The main points of Elisa operation are summarized, high-quality reagents, good equipment and correct operation are necessary conditions to ensure accurate and reliable ELISA test results. The distilled water or deionized water used in ELISA, including those used for washing, should be fresh and high-quality. The purified water required by our company has a conductivity of less than 1.5 μs / cm. 1. Collection and storage of specimens: Most ELISA tests use serum as the specimen. Serum samples can be collected according to conventional methods, and care should be taken to avoid hemolysis. When erythrocytes are lysed, substances with peroxidase activity will be released. In the ELISA assay marked with HRP, hemolysis samples may increase non-specific coloration. Serum specimens should be tested when fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, which will also produce false positive reactions. Generally speaking, serum samples measured within 5 days can be placed at 4 ° C. If the samples are stored in the refrigerator for too long, the serum IgG will polymerize, which will deepen the reagent background of the indirect method. Stored at -20 ℃ if measured over a week. After the frozen serum is thawed, the protein is locally concentrated and unevenly distributed. It should be thoroughly mixed to avoid air bubbles. Serum specimens that are cloudy or precipitated should be centrifuged or filtered, and then clarified before testing. Repeated freezing and thawing will make the antibody titer drop, so if the serum samples for antibody detection need to be stored for multiple tests, it should be stored in small quantities in ice. Attention should be paid to aseptic operation when storing serum since collection, and appropriate preservatives can also be added. Specimens with incomplete anticoagulation may cause false positives due to fibrinogen interference. It is recommended to avoid anticoagulation, especially heparin anticoagulants. 2. Add sample: When adding sample, add the substance to the bottom of the ELISA plate well, avoid adding to the upper part of the well wall, and pay attention not to spill. 3. Insulation: When establishing an ELISA method for reaction kinetics studies, the experiment showed that the two antigen-antibody reactions were generally at 37 ℃ for 1-2 hours, and the product formation reached the peak. In order to avoid evaporation, the board should be covered, and the hole of the board can also be covered with plastic sealing paper or cling film. The reaction boards should not be stacked to ensure that the temperature of each board can be quickly balanced. It should be noted that the temperature and time of incubation should be as accurate as possible. Since the incubation of the company's kits is done in an air bath, the use of a water bath will cause high values ​​or flower plates. In addition, there is an edge effect during incubation, and the value on the edge will be higher. It is recommended to place the quality control in a non-edge position for objective judgment. 4. Washing: Although washing is not a reaction step in the ELISA process, it determines the success or failure of the experiment. ELSIA is to separate free and bound enzyme labels by washing. Washing to remove substances remaining in the wells of the plate that could not bind to the solid-phase antigen or antibody, as well as interfering substances that were non-specifically adsorbed to the solid-phase carrier during the reaction. The adsorption of polystyrene and other plastics to proteins is universal, and this non-specifically adsorbed interfering substance should be washed down during washing. It can be said that in the ELISA operation, washing is the most important key technology, which should be highly valued by the operator, and the operator should wash strictly according to the requirements, not sloppy. The washing solution is mostly a neutral buffer solution containing a non-ionic detergent. The combination of the polystyrene carrier and the protein is hydrophobic. The non-ionic detergent contains both hydrophobic and hydrophilic groups. The hydrophobic group and the hydrophobic group of the protein are combined by hydrophobic bonds, thereby weakening the protein and the protein. The combination of the solid-phase carrier and the combination of the hydrophilic group and the water molecule restores the protein to the state of the aqueous solution, thereby leaving the solid-phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration can be between 0.05% -0.2%. When it is higher than 0.2%, it can desorb the antigen or antibody coated on the solid phase and reduce the test. Sensitivity. When washing the plates, be careful not to mix the washing solutions of the various kits. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used is preferably below 1.5us / cm. If the lotion is crystallized, it should be prepared after melting. Ensure that the washing plate soak time is about 40 seconds. The cleaner the liquid in the hole is absorbed by the washing machine, the better the washing effect. The manual washing of the plate prevents the washing liquid from forming bubbles in the hole. 5. Color development and color comparison: After TMB is exposed to HRP, the color development reaches its peak in about 40 minutes, and then gradually weakens. After 2 hours, it completely fades to colorless. There are many termination solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This type of terminator can still maintain the blue color for a long time (12-24 hours), and is a good terminator for visual judgment. In addition, all kinds of acidic terminating liquids will turn blue into yellow, at this time, the absorbance value can be measured with a specific wavelength (450nm). The microplate reader is abbreviated as the microplate reader, usually referring to the photometer dedicated to reading the absorbance of ELISA results. The main performance indicators of the microplate reader are: reading speed, reading accuracy, repeatability, accuracy and measurable range, linearity and so on. The reading of an excellent microplate reader can generally be accurate to 0.001, with an accuracy of ± 1% and a repeatability of 0.5%. The microplate reader should not be placed under the sunlight or strong light. The room temperature should be 15 ~ 30 ℃ during operation. The instrument should be preheated for 15-30 minutes before use. The reading result is more stable. The main points of Elisa operation summary When reading the A value, the sensitive absorption peak of the product should be selected, such as OPD with a wavelength of 492nm. Some microplate readers can use dual-wavelength reading, that is, each well is read twice in succession, the first time at the optimal wavelength (W1), the second time at the insensitive wavelength (W2), and the ELISA does not move between the two measurements The position of the board, the final measured A value is the difference between the two (W1-W2). Dual-wavelength reading can reduce light interference caused by scratches or fingerprints on the container.
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