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NT-proCNP ELISA

Cat. No.: BI-20812

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Therapeutic Areas
edta pl
heparin pl
app h pop
CNP structure
NT-proCNP standard curve
F T of samples
sampl matrix
Screenshot from 2019-03-22 15-36-38
Assay Characteristics
  • Category number

    BI-20812

  • Method

    Sandwich ELISA, HRP/TMB, 12×8-well detachable strips

  • Sample type

    Serum, EDTA plasma, citrate plasma, heparin plasma, urine, cell culture supernatant, non-human samples (rat, mouse, pig)

  • Assay time

    20 min / 3 h / 30 min

  • Sample volume

    20 µl / well

  • Sensitivity

    0.7 pmol/l (= 90 pg/ml)

  • Standard range

    0 – 128 pmol/l (= 0 – 638 pg/ml)

  • Conversion factor

    1 ng/ml = 0.201pmol/l (MW: 4.985kDa)

  • Specificity

    This assay recognizes recombinant and endogenous NT-proCNP.

  • Precision

    In-between-run (n=8): ≤ 7 % CV

    Within-run (n=5): ≤ 6 % CV

  • Validation Data

    See validation data tab for: precision, accuracy, diltuion linearity, values for healthy donors, etc.

  • Detection target

    Recombinant and endogenous NT-proCNP.

Product details

Product overview

The NT-proCNP immunoassay is a 4 hour, 96-well sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of NT-proCNP in human serum, plasma, urine, cell culture supernatants and non-human samples (rat, mouse, pig).

Principle of the Assay

This kit is a sandwich enzyme immunoassay for the quantitative determination of NT-proCNP in human serum, plasma, urine, cell culture supernatants and non-human samples (rat, mouse, pig).

The figure below explains the principle of a sandwich ELISA:

In a first step, assay buffer and standard/control/sample are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal sheep anti-NT-proCNP antibody, for a short incubation. Without the need of a washing step, conjugate (sheep anti-human NT-proCNP-HRP) is added into the wells. NT-proCNP present in the standrad/sample/control binds to the precoated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material is removed. After a final washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of NT-proCNP present in the sample. This color change is detectable with a standard microtiter plate ELISA reader.

The NT-proCNP ELISA kit uses highly purified, epitope-mapped antibodies. The antibodies utilized in the NT-proCNP ELISA (BI-20892) are as follows:

- Capture antibody: polyclonal sheep anti-NT-proCNP (epitope: AA62-68 of Uniprot ID P23582)

- Detection antibody: polyclonal sheep anti-NT-proANP (epitope: AA34-37 of Uniprot ID P23582)

Typical Standard Curve

The figure below shows a typical standard curve for the NT-proCNP ELISA. The immunoassay is calibrated against recombinant NT-proCNP peptide:



Kit Components

CONT

 KIT COMPONENTS

QUANTITY

PLATE

Polyclonal sheep anti-NT-proCNP antibody pre-coated microtiter strips in strip holder, packed in an aluminum bag with desiccant

12 x 8 tests

WASHBUF

Wash buffer concentrate 20x, natural cap

1 x 50 ml

STD

Standards 1-7, (0; 4; 8; 16; 32; 64; 128 pmol/l), recombinant NT-proCNP in human serum, white caps, lyophilized

7 vials

CTRL

Control A + B, yellow caps, lyophilized, exact concentrations see labels

2 vials

ASYBUF

Assay buffer, red cap, ready to use

1 x 8 ml

CONJ

Conjugate (polyclonal sheep anti-NT-proCNP-HRP), amber cap, ready to use

1 x 7 ml

SUB

Substrate (TMB solution), amber bottle, blue cap, ready to use

1 x 13 ml
STOP

Stop solution, white cap, ready to use

  1 x 7 ml

Storage instructions: All reagents of the NT-proCNP ELISA kit are stable at +4°C (+2-8 °C) until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage

Serum, EDTA plasma, heparin plasma, citrate plasma, cell culture supernatant and urine are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
 

Serum & Plasma

 

Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparin or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Do not freeze-thaw samples more than five times. 

