1

Human Monomeric CRP (mCRP) ELISA
Cat. No.: BA1026
Enzyme Immunoassay for the quantitative determination of monomeric CRP(mCRP) in human serum and plasma.
C-reactive protein (CRP) is a multipotent protein that undergoes
conformational changes between circulating native pentameric CRP (pCRP),pentameric symmetrical forms (pCRP*) and monomeric CRP (mCRP) forms.
mCRP exhibits strong pro-inflammatory activity and activates platelets,
leukocytes, and endothelial cells. Abundant deposition of mCRP in inflamedtissues plays a role in several disease conditions, such as
ischemia/reperfusion injury, Alzheimer’s disease, and cardiovascular disease.
Conversion of pCRP to mCRP induces inflammatory signalling, monoacyl
phosphatidylcholine generated by PLA2, or by oxidation lipid acyl chains,promotes binding and dissociation of pCRP to mCRP. mCRP gains functionallyactive neoepitopes that carry out highly pro-inflammatory and prothrombotic features, thus mCRP can bind cholesterol and enter plasmatic
membrane and activate pro-inflammatory responses.
Deposition of mCRP, which has a much lower aqueous solubility than pCRP,
has been shown in the brain in infarcted areas of Alzheimer diseases patientsand in regions with amyloid burden, in atherosclerotic plaques in vasculardisease and in other foci of inflammatory tissue injuries.
PRINCIPLE OF mCRP ELISA
The microtiter plate is coated with the antibody specifically binding themCRP. The human serum and plasma are incubated in the plate with thecapture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm) andare proportional to the concentration of mCRP in the specimen. The
concentration of mCRP in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

KIT CONTENTSItem
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution

Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

MATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves and protective
clothing
The substrate solution should remain colourless, keep it protectedfrom light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS••Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human mCRP Master Standard in Dilution Buffer, forthe volume information see the Certificate of Analysis. Let it rehydrate for 15
min The concentration of human mCRP Master Standard is
80 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard to produce a dilution series (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves as Blank.
Volume of Standard

v_02

Std1

Standard 80 ng/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2300 µL of Std1300 µL

40 ng/mL

Std3300 µL of Std2300 µL

20 ng/mL

Std4300 µL of Std3300 µL

10 ng/mL

Std5300 µL of Std4300 µL

5 ng/mL

Std6300 µL of Std5300 µL

2.5 ng/mL

Std7300 µL of Std6300 µL

1.25 ng/mLBlank-300 µL0 ng/mL

80 ng/mL23.

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls indeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:2 prior to use,
see Preparation of samples.

Accuracy

Dilution linearity
SampleDilution

Measuredconcentration(ng/mL)
39.4
18.7
9.5
4.7
18.6
96.5
46.6
22.6

Expected
concentration(ng/mL)
196.6
98.5
49.2
92.9
46.4
23.2

Yield(%)

Measuredconcentration(ng/mL)
3.27
20.0
11.6
7.2

Expected
concentration(ng/mL)
23.3
13.3
8.3

Yield(%)

1
Preparation of Wash Buffer 1x2x4x8x

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at
4oC for two weeks or at -20oC for long term storage.

22x4x8xPreparation of samples
Human serum and plasma may be used with this assay. For long-term storage
the samples should be frozen at minimum -70oC. Lipemic or haemolyticsamples may cause false results.
Recommended dilution of samples is 1:2, i.e., for singlets 80 µL of sample +80 µL of Dilution Buffer, for duplicates 150 µL of samples + 150 µL of Dilution
Buffer, respectively.

Spiking Recovery
SampleSpike(Ng/mL)
120
105ASSAY PROCEDURE
Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate forOVERNIGHT at 4oC ±2oC, NO shaking.
3. Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well. Incubatefor 1 hour at 25oC ±2oC, shaking at 350 rpm.
5. Wash the wells 5-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for 30
min at 25oC ±2oC, shaking at 300 rpm.
7. Wash the wells 5-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
8. Pipette 100 µL Substrate solution, incubate for 30 min
at 25oC ±2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450 or 450/630 nm within 15 min.

The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Human mCRP Standard Curve
43.5

31.5
11.25

2.5

5

10204080

Concentration (ng/mL)

Sensitivity

The limit of detection, defined as a concentration of human MCRP giving
absorbance higher than absorbance of blank + 3 standard deviations, is better
than 0.63 ng/mL of sample.

Intra-assay
Sample1220Samples used in the tests were diluted 1:2 as recommended and assayed. Theresults are multiplied by the dilution factor.

2.

2.5

0.5

PERFORMANCE CHARACTERISTICS

1.

868887

Typical standard curve

Absorbance (450/630 nm)

1.
2.

95
96
96
104
10097

POPULATION AND CLINICAL DATA
Typical mCRP values in human serum were obtained with BioLab Assays
Human Monomeric CRP (mCRP) ELISA kit.

Precision

Individuals with hsCRP 1-5mg/L
Individuals with pancreatic cancer

Mean (ng/mL)
2.1
2.4

SD
0.1
0.2

CV (%)
4.3
8.1

Inter-assay (Run – to – run)
SampleMean (ng/mL)
146.1
28.8

SD
6.7
0.4

CV (%)
14.55.0

Individuals with bacterial infection,
CRP over 50mg/L

1.2-4.8 ng/mL (median 2.6ng/mL)
18.3 – 73.9 ng/mL (median 36.5ng/mL)
24.1 – 98.3 ng/mL (median 47.7ng/mL)

According to the health status, mCRP next to mCRP/pCRP or mCRP/hsCRP
ratio should be taken into consideration to evaluate a risk value.
RESOURCE
1

Garcia-Lara E, Aguirre S, Clotet N, Sawkulycz X, Bartra C, Almenara-Fuentes L, et al.
Antibody Protection Against Long-Term Memory Loss Induced by Monomeric C-Reactive
Protein in a Kouse Model of Dementia. Biomedicines 9(7):828 (2021)

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