1. Intended Use
This Ultrasensitive 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
The ultra-sensitive 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 in 2.5 hours. 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 labeled with biotin. The signal will be further amplified by the binding of streptavidin-conjugated horseradish peroxidase (HRP-SPT). 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.

5. Reagents Supplied
E
ach kit is sufficient for one 96-well plate and contains the following components:
1. Antibody Coated 8-well Strips (12 strips, total 96 wells)
8-well strips coated with mouse anti-human CRP monoclonal antibody (with 2 holders and 2 plate-covers provided).
2. CRP calibrator Set
7 vials, with concentrations of 0, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25 and 2.5 ng/ml human CRP in phosphate-buffered saline-BSA solution.
3. Assay Diluent (10×, 10 ml)
4. Washing Buffer (10×, 20 ml)
5. Biotinylated Antibody Solution (100×, 3 vials, 40 ul/vial)
6. HRP-SPT Solution (100×, 3 vials, 40 ul/vial)
7. TMB Substrate Solutions A and B (6 ml each)
8. Stop Solution (6 ml)
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 desired amount of 10x concentrated Assay Diluent with distilled or deionized water to 1× working concentration.
2) 1x Wash buffer. Dilute the 10x concentrated Washing Buffer with distilled or deionized water to 1× working concentration.
3) 1x Biotinylated Antibody Solution. Dilute Biotinylated Antibody Solution, 100 ul of the 1x Biotinylated Antibody Solution is required per well. Prepare only as much 1x Detection antibody solution as needed. Return the 100x Biotinylated Antibody Solution to 2-8°C immediately after the necessary volume is removed.
4) 1x HRP-SPT Solution. Dilute HRP-SPT Solution, 100 ul of the 1x HRP-SPT Solution is required per well. Prepare only as much 1x HRP-SPT Solution as needed. Return the 100x HRP-SPT Solution to 2-8°C immediately after the necessary volume is removed.
5) 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 ul of serum to 198 ul of Assay Diluent in a 1.5 ml microtube and mix well by vortexing. This diluted sample is further diluted by 20× by adding 20 ul of the diluted sample to 380 ul of Assay Diluent in a 1.5 ml microtube and mix well by vortexing (i.e. the final dilution factor of the sample should be 2000×)
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 100 μl of CRP standards, diluted samples or controls into appropriate wells.
3. Incubate at room temperature (18-25 °C) for 30 minutes.
4. Discard the contents into a waste container and dispense 300 μl washing buffer into each well.
5. Discard the washing buffer and strike the strips on dry paper towels to remove residual buffer. Repeat the steps 4-5.
6. Dispense 100 μl diluted biotinylated antibody into each well and incubate at room temperature for 30 minutes.
7. Repeat steps 4-5 three times.
8. Dispense 100 μl diluted HRP-SPT into each well and incubate at room temperature for 20 minutes.
9. Repeat steps 4-5 for four times.
10. Dispense 100 μl TMB substrate mixture into each well and gently mix for 10 seconds.
11. Incubate at room temperature for 10 minutes.
12. Stop the reaction by adding 50 μl 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 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 2000.
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 independent assays.

10. Performance Characteristics
1) Sensitivity. The minimum detectable CRP level of this ELISA assay as determined by 2SD from the mean of 10 zero standard is estimated to be 0.02 ng/ml. The functional sensitivity was determined as 0.039 ng/ml.
2) Specificity. The assay was tested to have no cross-reactivity with mouse or rat CRP, or other cytokines or hormone molecules.
3) Precision.
a. Intra-assay variation – Within-run precision was determined by triplicate determination of CRP concentrations of five serum samples in one assay.
b. Inter-assay variation – Between-run precision was determined by replicate determination of CRP concentrations of five serum samples over a series of independent assays.
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
1
2. References
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.