ELISA test: ten methods of sample collection, storage and treatment
There are many kinds of samples in Lisa experiment, including serum, plasma, cell supernatant, cell lysate, urine, synovial fluid, cerebrospinal fluid, alveolar lavage fluid and tissue homogenate of various tissues; The collection and processing methods are different. In order to make customers better understand the sample collection of ELISA kit, our company will collect, process and save all kinds of samples in combination with the actual situation of the company. Most of the methods are from literature and kit instructions. Some methods have been verified by our company, but some methods have not been actually operated. Therefore, the following methods are only for reference.
1、 Collection, treatment and preservation of serum in ELISA experiment;
1. Collect 2ml fresh blood with vacuum blood collection needle and add it into sterile test tube;
2. After blood collection, let it stand at room temperature for 1 hour;
3. The collected blood was centrifuged at 4 ℃ and 2500rpm for 10min. The centrifuged homogenate was left in the supernatant and the precipitate below was discarded.
4. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
5. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
Most ELISA tests were based on serum. Serum samples can be collected by conventional methods. Attention should be paid to avoid hemolysis. Substances with peroxidase activity will be released during erythrocyte lysis. In the ELISA determination marked with HRP, hemolytic samples may increase nonspecific color development. In addition, bacterial contamination should be avoided, because the bacteria may contain endogenous HRP, which will also produce false-positive reaction. Serum samples should be tested when fresh. If it is stored in the refrigerator for too long, it can polymerize and deepen the background in indirect ELISA.
2、 Collection, treatment and preservation of plasma in ELISA experiment;
1. Collect 2ml fresh blood with vacuum blood collection needle and add it into sterile test tube;
2. Add anticoagulants (EDTA, sodium oxalate, heparin and sodium citrate) according to 1:9, and separate plasma on ice within 30 minutes to reduce platelet pollution; (it can also be collected directly with anticoagulant tube);
3. The collected blood was centrifuged at 4 ℃ and 2500rpm for 5min, and the centrifuged homogenate was left in the supernatant and the precipitate below was discarded.
4. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
5. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests. Please pay attention to whether there are special requirements for anticoagulants in the Index Manual. It is recommended to use EDTA. In addition to fibrinogen and anticoagulant, other components in plasma are equal to serum.
Precautions: the preparation of plasma samples needs the help of anticoagulant EDTA. The samples with incomplete anticoagulation cause false positive due to the interference of fibrinogen.
3、 Collection, treatment and preservation of urine in ELISA experiment;
1. Collect 500ul urine sample and add it into sterile test tube;
2. The collected blood is centrifuged at 4 ℃ and 2500 rpm for 10 minutes, and the centrifuged sample is left in the supernatant and the sediment below is discarded.
3. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
4. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
4、 Collection, treatment and preservation of cerebrospinal fluid in ELISA experiment;
1. Collect cerebrospinal fluid samples and add them into sterile test tubes;
2. The collected blood is centrifuged at 4 ℃ and 2500 rpm for 10 minutes, and the centrifuged sample is left in the supernatant and the sediment below is discarded.
3. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
4. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
5、 Collection, treatment and preservation of cell supernatant in ELISA experiment;
1. Collect 500ul of cell culture supernatant and add it into sterile test tube;
2. The collected blood was centrifuged at 4 ℃ and 2500rpm for 10min. The centrifuged homogenate was left in the supernatant and the precipitate below was discarded.
3. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
4. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
6、 Collection, treatment and preservation of cell lysate in ELISA experiment; (protein concentration needs to be measured)
1. Take the cell culture plate or cell suspension and wash the cell culture plate with PBS (ph7.2-7.4) for 1-2 times.
2. By repeatedly freezing and thawing or adding tissue protein extraction reagent (lysate), the cells are destroyed and intracellular components are released.
3. The collected blood was centrifuged at 4 ℃ and 2500rpm for 20min. The centrifuged homogenate was left in the supernatant and the sediment was discarded.
4. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
5. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
7、 Collection, treatment and preservation of alveolar lavage fluid in ELISA experiment;
1. 10% chloral hydrate solution, abdominal anesthesia.
2. Open the abdominal cavity, take another 10ml syringe to collect blood from the inferior vena cava, and drain the blood as much as possible.
3. The left lung was lavaged on ice. Take 5ml of 37 ℃ normal saline and lavage in three times: 2ml, 1.5ml and 1.5ml. The recovery rate is more than 70%.
4. The alveolar lavage fluid was centrifuged at 4 ℃ and 1500 rpm for 10 min.
5. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
6. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
8、 Collection, treatment and preservation of synovial fluid in ELISA experiment;
1. Oral joint: the patient takes the sitting position, after routine disinfection, perform local infiltration anesthesia under the skin and in the posterior area of the joint. Then ask the patient to open his mouth, use the syringe (No. 5 needle) equipped with 2ml normal saline for puncture in the supraarticular cavity, enter the needle behind the condyle, the needle tip direction is inclined forward, inward and upward, and slightly back after reaching the inclined plane behind the articular nodule. If light yellow liquid is found, it indicates that he has entered the supraarticular cavity, inject 2ml normal saline, and inject and withdraw it repeatedly for five times.
