The samples available to ELISA detection usually have various types, such as serum, plasma, urine, and cell culture supernatant or tissue homogenate. The pretreatment methods for different types of samples are also different. Appropriate sample treatment is the first step to ensure the correctness and accuracy of ELISA detection. Here we’ll introduce treatment methods for different sample types.
Serum is the most common sample type detected in ELISA, the treatment of which is also very simple.
First, use a test tube or centrifugal tube without pyrogen or endotoxin to collect blood samples. Then keep the test tube or centrifugal tube for 2 hours at room temperature or overnight at 4℃ to separate out the serum (It is recommended to tilt the test tube or centrifugal tube to expand the cross section of the liquid surface, so that the serum can be separated out at a larger degree). Afterwards, centrifuge the tube for 20 min at 1000×g at 4℃, collect the supernatant(serum) carefully and carry out the assay immediately. It is suggested to aliquot and store the collected serum at -20℃ or -80℃ to avoid repeated freeze-thaw cycles.
Avoid hemolysis in the process of collecting blood samples because the erythrocyte will release substances with peroxidase activity when it lyses. In this case, non-specific chromogenic reaction will appear in HPR-labeled ELISA, resulting in inaccuracy of the detection. Meanwhile, avoid of bacterial contamination since the bacteria may contain endogenic HRP, which may result in false positives of the detection.
First, use a blood-collection tube or centrifugal tube with anticoagulant to collect blood samples. Then centrifuge the samples for 15 min at 1000×g at 4℃ within 30 min of collection and collect the supernatant (plasma) carefully. It is suggested to aliquot and store the collected plasma at -20℃or -80℃ to avoid repeated freeze-thaw cycles. Avoid hemolysis or hyperlipidemia samples.
The common anticoagulants include EDTA, heparin sodium, sodium citrate and so on. Read the ELISA kit manual carefully before detection, ensure that whether the kit has special requirements for anticoagulants.
3. Cell culture supernatant
First, aspirate the cell culture supernatant to the centrifugal tube and centrifuge for 20min at 1000×g. Then remove the cell fragments and impurities, collect the supernatant and store at -20℃ or -80℃ to avoid repeated freeze-thaw cycles.
Repeated freeze-thaw method
1)Aspirate the medium in the culture plate and dissociate the cells by trypsin. Then add moderate amount of medium to flush the cells off the culture plate. As for suspension cell, this step can be omitted.
2)Collect the cell suspension into the centrifugal tube and centrifuge for 10 min at 1000×g. Then discard the medium and wash the cells for 3 times with pre-cooled PBS.
3)Add moderate amount of pre-cooled PBS（It is recommended to add some protease inhibitor into the PBS immediately before use）to keep the cells resuspended. In the 6-well culture plate, each well needs 150-250 μL of PBS to resuspend the cells.
4)Keep the samples at -20℃ or -80℃ to freeze and thaw them at room temperature successively. Repeat the freeze-thaw process for several times until the cells are lysed fully. You can also sonicate the suspension with an ultrasonic cell disrupter.
5)Centrifuge for 10 min at 10,000×g at 4℃. Then remove the cell fragments, collect the supernatant and store at -20℃ or -80℃ to avoid repeated freeze-thaw cycles.
5. Tissue homogenate
1)Rinse the tissues with pre-cooled PBS (0.01M, PH 7.4) to remove the residual blood or impurities on the tissue surface.
2)Tissue pieces should be weighed and then minced to pieces which should be cut as small as possible to be fully homogenized.
3)Add appropriate amount of pre-cooled PBS (the volume depends on the weight of the tissue. 9 mL PBS would be appropriate to 1 gram tissue pieces. After detection, the calculated sample concentration should multiply by the corresponding dilution ratio. It is recommended to add some protease inhibitor into the PBS immediately before use) and homogenize with a glass homogenizer on ice or in ice bath.
4)Aspirate the homogenates to the centrifugal tube and centrifuge for 5-10 min at 5000×g at 4℃. Collect the supernatant and store at -20℃ or -80℃ to avoid repeated freeze-thaw cycles.
6. Urine, saliva and other biological fluid samples
Centrifuge at 1000×g for 20 min, then collect the supernatant and carry out the assay immediately.
In general, as ELISA can only detect the content of soluble protein, the samples should be clear and transparent and be centrifuged to remove the sediment or suspended matter.
To ensure the accuracy of the detection, the samples stored at -20℃ or -80℃ should be detected within 1-6 months, and the samples stored at 4℃ should be detected within one week.
Besides, the samples should not contain NaN3 which will inhibit the activity of HRP and cause false negative results.