ELISA (Enzyme-linked Immunosorbent Assay)

Summary

This video explains the process of an Enzyme-Linked Immunosorbent Assay (ELISA), a technique used to detect and quantify soluble substances like proteins. It covers different ELISA types—direct, indirect, and sandwich—detailing the steps of antigen immobilization, antibody binding, enzyme conjugation, and color measurement. The sandwich ELISA is highlighted, demonstrating how primary antibodies capture the antigen, and enzyme-linked secondary antibodies produce a color change to indicate the presence of the target protein. This assay is commonly used in research and diagnostics for its precision in protein detection.

Takeaways

• 😀 ELISA stands for Enzyme-Linked Immunosorbent Assay, a plate-based technique for detecting and quantifying soluble substances like proteins.
• 😀 During an ELISA, the antigen (target molecule) is immobilized on a plate and specifically detected by enzyme-linked antibodies.
• 😀 The enzyme linked to the antibody reacts with a colorless substrate, converting it into a colorful product that can be measured.
• 😀 Antibodies are key components in ELISA, with one antibody attached to the well and another that binds to the target protein.
• 😀 The enzyme-linked antibody, when in contact with the substrate, produces a measurable color change that indicates the presence of the target protein.
• 😀 There are several types of ELISA: Direct, Indirect, and Sandwich, each varying in the number and type of antibodies used.
• 😀 In Direct ELISA, the antigen is immobilized on the plate, and the enzyme-linked antibody directly binds to it.
• 😀 Indirect ELISA involves a primary antibody binding the antigen, followed by a secondary antibody binding to the primary, which is enzyme-linked.
• 😀 Sandwich ELISA involves two antibodies that bind to the antigen, with one immobilized and the other enzyme-linked.
• 😀 All ELISA methods have one thing in common: the final antibody binding is conjugated to an enzyme to confirm binding.
• 😀 An example of Sandwich ELISA involves wells coated with primary antibodies for protein X, where a sample and control are tested to measure the antigen's presence.
• 😀 The color change in the Sandwich ELISA is used to quantify the antigen by spectrophotometry, with the control remaining colorless due to no enzyme activity.

Q & A

What does ELISA stand for?

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a plate-based technique used to detect and quantify soluble substances such as proteins.

How does an ELISA detect and quantify proteins?

In an ELISA, the target antigen (protein) is immobilized on a plate and detected by antibodies that are conjugated to an enzyme. When a colorless substrate is added, the enzyme produces a colorful product, which can be measured to quantify the protein.

What is the role of antibodies in an ELISA?

Antibodies in an ELISA are used to specifically bind to the target antigen (protein). One antibody is immobilized on the plate, while another is enzyme-conjugated to detect and confirm the presence of the antigen.

What is the purpose of the washing step in an ELISA?

The washing step removes any unbound substances from the wells, ensuring that only the specific bound antibodies and antigens remain, which helps increase the accuracy of the test.

What is the difference between direct, indirect, and sandwich ELISA?

In direct ELISA, the enzyme-linked antibody binds directly to the immobilized antigen. In indirect ELISA, a primary antibody binds to the antigen, and a secondary, enzyme-conjugated antibody binds to the primary antibody. In sandwich ELISA, two primary antibodies are used: one to capture the antigen and another to bind and confirm the antigen's presence.

What is typically used as the enzyme in ELISA tests?

Horseradish peroxidase (HRP) is commonly used as the enzyme in ELISA tests. This enzyme converts the colorless substrate into a measurable colorful product.

How can the results of an ELISA be measured?

The results of an ELISA can be measured using spectrophotometry. The colorful product produced by the enzyme reaction is quantified by measuring the absorbance at specific wavelengths.

What is the purpose of using a control in an ELISA experiment?

A control is used to verify the specificity of the test. In the provided example, a control without the target protein ensures that any color change observed in the experimental well is due to the antigen-antibody interaction and not other factors.

Why is it important that the last antibody binding in an ELISA is enzyme-conjugated?

The enzyme-conjugated antibody is essential for confirming the presence of the antigen, as it catalyzes the conversion of a colorless substrate into a colorful product, allowing for measurable results.

Can the same technique be applied to other types of molecules besides proteins?

Yes, while the most common use of ELISA is for detecting and quantifying proteins, the technique can also be applied to other molecules like hormones, viruses, or antibodies, depending on the specific design of the assay.