ELISA sample preparation and precautions
ELISA sample preparation and precautions
1. Collection, processing and preservation of serum
1. Vacuum blood collection needle collects fresh blood and is added to a sterile test tube;
2. After standing at room temperature for 1 hour;
3, the blood collection will be centrifuged at 4 ° C, 2500 rpm for 10 min;
4. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -80 ° C for 6 months;
5, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements.
Most ELISA tests use serum as a specimen. Serum samples can be collected according to conventional methods. Care should be taken to avoid hemolysis. When red blood cells are dissolved, substances with peroxidase activity are released. In ELISA assays marked with HRP, hemolyzed specimens may increase non-specific color development. In addition, bacterial contamination should be avoided, as the cells may contain endogenous HRP and may also produce false positive reactions. Serum samples should be tested fresh. If stored in the refrigerator for a long time, the polymerization can occur, and the background can be deepened in the indirect ELISA.
Second, the collection, processing and preservation of plasma in ELISA experiments
1. Collect 2ml fresh blood from the vacuum blood collection needle and add it to the sterile test tube;
2. Add anticoagulant (EDTA, sodium oxalate, heparin, sodium citrate) at 1:9, and separate the plasma on ice to reduce platelet contamination within 30 minutes; (can also be collected directly with anticoagulation tube);
3, the blood collection will be centrifuged at 4 ° C, 2500 rpm for 5 min;
4. Take the supernatant. Pack and store frozen,
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -80 ° C for 6 months;
5, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements. Please note that there are special requirements for anticoagulants in the indicator sheet. EDTA is recommended. In addition to fibrinogen and anticoagulant, the plasma is equivalent to serum. Preparation of plasma specimens requires the use of the anticoagulant EDTA, and specimens with incomplete anticoagulation cause false positives due to fibrinogen interference.
3. Collection, processing and preservation of urine in ELISA experiments
1. Collect 500ul of urine sample and add it to the sterile test tube;
2, the blood collection will be centrifuged at 4 ° C, 2500 rpm for 10 min;
3. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -80 ° C for 6 months;
4, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements.
4. Collection, processing and preservation of cerebrospinal fluid in ELISA experiments
1. Collect cerebrospinal fluid samples and add them to sterile test tubes;
2, the collected cerebrospinal fluid was centrifuged at 4 ° C, 2500 rpm for 10 min;
3. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -80 ° C for 6 months;
4, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements.
5. Collection, processing and preservation of cell supernatant in ELISA experiments
1. Collect the cell culture supernatant and add it to the sterile test tube;
2. The collected cell supernatant was centrifuged at 2 ° C for 10 min at 2500 rpm;
3. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
4, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements.
6. Collection, processing and preservation of alveolar lavage fluid in ELISA experiment
1, 10% chloral hydrate solution, abdominal anesthesia;
2, open the abdominal cavity, another 10ml syringe in the inferior vena cava blood collection, as much as possible to put the blood;
3. Lung lavage of the left lung on the ice. Take 5 ml of physiological saline at 37 ° C, and irrigate three times: 2 ml, 1.5 ml, 1.5 ml. The recovery rate is above 70%;
4, the collected alveolar lavage fluid was centrifuged at 4 ° C, 2500 rpm for 10 min;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
7. Collection, processing and preservation of joint synovial fluid in ELISA experiment
1, oral joints: patients take a seat, after routine disinfection, for subcutaneous and post-joint area local infiltration anesthesia. Then the patient was opened with a large mouth, using a syringe containing 2 ml of normal saline (5-gauge needle) for the supra-articular cavity puncture, and then the needle was inserted, and the direction of the needle tip was obliquely forward, inside, and up, and reached the posterior aspect of the joint nodule and then retreated. If you find a light yellow liquid, it means that you have entered the upper cavity of the joint, inject 2ml of normal saline, and repeatedly inject and withdraw five times;
2, knee joint synovial fluid: aseptic puncture to obtain 3ml of joint fluid;
3. The collected synovial fluid is centrifuged at 1500 rpm for 15 min at 4 ° C in a centrifuge;
4. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
5, according to your experimental needs, take appropriate amount of supernatant to carry out various ELISA measurements.
