Coating antigens refers to the process of attaching antigens to a solid surface, such as the wells of a microplate, for use in a binding assay.
Based on the provided reference, coating means the immobilization of antigen, antibodies or any other compound on the well surface, for the purpose of a binding assay. In the context of "coating antigens," this specifically means the immobilization of antigen onto the surface.
Understanding the Process
This immobilization step is fundamental in many immunological and biochemical techniques, most notably in the Enzyme-Linked Immunosorbent Assay (ELISA). The reference highlights that the coated plate is often part of a commercial ELISA kit or a self-made (home-brew) assay, and the term "ELISA coating" is frequently used.
Why Coat Antigens?
The primary purpose of coating antigens onto a surface is to create a solid phase target that specific binding partners, such as antibodies from a sample (like serum or plasma), can bind to. This allows for the detection and measurement of these binding partners.
- Detection: Enables the specific binding of antibodies present in a sample to the immobilized antigens.
- Wash Steps: Allows unbound components from the sample to be washed away easily, reducing background noise and ensuring that only specifically bound molecules are detected.
- Assay Foundation: Forms the crucial first layer for many multi-step binding assays like ELISA.
Where is Antigen Coating Used?
Antigen coating is a critical step in various laboratory techniques:
- ELISA: The most common application, used for detecting and quantifying antibodies in samples (e.g., testing for exposure to pathogens) or sometimes for detecting antigens.
- Immunosensing: Used in biosensors where immobilized antigens capture specific antibodies for detection.
- Protein Arrays: Creating arrays where multiple different antigens are spotted onto a surface for high-throughput screening of antibody binding.
How is Coating Performed?
Typically, antigens are dissolved in a suitable buffer and incubated in the wells of a microplate, commonly made of polystyrene. The proteins (antigens) passively adsorb to the plastic surface, primarily through hydrophobic interactions. After the incubation period, unbound antigen is washed away, and the remaining free binding sites on the plate surface are blocked with a blocking buffer (e.g., containing non-fat milk or bovine serum albumin) to prevent non-specific binding in subsequent steps.
In summary, coating antigens is a foundational step in numerous binding assays, involving the essential immobilization of antigen onto a solid support to facilitate specific interactions and enable detection.
