Human APPβ ELISA Kit,specification,price,image-Bio-Equip in China

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Product Specification

Usage

Specimen Requirements

1. Specimens should be extracted as soon as possible after collection, following the relevant literature. Experiments should be performed as soon as possible after extraction. If experiments cannot be performed immediately, specimens can be stored at -20°C, but repeated freeze-thaw cycles should be avoided.

2. Samples containing NaN3 cannot be assayed, as NaN3 inhibits horseradish peroxidase (HRP) activity.

Procedure

1. Dilution of Standard: This kit provides one standard at full strength. Users can dilute the standard in a small test tube according to the following chart.

20ng/ml	Standard No. 5	Add 150μl of original standard to 150μl of standard diluent
10ng/ml		Standard No. 4	150μl of Standard No. 5 was added to 150μl of Standard Diluent
5ng/ml	Standard No. 3	150μl of Standard No. 4 was added to 150μl of Standard Diluent
2.5ng/ml	Standard No. 2	150µl of Standard No. 3 was added to 150µl of Standard Diluent
1.25ng/ml	Standard No. 1	150µl of Standard No. 3 was added to 150µl of Standard Diluent
1.25ng/ml	Add 150 μl of Standard No. 2 to 150 μl of Standard Diluent.

2. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-linked reagent; all other steps remain the same), standard wells, and test sample wells. Accurately add 50 μl of the standard to the enzyme-linked coated plate. First, add 40 μl of sample diluent to the test sample wells, followed by 10 μl of the test sample (final sample dilution is 5-fold). Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix.

3. Incubation: Seal the plate with sealing film and incubate at 37°C for 30 minutes. 4. Preparation: Dilute the 30x concentrated wash buffer 30x with distilled water and set aside. 5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with wash buffer, let it sit for 30 seconds, then discard. Repeat this process 5 times and pat dry. 6. Add enzyme: Add 50µl of enzyme-labeled reagent to each well, excluding the blank well. 7. Incubation: Same as in 3. 8. Wash: Same as in 5. 9. Color development: First add 50µl of Color Developer A to each well, then add 50µl of Color Developer B. Gently shake to mix. Develop at 37°C in the dark for 10 minutes. 10. Stop: Add 50µl of Stop Buffer to each well to terminate the reaction (the blue color will immediately turn yellow).

11. Measurement: Use the blank well as the zero setting and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. Measurements should be performed within 15 minutes after adding the stop solution.

Calculation

Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to draw a standard curve on graph paper. Based on the OD value of the sample, find the corresponding concentration from the standard curve; then multiply by the dilution factor. Alternatively, use the standard concentration and OD value to calculate the linear regression equation of the standard curve. Substitute the sample OD value into the equation to calculate the sample concentration. Multiply by the dilution factor to obtain the actual sample concentration.

Species Reactivity

Human

Theory

This kit uses a double-antibody sandwich assay to measure the level of human amyloid precursor protein β (APPβ) in a sample. A microplate is coated with purified human amyloid precursor protein β (APPβ) antibody to form a solid-phase antibody. APPβ is then added sequentially to the antibody-coated microwells. This antibody then binds to an HRP-labeled APPβ antibody, forming an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the plate is then developed with the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of APPβ in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the concentration of human APPβ in the sample is calculated using a standard curve.

Detection Type

Used to determine the content of amyloid precursor protein β (APPβ) in human serum, plasma and related liquid samples

Composition

Serial number	Component name	96T	Serial number	Component name	96T
1	30 times concentrated washing liquid	20ml×1 bottle	6	2	Enzyme label reagent	6ml×1 bottle	7	Stop solution	6ml×1 bottle
3	Enzyme-labeled coated plate	12 Hole×8 strips	8	Standard product (40ng/ml)	0.5ml×1 bottle
4	Sample diluent	6ml×1 bottle	9	5	Developer A solution	6ml×1 bottle	10	Sealing film	2 photos

General Notes

1. After removing the kit from the refrigerator, allow it to equilibrate at room temperature for 1 hour before use. If the enzyme-coated plate is opened and not completely used, store it in a sealed bag. 2. Crystals may form in the concentrated wash buffer. Warming in a water bath can aid dissolution during dilution. This will not affect the results. 3. Use a pipette for each sample addition step and frequently calibrate its accuracy to avoid experimental error. The time for each addition should ideally be within 5 minutes. For large numbers of samples, using a dispenser is recommended. 4. Develop a standard curve with each measurement, preferably with replicates. If the analyte content in the sample is too high (the sample OD value is greater than the OD value of the first standard well), dilute the sample a certain number of times (n times) with sample diluent before measurement. When calculating the final dilution, multiply by the total dilution factor (×n×5).

5. The sealing film is for single use only to avoid cross contamination.

6. Please store the substrate in a dark place.

7. Strictly follow the instructions for operation. The test results must be determined based on the readings of the microplate reader.

8. All samples, washing solutions and various wastes should be treated as infectious materials.

9. Components of different batches of this reagent must not be mixed.

Storage Temp.

Unopened test kit, sealed and stored at 2-8℃, valid for 6 months

Test Range

0.6ng/ml - 20ng/ml