1. Intended Use
This One-Step Human C-Reactive Protein (CRP) ELISA kit is designed for quantification of Human C-Reactive Protein in serum, plasma. This assay kit is for in vitro and research use only.
2. Storage, Expiration
The kit should be stored at 2-8oC upon receipt, and should be equilibrated to room temperature before assay.
Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers.
Remove any unused strips from the human adiponectin microplate, return them to the foil pouch and keep at 4 oC.
3. Introduction
C-reactive protein (CRP) is a circulating protein mainly secreted from the liver. This acute phase protein consists of five identical non-glycosylated subunits of 23 kDa, that give rise to a symmetrically arranged globular protein with molecular weight of approximately 120 kDa.1 It has long been recognized that CRP is closely related to immunology, inflammation and host defense; as a result it has been used as an inflammatory marker. Normally CRP is presenting only in a trace amount in circulation (<1ug/ml)5,6 but can increase over 1,000-fold under acute inflammatory state. There is accumulating evidence suggesting that CRP plays an important role in the pathogenesis of cardiovascular diseases (CVD) and type 2 diabetes.2-4 Individual with blood CRP levels <1ug/ml, 1-3ug/ml and >3ug/ml is considered to have low, moderate and high risk, respectively, of CVD and myocardial infarction.7 Therefore, circulating CRP level has become a promising measure of CVD risks.8,9
4. Test Principle
Our Rapid Human CRP ELISA is based on a solid phase enzyme-linked immunosorbent assay using monoclonal antibodies that against human CRP. The assay system is designed for the quantitative detection of human CRP in plasma or serum within 1 hour. A mouse monoclonal antibody has been pre-coated onto 8-well strips which could be mould onto the strip holder provided. CRP molecules in the samples are sandwiched by another mouse monoclonal antibody conjugated with horseradish peroxidase (HRP). All unbound materials are washed away before the addition of HRP substrate. The color developed by the enzyme activity is determined after stopping the reaction by the addition of acidic solution. The color intensity represents the amount of CRP presents in the samples.
5. Reagents Supplied
ach kit is sufficient for one 96-well plate and contains the following components:
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 1. Micro-titer Strips (96 wells): coated with anti-human CRPP monoclonal antibody, sealed
2. Wash Buffer (5x): 25 ml
3. Assay Buffer (10x): 15 ml
 4. Detection Antibody (100x): a monoclonal antibody against human CRP conjugated with horseradish peroxidase, 0.06 ml
 5. CRP Standard Set (7 vials, 1.5 ml/vial, with concentrations of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 ng/ml).
6. Quality controls: High and Low, lyophilized
7. Substrate A: 6 ml
8. Substrate B: 6 ml
9. Stop Solution: 6 ml
10. Plate cover: x 1.
6. Preparation of Reagents
Pre-warm all reagents to room temperature (18-25°C) before the assay. All reagents should be freshly diluted before use. Diluted reagents are not stable under the storage condition suggested in this manual.
1) 1x Assay diluent. Dilute the 10x concentrated Assay Diluent with distilled or deionized water to 1× working concentration.
2) 1x Wash buffer. Dilute the 5x concentrated Washing Buffer with distilled or deionized water to 1× working concentration.
3) 1x Detection antibody solution. Dilute the 100x Detection Antibody with 1x Assay buffer, 50 ul of the 1x Detection antibody solution is required per well. Prepare only as much 1x Detection antibody solution as needed. Return the 100x Detection Antibody to 2-8°C immediately after the necessary volume is removed.
4) Substrate solution. Mix the desired amount of TMB Substrate Solutions A and B in a 1:1 ratio immediately before starting the reaction. Do not pre-mix the solutions during preparation of other reagents. Return Substrate A & B to 2-8°C immediately after the necessary volume is removed.
7. Preparation of Samples
 1. This kit is designed for quantitative detection of human CRP in plasma or serum samples. The intended use of this kit for detection of human CRP in other biological fluids or solutions was not tested.
 2. Blood should be collected using standard venipuncture techniques. After blood is clotted, centrifuge samples at 1,500 to 2,000g for 15 minutes and collect the supernatants.
 3. Dilute the serum 100× with Assay Diluent by adding 2 μl of serum to 198 μl of Assay Diluent in a 1.5 ml microtube (It is the recommended dilution factor ONLY. Users are suggested to determine the desirable dilution factor if the expected CRP concentration in the sample is lower than 0.156 ug/ml or higher than 10 ug/ml).
 4. Specimens cannot be assayed within 24 hours after collection should be frozen at -20 °C or below, and will be stable for up to 6 months.