8. Collection, processing and preservation of cells in ELISA experiments
1. Cell plate adherent cells are directly lysed: the cells are discarded and the supernatant is incubated, washed three times with PBS, and the appropriate amount of lysate is resuspended (excessive concentration will be reduced, it is important), ultrasonically disrupted or blown with a gun for 100 times;
2, cell pellet lysis: cell pellet plus 1ml PBS resuspended (be careful not to cause cell rupture too much), centrifuge at 1000rpm for 5 minutes, discard the supernatant, repeat 3 times, add appropriate amount of lysate to resuspend (excess will reduce the concentration, Very important), ultrasonically broken or blown with a gun for 100 times;
3. Suspension cell lysis: transfer to EP tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash PBS three times, add appropriate amount of lysate to resuspend (excess will reduce the concentration, it is important), sonicate or blow with a gun 100 times;
4, cleavage on ice for 30mins, centrifuged at 4 ° C, 12000 rpm for 10mins;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
Protease inhibitors are required if necessary depending on the indicator to be tested. If it is detected immediately after treatment, it can also be directly lysed without adding these inhibitors. The amount of lysate will vary according to different cell lines. The principle is that the total protein concentration after lysis is not less than 0.5 mg/ml.
9. Collection, processing and preservation of fragile soft tissues (brain, liver and kidney) in ELISA experiments
1. Weigh the tissue block (50mg-100mg) and add the lysate according to the mass to volume ratio of 1:9 (Example: 50mg tissue wet weight is added to 450ul lysate);
2. Cut the tissue block with an ophthalmic scissors in an ice water bath;
3. Electric homogenizer or ultrasonic crushing in ice water bath;
4, centrifuged at 4 ° C, 12000 rpm for 15mins;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
Protease inhibitors are required if necessary depending on the indicator to be tested. If detected immediately after treatment, it is also possible to directly lyse without these inhibitors.
X. Collection, processing and preservation of myocardium and muscle tissue in ELISA experiment
1. Weigh the tissue block (50mg-100mg) and add the lysate according to the mass to volume ratio of 1:9 (Example: 50mg tissue wet weight is added to 450ul lysate);
2. Cut the tissue block with an ophthalmic scissors in an ice water bath;
3. Grinding rod grinding or ultrasonication in an ice water bath;
4, centrifuged at 4 ° C, 12000 rpm for 15mins;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
Myocardium and muscle tissue cannot be homogenized by homogenizer, and the tissue block will be twisted into the drill bit to form a mass, which cannot be shredded by the homogenizer head, and needs to be fully broken by grinding or ultrasonic. Protease inhibitors are required if necessary depending on the indicator to be tested. If detected immediately after treatment, it is also possible to directly lyse without these inhibitors.
XI. Collection, processing and preservation of epithelial tissue (skin, bladder, intestine) and human or rat cornea in ELISA
1. Weigh the tissue block (50mg-100mg) and add the lysate according to the mass to volume ratio of 1:9 (Example: 50mg tissue wet weight is added to 450ul lysate);
2. Cut the tissue block with an ophthalmic scissors in an ice water bath;
3. Grinding rod grinding or ultrasonication in an ice water bath;
4, centrifuged at 4 ° C, 12000 rpm for 15mins;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
Epithelial tissue has strong toughness and is difficult to cut. It needs to increase the time of tissue breakage to fully break. Protease inhibitors are required if necessary depending on the indicator to be tested. If detected immediately after treatment, it is also possible to directly lyse without these inhibitors.
12. Collection, processing and preservation of small granular tissues (ganglion, mouse cornea, sclera) in ELISA experiments
1. Add 200 ul of lysate;
2. The tissue block is cut by micro-shearing in an ice water bath;
3. Grinding rod grinding or ultrasonication in an ice water bath;
4, centrifuged at 4 ° C, 12000 rpm for 15mins;
5. Take the supernatant. Pack and store frozen spare:
Store at 24-8 ° C for 24-48 hours;
Store at -20 ° C for 1 month;
Store at -70 ° C for 6 months;
6. According to your experimental needs, take appropriate amount of supernatant to perform various ELISA tests.
The amount of small particles is relatively small, and there is a risk that the target protein amount is lower than the detection limit. Note that the total protein concentration of the supernatant after centrifugation of the homogenate is greater than 0.5 mg/ml. Protease inhibitors are required if necessary depending on the indicator to be tested. If detected immediately after treatment, it is also possible to directly lyse without these inhibitors.
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