Analysis of common problems in experimental operation by ELISA kit(3)

(4) 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-based colorimeter is called a microplate reader, and usually 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, select the sensitive absorption peak of the product, such as OPD with 492nm wavelength. Some microplate readers can be used for dual-wavelength reading, that is, two readings per hole, * times in the appropriate wavelength (W1), and second in the insensitive wavelength (W2), the ELISA plate is not moved between the two measurements. The position, the final measured A value of Zui is the difference between the two (W1-W2). Dual-wavelength reading reduces light interference caused by scratches or fingerprints on the container. When the results are judged by the naked eye, the color development is not easy to observe, and the accuracy of the results is affected. It is necessary to use a microplate reader to ensure consistency of results.

(5) Influence of HooK effect

With the application of the one-step ELISA method, the antigen content in some specimens is too high, resulting in a HooK effect. Affecting the test results, the simultaneous dilution test or the use of a two-pair semi-quantitative method with a high linear range can reduce the occurrence of the HooK reaction.

(6) Influence of interfering substances

Some people think that about 40% of human serum samples contain non-specific interfering substances, which can affect the test results to varying degrees; common interfering substances are: rheumatoid factor, complement, heterophilic antibody, target antigen autoantibodies, iatrogenic induction Anti-mouse Ig (s) antibodies, cross-reactive substances and other substances. For example, the RF factor can be combined with the FC segment of the labeled secondary antibody to cause a false positive. Complement is activated from C1q, and the antibody molecule of the primary antibody and the secondary antibody is deformed. When the C1q binding point of the Fc is exposed, the complement C1q can be The two are connected to cause false positives. Inactivation of complement at 56 °C for 30 minutes can reduce the false positive rate. High concentrations of AFP (such as pregnant women) may form dimers during storage, which may cause the background to be too deep to affect the test results.

(7) The impact of drugs

The high-priced hepatitis B immunoglobulin will form a complex with HBsAg, which will affect the detection of HBsAg. Therefore, after HBsAg-positive patients are injected with hepatitis B immunoglobulin, HBsAg detection will be negative, resulting in the emergence of two pairs of rare patterns of hepatitis B, such as We often encounter pregnant women with hepatitis B and Sanyang. In order to block the vertical transmission of mother and baby of hepatitis B, routine injection of 200 mg of hepatitis B immunoglobulin in the 8th, 9th and 10th of pregnancy not only affects the detection of HBsAg in pregnant women; The newborns also have HBsAg-negative, HBeAg-positive and HBcAb-positive and other rare patterns. It may be that hepatitis B immunoglobulin IgG antibodies can enter the fetus through the placenta to form a complex with HBsAg in the fetal blood; then the newborn HBsAg detection Negative; treatment with 0.5 M hydrochloric acid for 1 hour increased the rate. In order to prevent hepatitis B, some HBsAg-negative people can detect HBsAg in serum within 1-2 weeks after vaccination with hepatitis B vaccine, forming a positive HBsAg positive. This may be the main component of hepatitis B vaccine is HBsAg. There are ways to It is detected; as in electrochemiluminescence, it is recommended that HBsAg should not be tested within 1 month after vaccination with hepatitis B vaccine.

(8) Antigen factor

The effect of fusion proteins on genetic engineering antigen specificity. Take the hepatitis C diagnostic kit as an example, because the coated genetically engineered antigen is a fusion protein, and some sequences derived from the expression vector can react with anti-E. coli factors in serum to produce suspicious specimens. A small number of HBV infections do not contain HbsAg in peripheral blood. HBsAg can be found in the peripheral blood of most infected people after HBV infection, and the content is between 5 ng and 600 μg/ml. According to reports in the literature, the low content of HBsAg carriers found in blood donors so far is 0.2 ng/ml. The high content can reach 2000μg/ml or more. However, a small number of HBV-infected patients were negative for serum HBsAg, such as fulminant hepatitis B and HBV S gene mutation. In acute severe hepatitis B, hepatocytes are mainly composed of synthetic HBcAg with little or no synthesis of HBsAg, so that there is no HBsAg in peripheral blood. The pre-S/S gene of HBV encodes HBsAg, which constitutes the outer membrane of the virus, and is divided into adw, adr, ayw and ayr according to the subtype determinants. The determinant is highly immunogenic, and natural infections in HBV or injection of HBsAg vaccine can cause anti-HBs responses. For example, when the S gene 145 codon variation causes its original glycine to be replaced by arginine, the antigenicity of the a determinant can be changed, so that the antibody produced by the body has no effect on the mutant strain, and can cause the serum of the HBV infected patient simultaneously. HBsAg and anti-HBs appear. At the same time, hepatitis B vaccination is not effective in preventing infection of such variant viruses. The variation of the hepatitis B virus S gene has natural variation and evades immune variation. The mutation can occur in multiple sites, and several mutations can exist at the same time. These mutations contribute to the persistence of the virus carrying state. Recently, studies have shown that the pre-S1 region loss mutation (amino acids 58-118) is an important cause of HBsAg-negative HBV infection. The S promoter is located in the pre-S1, which is a regulatory element for the synthesis of HBsAg, and the loss of pre-S1 mutations will affect The function of the S promoter, which in turn affects the synthesis of HBsAg.

In short, high-quality reagents, good-state instruments, interference with various influencing factors, and proper operation are necessary conditions to ensure accurate and reliable ELISA results.