Rat Wnt-2b protein (WNT2b) enzyme-linked immunosorbent assay kit instruction manual

Read this manual carefully before use. The enzyme-linked immunosorbent kit is based on the principle of double antibody sandwich technology to detect rat Wnt2B, which can only be used for research purposes and should not be used for medical diagnosis.

Uses : For the determination of Wnt2B in rat serum, plasma and related liquid samples.

Working principle The kit uses a biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of Wnt2B in the sample. Rat Wnt2B was added to the well-labeled wells pre-coated with rat Wnt2B monoclonal antibody, and incubated; after incubation, biotin-labeled anti-Wnt2B antibody was added. It is then combined with streptavidin-HRP to form an immune complex, which is then incubated and washed to remove unbound enzymes, then added to substrates A and B to produce a blue color, which is converted to a final form by acid. Yellow. The depth of the color was positively correlated with the concentration of rat Wnt2B in the sample.
Kit composition

Kit composition	48-hole configuration	96-well configuration	save
Instruction manual	1 serving	1 serving
Sealing film	2 pieces (48)	2 pieces (96)
sealed bag	1	1
Enzyme label coated plate	1×48	1×96	2-8 ° C preservation
Standard 160ng/L	0.5ml × 1 bottle	0.5ml × 1 bottle	2-8 ° C preservation
Standard dilution	3ml × 1 bottle	6ml × 1 bottle	2-8 ° C preservation
Streptavidin-HRP	3 ml × 1 bottle	6 ml × 1 bottle	2-8 ° C preservation
Biotinylated anti-WNT2B antibody	0.5ml × 1 bottle	1 ml × 1 bottle	2-8 ° C preservation
Developer A solution	3 ml × 1 bottle	6 ml × 1 bottle	2-8 ° C preservation
Developer B solution	3 ml × 1 bottle	6 ml × 1 bottle	2-8 ° C preservation
Stop solution	3ml × 1 bottle	6ml × 1 bottle	2-8 ° C preservation
Concentrated washing solution	(20ml × 20 times) × 1 bottle	(20ml × 30 times) × 1 bottle	2-8 ° C preservation

Reagents and equipment that are not provided

1. 37 ° C incubator.
2. Standard specification microplate reader.
3. Precision pipettes and disposable tips
4. Distilled water,
5. Disposable test tube
6. Absorbent paper

Precautions

1. The kit removed from 2-8 ° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme label is not used up after the plate is opened, the slats should be stored in a sealed bag.
2. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test error.
3. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
4. To avoid cross-contamination, avoid reusing the tips and closures in your hand.
5. Other reagents that are not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.
6. Substrate B is sensitive to light and avoids prolonged exposure to light.

Washing method
Manual washing method: smash the liquid in the microplate; pour a few layers of absorbent paper on the test bench, and take a few times with the microplate; push the diluted washing solution into the hole at least 0.35ml, soak 1 2 minutes. Repeat this process several times as needed.

Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Specimen requirements
1. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
2. 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.
Operating procedure

1. Dilution of standard: (This kit provides one original standard, which can be diluted in a small tube according to the following chart.)

80ng/L	Standard No. 5	Add 120 μl of standard dilution to 120 μl of standard dilution
40ng/L	Standard No. 4	Add 120 μl of Standard No. 5 to 120 μl of Standard Diluent
20 ng/L	Standard No. 3	Add 120 μl of Standard No. 4 to 120 μl of Standard Diluent
10ng/L	Standard 2	Add 120 μl of Standard No. 3 to 120 μl of Standard Diluent
5 ng/L	Standard No. 1	Add 120 μl of Standard 2 to 120 μl of Standard Diluent

2. Determine the number of slats required based on the number of samples taken and the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and the double hole can be used as much as possible.
3. Loading: 1) blank well, blank control well without sample, biotinylated anti-WNT2B antibody, streptavidin-HRP, only coloring agent A&B and stop solution, the other steps are the same; 2 Standard hole: add standard 50ul, streptavidin-HRP50ul (standard biotin antibody has been integrated in the standard, so it is not added); 3) test sample hole: add 40ul sample, then add anti-WNT2B 10 μl of antibody, streptavidin-HRP50ul, covered with a sealing membrane, gently shaken and mixed, and incubated at 37 ° C for 60 minutes.
4. Dosing: 30 times concentrated washing solution was diluted with distilled water 30 times and used.
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. Color development: Add A50ul of coloring agent to each well, then add 50μl of coloring agent, gently shake and mix, and develop color at 37°C for 15 minutes.
7. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow).
8. 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 10 minutes after the addition of the stop solution.
9. Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. It can also be calculated using various application software.

Summary of operating procedures:


Kit performance:
1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.92 or more.
2. Within and between batches should be less than 9% and 15% respectively

examination range:
Detection range: 2ng/L -90ng/L

Storage conditions and expiration date:

Storage: 2-8 ° C.
Validity: 6 months (2-8 ° C).

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