Enzyme-linked immunosorbent assay (ELISA) - Animation

Dr.G Bhanu Prakash Animated Medical Videos

11 Feb 201901:22

Summary

TLDRELISA, or enzyme-linked immunosorbent assay, is a highly sensitive method for detecting specific antibodies or antigens. The Direct ELISA tests for antigen presence by adsorbing an unknown antibody to a microtiter plate, followed by the addition of a sample and an enzyme-linked antibody. If the antigen is present, it binds and retains the enzyme, and a color change indicates its presence. The Indirect ELISA involves adding antigen to the plate, then serum suspected of containing antibodies, and an enzyme-linked antibody that reacts with the constant region of antibodies. A color change after substrate addition confirms the antibody's presence, offering a reliable way to identify immune responses.

Takeaways

🧪 ELISA is a highly sensitive test used to detect antibodies or specific antigens.
🔍 The Direct ELISA is used to test for the presence of an antigen by adsorbing an unknown antibody to the well of a microtiter plate.
💧 After rinsing to remove excess antibody, a sample suspected of containing the antigen is added to the well.
🔗 An enzyme-linked antibody that can react with the antigen is added, binding to the antigen if present, and is retained in the well.
🎨 The addition of a colorless substrate for the enzyme leads to color development, indicating the presence of the antigen.
🔄 The Indirect ELISA involves adding antigen to the microtiter plate well, allowing it to attach to the walls.
💧 Rinsing again removes excess antigen before adding serum suspected of containing antibodies.
🔗 An enzyme-linked antibody that reacts with the constant region of other antibodies is added in the Indirect ELISA.
🎨 The addition of a colorless substrate in the Indirect ELISA, followed by color development, indicates the presence of the identified antibody.
🔬 Both Direct and Indirect ELISA methods rely on the specific binding of antibodies to antigens for detection.
📈 The development of color in ELISA tests is a visual indicator of the presence of the target antigen or antibody, making it a qualitative and semi-quantitative assay.

Q & A

What is the enzyme-linked immunosorbent assay (ELISA)?
-ELISA is an extremely sensitive test used to detect antibodies or specific antigens in a sample.
What is the purpose of the direct ELISA test?
-The direct ELISA test is used to detect the presence of antigens by using an unknown antibody that is adsorbed to the well of a microtiter plate.
How is the antigen detected in a direct ELISA test?
-In a direct ELISA, the antigen is detected by adding a sample suspected of containing the antigen. If the antigen is present, an enzyme-linked antibody that can react with the antigen is added and binds to it, which is then retained in the well.
What role does the enzyme-linked antibody play in a direct ELISA test?
-The enzyme-linked antibody in a direct ELISA test binds to the antigen if it is present, indicating its presence through the retention in the well.
What is added after the enzyme-linked antibody in a direct ELISA test?
-A colorless substrate for the enzyme is added after the enzyme-linked antibody in a direct ELISA test.
How is the presence of the antigen indicated in a direct ELISA test?
-The presence of the antigen is indicated by the development of color after the addition of the colorless substrate for the enzyme.
What is the indirect ELISA test used for?
-The indirect ELISA test is used to detect the presence of antibodies by adding antigen to the microtiter plate well and allowing it to attach to the walls.
How does the indirect ELISA test differ from the direct ELISA test?
-In the indirect ELISA test, the antigen is added to the well first, followed by the serum suspected of containing antibodies, and then an enzyme-linked antibody that reacts with the constant region of other antibodies is added.
What is the final step in identifying the presence of antibodies in an indirect ELISA test?
-The final step in an indirect ELISA test is the addition of a colorless substrate, and the development of color indicates the presence of the antibody being identified.
Why is rinsing necessary in both direct and indirect ELISA tests?
-Rinsing is necessary to remove excess antibody or antigen, ensuring that only specific interactions are detected and avoiding false positives.
What does the development of color signify in both direct and indirect ELISA tests?
-The development of color in both direct and indirect ELISA tests signifies a positive result, indicating the presence of the antigen or antibody being tested for.

