ELISA PROTOCOL
Principle
The enzyme-linked immunosorbent assay (ELISA) is a solid-phase and plate-based method used to detect and quantify specific substances like antigens, antibodies, hormones, and proteins. In an ELISA, the antigen from the sample is immobilized in a microplate well either directly or by a specific antibody known as a capture antibody. Antigen-antibody complexes are formed when the primary detection antibody is added. Then the antigen-antibody complex is subsequently attached to a secondary antibody labeled with an enzyme (most commonly horseradish peroxidase, alkaline phosphatase, and glucose oxidase), which can catalyze substrates and produces a detectable signal, usually a color change.
ELISAs are commonly divided into four different types (competitive ELISA, sandwich ELISA, indirect ELISA, and directed ELISA) depending on how the antigen is immobilized and detected. Among them, we will focus on the most used indirect ELISA protocol.
Fig.1 Four general ELISA protocols.
Material
| Reagents | Instruments and Consumables | |
| Coating Buffer | TMB Solution | ELISA Reader |
| Coated Antigen | Stopping Buffer | Incubator |
| Blocking Buffer | Detect Antibody | Micropipette 20-200ul |
| Washing Buffer | Sample Buffer | Micropipette Multi-Channel 50-300ul |
| Positive Control | ||
| Negative Control | ||
| Enzyme-labeled Secondary Antibody | ||
Procedure
- Bind the antigen to well. Coat 96-well plate with 2 µg/mL capture antigen coating buffer, 100 µl/well, and incubate overnight at 4°C.
- Wash the plate. 300 μl PBS/well, wash 3 times (2 minutes each time).
- Block. 200 µl blocking buffer/well. Incubate at 37°C for 2 hours.
- Wash the plate. 300 μl PBS/well, wash 4 times (2 minutes each time).
- Add primary antibody. Add 100 µl of sample and control to the corresponding experimental wells. Incubate at 37°C for 1 hour. Wash the plate 4 times.
- Add 100 µl enzyme-labeled secondary antibody to each well. Incubate at 37°C for 1 hour. Wash the plate 4 times.
- Add TMB solution, 100 µl/well. Incubate at 37°C for 10-15 minutes.
- Add stop buffer, 50 µl/well, and mix thoroughly.
- Within 5 minutes after the reaction has stopped, read the plate at 450 nm.
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