Human soluble amyloid precursor protein ( supersensitive ) detection kit
(Japan IBL imported, article number: 27734)
Japan's IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
Introduction to the introduction
Alzheimer's disease (AD) was first published by German neuropathologist A. Alzheimer in 1907 and is recognized as a major cause of dementia.
principle
The solid phase ELISA kit uses two highly specific antibodies. The TMB substrate solution is used as a color developer. The intensity of color development is proportional to the amount of soluble amyloid precursor protein α (sAPP α).
examination range
0.78- 50ng/mL
expected usage
For research purposes, not for diagnostic procedures.
â– This IBL test kit can be used for quantitative detection of sAPP in human serum, EDTA plasma, cerebrospinal fluid, and cell culture supernatant.
â– Recommended serum samples, EDTA-plasma samples diluted 4-8 times.
â– Cerebrospinal fluid samples are more than 4 times.
â– If the cell culture supernatant contains serum such as FCS, there may be a cross reaction. It is recommended to set a negative medium quality control.
Kit composition
1 pre-coated plate: anti-human-sAPP α (2B3) mouse IgG monoclonal antibody, affinity purification 96T
2 concentrated enzyme-labeled antibody (30X) HRP-labeled anti-human APP (R101A4) mice
IgG monoclonal antibody, Fab' affinity purification 0.4 mL x 1
3 standard: recombinant human sAPPα protein 0.5mL x 2
5 enzyme conjugate dilution 1% BSA, 0.05% Tween 20 PBS 12mL x 1
8 concentrated washing solution: 40 × 0.05% Tween 20 phosphate buffer 50mL x 1
Instructions
1 required experimental equipment ( but not provided in this kit )
Microplate reader (450nm) micropipette and tip
Measuring cylinder and beaker
Refrigerator (4 ° C) coordinate paper (log / log)
Absorbent paper
Washing bottle
Disposable test tube for "2 concentrated enzyme-labeled antibody" and "6 substrate solution: TMB solution"
2 preparation
1) Preparation of washing liquid
2) Preparation of enzyme conjugates
"2 Concentrated enzyme-labeled antibody " is a 30-fold concentrate. Use one-time test tube to dilute "2 concentrated enzyme-labeled antibody" with "5 Enzyme Diluent" to prepare the desired enzyme conjugate. (Dilute 1:30 )
Example: If you are using 8-well, we need to configure 800ul of labeled antibody, 30ul of "2 Concentrated Enzyme-labeled Antibody" and 870ul of "5 Enzyme Diluent" to add 100ul per well.
3) Preparation of standard products
Dilute the bottle of "3 standard " with 0.5 mL of deionized water and mix to make a 100 ng/mL human sAPPα standard.
4) Dilution of standards
Prepare 8 tubes for standard dilution and number. Add 230 μL of “4EIA buffer†to each tube. Take 230 μL of the standard solution in tube 1 and mix. Then take 230 μL of solution from tube 1. In the test tube 2, according to this rule, the following standard is used to prepare a series of standard products. The eighth test tube is 0 ng/mL as a zero standard.
5) Dilution of the sample
The sample must be diluted with the “4EIA buffer†provided in the kit . If the concentration range of sAPPα in the sample is not clear, it is recommended to pre-form several different concentrations to determine the appropriate detection concentration.
Detection step
All reagents are equilibrated to the greenhouse (approximately 30 minutes) and mixed before use. Ensure that the reagents have no quality problems. Prepare a standard curve while testing the sample.
1 set reagent blank control. Add 100μL "4EIA buffer" in the microwell.
2 Add 100 μL sample blank (tube 8), series standard (tube 1-7), and sample in the corresponding holes.
3 Incubate overnight at 4 ° C after the cover
4 Wash the plate with the washing solution. Then add the washing buffer, no need to cover for 15-30 seconds, then completely remove the washing buffer. This step needs to be repeated at least 7 times. Finally, the residue is removed on the absorbent paper to remove the residual liquid. drop.
If the automatic washing machine is used, after the plate is washed 4 times on the washing machine, the above manual washing step still needs to be repeated 3 times.
5 add 100 μL of enzyme solution in each well.
6 cover and incubate at 4 ° C for 30 minutes
7 Wash the plate 9 times in step 4.
8 Add the required amount of “6 substrate solution: TMB solution†to a disposable tube. Then take 100 μL of substrate solution in each well. Note that the excess substrate in the disposable tube should not be returned. Avoid cross infection in the bottle.
9 Incubate the coated plate for 30 minutes at room temperature. After adding the substrate solution, the liquid in the well will turn blue.
10 Add 100 μL of “7 Stop Liquid†to each well, and mix the light-strip strips. After adding the stop solution, the liquid in the well will turn yellow.
11 Remove the dirt or droplets at the bottom of the plate to ensure no bubble formation. Measure the absorbance at 450 nm with a microplate reader within 30 minutes after the addition of the stop solution.
pay attention
1 The sample should be tested immediately after collection. If it is stored frozen, it needs to be thawed to avoid repeated freezing and thawing.
2 samples must be diluted with "4EIA buffer".
3 recommended sample and each standard for double detection
4 samples should be kept in the neutral pH range. Because the contamination of organic solvents will affect the test results
5 wash buffers provided with the kit can be used for washing plates. Insufficient washing may result in erroneous results.
6 Light the plate on the absorbent paper to remove residual droplets. Do not scratch the micropores with absorbent paper.
7 "6 substrate liquid" should be stored in the dark to avoid contact with metal.
8 plus "7 stop solution", reading must be taken within 30 minutes.
Result analysis and calculation
All measured absorbance values ​​(including standards, unknown test samples) need to subtract the absorbance value of the blank control. On the double logarithmic coordinate paper, the standard concentration is used as the abscissa and the corresponding absorbance is taken as the ordinate. The standard curve is drawn through smooth points. The concentration of the detected sample can be read directly in the standard curve.
The above standard curve is for demonstration purposes only and cannot be used for reading sample results. It is necessary to establish a standard curve for each test.
This translation is for reference only, please refer to the original for details.
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