4 Steps of ELISA (Enzyme-Linked Immunosorbent Assay Simply Explained)

Summary

The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used immunological test to measure antibodies, proteins, and glycoproteins in biological samples. It is conducted in 96-well plates, allowing for high-throughput analysis. The process involves four main steps: coating with a capture antibody, binding of the target antigen, addition of an enzyme-labeled detection antibody, and the conversion of a substrate into a measurable colored product. ELISA is valuable for determining antigen concentrations, with the results quantified using a standard curve and optical density readings.

Takeaways

• 😀 ELISA (Enzyme-Linked Immunosorbent Assay) is an immunological technique used to measure antibodies, proteins, and glycoproteins in biological samples.
• 😀 ELISA is typically carried out in 96-well plates, allowing multiple samples to be tested in one experiment.
• 😀 The process of ELISA can be illustrated through the sandwich ELISA method, which involves several key steps.
• 😀 The first step of sandwich ELISA is coating the plate with a capture antibody that binds to the target antigen.
• 😀 Excess unbound capture antibodies are washed away to prevent interference with the results.
• 😀 The sample is added next, and any target antigens in the sample bind to the capture antibody already on the plate.
• 😀 A detection antibody, labeled with an enzyme, is then added to bind to the target antigen bound to the capture antibody.
• 😀 Excess substances, including unbound detection antibodies, are washed away to ensure accuracy.
• 😀 A substrate is added, which is converted into a colored product by the enzyme on the detection antibody, enabling detection and quantification.
• 😀 The optical density (OD) of the resulting color is measured using a plate reader and is used to determine antigen concentration using a standard curve.
• 😀 ELISA is useful for determining antigen concentrations in samples and is highly sensitive and specific due to the use of antibodies.

Q & A

What is the enzyme-linked immunosorbent assay (ELISA)?

ELISA is an immunological assay used to detect and measure antibodies, proteins, and glycoproteins in biological samples.

Why is ELISA commonly used in scientific experiments?

ELISA is commonly used because it allows for the detection of specific proteins and antibodies in samples, providing valuable information for research and diagnostics.

How is ELISA typically performed?

ELISA is typically carried out on 96-well plates, allowing for the measurement of multiple samples in a single experiment.

What is the purpose of the capture antibody in ELISA?

The capture antibody is used to bind specifically to the target antigen, which is the molecule of interest in the sample.

What happens after the capture antibody is added to the plate?

After the capture antibody is added, any excess or unbound antibodies are washed away, ensuring only the antibody bound to the target antigen remains.

What is the role of the detection antibody in the ELISA process?

The detection antibody binds to the target antigen, which is already bound to the plate through the capture antibody. It is labeled with an enzyme to produce a measurable signal.

Why is washing done multiple times during the ELISA procedure?

Washing is done to remove any excess unbound substances, ensuring that only the desired antigen-antibody complexes remain for measurement.

How is the antigen concentration measured in an ELISA test?

The antigen concentration is measured by assessing the optical density (OD) of the colored product formed by the enzyme acting on a substrate. The OD is compared against a standard curve to determine the concentration.

What is the 'sandwich' structure in sandwich ELISA?

In sandwich ELISA, the target antigen is 'sandwiched' between the capture antibody and the detection antibody, facilitating the detection process.

How does the standard curve help in ELISA?

The standard curve, created using known antigen concentrations, allows for the calculation of the unknown antigen concentration in the sample based on its OD reading.