Analysis of common problems in ELISA standard operation and technical service

1. Key points of ELISA standard operation 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 the ELISA method for the reaction kinetics study, the experiment showed that the two antigen-antibody reactions were generally carried out at 37 ℃.
Within 1-2 hours, product formation reaches its 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 rendering and color comparison
After TRP is treated with HRP, the color development reaches its peak in about 40 minutes, and then gradually weakens. After 2 hours, it completely disappears to colorless. There are many termination solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can all terminate the reaction. This type of terminator can still maintain the blue color for a long time (12-24 hours), and it is a good terminator for visual judgment. In addition, all kinds of acidic stop solution will change the 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.

When measuring the value of A, the sensitive absorption peak of the product should be selected. For example, OPD uses 492nm wavelength. 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.

2. Background and Cause Analysis of False Positives

1. The difference between genetically engineered antigen and synthetic peptide antigen
1.1 Genetically engineered antigens are protein antigens expressed in prokaryotic or eukaryotic form of antigen genes in plasmid vectors, most of which use E. 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 the limitations of the process, the number of synthetics is limited, which 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. In the early days, the kit with synthetic peptide as the coating antigen has a validity period of only 3-4 months, and the after-effect period of the genetically engineered antigen is greatly extended.
c. Genetically engineered antigens fused and expressed specific antigenic determinant genes. The expression product contains more antigenic determinants, which can improve the sensitivity of the kit and increase the detection rate.
d. Difficult to purify. The purification technology of genetic engineering antigen is more difficult.
1.2 Synthetic peptide antigen is a peptide fragment artificially synthesized based on the amino acid sequence of an 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 epitope
c. High purity
d. Poor stability Due to the unparalleled superiority of genetically engineered antigens over synthetic peptide antigens, ELISA diagnostic reagents have undergone a transition from synthetic peptides to genetically engineered antigens. As far as the HCV ELISA kit is concerned, the first-generation products are synthetic peptide antigens, mainly peptide fragments of HCV-specific epitopes; the second-generation products are coated with both genetically engineered antigens and synthetic peptides. The genetically engineered antigens are incomplete and only include the core region fragments 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 has been greatly improved.
For historical reasons, people often measure the ELISA reaction kit by the quality of the reaction background, so some manufacturers use single-segment genetic engineering antigens and synthetic peptide coatings to maintain a good background. The popularity of such kits Disease sensitivity is not enough, and stability is also a problem. It is gratifying that some manufacturers insisted on the high epidemiological sensitivity of the kits and treated the 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. Taking the hepatitis C diagnostic kit as an example, because the coated genetically engineered antigen is a fusion protein and contains some sequences from the expression vector, it can react with anti-E. Coli factors in the serum to produce a suspicious specimen.