NT-proBNP ELISA kit (Human NT-proBNP) | SK-1204

Product details

Product overview
Principle of the Assay

Typical Standard Curve
ELISA kit Components

NT-proBNP ELISA Product Overview

The NT-proBNP ELISA kit is a 3.5 hour, 96-well sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of NT proBNP in human serum and EDTA plasma.

NT-proBNP ELISA Assay Principle

The NT pro BNP ELISA is a sandwich enzyme immunoassay for the quantitative determination of NT-proBNP in human serum and EDTA plasma.

The figure below explains the principle of a sandwich ELISA:

In a first step, sample and conjugate (sheep anti-human NT-proBNP-HRP) are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal sheep anti-NT-proBNP antibody. NT-proBNP present in the sample binds to the pre-coated antibody in the well and forms a sandwich with the conjugate (detection antibody). In the washing step all non-specific unbound material is removed. In a second step, the substrate (Tetramethylbenzidine; TMB) is pipetted into the wells. The enzyme catalyzed color change of the substrate is directly proportional to the amount of NT-proBNP present in the sample. This color change is detectable with a standard microtiter plate ELISA reader.

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

Capture antibody: AA 32-57 (polyclonal sheep anti-human NT-proBNP)
Detection antibody: AA 8-29 (HRP-labeled polyclonal sheep anti-human NT-proBNP)

NT-proBNP ELISA Typical Standard Curve

The figure below shows a typical standard curve for the human NT proBNP ELISA. The immunoassay is calibrated against recombinant NT-proBNP (1-76) peptide:

NT-proBNP ELISA Kit Components

Contents	Description	Quantity
PLATE	Polyclonal sheep anti-human NT-proBNP antibody pre-coated microtiter strips in a strip holder, packed in an aluminum bag with desiccant	12 x 8 tests
WASHBUF	Wash buffer concentrate 20x, clear cap	1 x 50 ml
STD	Standards 1-5, recombinant human NT-proBNP (0; 10; 40; 160; 640 pmol/l), white caps, lyophilized	5 vials
CTRL	Controls, yellow caps, lyophilized, exact concentrations see labels	2 vials
CONJ	Conjugate (polyclonal sheep anti-human NT-proBNP-HRP), red dye, brown cap, ready to use	1 x 22 ml
SUB	Substrate (TMB solution), amber bottle, blue cap, ready to use	1 x 22 ml
STOP	Stop solution, white cap, ready to use	1 x 7 ml

Storage instructions: All reagents of the human NT-proBNP 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
Reagent Preparation
Sample Preparation

Assay Protocol
Calculation of Results

Sample Collection & Storage

Serum and EDTA plasma 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 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. NT-proBNP is stable in whole blood at room temperature (18-26°C) for several hours. Nevertheless, we recommend separating serum or EDTA plasma by centrifugation as soon as possible. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or hemolyzed samples may give erroneous results. Do not freeze-thaw samples more than three 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

1.	Pipette 500 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The standard and control concentration are printed on the label of each vial.
2.	Leave at room temperature (18-26°C) for 10 min. Swirl gently.

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

Sample Preparation

Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend dublicate mesurements for all samples. Samples for which the optical density (OD) value exceeds the highest point of the standard range (STD5, 640 pmol/l) can be diluted with STD1 or NT-proBNP negative human serum.

NT-proBNP ELISA 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/SAMPLE/CTRL (Standard/Sample/Control) 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.	Add 50 µl STD/CTRL/SAMPLE in dublicates into the respective wells.
5.	Add 200 µl CONJ (Conjugate) into each well. Swirl gently.
6.	Cover the plate tightly and incubate for 3 hours at room temperature.
7.	Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel.
8.	Add 200 µl SUB (Substrate) into each well.
9.	Incubate for 30 min at room temperature in the dark.
10.	Add 50 µl STOP (Stop solution) into each well, swirl gently
11.	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 = 0.118 pmol/l (MW: 8.475 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 STD5 and the values of the CTRL are in range (target ranges see labels).

Background & Therapeutic Areas

NT-proBNP Protein

BNP is mainly expressed by ventricular myocardium in response to volume overload and increased filling pressure. The BNP gene encodes a 134-amino acid preproBNP precursor, which is converted to 108-amino acid proBNP by the proteolytic cleavage of a 26-amino acid signal peptide (26). ProBNP is further cleaved into the the N-terminal fragment of proBNP (NTproBNP, 1-76) and the C-terminal region biologically active BNP hormone (77-108) (30) also named BNP-32 and additional C-terminal fragments (cf. http://www.uniprot.org/uniprot/P16860#PRO_0000001532; (Kambayashi et al., 1990; Nakagawa et al., 2019; Sudoh et al., 1989).

