ELISA (enzyme-linked immunosorbent assay) uses immunological principles and chemical reaction color development. The samples to be tested are mostly serum, plasma, urine, cell or tissue culture supernatant, so tissue embedding, sectioning and other techniques are not used. This is the main difference from immunohistochemistry. At the beginning of the operation, antigens or antibodies need to be bound to the surface of a solid carrier, so that the antigen antibody enzyme substrate complex formed later adheres to the carrier. This is the meaning of "adsorption", and then the enzyme reacts with the substrate to produce a color reaction for quantitative detection. It is also one of the most sensitive techniques for detecting antigens or antibodies.
Simply put, the primary antibody captures the antigen, while the secondary antibody with its own enzyme captures the primary antibody, causing the substrate to react and emit light. Express the amount of antigen through metering (OD value). Of course, this is just the most commonly used indirect method.
There are three commonly used enzyme-linked immunosorbent assay methods:The most commonly used method for detecting antibodies is primarily for detecting antibodies against pathogens.Advantages: Secondary antibodies can enhance signals, and there are multiple options for different measurements and analyses. Primary antibodies without enzyme labeling can retain their maximum immunoreactivity.Disadvantage: The probability of interactive reactions occurring is relatively high2. Double antibody sandwich methodThe most commonly used method for detecting large molecule antigens.Advantages: High sensitivity, high specificity, antigen does not require prior purification.Disadvantage: The antigen must have two or more antibody binding sites.Detect small molecule haptens (drugs, hormones, etc.).Advantages: It can be applied to relatively impure samples and has high data reproducibility.Disadvantage: The overall sensitivity and specificity are poor.
After the experiment is completed, the corresponding OD value is measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve is established to calculate the concentration of the sample.OD stands for optical density, also known as absorbance. Absorbance refers to the use of a substance's unique absorption spectrum to identify or determine its content. In other words, a specific wavelength of light is passed through the analyte, which can absorb a portion of the light and calculate its concentration based on absorbance. Generally, the higher the concentration of the detected substance, the higher the absorbance value.Note that through ELISA experiments, we obtain individual sample concentration values. Therefore, the ELISA result graphs mentioned in the article are generally statistical analyses of these data, such as:
Of course, some also directly use the measured OD values for statistical analysis, such as:
Through statistical analysis, we can determine whether the differences in histone levels obtained under different experimental conditions are statistically significant, and thus obtain the conclusions we need.Now that we have shared the three giants of protein detection experiments, let's summarize the differences among them:Immunohistochemistry, Western blotting, and ELISA are the three major immunological tools used for localization, qualitative analysis, and quantitative analysis, respectively.1. Localization is the biggest advantage of immunohistochemistryThis method can accurately locate antigens in tissues and cells, allowing for simultaneous observation of the localization of different antigens in the same tissue or cell. This enables a combination of morphological and functional studies. Compared to other protein detection methods, immunohistochemistry has more direct and accurate localization, high qualitative sensitivity, and is the preferred method for localization detection and analysis. It is of great significance for in-depth research in the field of pathology, especially for the study of factor translocation.2.Compared with IHC technology, WB has more accurate quantificationOf course, WB can also be used for qualitative and localization (by extracting membrane proteins, nuclear proteins, and cytoplasmic proteins to detect their antigen content, indirectly reflecting their localization), but its sensitivity is much lower than IHC.3.Compared with immunohistochemistry, ELISA is the most accurate quantitative method and is one of the preferred methods for detecting secreted proteins.Especially useful for micro quantitative analysis of multiple samples, with very few reagents and sample sizes used, resulting in accurate results. IHC has strong specificity and usually uses tissues or cells as samples, while ELISA usually uses serum or tissue grinding solution. Immunohistochemistry can quickly detect the positivity or negativity of antibodies in samples, and is generally not used for quantitative detection.The above article is excerpted from the internet
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