Specimen Preparation and Storage for ELISA
2012-06-113223
Serum -Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 2-8°C. Centrifuge at approximately 1000 × g (or 3000 rpm) for 15 minutes. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.
Plasma -Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g (or 3000 rpm) at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS (0.02mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in a certain amount of PBS with a glass homogenizer on ice. The resulting suspension was subjected to ultrasonication or to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 15 minutes at 1500×g (or 5000 rpm). Remove the supernate and assay immediately or aliquot and store samples at -20°C or -80°C.
Cell lysates - Cells should be lysed according to the following directions.
1) Adherent cells should be detached with trypsin and then collected by centrifugation. Suspension cells can be collected by centrifugation directly.
2) Wash cells three times in PBS.
3) Cells were resuspended in PBS and subjected to ultrasonication for 3 times. Alternatively, freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.
4) Centrifuge at 1000×g (or 3000 rpm) for 15 minutes at 2-8 °C to remove cellular debris.
5) Assay immediately or store samples at -20°C or -80°C.
Cell culture supernatants and other body fluids - Centrifuge cell culture media at 1000 × g (or 3000 rpm) for 15 minutes to remove debris. Assay immediately or store samples at -20°C or -80°C.
NOTE:
1. Samples should be aliquoted and must be stored at -20°C (less than 3 months) or -80°C (less than 6 months) to avoid loss of bioactivity and contamination. If samples are to be run within 24 hours, they may be stored at 2- 8°C. Avoid repeated freeze-thaw cycles.
2. Prior to assay, the frozen sample should be brought to room temperature slowly and mixed gently.
3. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.
4. Samples containing a visible precipitate must be clarified prior to use in the assay. Care should be taken to minimize hemolysis. Do not use grossly hemolyzed or lipemic specimens.
5. Do not use heat-treated specimens.
Urine -Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge at 1000 × g (or 3000 rpm) for 15 minutes to remove particulate matter and assay immediately or store samples at -20°C or -80°C.
Saliva -Rinse the mouth at least twice with cool water. Then chew sugarless gum for 1-2 minutes, swallowing saliva as usual. Continue to chew the gum and collect saliva using sample tube. Centrifuge samples at 1000 × g (or 3000 rpm) for 15 minutes and assay immediately or store samples at -20°C or -80°C. Instruct the patient to breathe deeply and cough from deep down in the lungs. Instruct them that saliva and upper respiratory secretions are not sputum and are not acceptable specimens. For patients unable to bring up sputum, deep coughing may be induced by inhalation of an aerosol of warm, hypertonic (5%-15%) saline.
Bone Tissue - Extract demineralized bone samples in 4 M Guanidine-HCl and protease inhibitors. Dissolve the final sample in 2 M Guanidine-HCl.
Milk - Collect milk using sample tube. Centrifuge samples at 1000 × g (or 3000 rpm) for 15 minutes. Assay immediately or aliquot and store samples at -20°C or -80°C.