Molecular weight	8.475 kDa
Cellular localisation	Secreted
Post-translational modifications	Glycosylation, disulphide bond
Sequence similarities	Natriuretic peptide family
Alternative names	BNP, Natriuretic Peptide B, NPPB, BNPT,
Pubchem ID	4879 link: https://www.ncbi.nlm.nih.gov/gene/4879
Genecards	NPPB link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=NPPB&keywords=nppb
PDB	1YK1: https://www.ebi.ac.uk/pdbe/entry/pdb/1YK1 3N56: https://www.ebi.ac.uk/pdbe/entry/pdb/3N56
OMIM	600295 link: https://www.omim.org/entry/600295?search=nppb&highlight=nppb
Protein Atlas	NPPB link: https://www.proteinatlas.org/ENSG00000120937-NPPB/tissue
Uniprot ID	P16960 link: https://www.uniprot.org/uniprot/P16860

NT-proBNP Function

BNP has a key role in cardiovascular homeostasis with biological actions including natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion. A high concentration of BNP in the bloodstream is indicative of heart failure.

Heart and Cardiovascular Disease

Acute myocardial infarction

Cardiac impairment

Risk assessment of rheumatoid arthritis patients under nonsteroidal anti-inflammatory agents/analgesics (NSAIAs)

Congenital heart disease

Coronary artery disease

Heart failure

Secondary hypertension
Metabolic Disease

Diabetes

Obesity
Nephrology

Renal disease

Literature

Atrial and brain natriuretic peptides: Hormones secreted from the heart.
Nakagawa, Y., Nishikimi, T., Kuwahara, K., 2019. Peptides 111, 18–25.
PMID: 29859763

Isolation and sequence determination of human brain natriuretic peptide in human atrium.
Kambayashi, Y., Nakao, K., Mukoyama, M., Saito, Y., Ogawa, Y., Shiono, S., Inouye, K., Yoshida, N., Imura, H., 1990. FEBS Lett. 259, 341–345
PMID: 2136732

Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide.
Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K., Matsuo, H., 1989. Biochem. Biophys. Res. Commun. 159, 1427–1434
PMID: 2522777

Validation Data

Calibration
Detection Limit & Sensitivity
Precision
Accuracy
Dilution Linearity & Parallelism
Specificity

Cross Reactivity
Sample Stability
Sample Values
Matrix Comparison
Comparison with other Assays

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.

Validation literature

1. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology.
2. EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation.
3. Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018

Calibration

This immunoassay is calibrated against recombinant human NT-proBNP (1-76) peptide.

NT-proBNP ELISA Detection Limit & Sensitivity

To determine the sensitivity of the NT-proBNP 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 NT-proBNP 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%. To determine the LLOQ, standard 2, i.e. the lowest standards containing NT-proBNP, is diluted, measured three times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.

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

LOD	3.0 pmol/l
LLOQ	3.3 pmol/l

NT-proBNP ELISA 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

Two samples of known concentration were tested three 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

Mean NT-proBNP [pmol/l]

SD [pmol/l]

CV [%]

Sample 1

60.2

2.0

Sample 2

35.5

0.9

In-Between-Run Precision

Two 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.

In-Between Run Precision

Mean NT-proBNP [pmol/l]

SD [pmol/l]

CV (%)

Sample 1

52.1

1.7

Sample 2

108.1

7.9

NT-proBNP ELISA Accuracy

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

The recovery of the NT-proBNP ELISA was measured by adding recombinant NT-proBNP to human samples containing a known concentration endogenous NT-proBNP. 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-proBNP human ELISA in different sample matrices:

		% Recovery
Sample Matrix	n	+ 80 pmol/l		+ 320 pmol/l
Sample Matrix	n	Mean	Range	Mean	Range
Serum	4	99	93 - 109	108	105 - 113
EDTA plasma	4	94	91 – 97	93	87 - 101