Knee joint synovial fluid: obtain joint fluid by aseptic puncture.
2. The articular fluid was centrifuged with a centrifuge at 4 ℃ and 1500 rpm for 15 minutes. The centrifuged homogenate was left in the supernatant and the precipitate below was discarded.
3. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
4. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
9、 Collection, treatment and preservation of various tissue homogenates (easily homogenized tissues) in ELISA experiment; (protein concentration needs to be measured)
1. Collect the tissue, rinse it in cold normal saline, remove the blood, wipe dry the filter paper, weigh it, remove the attached connective tissue, and weigh the tissue block.
2. Use a pipette to measure 0.1M PBS or 0.86% cold normal saline. The total volume of homogenizing medium or normal saline should be 9 times the weight of tissue block. Use ophthalmic small scissors to cut the tissue block as soon as possible (the operation should be carried out in ice water bath in nature, and put the small beaker containing tissue into ice water).
3. There are many ways to homogenize:
a) Manual homogenization: pour the chopped tissue into the glass homogenization tube, wash the remaining 1 / 3 homogenization medium or normal saline, and pour it into the homogenization tube together for homogenization. Hold the homogenization tube in your left hand, insert the lower end into the vessel containing ice water mixture, insert the tamping rod vertically into the casing with your right hand, rotate it up and down for dozens of times (6-8 minutes), Fully grind to homogenize the tissue.
b) Machine homogenization: 10% tissue homogenization is made by grinding up and down with a tissue crusher at 10000 ~ 15000 R / min. it can also be prepared by internal cutting tissue homogenization mechanism (homogenization time 10 seconds / time, gap 30 seconds, continuous 3 ~ 5 times, in ice water). The homogenization time of skin and muscle tissue can be extended.
4. The prepared 10% homogenate was centrifuged with a centrifuge at 4 ℃ and 3000 rpm for 15 min, and the centrifuged homogenate was left with the supernatant and the sediment below.
5. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
6. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
10、 Collection, treatment and preservation of various tissue homogenates (tissues that are not easy to homogenate) in ELISA experiment; (protein concentration needs to be measured)
7. Collect the tissue, rinse it in cold normal saline, remove the blood, wipe dry the filter paper, weigh it, remove the attached connective tissue, and weigh the tissue block.
8. Use a pipette to measure 0.1M PBS or 0.86% cold normal saline. The total volume of homogenizing medium or normal saline should be 9 times the weight of tissue block. Use ophthalmic small scissors to cut the tissue block as soon as possible (the operation should be carried out in ice water bath in nature, and put the small beaker containing tissue into ice water).
9. There are many ways to homogenize:
a) Pour the remaining homogenate into the homogenate tube for 8 / 6 minutes, and then insert the remaining homogenate into the homogenate tube with the left hand into the homogenate tube for 3 / 6 minutes, and then wash the remaining homogenate tube with the right hand, Fully grind to homogenize the tissue.
b) Machine homogenization: 10% tissue homogenization is made by grinding up and down with a tissue crusher at 10000 ~ 15000 R / min. it can also be prepared by internal cutting tissue homogenization mechanism (homogenization time 10 seconds / time, gap 30 seconds, continuous 3 ~ 5 times, in ice water). The homogenization time of skin and muscle tissue can be extended.
c) Ultrasonic pulverization: use ultrasonic pulverizer for pulverization. Soniprep150 ultrasonic generator can be used for ultrasonic treatment with an amplitude of 14 microns for 30 seconds to break cells, or domestic ultrasonic generator can be used for 3 ~ 5 times with 40 amps, 5 seconds / time and 10 seconds gap.
10. The prepared 10% homogenate was centrifuged with a centrifuge at 4 ℃ and 3000 rpm for 15 min, and the centrifuged homogenate was left with the supernatant and the sediment below.
11. Take the supernatant. Repackaging, freezing and storage for standby,
At 2-8 ℃, it can be stored for 24-48 hours;
-At 20 ℃, it can be stored for 1 month;
-It can be stored for 6 months at 70 ℃;
12. According to your experimental needs, take an appropriate amount of Shangqing night for various ELISA tests.
Shanghai FanTai biology suggests:
1. It is only responsible for the kit itself, not for the sample consumption caused by the use of the kit. Please fully consider the possible use of samples and reserve sufficient samples before use.
2. The sample content shall be predicted before the experiment. If the sample concentration is too high, the sample shall be diluted to make the diluted sample meet the detection range of the kit, and then multiplied by the corresponding dilution multiple during calculation.
3. If the tested samples are not included in the samples listed in the instructions, it is recommended to conduct pre experiments to verify their effectiveness, and pay attention to the retention of samples.
4. The tissue homogenate or cell extract prepared with chemical lysate may lead to the deviation of ELISA results due to the introduction of some chemicals.
5. If the sample is cell culture supernatant, there may be undetectable conditions due to many interference factors of such samples, such as cell state, cell number, sampling time, etc.
6. Some natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection antibody and capture antibody used in this product.
7. It is recommended to use fresh samples. If they are kept for a long time, there may be protein degradation or denaturation, resulting in deviation of experimental results.