Principle of the Assay
This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti- Aβ42
antibody was pre-coated onto 96-well plates. And the biotin conjugated anti- Aβ42 antibody
was used as detection antibodies. The standards, test samples and biotin conjugated detectionantibody were added to the wells subsequently, and washed with wash buffer. HRP-
Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB
substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to
produce a blue color product that changed into yellow after adding acidic stop solution. The
density of yellow is proportional to the Aβ42 amount of sample captured in plate. Read the
O.D. absorbance at 450nm in a microplate reader, and then the concentration of Aβ42 can be
calculated.
Precautions
1. To inspect the validity of experiment operation and the appropriateness of sample dilution
proportion, pilot experiment using standards and a small number of samples is
recommended.
2. After opening and before using, keep plate dry.
3. Before using the Kit, spin tubes and bring down all components to the bottom of tubes.
4. Storage TMB reagents avoid light.
5. Washing process is very important, not fully wash easily cause a false positive.
6. Duplicate well assay is recommended for both standard and sample testing.
7. Don’t let Micro plate dry at the assay, for dry plate will inactivate active components on
plate.
8. Don’t reuse tips and tubes to avoid cross contamination.
9. Avoid using the reagents from different batches together.
Material Required but Not Supplied
1. Microplate reader (wavelength: 450nm)
2. 37°C incubator
3. Automated plate washer
4. Precision single and multi-channel pipette and disposable tips
5. Clean tubes and Eppendorf tubes
6. Deionized or distilled water
Manual Washing
Discard the solution in the plate without touching the side walls. Clap the plate on absorbent
filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and
soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent
filter papers or other absorbent material. Repeat this procedure two more times for a total of
THREE washes.
2Automatic Washing
Aspirate all wells, and then wash plate THREE times with 350ul wash buffer. After the final wash,
invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is
recommended that the washer shall be set for soaking 1 minute.
Sample Collection and Storage
Isolate test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot
and store at -20°C for long term. Avoid multiple freeze-thaw cycles.
Serum: Place whole blood sample at room temperature for 2 hours or put it at 4°C
overnight and centrifugation for 20 minutes at approximately 1000×g, Collect the
supernatant and carry out the assay immediately. Blood collection tubes should be
disposable, non-pyrogenic, and non-endotoxin.
Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15
minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and
carry out the assay immediately. Avoid hemolysis, high cholesterol samples.
Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to
remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4).
Mince tissue after weighing it and get it homogenized in PBS (the volume depends on the
weight of the tissue. Generally speaking, 9mL PBS would be appropriate to 1 gram tissue
pieces. Some protease inhibitors are recommended to add into the PBS) with a glass
homogenizer on ice. To further break the cells, you can sonicate the suspension with an
ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then
centrifuged for 5minutes at 5000×g to get the supernate.
Cell Culture supernate: Centrifuge supernatant for 20 minutes at 1000×g at 2 - 8°C to
remove insoluble impurity and cell debris. Collect the clear supernate and carry out the
assay immediately.
Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect
supernatant and carry out the assay immediately.
Sample Preparation: Samples shall be clear and transparent and remove suspended solids
by centrifugation.
Note: Samples to be used within 5 days can be stored at 4°C, besides that, samples must be
stored at -20°C (assay ≤1 month) or -80°C(assay≤2 months) to avoid loss of bioactivity and
contamination. Hemolyzed samples are not suitable for this assay.