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

		NT-proBNP [pmol/l]			% Recovery
Sample Matrix	ID	Reference	+ 80 pmol/l	+ 320 pmol/l	+ 80 pmol/l	+ 320 pmol/l
Serum	s1	1.6	88.5	347.2	109	108
Serum	s2	0.1	74.2	360.2	93	113
Serum	s3	1.7	80.9	338.7	99	105
Serum	s4	4.4	81.6	346.5	96	107
				Mean	99	108
				Min	93	105
				Max	109	113

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

		NT-proBNP [pmol/l]			% Recovery
Sample Matrix	ID	Reference	+ 80 pmol/l	+ 320 pmol/l	+ 80 pmol/l	+ 320 pmol/l
EDTA plasma	e1	0.8	73.4	276.8	92	87
EDTA plasma	e2	7.7	72.8	279.0	91	87
EDTA plasma	e3	9.3	77.2	324.1	97	101
EDTA plasma	e4	19.1	74.9	309.5	94	97
				Mean	94	93
				Min	91	87
				Max	97	101

NT-proBNP ELISA Dilution Linearity & Parallelism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing NT-proBNP 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. 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 and range of serum and plasma samples spiked with recombinant human NT-proBNP and serially diluted with assay buffer:

		% Recovery of recombinant NT-proBNP in diluted samples
Sample Matrix	n	1+1
		Mean	Range
Serum	3	117	112 - 119
EDTA plasma	2	84	83 - 84

Data showing dilution linearity of recombinant human NT-proBNP in human serum samples:

		NT-proBNP [pmol/l]
Sample matrix	ID	Reference	1+1	% Recovery
Serum	s1	338.7	202.2	119
Serum	s2	360.2	202.4	112
Serum	s3	278.5	165.7	119
			Mean	117
			Min	112
			Max	119

Data showing dilution linearity of recombinant NT-proBNP in human EDTA plasma samples:

		NT-proBNP [pmol/l]		% Recovery
Sample matrix	ID	Reference	1+1	1+1
EDTA plasma	e1	347	145	84
EDTA plasma	e2	277	115	83

Parallelism was assessed by serially diluting human samples containing endogenous NT-proBNP with assay buffer.

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

		% Recovery of endogenous NT-proBNP in diluted samples
Sample Matrix	n	1+1
		Mean	Range
Serum	3	82	79 - 86
EDTA plasma	2	84	83 - 85

Data showing paralellism of endogenous NT-proBNP in human serum samples:

		NT-proBNP [pmol/l]		% Recovery
Sample matrix	ID	Reference	1+1	1+1
Serum	s1	44.2	17.4	79
Serum	s2	51.3	20.4	80
Serum	s3	61.7	26.5	86
			Mean	82
			Min	79
			Max	86

Data showing paralellism of endogenous NT-proBNP in human EDTA plasma samples:

		NT-proBNP [pmol/l]		% Recovery
Sample matrix	ID	Reference	1+1	1+1
EDTA plasma	e1	44.2	17.4	79
EDTA plasma	e2	51.3	20.4	80
			Mean	84

NT-proBNP ELISA Specificity

This assay recognizes endogenous (natural) and recombinant human NT-proBNP (1-76).

Sample Stability

The stability of endogenous NT-proBNP was tested by comparing NT-proBNP measurements in samples that had undergone 3 freeze-thaw cycles.

For freeze-thaw experiments, samples were collected according to the supplier's instruction using blood collection devices and stored at -80°C. Reference samples were freete-thawed once. The mean recovery of sample concentration after 3 freeze-thaw cycles is 94%.

	NT-proBNP [pmol/l]				% Recovery 3 F/T vs ref
Sample ID Serum	Reference	1x	2x	3x	% Recovery 3 F/T vs ref
#S1	70	67	67	69	98
#S2	15	13	15	14	98
#S3	215	185	191	188	87
#S4	141	129	129	126	90
#S5	49	47	48	47	96
#E1	203	191	199	187	92
#E2	282	257	271	248	88
#E3	68	64	79	69	101
				Mean	94

Samples can undergo at least up to 3 freeze-thaw cycles.

Sample Values

NT-proBNP Values in Apparently Healthy Donors

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

Sample Matrix	n	Median [pmol/l]	Median [pg/ml]	Range [pmol/l]
Serum	70	5.8	49.1	1.1 – 541.5
EDTA plasma	28	5.6	47.4	0.6 – 32.0

We recommended establishing the normal range for each laboratory.

