Processing of Common Samples in Elisa

ELISA is a rapid, sensitive, accurate and reliable quantitative analysis method. Sample preparation plays a pivotal role in a successful ELISA experiment. At the beginning of collecting sample for ELISA, we might have a few problems. The common samples used for ELISA include blood (serum, plasma), tissue homogenates, cell lysates and cell culture supernatants, etc. Many factors will have influence on the results of ELISA experiment, such as sampling time, processing, storage, etc. Now we introduce sample processing methods for reference.

1. Blood

After collecting, blood should be processed as soon as possible to separate serum (plasma) from whole blood. What’s the difference between serum and plasma? Compared to plasma, serum lacks fibrinogen, other ingredients are totally the same. Plasma could be a better choice when we test some factors related to coagulation. However, due to plasma contains fibrinogen, serum is recommended when fibrinogen has influence on targets to be tested. Both of them are suitable for most ELISA experiments.

1) Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1,000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

2) Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1,000×g at 2-8℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

2.Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type.

1)Tissues were rinsed in ice-cold PBS to remove excess blood thoroughly and weighed before homogenization.

2)Minced the tissues to small pieces and homogenized them in fresh lysis buffer (catalog: IS007, different lysis buffer needs to be chosen based on subcellular location of the target protein) (w:v = 1:20-1:50, e.g. 1mL lysis buffer is added in 20-50mg tissue sample) with a glass homogenizer on ice (Micro Tissue Grinders woks, too).

3)The resulting suspension was sonicated with an ultrasonic cell disrupter till the solution is clarified.

4)Then, the homogenates were centrifugated for 5 minutes at 10,000×g. Collect the supernates and assay immediately or aliquot and store at ≤-20℃.

3. Cell Lysates

Cells need to be lysed before assaying according to the following directions.

1) Adherent cells should be washed by cold PBS gently, then detached with trypsin, and collected by centrifugation at 1,000×g for 5 minutes (suspension cells can be collected by centrifugation directly).

2)Wash cells three times in cold PBS.

3) Resuspend cells in fresh lysis buffer with concentration of 10⁷ cells/mL. If necessary, the cells could be subjected to ultrasonication till the solution is clarified.

4) Centrifuge at 1,500×g for 10 minutes at 2-8℃ to remove cellular debris. Assay immediately or aliquot and store at ≤-20℃.

4. Cell culture supernatants

Centrifuge samples for 20 minutes at 1,000×g. Collect the supernates and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

5. Feces

Weight 100mg (about 80-120mg) feces, add 5ml PBS and oscillate for 40 seconds. Then homogenize them for 25-30 minutes. Put the homogenates (1mL) into a new tube and centrifuge for 20 minutes at 10,000×g. Collect the supernates and assay immediately or aliquot and store at ≤-20℃ or -80℃. Avoid repeated freeze/thaw cycles.

6. Urine

Aseptically collect the first urine of the day (mid-stream) or 24-hour urine directly into a sterile container. Centrifuge samples for 15 minutes at 1,000×g. Collect the supernates and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze-thaw cycles.

7. Cerebrospinal fluid

Centrifuge samples for 15 minutes at 1,000×g. Collect the supernates and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze-thaw cycles.

Sample processing is aimed to collect target molecules, which is the premise of successful experiment. As proteins are generally easily denatured and degraded, so the process should be as mild as possible. The storage of samples is also very important, especially be assured not to go freeze/ thaw cycles. And samples could be stored in aliquot. The storage time should be less than one week at 4 ℃, or not exceed 1 month at -20 ℃, or not exceed 2 months at -80 ℃. Before assaying, bring samples to room temperature, and heating samples is forbidden.