NT-proBNP (Human) ELISA Kit

Transcription

1 NT-proBNP (Human) ELISA Kit Catalog Number KA assays Version: 10 Intended for research use only

2 Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 4 Materials Required but Not Supplied... 4 Precautions for Use... 5 Assay Protocol... 6 Reagent Preparation... 6 Sample Preparation... 6 Assay Procedure... 7 Data Analysis... 8 Calculation of Results... 8 Performance Characteristics... 9 Resources References Plate Layout KA / 11

3 Introduction Intended Use For quantitative detection of N-terminal Prohormone of Brain Natriuretic Peptide in human serum, plasma, urine, cell culture supernatant or tissue samples. Background The N-terminal prohormone of brain natriuretic peptide (NT-proBNP) which consists of 76 amino acids, is the N-terminal fragment of the prohormone of brain natriuretic peptide. NT-proBNP level in the blood is used for screening, diagnosis of acute congestive heart failure (CHF) and may be useful to establish prognosis in heart failure, as it is typically higher in patients with worse outcome. NT-proBNP 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. Principle of the Assay This kit was based on standard one-step sandwich enzyme-linked immune-sorbent assay technology. The purified anti-nt-probnp antibody was pre-coated onto 96-well plates. And the HRP conjugated anti-nt-probnp antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, mixed and incubated, then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the NT-proBNP amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of NT-proBNP can be calculated. KA / 11

4 General Information Materials Supplied List of component Component One 96-well plate pre-coated with anti-nt-probnp antibody Standard: (2250 pg/ml) Wash buffer (30x): Dilution: 1:30 Standard diluent buffer Sample diluent buffer HRP conjugated anti-nt-probnp antibody (RTU) Stop solution TMB chromogenic reagent A TMB chromogenic reagent B Plate sealer Hermetic bag Amount 96 (8x12) wells 0.5 ml 20 ml 1.5 ml 6 ml 6 ml 6 ml 6 ml 6 ml 2 slices 1 bag Storage Instruction Store at 2-8 C in dark for 6 months. Materials Required but Not Supplied 37 C incubator Microplate reader (wavelength: 450nm) Precise pipette and disposable pipette tips Automated plate washer ELISA shaker 1.5 ml of Eppendorf tubes Absorbent filter papers Distilled water Plastic or glass container with volume of above 1L KA / 11

5 Precautions for Use 1. This kit is suitable for serum and plasma samples and is for in vitro scientific research use only. 2. All samples, wash buffer and each kind of reject should be handled as potentially hazardous and capable of transmitting disease. 3. The user must strictly follow the protocols outlined in this insert to obtain reliable results. Estimate the results on the basis of the data read by the microplate reader. 4. Do not remove microplate from the storage bag until needed, and the unused strips should be stored at 2-8 C in their pouch with the desiccant provided. 5. It is recommended to analyze all standards, samples and control (zero) wells in duplicate, and use their mean values. 6. If the initially obtained data is greater than 2000 pg/ml, please dilute the sample properly with sample diluent buffer, and multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution. 7. The test range of this kit is 125 pg/ml pg/ml, please ignore the out of range data which obtained by calculating through standard curve extending. 8. Prolong the incubation time if the hypochromasia obtained. Heat the water in the water bath during diluting if the crystalloid appeared in Wash buffer. 9. To avoid cross contamination of standards, samples and controls, use the clean and separate pipette tip for each. Add HRP conjugated antibody, sample diluent buffer and substrate without touching the side walls. Do not use the sealer repeatedly. 10. Do not use expired components or mix components from different lots. 11. Avoid expose TMB chromogenic reagent B and HRP conjugated antibody to the light for a long time. 12. It is recommend to add the solutions within 5 min, use the multichannel pipettor if necessary. KA / 11

