Analysis of common problems in experimental operation by ELISA kit(1)

Analysis of common problems in experimental operation by ELISA kit (enzyme-linked immunoassay test method) (1)

Because of its high sensitivity and good specificity, ELISA (enzyme-linked immunosorbent assay)   has been widely used in the screening of various infectious diseases   such as hepatitis, AIDS, prenatal and postnatal care. Although washing the plate is only one of the important steps in the ELISA experiment, as a professional manufacturer of plate washing machines, we must have a certain understanding of the factors affecting the results of the ELISA experiment. High quality reagents, good instrumentation and proper operation are essential for accurate and reliable ELISA results. If you do not pay attention, it is easy to appear whiteboard, flower board, color weakness and false positives. In particular, the phenomenon of flower board (that is, blank, negative, positive control and indoor quality control hole results are normal, but the OD value of the sample hole is obviously high) is a problem often encountered in the debugging of washing machines of various manufacturers. The precautions in the ELISA experiment are summarized as follows, with a view to bringing some inspiration to everyone to improve the installation and debugging level of the washing machine and improve the quality of clinical testing.

(1) Specimens and collection, storage and transportation factors

Severe hemolysis, in the ELISA assay labeled with HRP, the hemoglobin remaining in the pores has peroxidase-like activity, which catalyzes the coloration of the substrate to cause false positives; the serum mixed with red blood cells is easy to precipitate or adhere to the polyethylene pores. Washing; if there is bacterial contamination, the bacteria may contain endogenous HRP, which may also produce false positive reactions; specimens are incompletely coagulated, sometimes in order to gain time for rapid detection, often forcibly centrifuged to separate serum when blood has not begun to coagulate. Some fibrinogen remains in the serum, and fibrin blocks visible to the naked eye can be formed during the ELISA assay, which is likely to cause false positive results; blood collection tube washing is not thorough, repeated use is easy to cross-contamination; plastic test tubes can adsorb antigenic substances, samples Prolonged exposure to plastic tubes can cause false negatives in the antigen content of the sample.

Serum samples should be tested at fresh times and severe hemolysis specimens should be disabled. In general, serum samples measured within 5 days can be placed at 4 ° C. The specimens are stored in the refrigerator for too long, resulting in the polymerization of serum IgG, which makes the indirect reagent background deeper. It should be stored at -20 °C for more than one week. After the frozen serum is melted, the protein is partially concentrated and unevenly distributed. It should be thoroughly mixed and avoid bubbles. Bloody or precipitated serum samples should be centrifuged or filtered before clarification. Repeated freezing and thawing will cause the antibody titer to fall. Therefore, if the serum sample of the test antibody needs to be stored for multiple tests, it is recommended to use a small amount of ice storage; use a disposable glass test tube or vacuum tube to collect blood vessels; and use non-anticoagulated specimens. Heparin anticoagulated plasma increases OD value, which may be related to the high negative concentration of heparin, which is sensitive to the lack of elution of enzyme label; EDTA and enzyme inhibitors (such as NaN3) can inhibit horseradish peroxidase activity in ELISA system. After the blood sample is collected, it must be fully coagulated before separating the serum, or the specimen is collected with a blood collection tube with a separation gel or a suitable coagulant is added to the blood collection tube.

(2) The influence of reagents

ELISA diagnostic reagents have undergone a transition from synthetic peptides to genetically engineered antigens. Due to historical reasons, people often measure the ELISA reaction kit by the background of the reaction background. Therefore, some manufacturers use single-segment genetic engineering antigens and synthetic peptide coatings to maintain a good background. The popularity of such kits is prevalent. The sensitivity of the disease is not enough and the stability is also a problem. There are also manufacturers who insist on the high epidemiological sensitivity of the kit and scientifically treat the results. Genetically engineered antigens have unparalleled superiority over synthetic peptide antigens. For HCV-ELISA kits, *generation products are synthetic peptide antigens, mainly peptide fragments of HCV-specific antigenic determinants; second-generation product coatings The antigens have both genetically engineered antigens and synthetic peptides, but the genetic engineering antigens are incomplete at the time, including only the core region fragments of HCV; the third generation products basically use genetically engineered antigens, and these antigens include more and more stable , a higher purity HCV-specific antigen. The sensitivity of the third generation of reagents has been greatly improved. The differences between genetically engineered antigens and synthetic peptide antigens are as follows:

The genetically engineered antigen is a protein antigen expressed by prokaryotic or eukaryotic expression of an antigen gene in a plasmid vector, and most of them are expressed by Escherichia coli or yeast. This type of antigen has the following characteristics compared with synthetic peptides:

a. The molecular weight is large. Synthetic peptides are prepared by chemical methods. Due to the limitation of the process, the amount of synthesis is limited, and can only reach hundreds of amino acids. The antigen prepared by genetic engineering has a larger molecular weight.

b. Good stability. The stability of the coated antigen ensures that the validity of the kit is guaranteed. In the early stage, the kit with the synthetic peptide as the antigen was only valid for 3-4 months, and the post-effect period of the genetically engineered antigen was greatly extended.

c. The genetically engineered antigen fuses and expresses the specific antigenic determinant gene, and the expression product contains more antigenic determinants, which can improve the sensitivity of the kit and increase the detection rate.

d. It is difficult to purify. The purification technology of genetic engineering antigens is difficult.

A synthetic polypeptide antigen is a polypeptide 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 antigenic determinant; c. high purity; d. poor stability.

There are many manufacturers of hepatitis B two-and-a-half reagents in China; there are some differences in the sensitivity and specificity of reagents produced by different manufacturers. The data show that the specificity and sensitivity of reagents from different manufacturers are 89.4%-99.3% and 78%-89% respectively. Large difference. Some manufacturers have a difference of A value of more than 15% between the wells of the microplates; the activity of the labeling enzyme and the stability of the coloring solution are relatively poor. The use of poor quality reagents necessarily leads to false positives or false negatives of the results. therefore. Choosing high quality reagents is one of the keys to ensuring accurate results.

• When selecting reagents, select excellent detection reagents with high sensitivity, good specificity, good stability, small batch difference, convenient operation and time saving. National reference laboratories evaluate the quality of each manufacturer's reagents every year and publish the evaluation results regularly. Can be used as the main basis for selecting reagents.

• Pay attention to the batch number and date of manufacture of the reagent, although the reagent is valid for one year. It is best to use the reagents that have just been shipped from the factory.

Reagents from different manufacturers cannot be mixed.

Different methodological detection reagents will make some difference between the two halves. For example, in the actual work, the ELISA detection of HBsAg results is negative, and the electrochemiluminescence test is positive. In addition to methodological sensitivity; there are differences in the use of monoclonal or polyclonal antibody reagents. There is a difference in the detection of variant antigens or subtypes of hepatitis B markers by monoclonal antibodies. It is recommended that reagent manufacturers should consider the subtype and concentration concentrations in the preparation of the agents.