ELISA (Enzyme-linked immunosorbent assay) is an experimental method commonly used in biomedical research and is widely used in the detection of antibodies, antigens, hormones, viruses, etc. Through ELISA testing, researchers can obtain quantitative or qualitative analysis results, which makes it irreplaceable in clinical diagnosis, disease surveillance and drug development.
1. Coating stageThe first step of ELISA detection is to fix the primary component to be tested on the coated plate. This process is mainly achieved through passive adsorption, and its effect depends on the properties of the coating substance. If the sample contains multiple antigens, selective coated plates need to be selected to avoid nonspecific binding. However, when detecting antibodies in the indirect ELISA method, purified antigen is used; or when using capture antibodies in the sandwich ELISA method, the coated plate is already selective. The material of the coated board is mostly polystyrene or its derivatives, and its binding characteristics vary according to the target substance and detection requirements. For certain substances that are difficult to adsorption, such as highly glycosylated proteins, carbohydrates, etc., it is recommended to use a special treatment coated plate that allows covalent ligation.
2. Incubation stageAfter the reagent is added to the coated plate, incubation is required to promote binding or reaction. The temperature and time of incubation vary according to the detection steps and operations. Typically, the samples are incubated for 1 hour at 37°C after application. However, certain steps, such as blocking, can be performed overnight in the refrigerator; while incubation during the assay is usually performed at a short time at room temperature.
3. Washing stageWashing is a crucial step in ELISA detection. It must be performed after each application of the test ingredients until the results are determined. During washing, phosphate buffered saline-20 (PBST) is usually used as buffer to remove unbound antigens, antibodies, or reagents. Washing can be done manually using a multi-channel pipette or an automatic plate washing machine. Inadequate washing can lead to high background signals, while excessive washing can lead to lower sample signals. Consistency of washing is crucial to ensure reliability of plate results.
IV. Blocking StageAfter the protein is coated with ELISA plates, blocking is usually required to prevent non-specific binding of the antibody in subsequent steps. When blocked, mixed proteins that are not related to the detection are added to the plate and incubated to occupy the nonspecific binding site. Commonly used blocking buffers include skim dry milk, bovine serum albumin (BSA), and casein. In addition, nonionic detergents such as Tween 20 or Triton X-100 can also be used as blocking agents, but their blocking effect is not as long-lasting as proteins. Ineffective blocking can lead to increased background noise, reducing detection sensitivity and specificity.
5. Antibody stageAntibody is a key component of ELISA detection, and choosing the right antibody is crucial. Both monoclonal antibodies and polyclonal antibodies can be used, each with its advantages and disadvantages. Monoclonal antibodies are highly specific but costly; while polyclonal antibodies can bind to target substances at multiple binding points, amplifying signals and improving sensitivity. Although the use of secondary antibodies increases the steps and detection time, it may lead to an increase in sensitivity. Therefore, when optimizing the detection, you need to find the appropriate antibody pair.
VI. Determination stageNo matter what type of ELISA test is used, the last step is the measurement. The most commonly used assay method is to use enzyme-mediated chemical reactions of visible color changes, followed by measurements by UV-vis spectrophotometry. An enzyme-binding antigen or antibody is added to the test well and bound to the target substance if it exists. When appropriate substrate is added, the enzyme causes color changes, which are proportional to the amount of the target substance. Horseradish peroxidase (HRP) is one of the commonly used conjugates and is usually used in conjunction with the substrate 3,3',5,5'-tetramethylbenzidine (TMB). Under the action of HRP, TMB changed from blue to yellow, and then the reaction was terminated by adding sulfuric acid solution. Finally, the absorbance value of each well was measured in the 450nm band using a microplate reader and corrected and calculated according to the detection design, such as subtracting the average of blank pores, the average of technical repetitions, or the ratio to standard calculation.
ELISA detection is a complex but very important experimental method, widely used in various medical research and diagnosis. Through understanding and mastering the ELISA testing process, scientific researchers can accurately and quickly obtain relevant data, providing scientific basis for early diagnosis of diseases and drug development.
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