Human amyloid  770 (APP770) ELISA kit
(Japan IBL imported, article number: 27736)
Japan's IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
Introduction
This ELISA kit is used for the detection of human APP770 in EDTA-plasma, cerebrospinal fluid and supernatant.
Experimental principle
This kit is a sandwich ELISA kit that uses two highly specific antibodies. TMB as a color developer, the color intensity of the final solution is proportional to the amount of APP770 in the sample.
examination range
0.10~6.2ng/mL
expected usage
For research purposes only, not for diagnosis
This kit is used for the quantitative detection of human APP770 in EDTA-plasma cerebrospinal fluid and cell supernatant.
Kit composition
1. Microplate, 96T, coated with anti-human APP OX2 (351) rabbit IgG antibody, affinity purification
2. Labeled antibody concentrate, 30X, 0.4ml, HRP-labeled anti-human APP (R101A4) mouse IgG monoclonal antibody Fab fragment
3. Standard, 0.5ml, 2, recombinant APP770
4. EIA buffer, 30ml, 1% BSA, PBS containing 0.05% Tween 20
5. Label antibody dilution, 12ml, 1% BSA, PBS containing 0.05% Tween 20
6. Colorant, TMB, 15ml
7. Stop solution, 1N sulfuric acid, 12ml
8. Wash buffer concentrate, 40X, 50ml, 0.005% Tween 20 phosphate buffer
Preparation step
The required materials were tested but the kit did not provide
l Microplate reader (450nm)
l graduated cylinder and beaker
l Incubator (37 °C ± 1 °C)
l absorbent paper
l Dilution test tube
l Micro pipettes and sampling tips
l deionized water
l coordinate paper (log/log)
l Test tube for dilution of standards
l washing bottle
Reagent preparation
1. Preparation of washing buffer: first equilibrate the washing concentrate to room temperature, mix well, then mix 50 ml of the concentrated solution with 1950 ml of deionized water to obtain. The prepared washing solution should be stored in the refrigerator and used up within 2 weeks.
2. Preparation of labeled antibody: The labeled antibody was diluted 30-fold with a labeled antibody dilution to obtain.
Example: If only one plate is measured, 8 wells, you need to label the antibody 800ul (30ul of labeled antibody + 870ul of labeled antibody dilution, mix, add 100ul per well)
This step is completed before the experiment begins
The remaining labeled antibody concentrate should be stored in a sealed bottle and stored at 4 ° C
3. Preparation of standard product: Pipette 0.5ml of deionized water into the standard bottle and mix well to obtain 12.4ng/ml human APP770 standard.
4. Dilution of standard: Prepare 8 tubes for standard dilution, add 230 ul EIA buffer to each tube
The concentration of each tube is as follows
No. 1 tube 6.2ng/ml
No. 2 tube 3.1ng/ml
Tube No. 3 1.55ng/ml
No. 4 tube 0.78ng/ml
No. 5 tube 0.39ng/ml
No. 6 tube 0.19ng/ml
Tube No. 7 0.10ng/ml
Tube 8 0ng/ml (test sample blank)
Pipette 230 ul of the standard product into the No. 1 tube, then pipette 230 ul from the No. 1 tube into the No. 2 tube and serially dilute it. The standard range is 6.2 to 0.10 ng/ml.
5. Sample dilution: Samples were diluted in EIA buffer. If the sample concentration has not been evaluated beforehand, we recommend that the sample be tested with several different dilution ratios to determine the optimal dilution ratio of the sample.
Experimental procedure
Before use, all samples should be equilibrated to room temperature for about 30 minutes, then thoroughly mixed to ensure no change in reagent quality. Standard curve and sample detection are performed simultaneously.
Reagent | Test sample | Standard | Test sample control well | Reagent control well |
Test sample 100ul | Dilution standard (1-7 tubes) 100ul | EIA buffer (8th tube) 100ul | EIA buffer 100ul |
Cover, incubate overnight at 4 ° C |
Wash the plate 7 times |
Enzyme-labeled antibody | 100ul | 100ul | 100ul | - |
Cover plate, incubate for 30 min at 37 ° C |
Wash the plate 9 times |
Reagent | 100ul | 100ul | 100ul | 100ul |
Incubate at room temperature for 30 min in the dark |
Stop solution | 100ul | 100ul | 100ul | 100ul |
Read the OD value at 450 nm within 30 min after adding the stop solution |
1) Determine the blank control wells and add 100 ul of EIA buffer to the corresponding wells.
2) Determine the sample control well, test the sample well and standard well, and then add 100 ul of the sample control, test sample and diluted standard to the corresponding microwell.
3) Cover, incubate overnight at 4 ° C
4) Wash the plate with a washing bottle, add the washing solution to the micropores for 15-30 seconds, and discard the washing solution. Repeat 7 times and finally pat dry on absorbent paper. If the plate is washed with a washer, wash it four times with a washer and then wash it three times with a wash bottle.
5) Add 100 ul of enzyme-labeled antibody to each well, except for the reagent blank control well
6) Cover plate, incubate for 30 min at 37 °C
7) Wash the plate 9 times according to step 4)
8) Add the required amount of developer to the test tube and then add 100 ul of developer to each well. To avoid contamination, do not pour the remaining reagent into the original reagent bottle.
9) Incubate at room temperature for 30 minutes in the dark, and the color of the solution will turn blue after adding the developer.
10) Add 100 ul of stop solution to each well, at which point the color of the solution will turn yellow.
11) Wipe off the stain or water droplets at the bottom of the microplate. Within 30 minutes after the stop solution is added, read at 450 nm at the microplate reader.
pay attention
1. The test sample should be tested or frozen immediately after collection. The frozen sample should avoid repeated freezing and thawing. It should be completely dissolved and mixed at low temperature before testing.
2. Test sample diluted with EIA buffer
3. Test samples and standards should be double tested
4. The test sample should be in the neutral pH range, and the contamination of organic solvents may affect the detection.
5. Only use the washing liquid provided by the kit to wash the plate, otherwise the test will fail.
6. Dry the microplate on the absorbent paper, do not wipe
7. The TMB substrate solution should be stored in the dark as it is sensitive to light and avoids contact with metals.
8. The microplate reader must be read within 30 minutes after the addition of the stop solution.
Result calculation
Before plotting the curve, all data should be subtracted from the test sample blank values, including standards and unknown samples. Draw a standard curve on the log-logarithmic scale paper with the OD value and concentration of the standard, and directly read the concentration of the unknown sample from the standard curve.
Attachment: Using automatic microplate reader detection, only need to set the relevant parameters of the microplate reader before the detection, such as coordinate parameters (linear, semi-logarithmic) and calculation mode parameters (three regression, four parameters or double logarithmic curve equation) , you can get the results directly
This translation is for reference only, please refer to the original for details.
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