Frequently Asked Questions （：Quesation ：Answer）
Instrumentation sample preparation and storage for ELISA assays
1. How to collect serum and plasma?
Reading at dual wavelengths is to correct the optical density contributed by the plate wells and other nonspecific interference. Our plates are chosen for their optical quality, Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 15 minutes at 1000×g at 2 - 8℃. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable, non-endotoxin.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8℃ within 30 minutes of collection. Collect the supernatant to carry out the assay. Hemolysis samples are not suitable for ELISA assay.
2. How to deal with tissue or cells?
Normally, the amount and ratio of each reagent a fully automated microplate ELISA analyzer needs are not the same as in our kit, so it’s not applicable. Whereas if you can tell Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells by trypsin. Collect the cell suspension into the centrifugal tube and centrifuge for 5 minutes at 1000×g. Discard the medium and wash the cells for 3 times with pre-cooled PBS. For each 1x106 cells, add 150-250uL of pre-cooled PBS (0.01M, pH=7.4) to keep the cells resuspended. Repeat the freeze-thaw process for several times until the cells are lysed fully. Centrifuge for 10minutes at 1500×g at 2 - 8℃. Remove the cell fragments, collect the supernatant to carry out the assay. Avoid repeated freeze-thaw cycle.
Cell culture supernatant: Collect samples and centrifuge for 20 minutes at 1000×g at 2 - 8℃. Collect the supernatant to carry out the assay.
Tissue homogenates: Generally, mince the tissues to small pieces and rinse them in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS volume (mL) =1:9) 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 5 minutes at 5000×g at 2 - 8℃, collect the supernatant to carry out the assay.
3. Saliva, Urine, milk, tear and other types of biological fluids
In general, use a sterile container to collect fluids, then centrifuge it at 1000-5000×g at 2 - 8℃ for 5-20min to remove particulates, collect the supernatant to carry out the assay.
Samples should be assayed within 7 days when stored at 2-8℃, otherwise samples must be divided up and stored at -20℃ (≤1 month) or -80℃ (≤3 months). Avoid repeated freeze-thaw cycles. Centrifuge again before assaying to remove any additional precipitates that may appear after storage.
1. Target analyte in tested samples
It’s recommended to test fresh samples by ELISA, which could avoid false positive or negative results caused by the degradation or decomposition of target analyte during long time storage.
2. Dilution factors and diluent
Dilution ratio: Refer to the specific protocol or published papers, or consult to our technical support.
Recommended diluent: Standard & Sample Diluent provided in the ELISA kit, we could provide more vials of it for free for specific application, just tell us when you place the order.
3. Assay procedure
Following the protocol strictly, here are some hints
a. Establish the complete Standard Curve for each assay.
b. Ensure the precise volume for each reagent addition
c. Try to finish the liquid addition for each reagent as soon as possible, better not over 10 min.
d. Keep constant speed for liquid addition to each well
e. To get more accurate experiment result, it’s recommended to conduct the ELISA assay as soon as possible once you receive the kit.
4. How to calculate/ deal with the raw datas?
After correct the average OD values, plot a four parameter logistic curve on log-log graph paper, with standard concentration on the x-axis and OD values on the y-axis, many softwares could also help to deal with it, such as Origin( recommended), CurveExpert.
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