 

Urine

 

Note: the experiments performed to measure NT-proCNP in urine samples did not undergo a full validation according to ICH guidelines. However, our performance check suggests that urine samples can be measured with this ELISA. For more information please see our validation data tab.

Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at -25°C or lower.

 

Cell Culture Supernatant

 

Note: the experiments performed to measure NT-proCNP in urine samples did not undergo a full validation according to ICH guidelines. However, our performance check suggests that urine samples can be measured with this ELISA. For more information please see our validation data file.

Remove particulates by centrifugation and assay immediately or aliquot and store samples at
-25°C or lower. Do not freeze-thaw samples more than five times.

Reagent Preparation

Wash Buffer

 

1.

Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature.

2.

Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay.

The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
 

Standards for Serum & Plasma Measurements

 

1.

Pipette 300 µl of distilled or deionized water into each standard (STDs) and control (CTRL) vial. The exact concentration is printed on the label of each vial.

2.

Leave at room temperature (18-26°C) for 15 min. Vortex gently.

Reconstituted STDs and CTRLs are stable at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for three freeze-thaw cycles.

Standards for Cell Culture Supernatant Measurements

 

For the preparation of cell culture-based standards always use the identical cell culture medium (CCM) as used for the experiment.

1.

Reconstitute standard 7 (STD7) in 300 µl deionized water. Leave at room temperature (18-26°C) for 15 min and mix well before making dilutions. Use polypropylene tubes.

2.

Mark tubes ccSTD6 to ccSTD1. Dispense 100 µl cell culture medium into each vial.

3.

Pipette 100 µl of STD7 into tube marked as ccSTD6. Mix thoroughly.

4.

Transfer 100 µl of ccSTD6 into the tube marked as ccSTD5. Mix thoroughly.

5.

Continue in the same fashion to obtain ccSTD4 to ccSTD2. CCM serves as the ccSTD1 (0 pmol/l NT-proCNP).

6.

Using the prepared standards, follow the protocol as indicated for serum, plasma and urine samples.

Attention: Supplied STD1-STD7 and controls are only valid for serum and should not be used for cell culture measurements.
 

Sample Preparation

Bring samples to room temperature and mix samples gently to ensure the samples are homogenous.
We recommend duplicate measurements for all samples.

Samples for which the OD value exceeds the highest point of the standard range can be diluted 1+1 with ASYBUF (assay buffer).  

Assay Protocol

Read the entire protocol before beginning the assay.

1.

Bring reagents and samples to room temperature (18-26°C). 

2.

Mark position for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. 

3.

Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until the expiry date stated on the label.

4.

Pipette 50 µl ASYBUF (assay buffer, red cap) into each well.

5.

Add 20 µl STD/CTRL/SAMPLE into the respective wells. Swirl gently.

6.

Cover the plate tightly and incubate for 20 minutes at room temperature.

7.

Add 50 µl CONJ (conjugate) into each well, swirl gently.

8.

Cover tightly and incubate for 3 hours at room temperature in the dark.

9.

Aspirate and wash wells 5x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel.

10.

Add 100 µl SUB (substrate, blue cap) into each well.

11.

Incubate for 30 min at room temperature in the dark.

12.

Add 50 µl STOP (stop solution, white cap) into each well, swirl gently.

13.

Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Calculation of Results

Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (reference wavelength 630 nm). Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.

Obtain sample concentrations from the standard curve. If required, pmol/l can be converted into pg/ml by applying a conversion factor (1 pg/ml = 0.201 pmol/l (MW: 4.985 kDa)). Respective dilution factors have to be considered when calculating the final concentration of the sample.

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit at production date. Data for OD obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.50 or more is obtained for the STD7 and the value of the CTRL is in range (target range see label).

