ELISA standard operation and common problem analysis

1. Key points of ELISA standard operation

High-quality reagents, good instruments and correct operation are necessary conditions to ensure the accuracy and reliability of ELISA test results. The distilled or deionized water used in ELISA, including those used for washing, should be fresh and high-quality purified water required by our company with a conductivity of less than 1.5μs/cm.

1. Collection and storage of specimens Most ELISA tests use serum as specimens. Serum samples can be collected according to conventional methods. Care should be taken to avoid hemolysis. When red blood cells are lysed, substances with peroxidase activity will be released. In ELISA assays marked with HRP, hemolyzed samples may increase non-specific coloration. Serum samples should be tested when they are fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, and false positive reactions may also occur. Generally speaking, serum specimens measured within 5 days can be placed at 4°C. If the specimens are stored in the refrigerator for too long, the serum IgG will polymerize, which will deepen the reagent background of the indirect method. It needs to be stored at -20°C if it is measured for more than one week. After the frozen serum is thawed, the protein is locally concentrated and unevenly distributed. It should be mixed thoroughly and avoid bubbles. Serum specimens with turbidity or precipitation should be centrifuged or filtered first, and then tested after clarification. Repeated freezing and thawing will cause the antibody titer to drop. Therefore, if the serum sample for antibody testing needs to be stored for multiple tests, a small amount of aliquots should be stored on ice. Attention should be paid to aseptic operation when preserving serum since collection, and appropriate preservatives can also be added. Specimens with incomplete anticoagulation may cause false positives due to the interference of fibrinogen. It is recommended that anticoagulants, especially heparin anticoagulants, are not used as much as possible.

2. Adding samples When adding samples, add the added substance to the bottom of the well of the ELISA plate, avoid adding to the upper part of the well wall, and be careful not to spill.

3. Incubation During the establishment of ELISA method for reaction kinetics research, experiments show that the two antigen-antibody reactions are generally at 37°C for 1-2 hours, and the product generation can reach the peak. In order to avoid evaporation, the plate should be covered, and the holes of the plate can also be covered with plastic paste sealing paper or cling film. 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, the use of a water bath will cause the value to be high or splattered. In addition, there is an edge effect during incubation, and the value on the edge will be too high. It is recommended that the quality control be placed in a non-edge position for objective judgment results.

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 purpose of separating free and bound enzyme markers. Washing is used to remove substances remaining in the wells that cannot bind to the solid-phase antigen or antibody, as well as interfering substances that are non-specifically adsorbed on the solid-phase carrier during the reaction. The absorption of proteins by plastics such as polystyrene is universal, and this non-specifically adsorbed interfering substance should be washed off during washing. It can be said that in the ELISA operation, washing is the most important key technology, which should arouse the attention of the operator, and the operator should wash strictly according to the requirements, and must not be sloppy. The washing liquid is mostly a neutral buffer containing non-ionic detergent. The combination of polystyrene carrier and protein is hydrophobic. Non-ionic detergent contains both hydrophobic group and hydrophilic group. The hydrophobic group and the hydrophobic group of the protein are combined by hydrophobic bond, thereby weakening the protein and The combination of the solid-phase carrier and the combination of the hydrophilic group and the water molecule make the protein return to the state of an 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% and 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. The sensitivity. When washing the plate, pay attention not to mix the washing solutions of various kits. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used is preferably under 1.5us/cm. If the lotion crystallizes, it should be prepared after it has melted. Ensure that the soaking time of the washing plate is about 40 seconds. The cleaner the liquid in the well is absorbed by the plate washer, the better the washing effect. Manual washing of the plate prevents the washing liquid from forming bubbles in the well.

