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The superiority of Elisa technology
There are many methods for measuring various trace organic substances in living organisms, and they can be roughly classified into three categories: physical, chemical, and immunological methods. In the physics analysis method, mainly by means of high-precision instruments such as ultraviolet spectrophotometer, mass spectrometer, gas chromatograph and high pressure liquid chromatography. The application of these instruments and equipment, although the detection of a small amount of organic matter in the body (can be detected in the range of 10-6mol / L ~ 10-12mol / L), but because of the complexity of operation, time-consuming, and high detection costs, it is not conducive to Promote application in production.
The chemical method is mainly carried out according to the chemical reaction of the analyte and other substances, and the color change. This method is the first basic method for quantitative determination of trace organic substances in living organisms. However, because of its low sensitivity (requires a lot of sampling), poor specificity, and troublesome operation, it is not conducive to popularization and application.
There are many methods for measuring common immunology. The biggest advantage is that the specificity is strong. The weak point is that the sensitivity is relatively poor, and the operation is troublesome, which is not conducive to large-scale measurement. The RIA technology developed in the 1960s has been developed in terms of sensitivity, and it can detect ultra-micro organic matter in the concentration of 10-6mol/L~10-12mol/L in vivo. Relatively speaking, RIA technology is one of the more advanced ones of these immunoassay technologies. Therefore, when evaluating the advantages and disadvantages of Elisa technology, RIA technology is often used as a reference.
The label used as an indicator in RIA technology is a radioisotope. The detection of radioisotopes requires expensive equipment and radiation protection equipment, and laboratories and radioactive waste treatment equipment specializing in radioisotope work are required. The health of operators is often Threatened by radiation exposure. In addition, in terms of RIA quality, although the sensitivity has reached the pg/ml level (ie, picograms per milligram of sample), it is unlikely that there will be much improvement, so its development prospects are small. In addition, as a radioactive isotope of the RIA indicator, due to the short half-life, such as 32P only 14.3 days, 33P only 25 days, 125I only 60 days, thus limiting the storage time of RIA reagents.
Compared with RIA technology, Elisa technology has the following advantages in the above aspects:
(1) High sensitivity
Although the sensitivity of the Elisa method for enzyme activity is currently not very satisfactory, the sensitivity of the assay is much higher than that of RIA in the enzymatic activity amplification of Elisa. According to the law of mass action. That is, the amount of immune complex formed by the immune reaction is proportional to the concentration of the reactant. It is presumed that the number of molecules to be detected is one. It is known that one molar concentration contains 6.02×1023 molecules, so the theoretical detection of the enzyme activity amplification Elisa method has a minimum detection limit of 1.7×10-24 mol/L. Although in practical applications, due to factors such as reaction conditions and reagent purity and instrument accuracy, this level is often not reached (greater than 104 molecules), but it shows that Elisa has great potential for improvement in sensitivity.
(2) Strong specificity
From the perspective of immune response, the specificity of Elisa and RIA should be consistent. However, in the Elisa method, the specificity of the reaction of the enzyme for detecting the indicator with its substrate increases the specificity of the method.
(3) Low requirements for equipment and equipment, low measurement cost
The Elisa assay can be performed in an ordinary laboratory. The commonly used instruments include a sampler, an incubator, and a special reader for the enzyme label. At the current price, the price of the microplate reader is only 1/6 to 1/4 of that of the liquid scintillation meter, so the cost per sample is less than 1/10 to 1/16 of the PIA. Certain qualitative Elisa methods can also be performed in the field and are very convenient to operate.
(4) The method is fast and simple
When the homogenase immunoassay method is operated, all the reagents are carried out in the same system without any separation step, and the operation is very simple and rapid, and the result can be obtained in a few minutes. Certain qualitative Elisa kits, such as the Elisa kit for the identification of certain cows and the diagnosis of pregnancy in foreign countries, can be completed in a few minutes.
(5) Longer reagent storage time
As enzyme and enzyme markers for detecting indicators, they are relatively stable under low temperature or dry conditions and can be stored for half a year to several years.
(6) High degree of automation
Because Elisa does not have radioisotope contamination, all the other steps can be automated by instrument, except that the sample to be tested needs manual operation. Under this automated condition, an average of more than 2,000 samples per person per day can be tested.
(7) There are many types of methods
Elisa technology can take advantage of the advantages of monoclonal antibodies and can be developed into many new methods using the effects of certain non-immune reagents (such as SPA, lectins, etc.). In addition, the development of Elisa technology can also start from both the enzyme and its substrate. This is because: First, the enzymes used in the Elisa technology are far more diverse than the radioisotopes that can be used as indicators of RIA detection. There are many enzymes in the biological world, and it is hopeful that many types of enzymes will be developed for Elisa and the corresponding Elisa method will be established. In contrast, the chemical elements of nature are limited and the types of radioisotopes are more limited. Second, due to the diversity of enzymes, there are various substrates for enzymes, and the corresponding color source or type of hydrogen donor has great development and development potential.
(8) No radioactive isotope pollution
Elisa technology uses enzymatic reaction as the basis for detecting the intensity of immune response. It is not necessary to contact radioisotopes in the terminal measurement, and there is no radioactive contamination problem (except for some enzyme immunoradiation substrates established to improve detection sensitivity).
In view of the superiority of Elisa technology and RIA technology in the above eight aspects, especially in terms of pollution and ease of operation, the advantages of Elisa technology are beneficial to the application of this technology in production and clinical practice. Statistics show that although RIA technology was born five years earlier than Elisa technology, its application in production practice is not as popular as Elisa technology. While promoting the application of nuclear technology, the IAEA is also promoting non-radioactive isotope labeling technology to narrow the scope of “nuclear diffusionâ€. It is expected that in the future, Elisa technology will gradually replace the trend of RIA technology.