Background & Therapeutic Areas

INFORMATION ON THE ANALYTE

NT-proCNP Protein

C–type natriuretic peptide (CNP) is a paracrine growth factor widely expressed in various tissues. The 103-amino acid propeptide is cleaved either between residues 50 and 51 or 81 and 82 to produce one of two biologically active peptides, carboxy-terminal proCNP (51–103) or carboxy-terminal proCNP (82–103), respectively, and an amino-terminal congener, N-terminal proCNP.



From: Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions. Potter LR et al., 2006; Endocrine Reviews 27(1):47–72. doi: 10.1210/er.2005-0014

Molecular Weight

4.985-kDa

Cellular localisation

Secreted

Post-translational modifications

Glycosylation, disulphide bonds

Sequence similarities

Natriuretic peptide family

Alternative Names

CNP2, CNP, Natriuretic Peptide Precursor Type C, C-type Natriuretic Peptide, Natriuretic Peptide C

Pubchem ID

4990 link: https://www.ncbi.nlm.nih.gov/gene/4880

Genecards

NPPC link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=NPPC

PDB

1JDP: https://www.uniprot.org/uniprot/P23582

OMIM

600296 link: https://www.omim.org/entry/600296

Protein Atlas

NPPC link: https://www.proteinatlas.org/ENSG00000163273-NPPC/tissue

Uniprot ID

P23582 link: https://www.uniprot.org/uniprot/P23582

NT-proCNP Function

C–type natriuretic peptide (CNP) is a paracrine growth factor widely expressed in tissues, including the vascular endothelium, where it is considered to provide vasoprotective functions. In endothelial cells and macrophages, it is secreted in response to several stimuli, including inflammatory mediators. CNP is rapidly degraded in tissues and negligible quantities enter the circulation. However, the N-terminal portion of the pro-hormone is not degraded at source and circulates in significantly higher concentrations than CNP. Therefore NT-proCNP is a valuable biomarker to determine CNP synthesis in tissues. CNP plays a critical role in linear growth. It is produced in the growth plate and signals through a paracrine mechanism. Recent studies have shown that the plasma concentrations of NT-proCNP correlate with linear growth velocity in all phases of skeletal growth and increase during rhGH (growth hormone) therapy (Olney et al., 2016). Furthermore, serum NT-proCNP levels increased after initiation of GH treatment in patients with achondroplasia/ hypochondroplasia (Kubota et al., 2016). Women with pregnancy complications, such as diminished fetal growth and pre-eclampsia show significantly increased NT-proCNP levels early in gestation (Espiner et al., 2015; Reid et al., 2014). NT-proCNP concentration at hospital admission has sufficient sensitivity and specificity to differentiate naturally occurring sepsis from non-septic systemic inflammatory response syndrome (SIRS) (Koch et al., 2012; Tomasiuk et al., 2014). Recently, Prickett and colleagues demonstrated in a cohort of over 2000 individuals, that in contrast to the close association of NT-proBNP with cardiac function, and predictive value for outcome after myocardial infarction, plasma NT-proCNP is highly correlated with renal function and is an independent predictor of mortality and cardiac readmission in individuals with unstable angina (Volpe et al., 2015).

Validation Data

All Biomedica ELISAs are validated according to international FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.

Calibration

This immunoassay is calibrated against synthetic human propeptide of CNP (AA 24-73 of Uniprot ID P23582, i.e. NT-proCNP 1-50).

Detection Limit & Sensitivity

To determine the sensitivity of the human total NT-proCNP ELISA, experiments measuring the lower limit of detection (LOD) and the lower limit of quantification (LLOQ) were conducted.

The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of NT-proCNP, with a confidence level of 99%. It is defined as the mean back calculated concentration of NT-proCNP-free sample (three independent measurements) plus three times the standard deviation of the measurements.

The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. The lowest concentration of NT-proCNP, which meets both criteria, is reported as the LLOQ.

The following values were determined for the NT-proCNP human ELISA:

LOD

0.7 pmol/l

LLOQ

0.5 pmol/l

Precision

The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).
 

Within-Run Precision

 
Samples of known concentration were tested five times to assess within-run precision, also called intra-assay precision. The experiment was performed by one operator using a single plate.