NT-proBNP Values in an Unselected Hospital Panel

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

Cohort	Sample Matrix	n	Median NT-proBNP [pmol/l]	Range [pmol/l]
Unselected hospital panel	Serum	117	46.5	0.0 – 621.1
Unselected hospital panel	EDTA plasma	40	28.6	0.3 – 489.2

NT-proBNP Values in Heart Failure Patients

NT-proBNP was measured in serum samples of patients from a heart failure cohort:

Cohort	Sample Matrix	n	Median NT-proBNP [pmol/l]	Range [pmol/l]
NYHA classification II	Serum	43	24.8	1.1 - 75.6
NYHA classification III	Serum	16	56.2	4.7 – 264.3
NYHA classification IV	Serum	7	166.8	84.1 – 399.3

The graph below shows a comparison of a heart failure cohort and apparently healthy donors:

Matrix Comparision

To assess whether plasma and serum matrix behave the same way in the NT-proBNP ELISA, concentrations of NT-proBNP were measured in EDTA-plasma and serum from apparently healthy donors. Each individual donated blood in all tested sample matrices.

Serum and plasma samples showed a correlation of R2=0.98.

The graph below shows the correlation of serum and plasma samples from apparently healthy individuals:

Comparison with other Assays

Comparison Between NT-proBNP Assays: Biomedica SK-1204 Versus Roche Cobas Generation Assay

Performance of the NT-proBNP ELISA was compared with the Roche Cobas Assay.

Data on serum values of a heart failure panel NYHA classification II-IV measured with the Biomedica NT-proBNP ELISA (#SK-1204) are shown below:

n=66	Biomedica pmol/l	Roche pmol/l	Biomedica pg/ml	Roche pg/ml
Mean	47.5	33.9	402.5	287.3
Max	399.3	398.7	3384.0	3378.9
Min	1.1	1.1	9.3	9.3

The figure below shows the correlation between the NT-proBNP ELISA and the Roche Cobas Assay in a heart failure panel NYHA classification II-IV panel:

Data on serum values of a heart failure panel NYHA classification IV measured with the Biomedica NT-proBNP ELISA (#SK-1204)

n=7	Biomedica pmol/l	Roche pmol/l	Biomedica pg/ml	Roche pg/ml
Mean	166.8	130.9	1413.6	1109.2
Max	399.3	398.7	3384.0	3379.0
Min	84.1	28.1	712.7	238.4

The figure below shows the correlation between the NT-proBNP ELISA and the Roche Cobas Assay in a heart failure panel NYHA classification IV panel:

Data on serum values of a heart failure panel NYHA classification III measured with the Biomedica NT-proBNP ELISA (#SK-1204) are shown below:

n=16	Biomedica pmol/l	Roche pmol/l	Biomedica pg/ml	Roche pg/ml
Mean	56.2	44.5	476.2	377.9
Max	264.3	200.1	2239.9	1696.0
Min	4.7	4.3	39.8	36.6

The figure below shows the correlation between the NT-proBNP ELISA and the Roche Cobas Assay in a heart failure panel NYHA classification III panel:

Data on serum values of a heart failure panel NYHA classification II measured with
the Biomedica NT-proBNP ELISA (#SK-1204) are shown below:

n=43	Biomedica pmol/l	Roche pmol/l	Biomedica pg/ml	Roche pg/ml
Mean	24.8	14.0	210.1	119,3
Max	75.6	44.7	640.7	379,1
Min	1.1	1.1	9.3	9,5

The figure below shows the correlation between the NT-proBNP ELISA (#SK-1204) and the Roche Cobas Assay in a heart failure panel NYHA classification II panel:

Citations

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Effect of various weight loss interventions on serum NT-proBNP concentration in severe obese subjects without clinical manifest heart failure. Hollstein T, Schlicht K, Krause L, Hagen S, Rohmann N, Schulte DM, Türk K, Beckmann A, Ahrens M, Franke A, Schreiber S, Becker T, Beckmann J, Laudes M. Sci Rep. 2021 May 12;11(1):10096. doi: 10.1038/s41598-021-89426-7. PMID: 33980890; PMCID: PMC8115663.
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NT-proBNP ELISA | SK-1204

NT-proBNP ELISA Product Overview

NT-proBNP ELISA Assay Principle

NT-proBNP ELISA Typical Standard Curve

NT-proBNP ELISA Kit Components