The Human Brain Natriuretic Peptide (BNP) ELISA, ECL Kit is a sensitive chemiluminescent sandwich enzyme immunoassay for the in vitro quantitative measurement of BNP in human serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids.
Test Principle:The microtiter plate provided in this kit has been pre-coated with an antibody specific to BNP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to BNP. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The addition of substrates A and B mixture generates glow light emission kinetics. Upon plate development, the intensity of the emitted light is proportional to the BNP level in the sample or standard.
Detection Range:6.86-5000pg/ml
Sensitivity:<2.56pg/ml
Precision:Intra-Assay: CV<10%Inter-Assay: CV<12%
Kit Components:*157442A: Microtiter Plate, 1x96 wells, Pre-coated with antibody; ready to use*157442B: Standard, 2x1vial 157442C: Standard Diluent, 1x20ml*157442D: Detection Reagent A, 1x120ul *157442E: Detection Reagent B, 1x120ul 157442F: Assay Diluent A (2x), 1x 6ml157442G: Assay Diluent B (2x), 1x 6ml157442H: Substrate A, 1x10ml157442K: Substrate B, 1x2ml157442L: Wash Buffer (30x), 1x20ml
Storage and Stability:Store *157442A, *157442B, *157442D and *157442E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Materials Required But Not Supplied:1. Luminometer capable of reading 96-well microplates with the following parameters: lag time 30.0secs; read time 1.0 sec/well . 2. Precision single or multi-channel pipettes and disposable tips
3. Eppendorf Tubes for diluting samples
4. Deionized or distilled water
5. Absorbent paper for blotting the microtiter plate
6. Container for Wash Solution
Sample Preparation and Storage:Serum:Use a serum separator and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
Plasma:Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
Tissue Homogenates:The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weighed before homogenization. The tissues were minced to small pieces and homogenized in 5-10ml of PBS with a glass homogenizer on ice (Micro Tissue Grinders works too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifuged for 5 minutes at 5000×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower.
Cell Lysates:Cells must be lysed before assaying according to the following directions. 1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). 2. Wash cells three times in cold PBS. 3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4X (or freeze cells at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) 4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris.
Cell Culture Superntants and Other Biological Fluids:Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles
Note:1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination
2. Sample hemolysis will influence the results so hemolyzed specimens should not be used
3. When performing the assay, bring samples to RT.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C
3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C
4. Aspirate and wash 3 times
5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C
6. Aspirate and wash 5 times
7. Add 100ul Substrate Solution. Incubate 10 minutes at 37°C
8. Read RLU immediately.
仕様
Size：96Tests
Source Antigen：Human
Specificity：This sandwich assay has high sensitivity and excellent specificity for detection of Brain Natriuretic Peptide (BNP). No significant cross-reactivity or interference between Brain Natriuretic Peptide (BNP) and analogues was observed.