 

Within-Run Precision

Sample

#1

#2

n

5

5

Mean NT-proCNP [pmol/l]

7.9

65.3

Standard deviation 

0.47

1.25

% CV

6

2

 
In-Between-Run Precision

 
Samples of known concentration were tested eight times to assess in-between-run precision, also called inter-assay precision. The experiment was performed by multiple operators using plates and kit components from two lots.

 

Within-Run Precision

Sample

#1

#2

n

8

8

Mean NT-proCNP [pmol/l]

8.2

64.1

Standard deviation 

0.54

1.42

% CV

7

2

Accuracy

The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.

The recovery of the total NT-proCNP ELISA was measured by adding recombinant NT-proCNP to human samples containing a known concentration endogenous NT-proCNP. The %recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.

This table shows the summary of the recovery experiments in the NT-proCNP ELISA in different sample matrices:

   

% Recovery

Sample Matrix

n

+12.8 pmol/l

+64 pmol/l

Mean

Range

Mean

Range

Serum

6

 102 91-115 101 96-105

EDTA plasma

6

 99 80-116 99 94-101

Citrate plasma

2

 100 94-106 100 97-103

Heparin plasma

2

 92 90-94 93 92-95
+ Individual measurements

Data showing recovery of NT-proCNP in human serum samples:

 

NT-proCNP [pmol/l] 

% Recovery

Sample Matrix

ID

Reference

+ 12.8 pmol/l

+ 64 pmol/l

+ 12.8 pmol/l

+ 64 pmol/l

Serum

S1

 32.4 42.3 83.6 103 105

Serum

S2

 34.4 42.6 81.1 91 100

Serum

S3

 20.5 30.7 74.8 95 101

Serum

S4

 24.0 34.4 73.8 100 96

Serum

S5

 28.5 39.6 79.9 109 103

Serum

S6

25.5

37.7

78.6

115

103

 

 

 

 

Mean

 102 101

 

 

 

 

Min

 91 96

 

 

 

 

Max

 115 105

Data showing recovery of NT-proCNP in human EDTA plasma samples:

 

NT-proCNP [pmol/l] 

% Recovery

Sample Matrix

ID

Reference

+ 12.8 pmol/l

+ 64 pmol/l

+ 12.8 pmol/l

+ 64 pmol/l

EDTA plasma

e1

 29.0 36.3 78.7 80 100

EDTA plasma

e2

 31.5 43.1 78.9 116 99
EDTA plasma

e3

 25.3 35.5 77.2 99 101
EDTA plasma

e4

 28.4 38.7 78.2 103 100
EDTA plasma

e5

 27.7 38.0 73.9 102 94
EDTA plasma

e6

 21.3 30.9 74.4 92 100

 

 

 

 

Mean

 99 99

 

 

 

 

Min

 80 94

 

 

 

 

Max

 116 101

Data showing recovery of NT-proCNP in human heparin plasma samples:

 

NT-proCNP [pmol/l] 

% Recovery

Sample Matrix

ID

Reference

+ 12.8 pmol/l

+ 64 pmol/l

+ 12.8 pmol/l

+ 64 pmol/l

Heparin plasma

h1

 28 37 75 90 95

Heparin plasma

h2

 30 39 74 94 92

 

 

 

 

Mean

 92 93

Data showing recovery of NT-proCNP in human citrate plasma samples:

   

NT-proCNP [pmol/l]

% Recovery

Sample Matrix

ID

Reference

+ 12.8 pmol/l

+ 64 pmol/l

+ 12.8 pmol/l

+ 64 pmol/l

Citrate plasma

c1

24

35

78

106

103

Citrate plasma

c2

29

38

77

94

97
       

Mean

100

100

Dilution Linearity & Parallelism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing NT-proCNP behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted human samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in human samples and provides evidence that the endogenous analyte behaves the same way as the recombinant one. For dilution linearity and parallelism are assessed for each sample type and are considered acceptable if the results are within 20% of the expected concentration.

