What is ELISA?
Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods of immunological assay in biopharmaceutical industry. ELISA can be defined as an assay used immune system and chemicals to detect an analyte with qualitative and quantitative analysis. ELISA is typically performed in 96-well plates and immobilize the reactants make it easy to separate the target analytes from the complex sample mixtures.
How does ELISA work?
There are various ELISA tests, but the basic mechanism can be described as that the target analyte is linked to antibody immobilized on the solid surface. Target analyte also can be linked to a protein (for example, streptavidin) if primary antibody is labeled with biotin. Direct detection occurs when the analyte has enzymatic activity. Indirect detection occurs when the analyte is linked with a secondary enzyme-labeled reagent (common used labels: HRP and AP). The final detection step is adding the substrate suitable for Colorimetric analysis.
How many types of ELISA?
There are four types of ELISA according to modifications of the basic procedure:
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
Direct ELISA
The target analyte is immobilized to the solid plate and then is incubated with labeled primary antibody. Labeled primary antibody is detected directly for analysis.
Indirect ELISA
The target analyte is immobilized to the solid plate and then is incubated with primary antibody. After that, labeled secondary antibody binds to the primary antibody and is detected for analysis.
Sandwich ELISA
Sandwich assay is the most useful and powerful method for ELISA detection. In this assay, the target analyte is bound between two primary antibodies that one used for immobilization and the other one used for detection.
Competitive ELISA
Competitive ELISA is only used when the antigen is small and has one epitope, or antibody binding site. Unlabeled antigen from samples and labeled antigen compete for binding to the immobilized antibody. Then the labeled antigen was detected for analysis.
How to choose ELISA method?
Deciding which ELISA method to use often depends on experiment purpose and analyte characteristics. Different procedures have their own advantages and proper methods should be chose.
Different Types of ELISA
| ELISA Tpye | Advantages | Disadvantages |
|---|---|---|
| Direct ELISA |
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| Indirect ELISA |
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| Sandwich ELISA |
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| Competitive ELISA |
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* reference see The United States Pharmacopeia (USP 35-NF 30)
Scientists from Creative Proteomics are experienced performing ELISA analysis and they can help you with specific requirements and sample treatments.
