Analysis of non-specific coloration in ELISA

【Abstract】 There are many reasons for influencing non-specific coloration in ELISA, such as kit specificity, detection of interferences containing enzyme markers in specimens, and problems during operation. 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 development

Since its inception in 1971, the enzyme-linked immunosorbent assay (ELISA) has been widely used in clinical diagnosis since its inception, with its high sensitivity, specificity, simple and safe operation, and has opened up a new era of immunological diagnosis. However, like other immunodiagnostic methods, the enzyme-free reagent often encounters non-specific problems in its research, production and use. This article has some reasons for the experiment and how to take some measures to eliminate non-specific color development. analysis.

1, kit specific factors

1, 1 selection of solid phase carrier. The solid phase carriers commonly used in ELISA are microtiter plates, beads and small test tubes, and 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 the plates and between the holes of the same plate. Polystyrene ELISA plates vary greatly in quality due to differences in raw materials and manufacturing processes. Therefore, when selecting a kit, the microplate model used for it should be certified and evaluated.

1, 2 the purity of the coating. In an enzyme-linked immunoassay, especially an indirect ELISA, the specificity of the reagent depends on the purity of the antigen used. At present, due to technical limitations, the purity of the coated antigen or antibody is unlikely to reach 100%, so some non-specific color development is inevitable, and only the purity and the specificity can be improved as much as possible. The coating antigens currently used are generally synthetic polypeptide antigens.

1, 3 coated antibody titer. Coated antibodies with high affinity and high specificity are important aspects that determine the specificity of the agent.

1, 4 closed. It is an important step in ELISA to block the gaps in the surface of the solid phase carrier that have not been occupied [2], reducing the interference of non-specific proteins in the subsequent steps.

2. Examine the interference object containing the enzyme label in the specimen.

2, 1 endogenous interferents: rheumatoid factor (RF), jaundice, etc., rheumatoid factor is an autoantibody that can act on a variety of animals and human IgG Fc segments, mostly IgM, can act as antigen components and solid phase and The enzyme-labeled antibody reacts to exhibit non-specific color development.

The blood sample of Astragalus membranaceus often contains endogenous peroxidase. If horseradish peroxidase is used as a marker, non-specific color development may occur.

2, 2 Exogenous interferences, often caused by sample collection, storage, improper handling, such as sample hemolysis, bacterial contamination, specimen agglutination and so on. Hemolysis specimens, red blood cells dissolve and rupture, release hemoglobin formation, and iron porphyrin in heme is a peroxidase analog. In ELISA assays labeled with HRP, hemolyzed specimens may increase non-specific coloration. If there is bacterial contamination, the bacteria may contain endogenous HRP (horseradish peroxidase) [2], and there are domestic reports that HBsAg reagent can cause false positives when detecting hemolyzed specimens [3]. If stored in the refrigerator for a long time, the IgG can be polymerized, and the background can be deepened in the indirect ELISA [2]. Therefore, blood specimens should be handled carefully during collection and processing, and it is not easy to store them in the refrigerator for too long.

When the specimen is agglutinated, some fibrinogen remains in the serum, which is also likely to cause false positives.

3. Problems during operation cause false positives

3, 1 sample 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 lead to a large relative error, so that the negative (or weakly positive) specimen is presented. Positive (or negative). When adding the sample, add the added substance to the bottom of the hole of the ELISA plate, avoid adding it to the upper part of the hole wall, and pay attention to not spilling, and no bubble can be generated. At present, most blood stations have used the automatic enzyme-free sample loading system to process specimens, which can better avoid the above errors.

3, 2 washing

Proper washing in an ELISA is a critical step in ensuring repeatable results and should be taken care of by the operator. Whether it is manual or machine operation, incorrect results are often associated with incorrect washing, which relies on washing to achieve separation of free and bound enzyme labels. By washing, substances which are not able to bind to solid antigens or antibodies remaining in the pores of the plate, and interfering substances which are non-specifically adsorbed to the solid phase carrier during the reaction are eliminated.

3, 3 incubation

Each reagent has its optimal reaction mode, with temperature and incubation time control being important factors. Due to the high incubation temperature and long reaction time, the whole plate has a high background and a high positive rate. The incubation usually uses a wet box or a water bath, and 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, and the reliability of the result is determined. First, the microplate reader should be regularly maintained, and the filter should be regularly calibrated. Secondly, the wavelength of the microplate reader should be set correctly. It is best to use dual wavelength, 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, it is best to wipe the bottom of the microplate and press the flat strip when reading with the microplate reader. Due to the different performance of various microplate readers, the instructions should be read in detail during use.

In summary, the lack of standardization of the enzyme-linked immunosorbent technology is currently technically available, although some standard serum or reference serum (serum plate) is currently available internationally and in China. However, due to limitations in methodological and technical conditions, some non-specific inevitability will occur in the enzyme-linked adsorption assay. However, we can reduce the specificity of detection by reducing the non-specific coloration to the lowest limit. Sex, and get more accurate and reliable experimental results.