The table below shows the mean recovery of recombinant NT-proCNP in serum and plasma serially diluted with STD1:

    % Recovery of recombinant NT-proCNP in diluted samples
Sample Matrix n 1+1 1+3 1+7 1+15
Mean  Range Mean  Range Mean  Range Mean  Range
Serum 6 93 89-97 89 78-97 86 78-100 99 89-118
EDTA plasma 6 99 95-102 95 89-102 93 84-101 97 85-111
Heparin plasma 2 102 94-111 100 94-105 92 84-99 93 90-96
Citrate plasma 2 95 94-97 93 89-96 87 84-90 99 98-100

Parallelism was assessed by serially diluting human samples containing endogenous NT-proCNP with STD1, i.e., human serum containing 0 pmol/l NT-proCNP.

The table below shows the mean recovery and range of serially diluted endogenous NT-proCNP in several sample matrices:

    % Recovery of endogenous NT-proCNP in diluted samples
Sample Matrix n 1+1 1+3
Mean  Range Mean  Range
Serum 6 99 96-102 98 93-119
EDTA plasma 6 103 98-110 98 92-104
Heparin plasma 2 96 94-98 92 86-98
Citrate plasma 2 100 98-103 100 97-102
+ Individual measurements

Data showing dilution linearity of endogenous NT-proCNP in human serum samples:

    NT-proCNP [pmol/l] % Recovery
Sample Matrix   Reference 1+1 1+3 1+1 1+3
Serum s1 32.4 15.6 7.6 96 94
Serum s2 34.4 17.2 8.0 100 93
Serum s3 20.5 10.1 4.9 99 94
Serum s4 24.0 11.5 5.6 96 94
Serum s5 28.5 14.1 6.8 99 96
Serum s6 25.5 13.0 7.6 102 119
        Mean 99 98
        Min 96 93
        Max 102
 119

Data showing dilution linearity of endogenous NT-proCNP in human EDTA plasma samples:

    NT-proCNP [pmol/l] % Recovery
Sample Matrix   Reference 1+1 1+3 1+1 1+3
EDTA plasma e1 29.0 14.3 6.9 99 95
EDTA plasma e2 31.5 17.4 8.2 110 104
EDTA plasma e3 25.3 12.8 6.1 101 97
EDTA plasma e4 28.4 13.9 6.5 98 92
EDTA plasma e5 27.7 15.2 7.1 110 102
EDTA plasma e6 21.3 10.7 5.2 101 98
        Mean 103 98
        Min 98 92
        Max 110 104

Data showing dilution linearity of endogenous NT-proCNP in human heparin plasma samples:

    NT-proCNP [pmol/l] % Recovery
Sample Matrix   Reference 1+1 1+3 1+1 1+3
Heparin plasma h1 28.1 13.7 6.8 98 97
Heparin plasma h2 29.7 15.3 7.6 103 102
        Mean 100 100

Data showing dilution linearity of endogenous NT-proCNP in human citrate plasma samples:

    NT-proCNP [pmol/l] % Recovery
Sample Matrix   Reference 1+1 1+3 1+1 1+3
Citrate plasma c1 24.1 11.4 6.9 94 86
Citrate plasma c2 28.8 14.1 5.2 98 98
        Mean 96 92

Specificity

This assay recognizes endogenous (natural) and synthetic human NT-proCNP.

Sample Stability

Sample preparation

Collect venous blood samples by using standardized blood collection tubes for serum or plasma. Perform serum and plasma separation by centrifugation according to supplier’s instructions of the blood collection devices as soon as possible.

The acquired serum or plasma samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower.

 

Freeze-Thaw of Serum Samples Containing Endogenous NT-proCNP

 
A set of samples (3 sera, 3 EDTA plasma, 2 citrate plasma) was aliquoted and freeze-thaw stressed. The reference samples are freeze-thawed once. The mean recovery of sample concentrations stressed by four freeze-thaw (F/T) cycles is 96%.

