NT-ProBNP ELISA Kit

Catalog No. ABIN1112769

The Human NT-ProBNP ELISA Kit (ABIN1112769) is a Colorimetric ELISA Kit designed to quantify Human NT-ProBNP.

Quick Overview for NT-ProBNP ELISA Kit (ABIN1112769)

Target: NT-ProBNP (Pro-Brain Natriuretic Peptide (NT-ProBNP) (NT-ProBNP))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 31.2-2000 pg/mL

Application: ELISA

Product Details NT-ProBNP ELISA Kit

Minimum Detection Limit: 31.2 pg/mL

Analytical Method: Quantitative

Sensitivity: 2 pg/mL

Components: 1. One 96-well plate pre-coated with anti-human NT-proBNP antibody 2. Human NT-proBNP standards: 2 tubes (20 µl / tube) (1 µg/ml) 3. Sample / Standard diluent buffer: 30ml 4. HRP conjugated anti-human NT-proBNP antibody (Concentrated): 150 µl.

Material not included: 1. 37 °C incubator 2. Microplate reader (wavelength: 450nm) 3. Precise pipette and disposable pipette tips 4. Automated plate washer 5. ELISA shaker 6. 1.5ml of Eppendorf tubes 7. Plate cover 8. Absorbent filter papers 9. Plastic or glass container with volume of above 1L

Application Details

Comment:

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti-NT-proBNP polyclonal antibody was pre-coated onto 96-well plates. The standards and test samples were added - the wells and any NT-proBNP present is bound by the immobilized antibody. After washing away any unbound substances the HRP conjugated anti-NT-proBNP monoclonal antibody was added - wells as detection antibodies. Following a wash - remove any unbound antibody-enzyme reagent the TMB substrates were added - visualize HRP enzymatic reaction. TMB was catalyzed by HRP - produce a blue color product that changed into yellow after adding stop solution. The density of yellow is proportional - the NT-proBNP amount of sample captured in plate. Read the O.D. absorbance at 450 nm in a microplate reader and then the concentration of NT-proBNP can be calculated.

Plate: Pre-coated

Reagent Preparation:

1. Before the experiment, centrifuge each kit component for several minutes to bring down all reagents to the bottom of tubes. 2. It is recommend to measure each standard and sample in duplicate. 3. Do NOT let the plate completely dry at any time! Since the dry condition can inactivate the biological material on the plate. 4. Do not reuse pipette tips and tubes to avoid cross contamination. 5. Do not use the expired components and the components from different batches. 6. To avoid the marginal effect of plate incubation for temperature differences (the marginal wells always get stronger reaction), it is recommend to equilibrate the ABC working solution and TMB substrate for at least 30 min at room temperature (37°C ) before adding to wells.The TMB substrate (Kit Component 8) is colorless and transparent before use, if not, please contact us for replacement.

Sample Preparation:

