In the detection work of automatic enzyme-linked immunosorbent assay (ELISA), there are various commonly used methods that are suitable for different experimental needs based on different detection principles. The following is a detailed introduction for you:
colorimetryIt is one of the most basic and commonly used detection methods for automatic enzyme-linked immunosorbent assay (ELISA). The principle is to use the absorption characteristics of substances towards specific wavelengths of light, and determine the concentration of the substance by measuring the absorbance of the solution. When light passes through the solution under test, some of the light will be absorbed by the substances in the solution, and the absorbance is proportional to the concentration of the substances within a certain range. Colorimetry can be divided into single wavelength colorimetry and dual wavelength colorimetry. Single wavelength colorimetry is suitable for samples with lighter colors and less interference, and the operation is simple and fast; Dual wavelength colorimetry can effectively eliminate the interference caused by turbidity, impurities, etc. in the sample, improve the accuracy of detection, and is widely used in the detection of complex samples such as serum and urine.
fluorescence methodWith its high sensitivity advantage, it plays an important role in trace substance detection. This method is based on certain substances emitting fluorescence under excitation light of specific wavelengths, and the fluorescence intensity is related to the concentration of the substance. Automatic enzyme-linked immunosorbent assay (ELISA) determines substance content by detecting the intensity of fluorescence. Fluorescence method is suitable for detecting trace components such as hormones, vitamins, and drug residues in living organisms. For example, when detecting pesticide residues in food, it can accurately capture extremely low concentrations of residual substances. However, the fluorescence method is easily affected by factors such as photobleaching and background fluorescence, and strict control of conditions is required during experimental operations.
ChemiluminescenceIt is a method of detecting using the luminescence phenomenon generated by chemical reactions. In the process of chemical reactions, certain substances absorb the energy released by the reaction and are in an excited state. When they return to the ground state, they emit photons, and the enzyme-linked immunosorbent assay (ELISA) reflects the concentration of the substance by detecting the number of photons. The chemiluminescence method has the advantages of high sensitivity, wide linear range, and fast detection speed, and is widely used in the field of immunoassay, such as the detection of infectious diseases and the screening of tumor markers. For example, in the detection of hepatitis B virus, chemiluminescence can detect the presence of the virus earlier and more accurately.
Time-resolved fluorescence methodIt is an improved form of fluorescence method that effectively solves the interference problem of background fluorescence in ordinary fluorescence methods. This method uses a marker with a long fluorescence lifetime, and after excitation light irradiation, waits for the background fluorescence to decay before detecting the fluorescence emitted by the marker. This method greatly improves the specificity and sensitivity of detection, and is commonly used in experiments such as neonatal screening and hormone testing that require high accuracy.
The homogeneous time-resolved fluorescence method combines the advantages of homogeneous detection and time-resolved fluorescence, without the need to separate free and bound labels. It is easy to operate and has high sensitivity and specificity. It has important applications in drug screening, protein interaction research, and other fields, and can quickly and accurately obtain experimental results.
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