FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Rat anti - Dog B type Natriuretic Peptide
Collect Sample – serum or blood plasma
Package size: 96 determinations Catalogue No.: QRCT –362514EIA \ UTL
Storage: 2 - 8 °C
BNP is released primarily by vascular endothelium and causes vasoconstriction. An endothelin precursor (big BNP or pro-BNP, 39 aa) is cleaved to BNP (21 aa) by endothelin converting enzyme (ECE) found on the endothelial cell membrane. BNP can then diffuse into the blood and circulate, then bind to ETA receptors found on vascular smooth muscle cells leading to calcium mobilization and smooth muscle contraction. The ETA receptor is coupled with a G-protein linked to phospholipase-C and the formation of IP3. BNP release is stimulated by angiotensin II (AII), antidiuretic hormone (ADH), thrombin, cytokines, reactive oxygen species, and shearing forces acting on the vascular endothelium. BNP release is inhibited by nitric oxide (NO), as well as by prostacyclin and atrial natriuretic peptide.This molecule has a number of other actions besides vasoconstriction: stimulation of aldosterone secretion, positive inotropy and chronotropy in the heart, decreasing renal blood flow and GFR, and release of atrial natriuretic peptide (ANP). BNP has been implicated in the pathogenesis of hypertension, vasospasm, and heart failure. In the latter condition, BNP is released by the failing myocardium where it cancontribute to calcium overload and hypertrophy. Experimentally, ETA receptor antagonists have been shown to decrease mortality and improve hemodynamics in heart failure.Overview Endothelin-1 (BNP), a peptide of 21 amino acid residues, is the most potent vasoconstrictor substance known.
. 1 、 Coated Microtitration Strips 96 wells.
2 、 Standard 25, 50, 100, 200, 400pg/ml,, 1 set.
3 、 Enzyme Conjugate Solution, 12 ml.
4 、 Substrate A, 6 ml.
5 、 Substrate B, 6 ml.
6 、 Stopping Solution (1N HCl), 6 ml.
7 、 Rinsing buffer,60 ml( x 20).
8 、 dilution,15ml(x5)
Materials required but not provided :
�
Precision pipettes: 0.10, 0.20, and 1.0 ml.
� able pipette tips.
�
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FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Rat anti - Dog B type Natriuretic Peptide
Collect Sample – serum or blood plasma
Package size: 96 determinations Catalogue No.: QRCT –362514EIA \ UTL
Storage: 2 - 8 °C
� V ortex mixer or equivalent.
� Absorbent paper or paper towels.
� Microtiter plate shaker
� Graph paper.
� A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450 nm .
Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. All reagents should be allowed to reach room temperature before use.
STEP1 Prepare the ringing buffer by diluting the ringing buffer 20×(example: 60ml+1140ml H
2
O), Prepare the Sample Diluent by diluting the
Diluent 5×(example: 15ml+60ml H
2
O)
STEP2 Dilute samples 1:100 distribing 5ul of sample into 500 ul of dilluent .
STEP3 Place 100ul of diluted sample /reagent blank/standard/controls to the wells of the strips. Incubate for 30 min at 37℃ 。 Wash 5 times 。
STEP4 Add 100ul of Enzyme Conjugate to each well 。 Incubate for 30 min at 37℃ 。 Wash 5 times 。
STEP5 Add 50ul
Substrate
A and
Substrate
B to each well 。 Incubate for 15 min at 37℃ 。
STEP6 Add 50ul of stop solution 。 read absorbance at 450nm within 15 min
The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. It is recommended that if manual pipetting is used, no more than 32 wells be used for each assay run, since pipetting of all standards, specimens and controls should be
2

FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Rat anti - Dog B type Natriuretic Peptide
Collect Sample – serum or blood plasma
Package size: 96 determinations Catalogue No.: QRCT –362514EIA \ UTL
Storage: 2 - 8 °C completed within 3 minutes. A full plate of 96 wells may be used if automated pipetting is available. Duplication of all standards and specimens, although not required, is recommended.
Calculate the average absorbance values (A
450
) for each set of reference standards, control, and samples. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration on linear graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis.Using the mean absorbance value for each sample, determine the corresponding concentration of in from the standard curve.
Results of a typical standard run with optical density readings at 450nm shown in the Y axis against concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.
The minimum detectable concentration of BNP in this assay is estimated to be 1.0 pg/ml.
Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. The results obtained from the use of this kit should be used only as an adjunct to other diagnostic procedures and information available to the physician.
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