Nt-proBNP Assay | BNP Fragment EIA | BI-20852W

Product details

Product Overview
Principle of the Assay

Typical Standard Curve
Kit Components

NT-proBNP Test Kit Product Overview

The NT-proBNP test kit is an overnight, 96-well competitive enzyme immunoassay (EIA). This NT-proBNP assay utilizes one antibody that binds to the amino acid region of 8-29 of the NT-proBNP peptide. Thus, the BNP fragment immunoassay is for the quantitative determination of NT-proBNP (1-76), including all NT-proBNP derived fragments containing the amino acid sequence between AA 8-29, in human serum and plasma.

NT-proBNP Test Kit Assay Principle

This NT pro BNP test kit is a competitive enzyme immunoassay (EIA) for the quantitative determination of NT-proBNP (1-76) and NT-proBNP fragments containing AA 8-29 in human serum, EDTA plasma, citrate plasma and heparın plasma.

The figure below explains the principle of the BNP fragment EIA:

In a first step, standard/control/sample and conjugate (synthetic NT-proBNP-HRP) are pipetted into the wells of the microtiter strips, which are pre-coated with anti-BNP fragment (AA8-29) antibody. BNP-fragment present in the standard/control/sample and the BNP fragment conjugate binds to the pre-coated antibody in the well. Since BNP fragments in the standard/control/sample and conjugate compete for the antibodies’ binding sites, the relative fraction of unlabeled BNP fragment is proportional to the concentration of BNP fragment in the standard/control/sample. In a washing step all non-specific unbound material is removed. In a second step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is inversely proportional to the amount of BNP fragment in the standard/control/sample. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of BNP fragment in the sample is determined directly from the dose response curve.

NT-proBNP Test Kit Typical Standard Curve

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

NT-proBNP Test Kit Components

CONT	KIT COMPONENTS	QUANTITY
PLATE	Polyclonal anti-BNP fragment (AA 8-29) antibody pre-coated microtiter strips in strip holder, packed in aluminum bag with desiccant	12 x 8 tests
WASHBUF	Wash buffer concentrate 20x, natural cap	1 x 50 ml
STD	Standards 1-7, (0; 200; 400; 800; 1,600; 3,200; 6,400 pmol/l), synthetic human NT-proBNP in human serum, white caps, lyophilized	7 vials
CTRL	Control, yellow cap, synthetic human NT-proBNP, lyophilized, exact concentration after reconstitution see label	1 vial
ASYBUF	Assay buffer, red cap, ready to use	1 x 20 ml
CONJ	Conjugate, (synthetic NT-proBNP-HRP), red dye, amber cap, ready to use	1 x 6 ml
SUB	Substrate (TMB solution), 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 BNP fragment EIA 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, EDTA plasma,citrate plasma and heparın plasma are suitable for use in this NT proBNP test. 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, heparın or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. BNP fragments are stable in whole blood, serum or plasma for several hours at room temperature or +4°C (+2-8°C). Nevertheless, perform serum and plasma separation by centrifugation according to 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. Samples can be subjected to five freeze-thaw cycles.

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 & Control for Serum and Plasma Measurements

1.	Pipette 200 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The standard and control concentrations are printed on the label of each vial.
2.	Leave at room temperature (+18-26°C) for 20 min. Swirl gently.

Reconstituted STDs and CTRL 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 duplicate measurements for all samples.

Samples for which the OD value is below highest point of the standard range can be diluted with STD 1 (0 pmol/l) or BNP fragment negative human serum.

NT-proBNP Test Kit Assay Protocol

Read the entire protocol before beginning the assay.

1.	Bring samples and reagents 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 (+2-8°C) in the aluminum bag. Strips are stable until the expiry date stated on the label.
4.	Pipette 150 µl ASYBUF (assay buffer, red cap) into each well.
4.	Add 30 µl STD/CTRL/SAMPLE in dublicates into the respective wells.
5.	Add 50 µl CONJ (conjugate, amber cap) into each well. Swirl gently.
6.	Cover the plate tightly and incubate overnight (16-25 h) at +4°C (+2-8°C) in the dark.
7.	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.
8.	Add 200 µl SUB (substrate, blue cap) into each well.
9.	Incubate for 20 min at room temperature in the dark.
10.	Add 50 µl STOP (stop solution, white cap) into each well.
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 (correction 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. 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 lot at production date. ODs obtained by customers may differ due to various influences including the normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an OD of 1.5 or higher is obtained for STD 1 and the value of the CTRL is within the target range (see label).

Background & Therapeutic Areas

BNP Fragment Protein

The natriuretic peptides are members of a family of structurally similar but genetically distinct peptide hormones, consisting of atrial-, brain-, and C-type (ANP, BNP, and CNP, respectively). ANP and BNP preferentially bind to a membrane-bound guanylyl cyclase (GC) receptor called GC-A or NPR1, whereas CNP is the physiological ligand for GC-B (NPR2). The natriuretic peptides play an important role in the regulation of cardiovascular and renal homeostasis and in the regulation of fatty acid metabolism and body weight.

