This kit is for research use only. Then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix. This is the actual concentration of the sample. Tri-layer Lamination,Three Layer Nonwoven Coated,Three Layer Laminates,Laminated Packaging Films,Breathable Lamination XINLE HUABAO MEDICAL PRODUCTS CO.,LTD. , https://www.golbaltravel.com
Detection range: 96T
3μg/L -80μg/L
purpose of usage:
This kit is used to determine brain natriuretic peptide (BNP) levels in human serum, plasma and related fluid samples.
Experimental principle This kit uses the double antibody sandwich method to determine the level of human brain natriuretic peptide (BNP) in the specimen. Purified human brain natriuretic peptide (BNP)
The antibody is coated with a microplate to prepare a solid phase antibody, and brain natriuretic peptide (BNP) is sequentially added to the microwell of the coated monoclonal antibody, and then HRP is added.
The labeled brain natriuretic peptide (BNP) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate.
TMB color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The depth of the color is positively correlated with brain natriuretic peptide (BNP) in the sample. Absorbance (OD) measured at 450 nm using a microplate reader
Value), the concentration of human brain natriuretic peptide (BNP) in the sample was calculated from the standard curve.
Kit composition
1 30 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 enzyme standard reagent 6ml × 1 bottle 8 standard (160μg / L) 0.5ml × 1 bottle
3 enzyme label coating plate 12 holes × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 sample dilution 6ml × 1 bottle 10 instructions 1 copy
5 developer A liquid 6ml × 1 bottle 11 sealing film 2 sheets
6 developer B liquid 6ml × 1 bottle 12 sealed bag 1 specimen request
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.
Add 150 μl of standard dilution to the original standard of 150 μl of 80 μg/L No. 5 standard
40μg/L No. 4 Standard 150μl of No. 5 Standard Add 150μl Standard Diluent
Add 20 μg/L No. 3 standard 150 μl of No. 4 standard to 150 μl of standard dilution
Add 10 μg/L No. 2 standard 150 μl of No. 3 standard to 150 μl of standard dilution
Add 5 μg/L No. 1 Standard 150 μl of No. 2 Standard to 150 μl Standard Diluent
2. Adding samples: set blank holes separately (the blank control wells are not added with samples and enzyme labeling reagents, the other steps are the same), standard holes,
Sample hole to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, and add 40 μl of the sample dilution to the sample well.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Dosing: Dilute 30 times concentrated washing solution with distilled water 30 times.
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A50 to each well, then add 50 μl of color developer B, gently shake and mix, and avoid coloration at 37 °C.
15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Calculate the concentration of the standard as the abscissa and the OD as the ordinate. Draw a standard curve on the coordinate paper, according to the sample.
The OD value is determined from the standard curve by the corresponding concentration; multiplied by the dilution factor; or the linear regression equation of the standard curve is calculated from the concentration of the standard and the OD value, and the OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied In dilution factor,
Precautions
1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, please multiply the total dilution by the total dilution. Multiple (×n×5).
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. In the case of an English manual, the English manual shall prevail.
Storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months
Human brain natriuretic peptide (BNP) enzyme-linked immunosorbent assay kit instruction manual