ELISA sample preparation for serum, plasma, supernatant and tissue

An ELISA measures what reaches the well. Proteins that degrade during collection, anticoagulants that interfere with binding, cell debris that adds background and matrix components that mask the target all change the number you report. The kit is validated for specific sample types under specific handling, so match both as closely as you can.

1. Collect blood in a plain or serum-separator tube.
2. Let it clot at room temperature, typically 30 minutes to 2 hours.
3. Centrifuge at about 1,000–2,000 × g for 10–20 minutes.
4. Transfer the serum without disturbing the clot, and aliquot.
5. Assay the same day, or freeze at -20 °C short term and -80 °C long term.

Haemolysed samples, which look pink or red, release haemoglobin and intracellular proteins that can raise background or interfere with the reaction. Note them and exclude them if the manual says so.

Collect blood into EDTA, heparin or citrate tubes and centrifuge within about 30 minutes at roughly 1,000 × g for 15 minutes. Then aliquot and store as for serum.

The anticoagulant matters. EDTA chelates metal ions; heparin can interfere with some antibody binding and with downstream PCR if you use the same sample; citrate dilutes the sample slightly. Use the anticoagulant listed in the kit manual, and keep it the same across a study.

Centrifuge the medium to remove cells and debris, typically 1,000–1,500 × g for 10 minutes, then aliquot and freeze if you are not assaying straight away.

1. Rinse tissue in ice-cold PBS to remove blood, then weigh it.
2. Homogenise on ice in PBS or a mild lysis buffer with protease inhibitors, at a fixed ratio such as 1 g of tissue per 5–10 mL of buffer.
3. For cells, lyse with the buffer the kit recommends, or freeze-thaw two or three times.
4. Centrifuge at about 10,000 × g for 10 minutes at 4 °C and keep the clear supernatant.
5. Measure total protein so you can report the target per mg of protein.

Strong ionic detergents such as SDS can denature the antibodies on the plate. The lysis buffer guide lists gentler options that keep proteins in their native form.

Centrifuge to remove cells and particles, then dilute as the manual directs. These fluids often contain the target at low levels, so check that your expected concentration is above the kit’s lowest detectable dose. For saliva, avoid food, drink and tooth brushing for 30 minutes before collection.

Thaw samples on ice or at room temperature, mix gently and centrifuge briefly before pipetting to remove any precipitate.

Run a pilot on a few representative samples at two or three dilutions before the full set. Choose the dilution that puts most samples near the middle of the standard curve.

Two checks show that your sample matrix is not distorting the result:

• Spike recovery. Add a known amount of standard to a sample and measure it. Recovery between about 80% and 120% means the matrix is not masking the target.
• Linearity of dilution. Dilute a high sample in series. Values corrected for dilution should agree within about 20%.