![<?echo $_SERVER['SERVER_NAME'];?>](/template/twentyseventeen/skin/images/header.jpg)
I. ELISA standard operating points
High quality reagents, good instrumentation and proper operation are essential for ensuring accurate and reliable ELISA results. The distilled water or deionized water used in the ELISA, including the fresh and high quality purified water used for washing, should have a conductivity of less than 1.5 μs/cm.
1. Take and save specimens
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 coloration. Serum samples should be tested when fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, which may also produce false positive reactions. In general, serum samples measured within 5 days can be placed at 4 ° C, and the specimens are stored in the refrigerator for too long, resulting in the polymerization of serum IgG, which makes the reagent background of the indirect method deeper. It should be stored at -20 °C for more than one week. After the frozen serum is melted, the protein is partially concentrated and unevenly distributed. It should be thoroughly mixed and avoid bubbles. Bloody or precipitated serum samples should be centrifuged or filtered before clarification. Repeated freezing and thawing will cause the antibody titer to fall. Therefore, if the serum sample of the antibody is to be stored for multiple tests, it should be stored in small amounts. Preservation of serum should be performed aseptically, and appropriate preservatives may be added.
Specimens with incomplete anticoagulation cause false positives due to fibrinogen interference. It is recommended that anticoagulation, especially heparin anticoagulant, be used as much as possible.
2. Sample loading
Add the sample to the bottom of the ELISA plate hole when loading, avoid adding it to the upper part of the hole wall, and be careful not to spill.
3. Insulation
When the ELISA method was established for the reaction kinetics study, the experiment showed that the two antigen-antibody reactions were generally carried out at 37 ° C.
The production of the product reaches its peak in 1-2 hours. In order to avoid evaporation, the board should be covered, and the plate holes can be covered with plastic sealing Paper or plastic wrap. The reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. It should be noted that the temperature and time of incubation should be as accurate as required. Since the company's kit incubation is done in an air bath, using a water bath can result in high values ​​or flower plates. In addition, there is an edge effect in the incubation, and the value on the side will be too high. It is recommended to put the quality control at the non-edge position for the objective judgment result.
4. Washing
Washing is not a reaction step in the ELISA process, but it determines the success or failure of the experiment. ELSIA relies on washing to achieve the separation of free and bound enzyme markers. The substance remaining in the pores of the plate which is not capable of binding to the solid phase antigen or antibody, and the interfering substance which is non-specifically adsorbed to the solid phase carrier during the reaction are removed by washing. The adsorption of proteins by plastics such as polystyrene is universal, and such non-specifically adsorbed interfering substances should be washed away 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. The operator should wash it strictly according to requirements, and should not be sloppy.
The washing solution is mostly a neutral buffer containing a nonionic detergent. The binding of the polystyrene carrier to the protein is hydrophobic. The nonionic detergent contains both a hydrophobic group and a hydrophilic group, and the hydrophobic group binds to the hydrophobic group of the protein through a hydrophobic bond, thereby weakening the protein and The solid phase carrier is combined, and by the combination of the hydrophilic group and the water molecule, the protein is returned to the aqueous solution state, thereby being separated from the solid phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration may be between 0.05% and 0.2%. When the concentration is higher than 0.2%, the antigen or antibody coated on the solid phase may be desorbed to reduce the test. Sensitivity.
When washing the plate, be careful not to mix the lotion of the various kits. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used should be below 1.5us/cm. If the crystallization is allowed to crystallize, it should be prepared after melting. The immersion time of the washing plate is guaranteed to be about 40 seconds, and the liquid in the hole is absorbed by the washing machine, and the washing effect is better. The hand washing plate prevents the washing liquid from forming bubbles in the hole.
5. Color development and colorimetry
After TMP was applied by HRP, the color peaked at about 40 minutes, then gradually weakened, and after 2 hours, it completely disappeared to colorless. There are a variety of stop solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This type of terminator still keeps the blue color for a long time (12-24 hours) and is a good terminator for visual judgment. In addition, various types of acidic stop solutions turn the blue color into yellow, at which point the absorbance can be read at a specific wavelength (450 nm). The enzyme standard colorimeter is referred to as a microplate reader, and generally refers to a photometer dedicated to reading the absorbance of an ELISA result. The main performance indicators of the microplate reader are: reading speed, accuracy of reading, repeatability, accuracy and measurable range, linearity and so on. Excellent microplate readers typically have a reading accuracy of 0.001, an accuracy of ±1%, and a repeatability of 0.5%. The microplate reader should not be placed under sunlight or strong light. The room temperature should be 15~30°C during operation. Preheat the instrument for 15-30 minutes before use. The test results are more stable.
When reading the A value, the sensitive absorption peak of the product should be selected, such as OPD with a wavelength of 492 nm. Some microplate readers can be used for dual-wavelength reading, that is, two readings per well, the first time at the optimum wavelength (W1), the second at the insensitive wavelength (W2), and the ELISA is not moved 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 reduces light interference caused by scratches or fingerprints on the container.
2. Analysis of the causes of background and false positives
1. The difference between genetically engineered antigen and synthetic peptide antigen
1.1 Genetically engineered antigen is a protein antigen expressed by prokaryotic or eukaryotic expression of an antigen gene in a plasmid vector, and Escherichia coli or yeast is used as an expression system. This type of antigen has the following characteristics compared with synthetic peptides:
a. The molecular weight is large. Synthetic peptides are prepared by chemical methods. Due to the limitation of the process, the amount of synthesis is limited, and can only reach hundreds of amino acids. The antigen prepared by genetic engineering has a larger molecular weight.
Soft Paper,Soft Tissue Paper,Softest Toilet Paper,Ultra Soft Toilet Paper
ShanDong YongFang Sanitary Products Co.,Ltd , https://www.sdyongfang.com