HRP conjugate - (12ml)
CRP standard (pre-diluted 1:10,000) - (1ml)
TMB/Peroxide Substrate - (12 ml)
CRP Specimen Diluent - (60ml)
Termination reagent - (12ml)
15X Wash buffer concentrate - (60ml)
Limitations of the Procedure:
No single assay should be used as the only basis for arriving at a
diagnostic conclusion.
For research use only.
Dynamic Range:
0.69mg/dL to 44.0mg/dL.
Reproducibility:
C.V.% 2-6% depending
upon the region of the standard curve.
Assay Procedure:
1:Dilute specimen 1:10,000 with the specimen diluent provided
2: Prepare serial two fold dilutions (at least 5 subsequent) of the standard to obtain a standard curve. (Standard should be run in at least duplicates)
3: Add 100uL of each of the diluted specimen or standard to each well.
4: Incubate at room temperature for one hour (22°- 25° C).
5: Decant and wash 5 times with diluted wash buffer (see "preparation of reagents").
6: Add 100uL of the HRP conjugated Anti-CRP to each well.
7: Incubate at room temperature for one hour.
8: Repeat washing as in step 5.
9: Add 100 uL of TMB/Peroxide to each well.
10: Incubate at room temperature for 30 minutes.
11: Add 100uL of 0.5N sulfuric acid to each well to terminate color development.
12: Read the optical density at 450nm using a standard microwell plate reader.