The table below shows NT-proCNP concentrations of samples after four freeze-thaw cycles:

 

NT-proCNP [pmol/l]

% Recovery

4 F/T vs ref

Sample ID

Reference

2x

3x

4x

#S1

16.0

15.9

16.1

15.6

98

#S2

55.1

52.3

52.2

51.3

93

#S3

21.0

20.5

18.9

20.8

99

#E1

14.2

14.6

14.3

13.3

94

#E2

10.7

10.8

10.4

9.6

90

#E3

15.1

17.4

15.6

16.8

112

#C1

14.6

13.0

13.1

13.4

92

#C2

16.2

15.3

15.6

15.4

95
       

Mean

96

According to our data, samples can undergo at least four freeze-thaw cycles.



Sample Values

NT-proCNP Values in Apparently Healthy Donors

 
Total NT-proCNP reference ranges were established using 24 serum and plasma samples from apparently healthy donors. No medical histories were available for the volunteers.

Sample Matrix

n

Median [pmol/l]

Range [pmol/l]

Serum

32

14.5

1.3 - 3.7

EDTA plasma

33

15.0

1.0 - 3.1

Heparin plasma

18

13.5

1.1 - 2.7

Citrate plasma

18

12.0

1.3 - 3.2



We recommended establishing the normal range for each laboratory.
 

NT-proCNP Values in an Unselected Hospital Panel

 
NT-proCNP was measured in plasma samples of patients from an unselected hospital panel:

Cohort

Sample Matrix

n

Median NT-proCNP [pmol/l]

Range [pmol/l]

Unselected hospital panel

Citrate plasma

8

14

9 - 17

Unselected hospital panel

EDTA plasma

8

19

15 - 26

Unselected hospital panel

Heparin plasma

8

16

11 - 28

The figures below show the distribution of NT-proCNP concentrations in an unselected hospital panel compared to samples from apparently healthy donors:

Matrix Comparison

Human NT-proCNP sample concentrations were compared between serum, EDTA plasma, and citrate plasma from an apparently healthy cohort (n=16):

 

NT-proCNP [pmol/l]

Donor ID

EDTA plasma

Citrate plasma

Heparin plasma

Serum

#1

17

14

16

20

#2

17

16

18

21

#3

13

11

12

16

#4

14

12

12

15

#5

16

17

18

22

#6

22

19

21

24

#7

15

13

14

19

#8

14

12

13

16

#9

15

11

12

16

#10

11

9

9

14

#11

24

20

22

28

#12

14

11

12

15

#13

13

10

10

14

#14

10

8

8

11

#15

28

23

26

34

#16

29

25

27

34

The measurements show that values of human NT-proCNP are higher in serum compared to plasma. In previous studies, it was shown that sample values for serum and plasma can differ for the measurement of chemistry analytes.

 

NT-proCNP [pmol/l]

% CV

Donor ID

Serum

EDTA plasma

Citrate plasma

Heparin

plasma

All matrices

Only plasma

#1

2.3

1.6

1.8

2.3

16

16

#2

2.6

1.8

1.9

2.4

15

11

#3

2.0

1.1

1.5

1.7

22

19

#4

1.9

1.2

1.3

1.7

19

15

#5

2.0

1.6

1.8

1.9

8

6

#6

3.7

2.6

2.7

3.2

14

8

#7

1.5

1.2

1.2

1.4

8

6

#8

1.5

1.0

1.4

1.6

16

19
       

Mean

15

13

Graph showing NT-proCNP levels dependence of sample concentrations on sample matrix:


 

Measurement of NT-proCNP in Cell Culture Supernatant

Note: the experiments performed to measure NT-proCNP in cell culture supernatants are not a full validation but are merely a performance check.

Cell culture medium (ccm: RPMI1640 containing 10% fetal calf serum) was tested undiluted and spiked with a final concentration of 64 pmol/l synthetic human NT-proCNP. The spiked solution was diluted 1+1, 1+3, 1+7 with the cell culture medium.

As a comparison, the spike recovery and dilution linearity of the standard matrix (=STD1) and the dilutions with assay buffer is shown.

