Abeta 1-42 ELISA 试剂盒

• Sample analyzed: IDE activator-treated 7PA2 cells (passage #6). A total of 18 samples were used. Observations were obtained in triplicated samples of 6 different treatment groups, and their average values were taken as the response variable.
• Cell culture and treatment protocol:
  • 3 x 10^5 7PA2 cells were seeded into 6-well plates and grown in Dulbecco’s modified medium with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 200 µg/ml G418 (gentamycin), and 2 mM L-glutamine.
  • When cells reached 90-95% confluency, the old media was aspirated, and cells were treated with 50 µM of IDE activators dissolved in a serum-free media.
  • Control cells were treated with DMSO since the solvent of the chemicals was DMSO (not exceeding 0.5% of the total volume of the media).
  • Cells were incubated for 72 hours at 37°C in a 5% CO2 incubator.
• Sample preparation:
  • Cells were collected by scraping in PBS and centrifuged at 4°C, 10000 rpm for 10 min.
  • The supernatant was removed, and 120 µl of lysis buffer (KCl 150 mM, HEPES 20 mM, MgCl2 5 mM, and DTT 0.5 mM) was added to the pellet.
  • Tubes with samples were kept in the ice bucket for 15 min, and cells were disrupted by pipetting.
  • Tubes were centrifuged at 4°C, 10000 rpm for 10 min.
  • The supernatant was transferred into a new tube, and a portion of the supernatant was used for protein determination.
  • The protein amount was determined by BCA assay, the total protein amount was calculated based on the albumin standard curve, and 100 µl of lysate was used for ELISA assay.
• Reagent preparation:
  • All reagents and samples were brought to ambient temperature for 20 minutes before use.
  • Wash buffer:
    • The washing buffer might have crystals; to melt them down, it was kept in the water bath at 40℃.
    • To prepare the washing buffer, 2 mL of 25x wash buffer was mixed with 48 mL of DW.
    • The unused solution was kept at 4 °C.
  • Standards:
    • 1 mL of sample dilution buffer was added into one standard tube (labeled as a 0 tube); it was kept at room temperature for 10 minutes and mixed thoroughly.
    • Seven tubes were labeled from 1 to 7.
    • 300 µL of sample dilution buffer was added into each tube.
    • 300 µL of the standard was transferred from the 0 tube to the 1st tube and mixed thoroughly.
    • 300 µL of sample from the 1st tube was added to the 2nd tube and mixed thoroughly.
    • This serial dilution was repeated until the 7th tube, and we got samples diluted at 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64.
    • Sample dilution buffer was added to the 7th tube and used as a blank.
  • Biotin-labeled antibody working solution:
    • The solution was prepared 1 hour before usage.
    • The biotin-detection antibody was diluted with antibody dilution buffer at a 1:100 ratio.
    • The mixture was prepared for 35 samples (1 extra for volume loss).
    • 35 µL of biotin-labeled antibody was added to 3465 µL of antibody dilution buffer and mixed thoroughly.
  • HRP-Streptavidin Conjugate Working Solution (SABC):
    • The solution was prepared 30 minutes before usage.
    • SABC was diluted with SABC dilution buffer at a 1:100 ratio.
    • The mixture was prepared for 35 samples (1 extra for volume loss).
    • 35 µL of SABC antibody was added to 3465 µL of SABC dilution buffer and mixed thoroughly.
• Assay procedure:
  1. Standards, test samples, and control (blank) were set on the pre-coated plate, respectively, and their positions were recorded.
  2. 100 µL of standard, sample, and blank were loaded into designated wells.
  3. The plate was sealed with a cover and incubated at room temperature for 120 minutes by slightly shaking at 300 rpm.
  4. The cover was removed, and the plate content was discarded by aspiration in a low vacuum pressure. Each well was washed twice with 300 µL of washing buffer.
  5. 100 µL of biotin-labeled antibody working solution was added into wells, and the plate was covered and incubated at room temperature for 60 minutes by slightly shaking at 300 rpm.
  6. The cover was removed, and plate content was discarded by aspiration in a low vacuum pressure. Each well was washed three times with 300 µL of washing buffer. Washing buffer stayed in the wells for 1-2 minutes each time.
  7. 100 µL of SABC working solution was added into each well; the plate was covered and incubated at room temperature for 30 minutes at 300 rpm.
  8. The cover was removed, and plate content was discarded by aspiration in a low vacuum pressure. Each well was washed five times with 300 µL of washing buffer. Washing buffer stayed in the wells for 1-2 minutes each time.
  9. Before adding TMB into wells, it was kept at 37°C for 30 minutes for equilibration. 90 µL of TMB substrate was added into each well; the plate was covered and incubated at 37°C in a dark place (the plate was wrapped with aluminum foil) for 20 minutes. The blue color was developed in the wells.
  10. The reaction was terminated by adding 50 µL of stop solution. The color turned yellow immediately. The adding order of the Stop solution was the same as the loading order of the TMB substrate solution.
  11. The plate was read immediately by a microplate reader at 450 nm wavelength.
• Calculation:
  • Data analysis was performed in GraphPad Prism 10.3.1 software.
  • Relative O.D.450 for samples was calculated using the following equation: The relative O.D.450 = The O.D.450 of each well - The O.D.450 of the blank well.
  • The standard curve was plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X) (4PL regression).
  • The target concentration of the samples was interpolated from the standard curve.

The aim of this experiment was the validation of ELISA kits, which determine the amount of β-amyloid 1-40 and 1-42 in biological samples. 7PA2 cells were used as a test sample after being treated with different IDE activators. 7PA2 cells stably express human APP751 bearing the V717F familial AD mutation and secrete significant amounts of APP products, and would be a perfect sample to validate β-amyloid ELISA kits.To generate a standard calibration curve, a wide range of concentrations were used for β-amyloid (1-40) and β-amyloid (1-42), between 7.8125-500 pg/ml for β-amyloid (1-40), and between 4.6875-300 pg/ml for β-amyloid (1-42). Blank O.D. values were subtracted from the standard sample and test O.D. values to remove the background signal. β-amyloid (1-40) displayed a higher background signal in comparison to the β-amyloid (1-42) ELISA kit, which was also the reason for the high deviation in CV% of the lowest concentrations of β-amyloid (1-40) standards, which means the usage of these two points might increase the variability of the actual amount of β-amyloid (1-40) in the test samples.

Standard sample concentrations of β-amyloid (1-42) showed low CV deviation, which led to building a good standard curve. A total of 18 test samples were analyzed (6 groups in triplicated samples), and the amount of β-amyloid (1-40) and β-amyloid (1-42) was determined by using the 4PL regression model curve by GraphPad Prism software. The amount of β-amyloid (1-40) and β-amyloid (1-42) in test samples was in the ranges of the calibration curve.