For quantitative detection of human APP in cell culture supernates.

Typical Data Obtained from Human APP

Typical Human APP ELISA Kit Standard Curve

Storage

Principle

Kit Components

Material Required But Not Provided

Notice for Application of Kit

8. In order to avoid marginal effect of plate incubation due to temperature difference (reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37°C for 30 min before using.

Preparation1. Sample Preparation and StorageStore samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot and freeze samples at -20°C. Avoid repeated freeze-thaw cycles. Cell culture supernates: Remove particulates by centrifugation, assay immediately or aliquot and storesamples at -20°C. 2. Sample Dilution Guideline The user needs to estimate the concentration of the target protein in the sample and select a proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve. Dilute the sample using the provided diluent buffer. The following is a guideline for sample dilution. Several trials may be necessary in practice. The sample must be well mixed with the diluents buffer.High target protein concentration (200-2000ng/ml). The working dilution is 1:100. i.e. Add 1μl sampleinto 99μl sample diluent buffer. Medium target protein concentration (20-200ng/ml). The working dilution is 1:10. i.e. Add 10μl sampleinto 90μl sample diluent buffer. Low target protein concentration (312-20,000pg/ml). The working dilution is 1:2. i.e. Add 50μl sampleto 50μl sample diluent buffer. Very low target protein concentration (≤312pg/ml). No dilution necessary, or the working dilution is 1:2.3. Reagent Preparation and Storage A. Reconstitution of the human APP standard: APP standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes of APP standard (20ng per tube) are included in each kit. Use one tube for each experiment. a. 20,000pg/ml of human APP standard solution: Add 1ml sample diluent buffer into one tube, keep the tube at room temperature for 10 min and mix thoroughly. b. 10,000pg/ml→312pg/ml of human APP standard solutions: Label 6 Eppendorf tubes with 10,000pg/ml, 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312pg/ml respectively. Aliquot 0.3ml of the sample diluent buffer into each tube. Add 0.3ml of the above 20,000pg/ml APP standard solution into 1st tube and mix. Transfer 0.3ml from 1st tube to 2nd tube and mix. Transfer 0.3ml from 2nd tube to 3rd tube and mix, and so on. Note: The standard solutions are best used within 2 hours. The 20ng/ml standard solution should be storedat 4°C for up to 12 hours, or at -20°C for up to 48 hours. Avoid repeated freeze-thaw cycles.B. Preparation of biotinylated anti-human APP antibody working solution: The solution should be prepared no more than 2 hours prior to the experiment. a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume) b. Biotinylated anti-human APP antibody should be diluted in 1:100 with the antibody diluent buffer and mixed thoroughly. (i.e. Add 1μl Biotinylated anti-human APP antibody to 99μl antibody diluent buffer.) C. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared no more than 1 hour prior to the experiment. a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume) b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the ABC dilution buffer and mixed thoroughly. (i.e. Add 1μl ABC to 99μl ABC diluent buffer.) Assay ProcedureThe ABC working solution and TMB color developing agent must be kept warm at 37°C for 30 min before use. When diluting samples and reagents, they must be mixed completely and evenly. Standard APP detection curve should be prepared for each experiment. The user will decide sample dilution fold by crude estimation of APP amount in samples.1. Aliquot 0.1ml per well of the 20,000pg/ml, 10,000pg/ml, 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312pg/ml human APP standard solutions into the precoated 96-well plate. Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add 0.1ml of each properly diluted sample of human cell culture supernates to each empty well. See “Sample Dilution Guideline” above for details. It is recommended that each human APP standard solution and each sample be measured in duplicate. 2. Seal the plate with the cover and incubate at 37°C for 90 min. 3. Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time. 4. Add 0.1ml of biotinylated anti-human APP antibody working solution into each well and incubate the plate at 37°C for 60 min. 5. Wash plate 3 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (Plate Washing Method: Discard the solution in the plate without touching the side walls. Blot the plate onto paper towels or other absorbent material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat this process two additional times for a total of THREE washes. Note: For automated washing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfilling wells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent material.) 6. Add 0.1ml of prepared ABC working solution into each well and incubate the plate at 37°C for 30 min. 7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (See Step 5 for plate washing method). 8. Add 90μl of prepared TMB color developing agent into each well and incubate plate at 37°C in dark for 20-25 min (Note: For reference only, the optimal incubation time should be determined by end user. And the shades of blue can be seen in the wells with the four most concentrated human APP standard solutions; the other wells show no obvious color). 9. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow immediately. 10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding the stop solution.