ELISA is an important analytical test in molecular diagnostics, medicine, plant pathology, and basic life science research. ELISA is usually described as the “gold standard” for macromolecule assessment in biological fluids due to its exceptional sensitivity and specificity.
The composition, processing, and preservation of the sample being tested are all major elements in enhancing ELISA’s accuracy and sensitivity. Unlike Immunoassays, ELISA provides non-denaturing conditions to identify proteins in their natural state in solution. Because ELISA can accommodate a wide range of aqueous biological matrices, the researcher may run into a number of possible stumbling blocks when obtaining specimens of unknown type. How should fresh biopsy tissue be removed to keep the target protein in its original state? To avoid matrix effects in the experiment, how should bodily fluids be prepared and stored? The most frequent sample types used with ELISA will be described here, along with sample preparation and storage guidelines.
ELISA sample preparation for plasma and serum:
- Blood
Many components can be detected in whole blood samples (for further information, please see our composition of blood page). The blood cells (red blood cells, white blood cells, and platelets) and the liquid component of blood are the most important components. The blood cells can be isolated from the blood liquid by samples were centrifuged whole blood. The resulting liquid will be serum or plasma, depending on how the blood is prepared.
- Serum
Serum is a blood element that is free of blood cells and clotting factors. This is almost similar to plasma, but lacking the fibrinogens. Serum without concept is produced when blood is coagulated (clotted), however some clotting factors may remain.
- Prepare a tube with entire blood and no additives.
- Allow 30 minutes for the blood to come to room temperature.
- Centrifuge for 10 minutes at 3,000 rpm and 4°C.
- Assay instantly, or aliquot a supernatant (serum) and store it at -80°C (avoid freeze/thaw cycles).
- Plasma
The extracellular matrix that supports blood cells is composed of plasma, which is a component of blood (red blood cells, white blood cells and platelets). Plasma is produced when blood is anticoagulated (not clotted), and it contains fibrinogens and clotting factors. If the target element of interest is a coagulating factor, plasma rather than serum must be used as the sample type.
- Fill a tube with whole blood and an anti-coagulant (Different proteins may require different anticoagulants; read the datasheet for more information.).
- Centrifuge for 10 minutes at 3,000 rpm and 4°C.
- Assay instantly, or aliquot supernatant (plasma) and keep it frozen at -80°C (avoid freeze/thaw cycles).
- Urine
- Collect urine using a metabolic cage.
- Remove any impurities by aspirating at 1000x g for 15 minutes at 2–8°C and test immediately, or aliquot and store samples at -20°C or -80°C.
- To extract any further precipitates that may form during storage, centrifuge once more before assaying.
- Tissue Homogenates
100 mg of tissue was rinsed in 1X PBS, homogenized in 1 mL of 1X PBS, and stored at -20°C overnight.
- The homogenates were centrifuged for 5 minutes at 5000 x g, 2 – 8°C, after two freeze-thaw cycles to destroy the cell membranes.
- The supernate was evaluated and removed as soon as possible.
- Alternately, aliquot the samples and store them at -20°C or -80°C.
- Before the assay, centrifuge the sample again once it has thawed.
- Cell Culture Supernatant
- Centrifuge cell culture media at 1,500 rpm and 4°C for 10 min.
- Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).
- Cell Lysates
Remove the fluid and provide the cells an ice-cold PBS rinse (pH 7.0-7.4).
- Scrape the cells from the plate and put them in a tube.
- Dilute the cell suspension with 1xPBS (pH 7.0-7.4) until the cell concentration is 100 million/ml, then store at -20°C overnight.
- The cell lysates were centrifuged for 5 minutes at 5000 g, 2 – 8°C, after two freeze-thaw cycles to break up the cell membranes.
- Collect the supernatant and put it aside.
- Cell lysates should be evaluated instantly or aliquoted and stored at -20°C.
- Before the assay, centrifuge the sample again once it has thawed.
- Milk
- Centrifuge for 15 minutes at 1500 x g and 4°C.
- Collect the liquid sample and repeat the process 3 times.
- Then use a 0.2 m filter, process the data.
- Assay immediately, or aliquot supernatant and store at -80°C (no freeze/thaw cycles).
- Saliva
- Collect saliva with a collection device which is not effective of protein binding or filtering (e.g. Salivette).
- Centrifuge for 2 minutes at 10,000 x g and 4°C.
- Assay immediately, or aliquot supernatant and store at -80°C (no freeze/thaw cycles).
ELISA SAMPLE HANDLING – TIPS & TRICKS
Tip 1: Handle Samples with Care. Proper sample handling can reduce variability between replicates and between tests. Be mindful when storing, thawing and pipetting samples. Some kits have special sample handling requirements, so always review the assay’s instructions for use before receiving samples and starting the procedure.
Tip#2: Follow these tips for proper sample handling technique.
Thawing- Thaw samples at room temperature.
Storing- Research the stability of your analyte/biomarker of interest and follow any special sample handling recommendations provided in your kit’s instructions for use. Limit time at room temperature and in the refrigerator. Store samples at ≤ -70°C whenever possible to prevent degradation of the analyte.
Aliquot samples to avoid freeze-thaw cycles.
Tip#3: Inspect the pipette tip after drawing sample to ensure there are no droplets or sample adhering to the exterior. Any droplets should be removed with a wipe, taking care to not touch the open end of the pipette tip. This will ensure excess sample is not transferred and minimizes variability between replicates.