5. Color development and colorimetry After HRP is used for TMB, the color development reaches its peak in about 40 minutes, then gradually weakens, and after 2 hours, it will completely fade to colorless. There are many kinds of stop solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can all stop the reaction. This type of terminator can still maintain the blue color for a long time (12-24 hours) without fading, and is a good terminator for visual judgment. In addition, various acidic stop solutions will turn blue into yellow. At this time, the absorbance can be measured at a specific wavelength (450nm). The microplate colorimeter is abbreviated as a microplate reader, and usually refers to a photometer dedicated to measuring the absorbance of ELISA results. The main performance indicators of the microplate reader are: reading speed, reading accuracy, repeatability, precision and measurable range, linearity and so on. The reading of a good microplate reader is generally accurate to 0.001, the accuracy is ±1%, and the repeatability is 0.5%. The microplate reader should not be placed under sunlight or strong light. The room temperature should be 15~30℃ during operation, and the instrument should be warmed up for 15-30 minutes before use. The reading results will be more stable. When measuring the A value, the sensitive absorption peak of the product should be selected, such as the wavelength of 492nm for OPD. Some microplate readers can be used for dual-wavelength reading, that is, each well is measured and read twice, the first time is at the optimal wavelength (W1), the second time is 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 can reduce light interference caused by scratches or fingerprints on the container.

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2. Background and analysis of the causes of false positives

1. The difference between genetic engineering antigen and synthetic peptide antigen

1.1 Genetically engineered antigens are protein antigens in which antigen genes are expressed in prokaryotic or eukaryotic plasmid vectors, mostly using Escherichia coli or yeast as the expression system. Compared with synthetic peptides, this type of antigen has the following characteristics:

 a. The molecular weight is large. Synthetic peptides are prepared by chemical methods. Due to technological limitations, the number of synthesis is limited and can only reach hundreds of amino acids; while the antigens prepared by genetic engineering have a larger molecular weight.

b. Good stability. The stability of the coated antigen can ensure the validity period of the kit. The validity period of the kit using synthetic peptide as the coating antigen in the early stage is only 3-4 months, and the validity period of the genetically engineered antigen is greatly prolonged.

c. The genetic engineering antigen fusion expresses specific epitope genes, and the expression product contains more epitopes, which can improve the sensitivity of the kit and increase the detection rate.

d. The purification is difficult. The purification technology of genetically engineered antigens is more difficult.

1.2 Synthetic polypeptide antigens are artificially synthesized polypeptide fragments based on the amino acid sequence of a certain antigenic determinant of a protein antigen molecule. Synthetic peptide antigens have the following characteristics:

a. The molecular weight is too small

b. Generally contains only one antigenic determinant

c. High purity

d. Poor stability Due to the unparalleled superiority of genetic engineering antigens over synthetic peptide antigens, ELISA diagnostic reagents have undergone the transition from synthetic peptides to genetic engineering antigens.

As far as HCV ELISA kits are concerned, the first-generation products are synthetic peptide antigens, mainly peptide fragments of HCV-specific epitopes; the second-generation product-coated antigens include both genetic engineering antigens and synthetic peptides. Genetically engineered antigens are not complete and only include fragments of the core region of HCV; the third-generation products basically use genetically engineered antigens, and these antigens include more, more stable, and higher purity HCV specific antigens. The sensitivity of the third-generation reagent is greatly improved. Due to historical reasons, people often judge ELISA reaction kits by the quality of the reaction background. Therefore, in order to maintain a better background, some manufacturers use single-segment genetic engineering antigens and synthetic peptide coatings. The popularity of such kits Pathological sensitivity is insufficient, and stability is also a problem. It is gratifying that some manufacturers insist on the high epidemiological sensitivity of the kit and treat the reaction results scientifically.

2. Causes of false positive background

2. 1 Antigenic factors 2.1.1 The influence of fusion protein on the specificity of genetic engineering antigens. Take the hepatitis C diagnostic kit as an example. Because the coated genetically engineered antigen is a fusion protein that contains some sequences from the expression vector, it can react with the anti-E. coli factor in the serum to produce a suspicious specimen.

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