Preparation of sample and reagents The concentrated reagents should be brought to room temperature and should be diluted before starting the test procedure. If crystals have formed in the reagents, warm them gently until they have completely dissolved. 1. Sample Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20 °C for long term. Avoid multiple freeze-thaw cycles. Cell culture supernatants: Centrifuge to remove particulates, analyze immediately or aliquot and store at -20 °C . Avoid multiple freeze-thaw cycles. Serum: Coagulate the serum at room temperature (about 2 hours). Centrifuge at approximately 1000 × g for 20 min. Analyze the serum immediately or aliquot and store at -20 °C . Avoid multiple freeze-thaw cycles.
Plasma: Collect plasma with EDTA or heparin as the anticoagulant. Centrifuge for 20 min at 1000 x g within 30 min of collection. Analyze immediately or aliquot and store at -20 °C. Avoid multiple freeze-thaw cycles. Citrate can not be used as anticoagulant here. Note: 1. Coagulate blood samples completely, then, centrifuge, and avoid hemolysis and particle. Grossly hemolyzed or lipemic samples may not be suitable for measurement of human NT-proBNP with this assay. 2. NaN3 can not be used as test sample preservative, since it is the inhibitor for HRP. 2. Wash buffer Pour entire contents (30 ml) of the Wash buffer (20x) (Kit Component 8) into a clean 600 ml graduated cylinder. Bring to final volume of 600 ml with distilled or deionized water. Mix gently to avoid foaming. Then, transfer to a clean wash bottle and store at 2-25°C. Wash buffer (1x) can be stable for 30 days. 3. Standard Standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes (20µl / tube) of standards are included in each kit. Use one tube for each experiment. After usage, the remaining standard has to be discarded. a. 2000 pg/ml of standard solution: Add 10 ml of Sample / Standard diluent buffer (Kit Component 3) into one Standard (Kit Component 2) tube, keep the tube at room temperature for 10 min and mix thoroughly. b. 1000 pg/ml ĺ 32.2 pg/ml of standard solutions: Label 6 tubes with 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, respectively. Aliquot 225 Pl of the Sample / Standard diluent buffer (Kit Component 3) into each tube. Add 225 Pl of the above 2000 pg/ml standard solution into 1st tube and mix thoroughly. Transfer 225 Pl from 1st tube to 2nd tube and mix thoroughly. Transfer 225 Pl from 2nd tube to 3rd tube and mix thoroughly, and so on. Note: The standard solutions are best used within 2 hours. The 2000 pg/ml standard solution should be used within 12 hours. Or store at -20 °C for up to 48 hours. Avoid repeated freeze-thaw cycles. 4. Preparation of HRP conjugated anti-human NT-proBNP antibody (Kit Component 4) working solution: the HRP conjugated antibody should be used within 30 min after diluting. a. Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume) b. Dilute the HRP conjugated anti-human NT-proBNP antibody (Kit Component 4) with Antibody diluent buffer (Kit Component 5) at 1:100 and mix thoroughly. i.e. Add 1 ȝl of HRP conjugated anti-human NT-proBNP antibody into 99 ȝl of Antibody diluent buffer. z Assay procedure 1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommend to measure each sample, standard, zero and optional control sample in duplicate. Remove extra microwell strips from holder and store them in foil bag at -20 °C° C. 2. Aliquot 0.1ml of 2000 pg/ml, 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml standard solutions into the standard wells. 3. Add 0.1 ml of Sample / Standard diluent buffer (Kit Component 3) into the control (zero) well. 4. Add 0.1 ml of properly diluted sample (human serum, plasma or cell culture supernatants) into test sample wells. Here is an example of the arrangement of sample, standard and zero in the plate wells: 1 2 3 4 A Standard 1 (2000.0 pg/ml) Standard 1 (2000.0 pg/ml) Sample 1 Sample 1 B Standard 2 (1000.0 pg/ml) Standard 2 (1000.0 pg/ml) Sample 2 Sample 2 C Standard 3 (500.0 pg/ml) Standard 3 (500.0 pg/ml) Sample 3 Sample 3 D Standard 4 (250.0 pg/ml) Standard 4 (250.0 pg/ml) Sample 4 Sample 4 E Standard 5 (125.0 pg/ml) Standard 5 (125.0 pg/ml) Sample 5 Sample 5 F Standard 6 (62.5 pg/ml) Standard 6 (62.5 pg/ml) Sample 6 Sample 6 G Standard 7 (31.25 pg/ml) Standard 7 (31.25 pg/ml) Sample 7 Sample 7 H Zero / Blank Zero / Blank Sample 8 Sample 8 5. Seal the plate with a cover and incubate at 37 for 90 min° C or at room temperature for 2 hours. 6. Remove the cover, aspirate the plate content and wash plate 5 times with Wash buffer (Kit Component 8) using one of the following methods: 1000 500 250 125 62.5 31.2 225 µl 225 µl 225 µl 225 µl 225 µl 225 µl NT-proBNP Protein Standards [pg/ml] 2000 pg/ml h.NT-proBNP Solution 2000 4 Manual Washing: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with Wash buffer (Kit Component 8) and vortex mildly on ELISA shaker for 2 min, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material. Repeat this procedure four more times for a total of FIVE washes. Automated Washing: Aspirate all wells, then wash plate FIVE times with Wash buffer (Kit Component 8) (overfilling wells with the buffer, about 400µl). After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer be set for a soaking time of 1 min or shaking. 7. Add 0.1 ml of HRP conjugated anti-human NT-proBNP antibody work solution into each well. Add the solution at the bottom of each well without touching the side wall. 8. Seal the plate with a new cover and incubate at 37 for 30 min or at room temperature for 60 min. 9. Remove the cover, aspirate the plate content and wash plate 5 times with Wash buffer (Kit Component 8), and each time let the wash buffer stay in the wells for 1-2 min. (Repeat Step 6). 10. Add 0.1 ml of TMB substrate (Kit Component 6) into each well, cover the plate and incubate at room temperature (18-25° C) in dark for about within 30 min. (Note: The color development on the plate should be monitored and the substrate reaction stopped before positive wells are no longer properly recordable. Determination of the ideal time period for color development has to be done individually for each assay. It is recommended to add the stop solution when the highest standard has developed a dark blue color.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated human NT-proBNP standard solutions), the other wells show no obvious color. 11. Add 0.1 ml of Stop solution (Kit Component 7) into each well and mix thoroughly. The color changes into yellow immediately. 12. Read the O.D. absorbance at 450 nm in a microplate reader within 30 min after adding the stop solution. Note: If the incubation without shaking, the obtained O.D. Values may be lower than the typical data, but the results are still valid. For calculation, average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. (The relative O.D.450) = (the O.D.450 of each well) - (the O.D.450 of Zero well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The human NT-proBNP concentration of the samples can be interpolated from the standard curve. Note: 1. If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution. 2. Calculation of samples with a concentration exceeding 2000pg/ml may result in incorrect, low human NT-proBNP levels. Such samples require further external pre-dilution according to expected human NT-proBNP values with Sample / Standard diluent buffer (Kit Component 3) in order to precisely quantitate the actual human NT-proBNP level. 5

Restrictions: For Research Use only

Handling

Preservative: Sodium azide, Thimerosal (Merthiolate)

Target Details for NT-ProBNP

Target: NT-ProBNP (Pro-Brain Natriuretic Peptide (NT-ProBNP) (NT-ProBNP))

Alternative Name: NT-ProBNP

Background: The N-terminal prohormone of brain natriuretic peptide (NT-proBNP) is a 76 amino acid N-terminal fragment of brain natriuretic peptide. Both BNP and NT-proBNP levels in the blood are used for screening, diagnosis of acute congestive heart failure (CHF) and may be useful to establish prognosis in heart failure, as both markers are typically higher in patients with worse outcome.BNP may be a useful screening tool for left ventricular dysfunction in patients with history suggestive of heart disease and be used to assist in forming a pretest probability, which in turn could greatly assist in appropriateness of patient referral and in optimization of drug therapy.