Instructions for use

CRP ELISADM E-4600R

CRP ELISAINTENDED USE
For the determination of C-Reactive Protein by enzyme immunoassay in human serum.
PRINCIPLE OF THE TESTThe principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ typeassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for
CRP is immobilized onto the microwell plate and another monoclonal antibody specific for a different region ofCRP is conjugated to horse radish peroxidase (HRP). CRP from the sample and standards are allowed to bind tothe plate, washed, and subsequently incubated with the HRP conjugate. After a second washing step, theenzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The
absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic
reaction is directly proportional to the concentration of CRP in the sample.
A set of standards is used to plot a standard curve from which the amount of CRP in samples and controls canbe directly read.
PROCEDURAL CAUTIONS AND WARNINGS
1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable
performance will only be attained by strict and careful adherence to the instructions provided.
2. Control materials or serum pools should be included in every run at a high and low level for assessing thereliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit
reagents and human specimens.
5. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
6. A standard curve must be established for every run.
7. The controls should be included in every run and fall within established confidence limits.
8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent
storage may be indicated when assay values for the controls do not reflect established ranges.
9. When reading the microplate, the presence of bubbles in the microwells will affect the optical densities(ODs). Carefully remove any bubbles before performing the reading step.
10. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored.
Instability or contamination may be indicated by the development of a blue colour, in which case it should
not be used.
11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,
sample, standard and control.
13. Do not mix various lot numbers of kit components within a test and do not use any component beyond theexpiration date printed on the label.
14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of CRP in human serum. The kit is
not calibrated for the determination of CRP in saliva, plasma or other specimens of human or animal origin.
2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum.
3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may leadto false results.
4. Only Standard A may be used to dilute any high serum samples. The use of any other reagent may lead tofalse results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found tobe non-reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCVand Human Immunodeficiency Virus (HIV) and found to be negative. However no test method can offer
complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagentsshould be considered a potential biohazard and handled with the same precautions as applied to any blood
specimen.
CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of thesereagents, wash with plenty of water. TMB is a suspected carcinogen.

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SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 ml of serum is required per duplicate determination. Collect 4-5 ml of blood into an
appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4oC
for up to 24 hours or at -10oC or lower if the analyses are to be done at a later date. Consider all humanspecimens as possible biohazardous materials and take appropriate precautions when handling.
SPECIMEN PRETREATMENT
Dilute serum samples 1:20 with Standard A before use.
Example: To 190 μl of Standard A add 10 μl of serum sample (1:20).
Do not dilute the standards and controls, they are ready for use.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
 Precision pipettes to dispense 10,20,50, 100, 190, 200 and 300 μl
 Disposable pipette tips
 Distilled or deionized water Plate shaker
 Microplate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater*(see assay procedure step 13).
REAGENTS PROVIDED
1. AA E-0030

Wash Buffer Concentrate – Requires Preparation X10

Contents:

One bottle containing buffer with a non-ionic detergent and a non-mercury preservative.

Volume:50 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute 50
ml of the wash buffer concentrate in 450 ml of water.
TMB Substrate - Ready To Use.

2. AA E-0055
Contents:

One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO
containing buffer.

Volume:16 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Stopping Solution - Ready To Use.

3. AA E-0080
Contents:

One vial containing 1M sulfuric acid.

Volume:6 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolStandards

Concentration

Volume/Vial
16 ml

DM E-4601

Standard A

0 ng/ml

DM E-4602

Standard B

100 ng/ml

0.5 mlDM E-4603

Standard C

400 ng/ml

0.5 mlDM E-4604

Standard D

1000 ng/ml

0.5 mlDM E-4605

Standard E

4000 ng/ml

0.5 mlDM E-4606

10,000 ng/ml

0.5 mlDM E-4651

Standard F
Control 1

DM E-4652

Control 2

Refer to vial labels for
expected value and acceptable
range!

0.5 ml
0.5 mlContents:

CRP in a protein-based buffer with a non-mercury preservative. Prepared by spikingbuffer with a defined quantity of CRP. Calibrated against World Health Organization(WHO) 1st IS 85/506.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months in unopened vials or as indicated on label. Once opened, the standards should
be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing andthawing cycles.

5. DM E-4613
Contents:

Assay Buffer - Ready To Use.
One vial containing a protein-based buffer with a non-mercury preservative.

Volume:40 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

6. DM E-4631

Mouse Anti-CRP Antibody Coated Microwell Plate-Break Apart Wells Ready To Use.