BNP is mainly expressed by ventricular myocardium in response to volume overload and increased filling pressure. BNP has a cleavable signal sequence. Mature BNP consists of 108 amino acids (proBNP or BNP-108), and undergoes cleavage resulting in physiologically active BNP-32 and additional C-terminal fragments (cf. http://www.uniprot.org/uniprot/P16860#PRO_0000001532), along with a physiologically inactive N-terminal peptide comprising amino acids 1-76 (NT-proBNP), which is further degraded proteolytically.

From Alpco website https://www.alpco.com/physiological-actions-of-natriuretic-peptides

Molecular weight	8.475 kDa
Cellular localisation	Secreted
Post-translational modifications	Glycosylation, disulfide 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	NPPC link: https://www.proteinatlas.org/ENSG00000120937-NPPB/tissue
Uniprot ID	P16960 link: https://www.uniprot.org/uniprot/P16860

BNP Fragment 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.

The discovery of natriuretic peptides identified an endocrine system that contributes to diuresis and vascular tone. The biology, biochemistry and the pathophysiological role of natriuretic peptides are described in several reviews.

Heart & 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

Validation Data

Calibration
Detection Limit & Sensitivity
Precision
Accuracy

Dilution Linearity
Specificity
Sample Stability
Sample Values

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

Show validation literature

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

Calibration

This NT-proBNP test kit is calibrated against recombinant NT-proBNP peptide.

NT-proBNP Test Kit Detection Limit & Sensitivity

To determine the sensitivity of the human total BNP fragment EIA, experiments measuring the lower limit of detection (LOD) were conducted.

LOD

3.0 pmol/l

NT-proBNP Test Kit Precision

The precision of an immunoassay 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.

Mean

BNP Fragment [pmol/l]

SD [pmol/l]

CV [%]

Sample 1

763

Sample 2

3236

251

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 different operators using plates and kit components from two lots.

Mean

BNP Fragment [pmol/l]

SD [pmol/l]

CV [%]

Sample 1

781

Sample 2

3199

236

NT-proBNP Test Kit Accuracy

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

The recovery of the BNP fragment EIA was measured by adding recombinant NT-proBNP to human samples containing a known concentration endogenous BNP fragment. The % recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value. All our ELISAs are expected to have % recovery rates within 25% of the nominal value of the sample.

This table shows the summary of the recovery experiments in the BNP fragment human EIA in different sample matrices:

		% Recovery
Sample Matrix	n	+ 533 pmol/l		+ 2 133 pmol/l		+ 4 889 pmol/l
Sample Matrix	n	Mean	Range	Mean	Range	Mean	Range
Serum	4	107	84 - 125	94	78 - 113
Citrate plasma	9					108	90 - 118

Show individual measurements

Data showing recovery of BNP fragment in human serum samples:

		BNP fragment [pmol/l]			% Recovery
Sample Matrix	ID	Reference	+ 533 pmol/l	+ 2 133 pmol/l	+ 533 pmol/l	+ 2 133 pmol/l
Serum	s1	1 241	1 709	3 128	96	93
Serum	s2	2 069	2 194	3 258	84	78
Serum	s3	66	746	2 091	125	95
Serum	s4	0	645	2 403	121	113
				Mean	107	94
				Min	84	78
				Max	125	113

Data showing recovery of BNP fragment in human citrate plasma samples:

		BNP fragment [pmol/l]		% Recovery
Sample Matrix	ID	Reference	+ 4 889 pmol/l	+ 4 889 pmol/l
Citrate plasma	c1	221	4,816	95
Citrate plasma	c2	528	6,061	112
Citrate plasma	c3	312	6,057	117
Citrate plasma	c4	512	6,052	112
Citrate plasma	c5	462	5,911	111
Citrate plasma	c6	448	5,613	106
Citrate plasma	c7	285	5,768	112
Citrate plasma	c8	485	6,339	118
Citrate plasma	c9	1,021	5,319	90%
			Mean	108
			Min	90
			Max	118

NT-proBNP Test Kit Dilution Linearity

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing NT-proBNP (and fragments of NT-proBNP containing AA sequence 8-29) 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.

The table below shows the mean recovery and range of serially diluted recombinant BNP fragment in serum:

		% Recovery of recombinant BNP fragment in diluted samples
Sample Matrix	n	1+1		1+3
		Mean	Range	Mean	Range
Serum	7	105	94 – 113	131	98 - 154

Show individual measurements

Data showing dilution linearity of recombinant BNP fragment in human serum samples:

		BNP fragment [pmol/l]			% Recovery
Sample matrix	ID	Reference	1+1	1+3	1+1	1+3
Serum	s1	1,384	661	661	96	98
Serum	s2	2,388	1,236	1,236	104	131
Serum	s3	2,214	1,216	1,216	110	141
Serum	s4	3,387	1,588	1,588	94	114
Serum	s5	2,642	1,464	1,464	111	154
Serum	s6	2,482	1,360	1,360	110	143
Serum	s7	2,451	1,382	1,382	113	137
				Mean	105	131
				Min	94	98
				Max	98	154

NT-proBNP Test Kit Specificity

The BNP fragment EIA utilizes one antibody that binds to the amino acid region of 8-29 of the NT-proBNP peptide. Thus, the BNP fragment immunoassay is for the quantitative determination of NT-proBNP (1-76) (natural and recombinant), including all NT-proBNP derived fragments containing the amino acid sequence between AA 8-29.

