CRP Rev.

1RC
3. Recovery
Known quantities of CRP were added to a serum that contained a low concentration of CRP.

Expected Value(mg/L) Recovered (mg/L) Percentage of Recovery

0.005 0.0053 106
0.0125 0.0102 82
0.025 0.0236 94

4. Linearity
HIGH SENSITIVITY C-REACTIVE PROTEIN
Two different patient samples were diluted with the “0” calibrator to 1:2, 1:4 and 1:8. CRP values were assayed
and results were corrected with the dilution factor. The results of these dilution tests are as follows:
(CRP) ELISA
Catalog No.: CR120C (96 Tests)
Serum Original Value (mg/L) Percentage of Recovery INTENDED USE
1:2 1:4 1:8 The Calbiotech, Inc. (CBI) C-Reactive Protein Ultra Sensitive ELISA Kit is intended for the quantitative determination
1 0.032 94 100 100 of C-reactive protein (CRP) in human serum or plasma.
2 0.041 93 88 117
SUMMARY AND EXPLANATION
3 0.095 84 84 82
C-Reactive protein (CRP) is an alpha globulin with a molecular mass of approximately 110,000 to 140,000 daltons,
and is composed of five identical subunits, which are noncovalently assembled as a cyclic pentamer. CRP is
REFERENCES
1. Schultz, D.R., and Arnold P.I.: “Properties of four acute phase proteins: C-reactive protein, serum amyloid A protein, glycoprotein,
synthesized in the liver and is normally present as a trace constituent of serum or plasma at levels less than 0.3 mg/dl.
and fibrinogen.” Seminars in Arthritis and Rheumatism 20: 129-147, 1990. CRP is one of the acute-phase proteins, the serum or plasma levels of which rise during general, nonspecific
2. Kindmark, C.O.: The Concentration of C-reactive protein in Sera from Healthy Individuals. Scand J Clin Lab Invest, 29: 407- 411, response to a wide variety of diseases. Although the detection of elevated levels of CRP in the serum is not specific
1972. for any particular disease, it is a useful indicator of inflammatory processes. Additionally, measurement of CRP by
3. Dowling, P., and Cook, S.: Immune events in demyelinating disease. In Wolfgang, F., Ellison, G.W., Stevens J.G., and Andrew, high-sensitivity CRP assays may add to the predictive value of other cardiac markers (myoglobin, creatine-kinase-MB,
J.M. (eds.): Multiple sclerosis. Academic Press Inc., New York, 269-277, 1972. troponin I and T), which are used to assess the risk of cardiovascular and peripheral vascular disease. Inflammation in
4. YUDKIN, J.S., ET. AL.: C-REACTIVE PROTEIN IN HEALTHY SUBJECTS: ASSOCIATION WITH OBESITY, INSULIN RESISTANCE, AND ENDOTHELIAL
the arteries may play a role in heart disease and HS-CRP can determine heart disease risk in those with undetected
DYSFUNCTION. A POTENTIAL ROLE FOR CYTOKINES ORIGINATING FROM ADIPOSE TISSUE? ARTERIOSCLER THROMB VASC BIOL 19:972-8,
1999. heart disease and risk of complications for those who have already had a heart event
5. Kushner, I., Rzewnicki, D.L.: The acute phase response: General aspects. Bailliere’s Clinical Rheumatology 8: 513-530, 1994.
6. Macy, E.M., Hayes, T.E., and Tracy, R.P.: Variability in the measurement of C-reactive protein in healthy subjects: implications PRINCIPLE OF THE ASSAY
for reference interval and epidemiological applications. Clin Chem, 43;1:52-58, 1997. The CRP ELISA kit is a solid phase direct sandwich method. The samples and anti-CRP-HRP conjugate are added to
7. Hedlund, P.: Clinical and experimental studies on C-reactive protein (acute phase protein). Thesis Acta Med Scand, 128 (Suppl, the wells coated with MAb to CRP. CRP in the patient’s serum binds to anti-CRP MAb on the well and the anti-CRP
361):1-71, 1961. second antibody then binds to CRP. Unbound protein and HRP conjugate are washed off by wash buffer. Upon the
8. Hedlund, P.: The appearance of acute phase protein in various diseases. Acta Med Scand, 128, (Suppl,196): 579-601, 1947.
addition of the substrate, the intensity of color is proportional to the concentration of CRP in the samples. A standard
9. Morley, J.J., Kushner, I.: Serum C-reactive Protein Levels. in: Kushner, I., Volanakis, J.E., and Gerwutz, H., eds., C-Reactive
Protein and the Plasma Protein Response to Tissue Injury. Annals of N.Y. Acad Sci, 389: 406-417, 1982. curve is prepared relating color intensity to the concentration of the CRP
2008-12-18
MATERIALS PROVIDED 96 Tests
1. Microwells coated with CRP MAb 12x8x1
2. CRP Standard: 6 vials ( ready to use) 0.7ml
For Research Use Only. Not for use in Diagnostic Procedures. 3. CRP Enzyme Conjugate: 1 bottle (ready to use) 12 ml
4. TMB Substrate: 1 bottle (ready to use) 12ml
5. Stop Solution: 1 bottle (ready to use) 12ml
6. Sample Diluent 50 ml
7. 20X Wash concentrate: 1 bottle 25ml

