Antibodies-Online/Pro-Brain Natriuretic Peptide (NT-ProBNP) ELISA Kit/ABIN2641941/10 x 96 tests
Antigen Epitope N-Term Reactivity Human Alternatives Method Type Sandwich ELISA Detection Range 0.312-20 ng/mL Minimum Detection Limit 0.312 ng/mL Application ELISA Options Supplier Supplier Product No. ![\"Independent](\"https://www.ebiomall.com/images/antibodies_online/201710/029606_validation_logo_scale_105x150_029606_validation_logo.png\" "\\"Successfully") | - Successfully validated (ELISA)
- by Alamo Laboratories
- No. #029606
- Date 02/09/2014
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- Read full report (PDF)
- Click to verify
| ![\"Pro-Brain](\"https://www.ebiomall.com/images/antibodies_online/201710/Pro-Brain+Natriuretic+Peptide+NT-ProBNP+ELISA+Kit_aspect_300x200_abimg497193.png\") ![\"Pro-Brain](\"https://www.ebiomall.com/images/antibodies_online/201710/Pro-Brain+Natriuretic+Peptide+NT-ProBNP+ELISA+Kit_aspect_300x200_abimg497194.png\") |
| Lot Number | U05095912 |
| Method validated | ELISA |
| Positive Control | Human serum |
| Negative Control | Goat serum |
| Notes | Signal was detected in positive control sample and not in negative control sample. |
| Primary Antibody | - Antigen: Human Pro-Brain Natriuretic Peptide (NT-ProBNP)
- Catalog number: ABIN368630
- Supplier: Log in to see
- Supplier catalog number: Log in to see
- Lot number: U05095912
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| Controls | - Positive control: Serum from normal adult human (specimen known to contain the target protein).
- Negative control: Serum from normal goat (specimens known to not contain the target protein).
- Standard curve: Serial two-fold dilutions from 20 ng/ml [20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0] were generated from the standard provided in the kit using standard/sample diluent buffer.
- Spike control: Standard diluted in standard/PBS diluent buffer [2.5 and 0].
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| Protocol | - All reagents in the ELISA kit were brought up to room temperature (RT) before use.
- 100 µL of standard or sample were added to wells in ELISA plate pre-coated with capture antibody. All samples and standards were assayed in triplicate.
- The plate was covered with sealer (provided in kit) and incubated for 2 hours at 37°C. Unbound material was aspirated but the wells were NOT Washed.
- 100 µL of Biotin-Antibody (diluted 1:100 in \"Biotin-Antibody Diluent”) was added to each well. Plate was covered with sealer (provided in kit) and incubated for 1 hour at 37°C. Unbound Biotin-Antibody was removed from each well and plate was washed three times with 350 µL of wash buffer (provided in the kit). After the last wash the plate was inverted against clean absorbent paper to remove any remaining liquid.
- 100 µL of HRP-Avidin Conjugate (diluted 1:100 in \"HRP-Avidin Diluent”) was added to each well. Plate was covered with sealer (provided in kit) and incubated for 1 hour at 37°C.
- Unbound HRP-Avidin was removed by washing five times with 350 µL of wash buffer (provided in the kit). After the last wash the plate was inverted and blotted against clean absorbent paper to remove any remaining liquid.
- 90 µL of TMB substrate was added to wells and the plate was covered with a new plate sealer. The plate was gently tapped to ensure mixing and incubated for 30 min at 37°C in the dark.
- After 30 min, when an apparent gradient appeared in the standard wells, the reaction was terminated by adding 50 µL of Stop Solution to each well.
- The optical density (OD value) of each well was read using a microplate reader set to 450 nm.
- The triplicate readings for each sample were averaged and the average zero standard optical density subtracted to yield ‘corrected absorbance at 450 nm’. A standard curve was generated by plotting the mean OD value for each standard on the X-axis against the concentration on the Y-axis using Excel. Standard curve was generated by regression analysis with four-parameter logistic.
- An equation ((y = -44.108x4 + 71.899x3 - 11.889x2 + 17.555x + 0.0627) was derived from the standard curve and used to calculate NT-proBNP concentrations in samples based on their Average Absorbance values.
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| Experimental Notes | None |
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