Natriuretic Peptides B (NPPB) ELISA Kit

Details for Product No. ABIN1979095, Supplier: Log in to see
Antigen
  • BNP1
  • NPPB
  • BNP
  • Bnf
  • AA408272
  • brain natriuretic peptide
  • natriuretic peptide B
  • natriuretic peptide type B
  • BNP
  • NPPB
  • Nppb
Alternatives
Dog (Canine) Natriuretic Peptides B ELISA Kit
Reactivity
Human, Mouse (Murine), Rat (Rattus)
Alternatives
Kits with alternative reactivity to:
12
10
7
4
4
1
1
1
1
Method Type
Competition ELISA
Detection Range
0.1-1.000 pg/mL
Minimum Detection Limit
0.1 pg/mL
Application
ELISA
Options
Supplier
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Supplier Product No.
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Purpose Human/Mouse/Rat Brain Natriuretic Peptide EIA Kit optimized for serum and cell culture medium. Competition-based ELISA on a 96-well strip plate.
Sample Type Cell Culture Supernatant, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Specificity Cross Reactivity: This EIA kit shows no cross-reactivity with any of the adipokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC.
Cross-Reactivity (Details) This ELISA kit shows no cross-reactivity with any of the adipokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC.
Sensitivity 1.66 pg/mL
Characteristics
  • Strip plates and additional reagents allow for use in multiple experiments
  • Quantitative protein detection
  • Establishes normal range
  • The best products for confirmation of antibody array data
Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Standard Peptide
  • Assay Diluent(s)
  • Biotinylated Peptide
  • HRP-Streptavidin
  • TMB One-Step Substrate
  • Stop Solution
  • Assay Diagram
  • Positive Control Sample
  • Capture Antibody
  • User Manual
Material not included
  • Distilled or deionized water
  • Precision pipettes to deliver 2 μL to 1 mL volumes
  • Adjustable 1-25 mL pipettes for reagent preparation
  • 100 mL and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Orbital shaker
  • Aluminum foil
  • Saran Wrap
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
Alternative Name BNP (NPPB ELISA Kit Abstract)
Background Brain natriuretic peptide (BNP)
Gene ID 4879
UniProt P16860
Research Area Cardiovascular, Hypertrophy, Hormones
Pathways Hormone Activity
Application Notes Recommended Dilution for serum and plasma samplesHuman: 2X / Mouse: 2X / Rat: 2X
Sample Volume 100 μL
Assay Time 5 h
Plate Pre-coated
Protocol
  1. Prepare all reagents, samples and standards as instructed.
  2. Add 100 μL detection antibody to each well.
  3. Incubate 1.5 h at RT or O/N at 4 °C.
  4. Add 100 μL standard or sample to each well.
  5. Incubate 2.5 h at RT.
  6. Add 100 μL prepared streptavidin solution.
  7. Incubate 45 min at RT.
  8. Add 100 μL TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 μL Stop Solution to each well.
  11. Read plate at 450 nm immediately.
Reagent Preparation
  1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
    2. Briefly centrifuge the Anti-BNP Antibody vial (Item N) and reconstitute with 5 µL of ddH2O before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
    3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-BNP antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
    4. Briefly centrifuge the vial of biotinylated BNP peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL of the 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated BNP will be 10 pg/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
    5. Preparation of Standards: Label 6 microtubes with the following concentrations: 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated BNP solution into each tube, except for the 1000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated BNP is 10 pg/mL in all standards. a. Briefly centrifuge the vial of standard BNP peptide (Item C) and reconstitute with 10 µL of ddH2O. In the tube labeled 1000 pg/mL, pipette 8 µL of Item C and 792 µL of 10 pg/mL biotinylated BNP solution (prepared in step 4 above). This is your BNP stock solution (1000 pg/mL BNP, 10 pg/mL biotinylated BNP). Mix thoroughly. This solution serves as the first standard. b. To make the 100 pg/mL standard, pipette 50 µL of BNP stock solution the tube labeled 100 pg/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated BNP and 50 mL of the prior concentration until 0.1 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL BNP, 10 pg/mL biotinylated BNP) serves as the zero standard (or total binding).
    6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the 1X Assay Diluent E. This solution will be used in steps 7 and
    9.
    7. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 2 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated BNP is 10 pg/mL.
    8. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer. 1000 100 10 1 0.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL
    9. Sample Preparation: Use 1X Assay Diluent E + biotinylated BNP to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated BNP is 10 pg/mL in every sample.
    Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated BNP to a final concentration of 10 pg/mL.
    Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
    10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 200- fold with 1X Assay Diluent E.
Sample Preparation

Use 1X Assay Diluent E + biotinylated BNP to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated BNP is 10 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated BNP to a final concentration of 10 pg/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.