OD values of spiked and diluted cell culture medium sample and standard matrix (STD1):

 

 

 OD

Dilution medium

Sample ID

Reference

+ 64 pmol/l

1+1

1+3

1+7

1+15

ccm

ccm

0.028

1.355

0.627

0.290

0.125

0.072

ASYBUF

STD1

0.031

1.271

0.601

0.318

0.156

0.090

Graph showing dilution of cell culture medium (ccm) and a comparison to the Standard (STD1), both spiked with the same amount of synthetic NT-proCNP (64 pmol/l).



Measurement of NT-proCNP in Urine

The ELISA has not been fully validated for the measurement of human NT-proCNP in urine samples but can be used for this sample matrix.

Undiluted human urine samples from apparently healthy subjects and from patients with kidney disease were tested.

Summary


Urine samples (n=48) were assayed with the Biomedica NT-proCNP ELISA (BI-20812) following the standard protocol using undiluted urine.

- Endogenous NT-proCNP was detectable in samples from kidney patients.

- Urine samples can be spiked – the average recovery of 8 human urine samples is 109%.

- If required, dilute urine samples 1+1 with assay buffer. Dilution linearity of samples containing high values of endogenous NT-proCNP (n=2) with assay buffer is 103%.

Specificity was assessed by adding the coating antibody utilized in the NT-proCNP ELISA assay to urine samples containing elevated endogenous NT-proCNP levels (n=2). The samples showed a competition of 93%.
 

Recovery

 
Recovery was assessed by adding STD7 (final concentration 64 pmol/l synthetic human NT-proCNP) directly to eight different human urine samples (ratio 1+1).

Data showing spike/recovery of human urine samples:

 

NT-proCNP c [pmol/l]

 

Sample ID

Reference

+ 64 pmol/l

% Recovery

#U1

1

68

106

#U2

59

111

128

#U3

1

67

105

#U4

1

67

104

#U5

0

63

98

#U6

80

116

118

#U7

0

64

100

#U8

1

70

109
   

Mean

109

Linearity

 
Dilution linearity was assessed by diluting urine samples containing endogenous NT-proCNP with ASYBUF (assay buffer supplied in the kit).

Data showing the dilution of endogenous NT-proCNP in urine samples:

 

NT-proCNP [pmol/l]

% Recovery

Sample ID

Reference

1+1

#U1

69

33

95

#U2

92

51

111
   

Mean

103

Competition

 
Specificity was assessed by adding the coating antibody utilized in the NT-proCNP ELISA assay to urine samples containing elevated endogenous NT-proCNP levels. 

Data showing the competition of the signal:

 

NT-proCNP [pmol/l]

  

Sample ID

Reference

Competition

% Recovery

#U1

69

6

91

#U2

92

4

96
   

Mean

93

Comparison of panels from various donors:

Cohort

n

Median NT-proCNP [pmol/l]

Range [pmol/l]

Apparently healthy

4

1

1 - 2

Kidney cohort I

24

1

0 - 92

Kidney cohort II

20

0

0 - 2

Measurement of NT-proCNP in Non-Human Samples

The sequence homology of NT-proCNP is very conserved among different species. We therefore assessed if the assay which is validated for human samples can be used in rat, mouse and pig samples. Other species types that share a high homology to human NT-proCNP can likely be measured with this assay (e.g. monkey, cats, dogs).

Note: the experiments performed for non-human species are not a full validation but are merely a performance check.

Uniprot ID

Species

% Sequence homology

P23582

C-type natriuretic peptide Homo sapiens (Human)

100

H2QJL6

PANTR - Uncharacterized protein - Pan troglodytes (Chimpanzee)

98

H2P8X1

PONAB - Uncharacterized protein - Pongo abelii (Sumatran Orangutan)

98

M3WH43

FELCA - Uncharacterized protein - Felis catus (Cat)

96

E2R4X2

CANLF - Uncharacterized protein - Canis lupus family

96

P18104

PIG - C-type natriuretic peptide Sus scrofa (Pig)

94

P55207

RAT - C-type natriuretic peptide - Rattus norvegicus (Rat)

92

Q9D288

MOUSE - Putative uncharacterized protein Mus musculus (Mouse)

86

Q544K5

MOUSE - C-type natriuretic peptide (Mus musculus)

86

Q61839

MOUSE - C-type natriuretic peptide Mus musculus (Mouse)

86

A0A1L1WKH8

RAT - Natriuretic peptide C - Rattus norvegicus

93

Measurement of NT-proCNP in Rat Samples

Rat NT-proCNP shares a 92% homology to human NT-proCNP.