6 Assay Protocol Reagent Preparation Wash buffer Dilute concentrated wash buffer 30-fold (1:30) with distilled water (i.e. add 20 ml of concentrated wash buffer into 580 ml of distilled water). Standard Dilute the standard: standard solution should be prepared no more than 2 hours prior to the experiment. 2,000 pg/ml of standard solution: Add ml of Sample / Standard diluent buffer into one Standard tube, keep the tube at room temperature for 10 min and mix thoroughly pg/ml 125 pg/ml of standard solutions: Label 4 Eppendorf tubes with 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, respectively. Aliquot 0.2 ml of the Sample / Standard diluent buffer into each tube. Add 0.2 ml of the above 2,000 pg/ml standard solution into 1st tube and mix thoroughly. Transfer 0.2 ml from 1st tube to 2nd tube and mix thoroughly. Transfer 0.2 ml from 2nd tube to 3rd tube and mix thoroughly, and so on. Sample Preparation Serum: Coagulate at room temperature for min, then, centrifuge at the speed of r.p.m. for 20 min to collect supernatant. If precipitation appeared, centrifuge again. Plasma: Collect plasma using EDTA or citrate plasma as an anticoagulant, and mix for min, centrifuge at the speed of r.p.m. for 20 min of collection. If precipitation appeared, centrifuge again. Urine: Collect urine using a sterile container, centrifuge at the speed of r.p.m. for 20 min to collect supernatant. If precipitation appeared, centrifuge again. For collection of hydrothorax and cerebrospinal fluid, take reference to this operation. Cell culture supernatant: Secretory components: use a sterile container to collect. Centrifuge at the speed of r.p.m. for 20 min to collect supernatant. Intracellular components: Dilute cell suspension with PBS (ph ) to make the cell concentration reached 1 million/ml. Damage cells and release of intracellular components through repeated freeze-thaw cycles. Centrifuge at the speed of r.p.m. For 20 min to collect supernatant. If precipitation appeared, centrifuge again. Tissue samples: Cut samples and weight, add certain volume of PBS (ph7.4), rapidly frozen with liquid nitrogen. After melting, store samples at 2-8 C. Add certain volume of PBS (ph7.4), homogenize thoroughly, centrifuge at the speed of r.p.m. for 20 min to collect supernatant. Note: 1. Coagulate blood samples completely, then, centrifuge, and avoid hemolysis and particle. 2. NaN 3 can not be used as test sample preservative, since it is the inhibitor for HRP. KA / 11

7 3. After collecting samples, analyze immediately or aliquot and store frozen at -20 C. Avoid repeated freeze-thaw cycles. Assay Procedure 1. Equilibrate kit components for min at room temperature. 2. Set test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. Do not add sample and HRP conjugated antibody to control (zero) well. 3. For test sample wells, add 40 µl of Sample diluent buffer first, then, add 10 µl of sample. Add the solution at the bottom of each well without touching the side wall. Shake the plate mildly to mix thoroughly. 4. Cover the plate with Plate sealer and incubate at 37 C for 30 min. 5. Remove the sealer, and wash plate using one of the following methods: Manual Washing: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers. Fill each well completely with Wash Buffer (1x) and vortex mildly on ELISA shaker for 2 min, then aspirate contents from the plate, and clap the plate on absorbent filter papers. Repeat this procedure four more times for a total of FIVE washes. Automated Washing: Aspirate all wells, then wash plates FIVE times using Wash Buffer (1x). After the final wash, invert plate, and clap the plate on absorbent filter papers until no moisture remained. It is recommended that the washer be set for a soaking time of 10 seconds or shaking. 6. Add 50 µl of HRP conjugated anti-nt-probnp antibody to each well (except control well). 7. Cover the plate with Plate sealer and incubate at 37 C for 30 min. 8. Remove the sealer, and wash the plate. (See Step 5) 9. Add 50 µl of TMB chromogenic reagent A into each well, and then, add 50 µl of TMB chromogenic reagent B, vortex gently the plate on ELISA shaker for 30 seconds (Or shake gently by hand for 30 seconds), and incubate in dark at 37 C for 15 min. The shades of blue can be seen in the wells. 10. Add 50 µl of stop solution into each well and mix thoroughly. The color changes into yellow immediately. 11. Read the O.D. absorbance at 450 nm in a microplate reader within 15 min after adding the stop solution. KA / 11

8 Data Analysis Calculation of Results 1. For calculation, (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: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution. 2. Cutoff value for reference: years old: <125 pg/ml; above 75 years old: <450 pg/ml. Take cutoff value of years old as an example for results analysis, if the relative O.D.450 <125 pg/ml, the results are negative, conversely, the relative O.D.450 >125 pg/ml, the resultss are positive, the clinical status of heart failure can be estimated according to these data. NT-proBNP detection concentration & NYHA heart failure classification correlation NYHA Classification NT-proBNP Detection Concentration (pg/ml) Normal (18-74 years old) <125 I 125~450 II 450~1700 III 1700~4200 IV >4200 Typical Data & Standard Curve Results of a typical standard run of a NT-proBNP (Human) ELISA Kit are shown below. This standard curve was generated for demonstration purpose only. Each user should obtain their own standard curve as per experiment. (N/A=not applicable) X pg/ml Y OD O.D. Concentration (pg/ml) KA / 11

9 Performance Characteristics Range 125 pg/ml-2000 pg/ml KA / 11

10 Resources References 1. Bhalla V, Willis S, Maisel AS (2004). "B-type natriuretic peptide: the level and the drug--partners in the diagnosis of congestive heart failure". Congest Heart Fail 10 (1 Suppl 1): Atisha D, Bhalla MA, Morrison LK, Felicio L, Clopton P, Gardetto N, Kazanegra R, Chiu A, Maisel AS (September 2004). "A prospective study in search of an optimal B-natriuretic peptide level to screen patients for cardiac dysfunction". Am. Heart J. 148 (3): KA / 11

11 Plate Layout A B C D E F G H KA / 11