Contents:

One 96 well (12x8) polyclonal antibody-coated microwell plate in a resealable pouch withdesiccant.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Mouse Anti-CRP Antibody-Horseradish Peroxidase (HRP) Conjugate
Concentrate – Requires Preparation X80

7. DM E-4640
Contents:

Anti-CRP monoclonal antibody-HRP conjugate in a protein-based buffer with a nonmercury preservative.

Volume:0.3 ml/vial

Storage:

Refrigerate at 2 - 8oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:80 in assay buffer before use (eg. 25 μl of HRP in 2 ml of assay buffer). If thewhole plate is to be used dilute 150 μl of HRP in 12 ml of assay buffer. Discard any that isleft over.

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ASSAY PROCEDURE
All reagents must reach room temperature before use. Standards controls and specimen samples should beassayed in duplicate. Once the procedure has been started, all steps should be completed without interruption.
Dilute serum samples 1:20 with Standard A before use.
1.

Prepare working solutions of the anti-CRP-HRP conjugate and wash buffer.

2.

Remove the required number of well strips. Reseal the bag and return any unused strips to therefrigerator.

3.

Pipette 20 μl of each standard, control and diluted specimen samples into the correspondingly
labelled wells in duplicate.

4.

Pipette 200 μl of assay buffer into each well.
(We recommend using a multichannel pipette).

5.

Incubate on a plate shaker (approximately 200 rpm) for 30 minutes at room temperature.

6.

Wash the wells 3 times with 300 µl of diluted wash buffer per well and tap the plate firmly against
absorbent paper to ensure that it is dry (The use of a washer is recommended).

7.

Pipette 100 μl of the conjugate working solution into each well.
(We recommend using a multichannel pipette).

8.

Incubate on a plate shaker (approximately 200 rpm) for 15 minutes at room temperature.

9.

Wash the wells 3 times with 300 µl of diluted wash buffer per well and tap the plate firmly against
absorbent paper to ensure that it is dry (The use of a washer is recommended).

10.

Pipette 100 μl of TMB substrate into each well at timed intervals.

11.

Incubate on a plate shaker for 10-15 minutes at room temperature.
(or until Standard F attains dark blue colour for desired OD).

12.

Pipette 50 μl of stopping solution into each well at the same timed intervals as in step 10.

13.

Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the stopping
solution.
If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a 405 or 415nm filter may
be substituted. The optical densities will be lower, however, this will not affect the results of donor/control
samples.

CALCULATIONS
1. Calculate the mean optical density of each standard duplicate.
2. Calculate the mean optical density of each unknown duplicate.
3. Subtract the mean absorbance value of the Standard A from the mean absorbance values of the standards,controls and serum samples.
4. Draw a standard curve on log-log paper with the mean optical densities on the Y-axis and the standard
concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curveis recommended.
5. Read the values of the unknowns directly off the standard curve.
6. If a sample reads more than 10,000 ng/ml then dilute it with Standard A at a dilution of no more than 1:10
from the original 1:20 diluted serum (or 1:200 from neat serum). The result obtained should be multipliedby the dilution factor.
TYPICAL TABULATED DATA

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StandardOD 1

OD 2

Mean OD

A

0.055

0.053

0.054

Value(ng/ml)
0B

0.105

0.103

0.104100C

0.271

0.276

0.274

400

D

0.607

0.633

0.620

1000

E

1.964

1.894

1.929

4000

F

2.829

2.827

2.828

10,000Unknown

1.035

1.048

1.042

1737

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.

OD 450nm

101

0.1
100

400

1000

4000

10000

CRP (ng/ml)

PERFORMANCE CHARACTERISTICSSENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting concentration ofthe mean OD of Standard A (based on 10 replicate analyses) plus 2 SD. Therefore, the sensitivity of the hs-CRP
ELISA kit is 10 ng/ml.
SPECIFICITY (CROSS REACTIVITY)
The specificity of the hs-CRP ELISA kit was determined by measuring the apparent CRP value of samples spiked
with the following compounds:
Substance
Apparent CRP
Value (ng/ml)
Human Albumin
Not Detected
Human Globulin
Not Detected
INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in ng/ml) are tabulated
below:SampleMeanSD
CV%
1
205.8
31.2
15.2
2769.238.4
5.038437.8
700.4
8.3
INTER-ASSAY PRECISION
Three samples were assayed ten times over a period of four weeks. The results (in ng/ml) are tabulated below:
Sample
123

