There are many reasons that affect the non-specific color development in ELISA, such as the specificity of the kit, the interference of the enzyme label in the test specimen, and problems in the operation process. This article makes a brief analysis of this reason, so that the above measures can be used to minimize non-specific color development, thereby improving the specificity of detection and obtaining more accurate and reliable experimental results.
【Key words】 ELISA non-specific color rendering
Since its introduction in 1971, the enzyme-linked immunosorbent assay (ELISA) has been widely used in clinical diagnosis due to its high sensitivity, strong specificity, easy operation and safety, and has opened a new era of immunological diagnosis. However, like other immunodiagnostic methods, enzyme-free reagents often encounter non-specific problems in its research, production and use. analysis.
1. Kit-specific factors
1. The choice of solid carrier. There are three kinds of solid-phase carriers commonly used in ELISA: microtiter plates, beads and test tubes. Microtiter plates are the most common [1]. It has good adsorption performance, high transparency at the bottom of the hole, low blank value, and similar performance between each plate and each hole of the same plate. Due to the different raw materials and the manufacturing process of polystyrene ELISA board, the quality of each product varies greatly. Therefore, when selecting a kit, the microplate model used must be certified and evaluated.
1, 2 The purity of the coating. In enzyme-linked immunoassays, especially indirect ELISA, the specificity of the reagent depends on the purity of the antigen used. At present, due to the limitation of technical conditions, the purity of the coating antigen or antibody cannot reach 100%, so some non-specific color development is inevitable, and the purity and specificity can only be improved as much as possible. The coating antigen currently used is generally a synthetic polypeptide antigen.
1, 3 titer of coated antibody. Coating antibodies with high affinity and high specificity are important aspects that determine the specificity of reagents.
1, 4 closed. It is an important step in ELISA, the purpose is to close the gaps on the surface of the solid phase carrier that have not been occupied [2], to reduce the interference of non-specific proteins in the subsequent steps.
2. Examine the interfering substance in the specimen containing the enzyme marker
2.1 Endogenous interfering substances: rheumatoid factor (RF), jaundice, etc. Rheumatoid factor is an autoantibody that can act on IgGFc segments of various animals and humans, most of which are IgM class, which can act as antigen components and solid phase and The enzyme-labeled antibody reacts, thereby displaying non-specific color development.
Jaundice blood specimens often contain endogenous peroxidase. If horseradish peroxidase is used as a marker, non-specific coloration may occur.
2. 2 Exogenous interfering substances are often caused by improper sample collection, storage, and handling, such as hemolysis of the sample, contamination by bacteria, and incomplete agglutination of the specimen. In hemolysis specimens, erythrocytes are lysed and ruptured to release heme, and the iron porphyrin in heme is an analog of peroxidase. In ELISA assays labeled with HRP, hemolysis specimens may increase non-specific coloration. If there is bacterial contamination, the bacteria may contain endogenous HRP (horseradish peroxidase) [2]. There are domestic reports that enzyme-free HBsAg reagents can cause false positives when detecting hemolytic specimens [3]. If stored in the refrigerator for too long, the IgG in it can polymerize, which can deepen the background in the indirect ELISA [2]. Therefore, blood samples should be taken care of during collection and processing, and it is not easy to store them in the refrigerator for too long.
When the specimen is not fully agglomerated, some fibrinogen will remain in the serum, which is also likely to cause false positives.
3. Problems during operation cause false positives
3, 1 loading
Indirect ELISA specimens are generally diluted. If the sample is not accurate, it will cause errors. Especially when the dilution factor is large, a small absolute error will result in a large relative error, making the negative (or weak positive) specimens present. Positive (or negative). When adding the sample, the added substance should be added to the bottom of the well of the ELISA plate, to avoid adding to the upper part of the well wall, and be careful not to splash out or generate bubbles. At present, most blood stations have used automatic enzyme-free sample addition systems to process specimens, which can better avoid the above errors.
3, 2 washing
Correct washing in ELISA is a crucial step to ensure reproducible results and should be paid attention to by the operator. Whether it is manual operation or machine operation, incorrect results are often related to incorrect washing. ELISA is to separate free and bound enzyme labels by washing. Washing to eliminate the substances remaining in the wells of the plate that could not bind to the solid antigen or antibody, and the interfering substances that were non-specifically adsorbed to the solid phase carrier during the reaction.
3. 3 Incubation
Each reagent has its optimal reaction mode, and temperature and incubation time control are important factors. Due to high incubation temperature and long reaction time, the background of the whole plate will be high and the positive rate will be high. Incubation generally uses a wet box or a water bath. The reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. To avoid evaporation, the plate should be covered.
3, 4 microplate reader interpretation
As an instrument for recording the measurement results, the performance of the microplate reader is stable or not, which determines the reliability of the results. First, the microplate reader should be maintained regularly, and the filter should be calibrated regularly; secondly, the wavelength setting of the microplate reader should be correct. It is best to use dual wavelengths, one detection wavelength and one reference wavelength to eliminate scratches on the bottom of the microplate, Light interference caused by unevenness, fingerprints or differences in liquid level. In addition, when reading with a microplate reader, it is best to wipe the bottom of the microplate and squeeze the flat strip. Because the performance of various microplate readers is different, you should read the instructions in detail
In summary, due to the lack of standardization of enzyme-linked immunosorbent assay technology, although there are some standard serum or reference serum (serum plate) in the world and in China. However, due to the limitations of methodology and technical conditions, sometimes certain non-specificity will inevitably occur in the enzyme-linked adsorption assay, but we can reduce the non-specific color development to the minimum by the above measures, thereby improving the specificity of detection Sex, and get more accurate and reliable experimental results.

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