According to our data, rat serum samples (n=8) showed a recovery of 97% and a linearity of 87%. The samples tested contained endogenous NT-proCNP concentrations between 9-38 pmol/ml. Competition of endogenous NT-proCNP concentrations from rat samples is 100%.

 

Linearity

 
Eight undiluted rat serum samples were tested in the NT-proCNP ELISA (note: standards contain synthetic human NT-proCNP spiked in a human serum matrix).

For linearity experiments, rat samples were diluted 1+1 with ASYBUF. The table below shows the recoveries relative to the expected concentrations of eight rat samples:

 

NT-proCNP

 

Sample ID

+ 64 pmol/l

% Recovery

#R1

68

94

#R2

76

95

#R3

76

95

#R4

68

99

#R5

78

101

#R6

81

97

#R7

74

97

#R8

66

97

 

Mean

97

Recovery

 
For recovery experiments, STD7 was added to the rat serum samples in a ratio 1+1 (final concentration 64 pmol/l).

The table below shows the calculated concentrations and spike recovery of eight rat samples:

 

NT-proCNP [pmol/l]

 

Sample ID

Reference

1+1

% Recovery

#R1

16

8

95

#R2

32

13

83

#R3

31

11

72

#R4

10

5

101

#R5

26

11

84

#R6

38

15

79

#R7

23

10

85

#R8

9

4

93
   

Mean

87

Competition

 
Specificity was assessed by adding the coating antibody utilized in the human NT-proCNP ELISA assay to the rat serum samples.

Data showing the competition of the signal:

 

NT-proCNP [pmol/l]

 

Sample ID

Reference

+ Coating AB

% Competition

#R1

15

0

100

#R2

28

0

100

#R3

29

0

100

#R4

10

0

100

#R5

27

0

100

#R6

34

0

100

#R7

22

0

100

#R8

9

0

100
   

Mean

100

Measurement of NT-proCNP in Mouse Samples

Mouse NT-proCNP shares an 86% homology to human NT-proCNP.

According to our data, mouse serum samples (n=8) showed an average recovery of 45% when spiked with STD7 in a ratio 1+1 (final concentration 64 pmol/l). The mouse samples tested did not contain endogenous NT-proCNP concentrations. Linearity was not tested.

Measurement of NT-proCNP in Pig Samples

Pig NT-proCNP shares a 94% homology to human NT-proCNP.

According to our data, pig serum samples (n=8) showed a recovery of 83%. The pig samples tested did not contain endogenous NT-proCNP concentrations. Linearity was not tested.

 

Recovery

 

Eight undiluted pig serum samples were tested in the NT-proCNP ELISA (note: standards contain synthetic human NT-proCNP spiked in a human serum matrix).

For Recovery experiments: STD7 was added to the pig serum samples in a ratio 1+1 (final concentration 64 pmol/l).

Calculation of pig sample concentrations, and spike recovery:

 

NT-proCNP [pmol/l]

 

Sample ID

Reference

+ 64 pmol/l

% Recovery

#P1

0

58

91

#P2

0

57

89

#P3

0

55

85

#P4

0

55

86

#P5

0

52

81

#P6

0

51

80

#P7

0

48

75

#P8

0

50

78
   

Mean

83

Comparison of NT-proCNP ELISA BI-20812 (2nd generation) vs BI-20872

The two NT-proCNP assays show a poor correlation due to the use of different reagents (e.g standard material, highly purified antibodies, and buffer solutions).

Citations

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