8. Assay Procedure
 1. Secure the desired number of coated strips into the holder and seal unused strips in the tin foil bag.
 2. Dispense 50ul of CRP standards, diluted samples or controls into appropriate wells.
 3. Dispense 50ul of Assay Diluent for the zero standard (blank),
 4. Dispense 50ul of HRP-conjugated secondary antibody into each well.
 5. Mix thoroughly by gently tapping on the sides of the holder for 30 seconds.
 6. Incubate at room temperature (18-25 °C) for 30 minutes in dark.
 7. Discard the contents into a waste container and dispense 300 μl washing buffer into each well and incubate for 30 seconds.
 8. Discard the washing buffer and tape the strips on dry paper towels to remove residual buffer.
 9. Repeat steps 7-8 for four times.
 10. Dispense 100ul TMB substrate mixture into each well and gently mix for 10 seconds.
 11. Incubate at room temperature for 8 minutes.
 12. Stop the reaction by adding 50ul of 2 N H2SO4 into each well and gently mix for 10 seconds.
 13. Read the absorbance at 450 nm with a micro-plate reader.

Note:
 1. Pre-warmed all coated strips, standards, diluted samples and controls to room temperature for 15 minutes prior to assay.
 2. It is recommended to read the absorbance within 10 minutes after the addition of stop solution.

9. Calculations
 1. Subtract the absorbance of the blank from that of the standards, samples and controls.
 2. Calculate the mean absorbance values of each standard. Generate a standard curve by plotting the absorbance obtained (y-axis) against CRP concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Any curve of 4-parameter or log-log curve fitting can be used for calculation.
 3. Determine CRP concentration of samples from standard curve and multiply the value by the dilution factor of 200.

Example of standard curve
NOTE: This standard curve is for the purpose of illustration only, and should not be used for calculating unknown CRP concentration. Users should construct their own standard curves for independentassays.
 
CRP, ng/ml  Absorbance, 450 nm  Blanked absorbance  
0  0.068  0  
0.78  0.141  0.073  
1.56  0.200  0.132  
3.12  0.323  0.255  
6.25  0.511  0.443  
12.5  0.912  0.844  
25  1.691  1.623  
50  2.588  2.52  

10. Performance Characteristics
1) Sensitivity. The minimum detectable CRP level of this ELISA assay, as determined by adding 2 standard deviation to the mean value of 10 zero standard replicates and calculating concentrations from standard curve, is estimated to be 0.5 ng/ml. The functional sensitivity was determined as 0.78ng/ml.
2) Specificity. The assay was tested to have no cross-reactivity with mouse or rat CRP, or other cytokines or hormone molecules, including TNFα, MCP-1, insulin, resistin and adiponectin et al.
3) Precision.
 a. Intra-assay variation – Within-run precision was determined by determination of CRP concentrations of five serum samples in one assay for 10 times (Table 1).
 b. Inter-assay variation – Between-run precision was determined by replicate determination of CRP concentrations of five serum samples in 10 independent assays.

Serum sample  Mean CRP (μg/ml)  Intra-assay
C. V. (%)  Inter-assay
C. V. (%)  
1  0.25  6.4  4.7%  
2  1.21  5.3  5.8  
3  2.64  4.2  4.7  
4  4.32  3.9  5.1  
5  6.46  5.8  6.4  

11. Troubleshooting and FAQs
1/ Weak signal in all wells
Possible explanations:
 - Omission of a reagent or a step
 - Improper preparation or storage of a reagent
 - Assay performed before reagents were allowed to come to room temperature
2/ High signal and background in all wells
Possible explanations:
 - Improper or inadequate washing
 - Overdeveloping; incubation time should be decreased before addition of Stop Solution
3/ High coefficient of variation (CV)
Possible explanation:
- Improper or inadequate washing
2. References
1
 1. Thompson D., Pepys M.B. and Wood S.P. (1999) Structure, 7, 169-177.
 2. Festa A, D’Agostino R. Jr., Tracy R.P. and Haffner S.M. (2002) Diabetes, 51, 1131-1137.
 3. Verma S. and Yeh E.T. (2003) Am J Physiol, 285, R1253-R1258.
 4. Jialal I., Devaraj S. and Venugopal S.K. (2004) Hypertension, 44, 6-11.
 5. Kindmark C.O. (1972) Scand J Clin Lab Invest, 29, 407-411.
 6. Macy E.M., Hayes T.E. and Tracy R.P. (1997) Clin Chem, 43, 52-58.
 7. Ridker P.M. (2004) Am Heart Hosp J, 2 (4 Suppl 1), 4-9.
 8. Benzaquen l.R., Yu H. and Rifai N. (2002) Crit Rev Clin Lab Sci, 39, 459-497.
9. Pearson T.A. et al., (2003) Circulation, 107, 499-511