Mean
227.0
1022.28791.8

SD
22.4
97.2
685.8

CV%
9.9
9.5
7.8

RECOVERY
Spiked samples were prepared by adding defined amounts of CRP to three serum samples. The results (inng/ml) are tabulated below:
Sample1 Unspiked+358+1430+5720
2 Unspiked+358+1430+5720
3 Unspiked+358+1430+5720

Version: 6.0-rObs. Result26376018206520
1352
1880
3020
7720
5546
6107
6169
10400

Exp. Result
621
1693
5983
1710
2782
7072
5904
6976
11266

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Recovery %
122.4
107.5
109.0
109.9
108.6
109.2
103.4
88.4
92.3

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LINEARITY
Three serum samples were diluted with Standard A. The results (in ng/ml) are tabulated below:
Sample
1
1:5
1:25
1:50
2
1:4
1:16
1:64
3
1:4
1:16
1:64

Obs.
Result
3662
894
136
62
6120
1922
428
110
8800
2472
614
148

Exp. Result

Recovery %

732.4
146.5
73.2
1530
382.5
95.6
2200
550
137.5

122.1
92.8
84.7
125.6
111.9
115.0
112.4
111.6
107.6

HIGH DOSE HOOK EFFECTThe hs-CRP ELISA kit did not experience a high dose hook effect when it was tested up to a CRP concentrationof 160,000 ng/ml.
EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values. All values are inng/ml.
MalesFemales Combined
N
43
45
88
Age
17-87
12-79
12-87
Abs.
733434-63,680
Range
63,68039,2402.5th132
139
135
Percentile
50th1197
1033
1104
Percentile
97.5th9710
6578
8910
Percentile
REFERENCES
1.
Wilkins J, et al., Rapid Automated High Sensitivity Enzyme Immunoassay of C-reactive Protein. Clin
Chem. 1998; 44(6 Pt 1): 1358-61.
2.
Ledue TB,et al. Analytical Evaluation of Particle- enhanced Immunonephelometric Assay for C-reactive
Protein, Serum Amyloid A and Mannose-binding Protein in Human Serum. Ann. Clin. Biochem. 1998; 35(Pt6): 745-53.
3.
Macy EM, et al. Variability in the Measurement of C-reactive Protein in healthy Subjects: Implications forReference Intervals and Epidemiological Aplications. Clin. Chem. 1997;43(1):52-8.
4.
Käpyaho K, et al. Rapid Determination of C-reactive Protein by Enzyme Immunoassay Using Two
Monoclonal Antibodies. Scand. J. Clin. Lab. Invest. 1989; 49(4):389-93.
5.
Eda S, et al. A New Method of Measuring C-reactive Protein, with a Low Limit of Detection, Suitable for
Risk Assessment of Coronary Heart Disease. Scand. J. Clin. Lab. Invest. 1999; 230:32-5
6.
Borque L, et al. Development and Validation of an Automated and Ultrasensitive Immunoturbidimetric
Assay for C-reactive Protein, Clin Chem. 2000; 46(11):1839-42.
7.
Roberst WL, et al. Evaluation of Nine Automated High-Sensitivity C-reactive Protein Methods:
Implications for Clinical and Epidemiological Applications. Part 2. Clin. Chem. 2001; 47(3):418-25.
8.
Rifai N, Ridker PM. High-sensitivity C-reactive Protein: a Novel and Promising Marker of Coronary Heart
Disease. Clin. Chem. 2001; 47:403-11
9.
Libby P, Ridker PM. Novel Inflammatory Markers of Coronary Risk: Theory Versus Practice. Circulation.
1999; 100:1147-50.
10.
Ross R. Atherosclerosis – An Inflammatory Disease. N Engl. J. Med. 199; 340(2):115-26.
11.
Libby P. Molecular Bases of the Acute Coronay Syndromes. Circulation. 1995; 91:2844-50
12.
Ridker PM, et al. C-reactive Protein and Other Markers of Inflammation in the Prediction of Cardiovasculardisease in Women. N. Engl J Med. 2000; 342:826-43.
13.
Ridker PM, et. al. Plasma Concentration of C-reactive Protein and Risk of Developing Peripheral Vascular
Disease. Circulation. 1998; 97:425-8.

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Symbols:

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Storagetemperature

Manufacturer

Expiry dateBatch code

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for useContentCautionCatalogue
numberEffective: 2018-12-12

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