Assay Procedure
  1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
    2. Add 100 µL anti-BNP antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C. 0
    3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each), Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    4. Add 100 µL of each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
    5. Discard the solution and wash 4 times as directed in Step
    3.
    6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes with gentle shaking at room temperature. It is recommended that incubation time should not be shorter or longer than 45 minutes.
    7. Discard the solution and wash 4 times as directed in Step
    3.
    8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec).
    9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

Assay Precision Intra-Assay: CV < 10 %
Inter-Assay: CV < 15 %
Restrictions For Research Use only
Handling Advice Avoid repeated freeze/thaw cycles.
Storage -20 °C
Storage Comment Standard, Biotinylated Brain Natriuretic peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
Expiry Date 6 months
Supplier Images
ELISA image for Natriuretic Peptides B (NPPB) ELISA Kit (ABIN1979095) Natriuretic Peptides B (NPPB) ELISA Kit
Product cited in: Li, Xuan, Liu, Dong, Luo, Sun: "Short‑term vagal nerve stimulation improves left ventricular function following chronic heart failure in rats." in: Molecular medicine reports, Vol. 12, Issue 2, pp. 1709-16, 2015 (PubMed).

Kazakov, Meier, Werner, Hall, Klemmer, Körbel, Lammert, Maack, Böhm, Laufs: "C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload." in: Stem cell research, Vol. 15, Issue 3, pp. 700-11, 2015 (PubMed).

Martín, Cordova, San Román, Gutierrez, Cachofeiro, Nieto: "Oleanolic acid modulates the immune-inflammatory response in mice with experimental autoimmune myocarditis and protects from cardiac injury. Therapeutic implications for the human disease." in: Journal of molecular and cellular cardiology, Vol. 72, pp. 250-62, 2014

Martín, Cordova, San Román, Gutierrez, Cachofeiro, Nieto: "Oleanolic acid modulates the immune-inflammatory response in mice with experimental autoimmune myocarditis and protects from cardiac injury. Therapeutic implications for the human disease." in: Journal of molecular and cellular cardiology, Vol. 72, pp. 250-62, 2014 (PubMed).

Jara, Benner, Sim, Liu, List, Householder, Berryman, Kopchick: "Elevated systolic blood pressure in male GH transgenic mice is age dependent." in: Endocrinology, Vol. 155, Issue 3, pp. 975-86, 2014 (PubMed).

Tang, Chen, Liang, Yao, Wu: "Ellagic acid prevents monocrotaline-induced pulmonary artery hypertension via inhibiting NLRP3 inflammasome activation in rats." in: International journal of cardiology, Vol. 180, pp. 134-41, 2014 (PubMed).

Shah, Kulkarni, Ferris, Amiji: "Analgesic Efficacy and Safety of DALDA Peptide Analog Delivery to the Brain Using Oil-in-Water Nanoemulsion Formulation." in: Pharmaceutical research, 2014 (PubMed).

Aurich, Niemann, Pan, Gruenler, Issa, Silber, Rohrbach: "Age-dependent effects of high fat-diet on murine left ventricles: role of palmitate." in: Basic research in cardiology, Vol. 108, Issue 5, pp. 369, 2013 (PubMed).

Bordicchia, Liu, Amri, Ailhaud, Dessì-Fulgheri, Zhang, Takahashi, Sarzani, Collins: "Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes." in: The Journal of clinical investigation, Vol. 122, Issue 3, pp. 1022-36, 2012 (PubMed).

Martín, Miana, Jurado-López, Martínez-Martínez, Gómez-Hurtado, Delgado, Bartolomé, San Román, Cordova, Lahera, Nieto, Cachofeiro: "DIOL triterpenes block profibrotic effects of angiotensin II and protect from cardiac hypertrophy." in: PLoS ONE, Vol. 7, Issue 7, pp. e41545, 2012 (PubMed).

Ku, Chen, Su: "DPP4 deficiency preserves cardiac function via GLP-1 signaling in rats subjected to myocardial ischemia/reperfusion." in: Naunyn-Schmiedeberg's archives of pharmacology, Vol. 384, Issue 2, pp. 197-207, 2011 (PubMed).

Background publications Liu, Aguirre, Evering, Hirakawa, May, Palacio, Wang, Zhang, Stevens: "miR-208a as a Biomarker of Isoproterenol-induced Cardiac Injury in Sod2+/- and C57BL/6J Wild-type Mice." in: Toxicologic pathology, Vol. 42, Issue 7, pp. 1117-29, 2014 (PubMed).

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