Sample Stability

Sample Preparation

BNP fragments are stable in whole blood, serum or plasma for several hours at room temperature or +4°C (2-8°C). Nevertheless, we recommend separating plasma or serum by centrifugation as soon as possible, e.g. 20 min at 2,000 x g, preferably at +4°C (2-8°C). Aliquot the acquired plasma or serum samples and store them at -25°C or lower. Samples can be subjected to five freeze-thaw cycles without any loss of immune reactivity. Serum samples can be stored for ≥ 2 years at -80°C.

Overnight Storage

BNP fragment concentrations of samples after overnight storage at room temperature:

	BNP fragment [pmol/l]		% Recovery
Sample ID	Reference	O.N. at RT	% Recovery
#S1	803	732	91
#S2	1 247	1 392	112
#S3	2 480	2 477	100
		Mean	101

The recovery of human serum samples after an overnight storage at room temperature is 101%.

Sample Values

BNP Fragment Values in Apparently Healthy Donors

Total BNP fragment reference ranges were established using 76 serum samples from apparently healthy donors. No medical histories were available for the volunteers.

Sample Matrix	n	Median [pmol/l]	Range [pmol/l]
Serum	76	392	150 – 1 631

We recommended establishing the normal range for each laboratory.

Show individual measurements

Sample ID	BNP fragment [pmol/l]	Sample ID	BNP fragment [pmol/l]	Sample ID	BNP fragment [pmol/l]
#1	166	#26	492	#51	359
#2	408	#27	420	#52	192
#3	576	#28	416	#53	341
#4	516	#29	614	#54	548
#5	351	#30	271	#55	342
#6	548	#31	335	#56	244
#7	348	#32	327	#57	398
#8	348	#33	428	#58	317
#9	250	#34	421	#59	253
#10	267	#35	598	#60	467
#11	270	#36	424	#61	611
#12	250	#37	337	#62	769
#13	895	#38	1,172	#63	269
#14	789	#39	348	#64	243
#15	407	#40	383	#65	150
#16	270	#41	321	#66	300
#17	162	#42	415	#67	244
#18	341	#43	523	#68	1,631
#19	383	#44	431	#69	431
#20	265	#45	453	#70	462
#21	364	#46	1,102	#71	784
#22	704	#47	455	#72	503
#23	243	#48	321	#73	1 045
#24	627	#49	578	#74	313
#25	287	#50	385	#75	629
				#76	606

BNP Fragment Values in Two Dialysis and a Heart Failure Panel

BNP fragment was measured in serum samples of patients from two dialysis, an NYHA class 3 (heart failure) and an unselected hospital panel:

Cohort	Sample Matrix	n	Median BNP fragment [pmol/l]	Range [pmol/l]
Dialysis panel 1	Serum	16	3,426	2,280 – 3 426
Dialysis panel 2	Serum	16	3,348	698 - >6 400
NYHA class 3	Serum	95	775	49 – 12 800
Unselected hospital panel	Serum	32	574	171 – 5 438

The figure below shows the comparison of concentrations of BNP fragment in several patient panels with apparently healthy donors:

Show individual measurements

Concentrations of 16 human serum samples from a dialysis panel 1 derived from a hospital lab:

Sample ID	BNP fragment [pmol/l]
#1	3 200
#2	6 081
#3	2 280
#4	>6 400
#5	3 070
#6	>6 400
#7	4 178
#8	>6 400
#9	3 863
#10	4 036
#11	2 806
#12	4 142
#13	3 653
#14	2 374
#15	2 893
#16	>6 400

Concentrations of 16 human serum samples from a dialysis panel 2 derived from a hospital lab:

Sample ID	BNP fragment [pmol/l]
#1	3 375
#2	6 120
#3	>6 400
#4	1 197
#5	4 331
#6	5 146
#7	781
#8	698
#9	3 321
#10	>6 400
#11	1 386
#12	3 549
#13	4 370
#14	2 662
#15	2 072
#16	1 404

Matrix Comparison

Correlation of Serum and Plasma Samples from Apparently Healthy Individuals

Measurement of BNP Fragment in 3 different sample matrices from 4 samples showed a mean CV of 17%. All 3 matrices can be tested in the assay.

Data showing the effect of the sample matrix:

	BNP fragment [pmol/l]
Sample ID	Serum	Heparın plasma	Citrate plasma	Mean	CV
#1	545	770	523	613	22%
#2	10,408	9,708	8,792	9,636	8%
#3	577	664	462	567	18%
#4	568	640	435	547	19%
				Mean	17%

Figure showing matrix comparison of BNP fragment sample concentrations between serum, heparın plasma, and citrate plasma in an apparently healthy cohort (n=4).

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BNP Fragment EIA | BI-20852W

NT-proBNP Test Kit Product Overview

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