MATERIALS NOT PROVIDED

1. Distilled or deionized water
2. Precision pipettes
Cat#: CR120C (96 Tests) 3. Disposable pipette tips
For Order and Inquiries, please contact 4. ELISA reader capable of reading absorbance at 450nm
Calbiotech Inc., 5. Absorbance paper or paper towel
10461 Austin Dr, Spring Valley, CA, 91978 6. Graph paper
Tel (619) 660-6162, Fax (619) 660-6970,
www.calbiotech.com STORAGE AND STABILITY
1. Store the kit at 2 - 8° C.
8°
2° 2. Keep microwells sealed in a dry bag with desiccants.
CEpartner4U, 3951DB; 13. NL. 3. The reagents are stable until expiration of the kit.
tel: +31 (0)6.516.536.26) 4. Do not expose test reagents to heat, sun, or strong light.
 CRP Rev.1EC
WARNINGS AND PRECAUTIONS Example of a Standard Curve
1. Potential biohazardous materials: OD 450
The calibrator and controls contain human source components which have been tested and found non-reactive nm Conc. mg/L
for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test Std 1 0.02 0
method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. Std 2 0.23 0.005
These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Std 3 0.49 0.01
Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984. Std 4 1.01 0.025
2. This test kit is designed for research use only.. Std 5 1.66 0.05
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are Std 6 2.40 0.1
handled.
5. The components in this kit are intended for use as an integral unit. The components of different lots should not EXPECTED VALUES
be mixed. It is recommended that each laboratory establish its own normal range based on the patient population. However,
6. It is recommended that standards, control and serum samples be run in duplicate. based on published literature healthy individuals are expected to have CRP values as follows: the CRP level in
7. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as normal human serum ranges from 0.2 to 10 mg/L, where 90% of apparently healthy individuals have CRP levels <3
following the exact time and temperature requirements prescribed are essential. Any deviation from this may mg/L and only 1% have levels >10 mg/L.
yield invalid data.
LIMITATIONS OF THE TEST
SPECIMEN COLLECTION HANDLING 1. The test results obtained using this kit serve only as an aid to diagnosis and should be interpreted in relation to
1. Collect blood specimens and separate the serum immediately. the patient’s history, physical findings and other diagnostic procedures.
2. Specimens may be stored refrigerated at (2-8° C) for 5 days. If storage time exceeds 5 days, store frozen at (- 2. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
20° C) for up to one month.
3. Avoid multiple freeze-thaw cycles. PERFORMANCE CHARACTERISTICS
4. Prior to assay, frozen sera should be completely thawed and mixed well. 1. Correlation with a Reference ELISA kit:
5. Do not use grossly lipemic specimens. A total of 84 sera were tested by this ELISA and a reference ELISA kit. Results were as follows:

REAGENTS PREPARATION
Correlation Slope Intercept
1X Wash Buffer: Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or
0.93 0.72 0.011
deionized water. Store at room temperature (18-26° C).

ASSAY PROCEDURE 2. Precision

Intra-Assay
Prior to assay, allow reagents to stand at room temperature (18-25°C). Gently mix all reagents before use.
1. Place the desired number of coated strips into the holder No. of Mean Standard Coefficient of
Serum
2. Dilute patient samples and controls 1:100 by adding 5 µl of samples to 495 µl of sample Diluent (STANDARDS Replicates (mg/L) Deviation Variation (%)
ARE READY TO USE). 1 16 0.004 0.0002 5.06
3. Dispense 10 µL of standard, diluted samples and controls into the appropriate wells 2 16 0.021 0.0011 5.28
4. Add 100 µl of enzyme conjugate to all wells. Tap the holder to remove air bubbles from the liquid and mix well. 3 16 0.008 0.0008 9.59
5. Incubate for 60 minutes at room temperature (18-26° C).
6. Remove liquid from all wells. Wash wells three times with 300 µl of 1X wash buffer. Blot on absorbent paper Inter-assay
towels. No. of Mean Standard Coefficient of
Serum
7. Add 100 µl of TMB substrate to all wells. Replicates (mg/L) Deviation Variation (%)
8. Incubate for 15 minutes at room temperature. 1 10 0.004 0.0003 8.51
9. Add 50 µl of stop solution to all wells. Shake the plate gently to mix the solution. 2 10 0.009 0.0007 8.34
10. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution. 3 10 0.020 0.0016 7.95

CALCULATION OF RESULTS
The standard curve is constructed as follows:
1. Check CRP standard value on each standard vial. This value might vary from lot to lot. Make sure you check
the value on every kit. See example of the standard attached.
2. To construct the standard curve, plot the absorbance for the CRP standards (vertical axis) versus the CRP
standard concentrations (horizontal axis) on a linear graph paper. Draw the best curve through the points.
3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control
or unknown sample.
4. The obtained values of the patient samples and control sera should be multiplied by the dilution factor of 100 to
obtain CRP results in mg/l.
5. Patient samples with CRP concentrations greater than 10 mg/l should be further diluted 10-fold after the initial
100-fold dilution (total dilution 1:1,000), and the final CRP values should be multiplied by 1,000 to obtain CRP
results in mg/l.