Outlines

00:00

🧪 Understanding ELISA: Sensitivity and Detection Methods

The paragraph introduces the enzyme-linked immunosorbent assay (ELISA) as a highly sensitive test for detecting the presence of antibodies or specific antigens. It explains the direct ELISA method, where an unknown antibody is used to capture antigens in a microtiter plate. The process involves adding a sample suspected of containing the antigen, followed by an enzyme-linked antibody that can bind to it if present. The addition of a colorless substrate and the development of color confirm the antigen's presence. The indirect ELISA is also described, where the antigen is first added to the microtiter plate, followed by a serum sample suspected of containing antibodies. An enzyme-linked antibody that reacts with the constant region of other antibodies is then added, with color development indicating the presence of the specific antibody.

Mindmap

Keywords

💡Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a biochemical test that detects and measures the presence of antibodies or antigens in a sample. It is highly sensitive and is widely used in medical and research fields. In the video's context, ELISA is the main topic, and the script explains how it is used to identify specific antigens or antibodies, which is central to understanding the video's theme.

💡Antibodies

Antibodies are proteins produced by the immune system to identify and neutralize foreign objects such as bacteria and viruses. In the script, antibodies are mentioned as the substances that the ELISA test can detect, highlighting their importance in immunological reactions and disease diagnosis.

💡Antigen

An antigen is any substance that induces an immune response in the body, including proteins from pathogens. The script describes how ELISA tests can be used to detect specific antigens, which is crucial for diagnosing infections or conditions where the presence of an antigen is indicative of disease.

💡Direct ELISA

Direct ELISA is a variation of the test where the antigen is already known and is detected directly in the sample. The script explains that in this method, an unknown antibody is used to capture the antigen, and the presence of the antigen is confirmed by the binding of an enzyme-linked antibody, as illustrated in the procedure described.

💡Microtiter Plate

A microtiter plate is a type of laboratory plasticware used for holding and organizing small volumes of liquid. In the context of the script, it is the vessel where the ELISA reaction takes place, with antibodies or antigens being adsorbed to the well's surface for testing.

💡Enzyme-Linked Antibody

This refers to an antibody that is conjugated with an enzyme, allowing for a colorimetric or fluorescent signal when the antibody binds to its target antigen. The script mentions the addition of an enzyme-linked antibody to the ELISA procedure, which is key to detecting the presence of the antigen.

💡Substrate

In the context of ELISA, a substrate is a chemical compound that is acted upon by an enzyme to produce a detectable signal, such as a color change. The script describes the addition of a colorless substrate that, upon reaction with the enzyme linked to the antibody, indicates the presence of the antigen.

💡Indirect ELISA

Indirect ELISA is a method where the primary antibody is not directly linked to an enzyme. Instead, a secondary enzyme-linked antibody that recognizes the primary antibody is used. The script explains that in this method, the antigen is first attached to the microtiter plate, followed by the addition of a serum sample and then an enzyme-linked antibody that reacts with the constant region of other antibodies.

💡Constant Region

The constant region of an antibody is the part of the protein that has the same amino acid sequence in all antibodies of the same class. In the script, the constant region is mentioned in the context of the indirect ELISA, where an enzyme-linked antibody that can react with this region is added to detect the presence of specific antibodies.

💡Color Development

Color development in ELISA refers to the visible change that occurs when the enzyme acts on its substrate, indicating a positive result. The script uses this term to describe the final step in both direct and indirect ELISA, where the presence of color confirms the presence of the antigen or antibody being tested.

Highlights

Enzyme-linked immunosorbent assay (ELISA) is an extremely sensitive test.

ELISA is used to detect antibodies or specific antigens.

Direct ELISA tests for the presence of antigen.

In Direct ELISA, unknown antibody is adsorbed to the well in a microtiter plate.

After rinsing, the sample suspected of containing the antigen is added.

An enzyme-linked antibody that can react with the antigen is added next.

Presence of antigen in the well causes the enzyme-linked antibody to bind and be retained.

A colorless substrate for the enzyme is then added.

Development of color indicates the presence of the antigen.

Indirect ELISA involves adding antigen to the microtiter plate well.

Antigen attaches to the walls of the microtiter plate in Indirect ELISA.

Serum suspected of containing antibodies is added after rinsing to remove excess antigen.

Enzyme-linked antibody capable of reacting with the constant region of other antibodies is added.

A colorless substrate is added followed by development of color indicating the presence of the antibody.

Indirect ELISA identifies the presence of specific antibodies in the serum.

Both Direct and Indirect ELISA techniques are valuable for detecting antigens and antibodies.

ELISA's sensitivity makes it a widely used method in diagnostics and research.

The process of ELISA involves specific steps to ensure accurate detection of antigens or antibodies.