Sandwich ELISA assay

Summary

This video tutorial delves into the specifics of sandwich ELISA, a highly sensitive technique for detecting antigens in biological samples. The process involves using two distinct monoclonal antibodies to 'sandwich' the antigen, resulting in a more precise and sensitive detection method compared to direct or indirect ELISA. The tutorial explains the steps of the sandwich ELISA, from fixing the first antibody to the addition of the sample, washing, and the use of a secondary enzyme-linked antibody to produce a colorimetric reaction indicating the presence of the antigen. Advantages include higher sensitivity and the ability to use crude samples without purification, while the challenge lies in the need for a deep understanding of the antigen and the design of specific antibodies for accurate detection.

Takeaways

• 🧪 Sandwich ELISA is a specific branch of ELISA techniques that is more sensitive, being two to five times more sensitive than direct or indirect ELISA.
• 🔍 The purpose of ELISA is to detect antigens, which are indicative of infections or infective agents in the body.
• 📚 Antigens are fragments of bacterial cells or viral particles, and their detection can help identify specific diseases.
• 🤝 In Sandwich ELISA, the antigen is 'sandwiched' between two different antibodies, hence the name.
• 🧬 Two specific monoclonal antibodies are required for Sandwich ELISA, each binding to different epitopes on the same antigen.
• 🛠️ The ELISA process involves using microplates with wells where one antibody is fixed at the bottom, and the antigen is detected through a series of steps.
• 🚿 Washing is a crucial step in ELISA to remove unbound antigens or antibodies, ensuring accurate results.
• 🔬 The detection process uses a primary antibody to bind the antigen and a secondary antibody linked to an enzyme for signal detection.
• 🎨 The addition of a substrate results in a color change, indicating the presence of the antigen of interest in the sample.
• 🚀 Sandwich ELISA offers advantages like higher sensitivity and does not require sample purification, making it more efficient.
• ⚠️ The main disadvantage of Sandwich ELISA is the complexity in designing the experiment, requiring a good understanding of the antigen and the design of two specific antibodies.

Q & A

What is the main focus of the video tutorial?

The video tutorial focuses on explaining the sandwich ELISA technique, a branch of the ELISA technique, which is more sensitive compared to direct or indirect ELISA.

What is the purpose of performing an ELISA process?

The ELISA process is performed for the detection of antigens present in the body, which can indicate the presence of infections or infective agents, thus helping in diagnosing diseases.

Why is the technique called 'sandwich ELISA'?

The technique is called 'sandwich ELISA' because the antigen is trapped between two antibodies, resembling a sandwich with something in the middle and two things on top.

How many antibodies are required in a sandwich ELISA technique?

Two different specific antibodies are required in a sandwich ELISA technique, each binding to a different epitope of the same antigen.

What is the role of the first antibody in the sandwich ELISA technique?

The first antibody, also known as the fixed antibody, is attached to the bottom of the well in the ELISA plate and is specific to bind with the antigen of interest.

What is the purpose of the wash step in sandwich ELISA?

The wash step is crucial to remove any unbound antigens and antibodies, ensuring that only the bound antigens and antibodies contribute to the final result, thus avoiding false positives.

What is the function of the secondary antibody in sandwich ELISA?

The secondary antibody in sandwich ELISA binds to the primary antibody that is attached to the antigen and is linked to an enzyme, which is necessary for the detection process by breaking down a substrate into a colored product.

Why is sandwich ELISA considered more sensitive than direct or indirect ELISA?

Sandwich ELISA is more sensitive because it uses two antibodies to capture the antigen, which increases the signal and allows for detection of the antigen at lower concentrations.

What are the advantages of sandwich ELISA over other types of ELISA?

Sandwich ELISA offers higher sensitivity, does not require sample purification, and is very specific for the target antigen, making it suitable for complex mixtures.

What is a potential disadvantage of sandwich ELISA compared to other types of ELISA?

A potential disadvantage is the difficulty in designing the experiment, as it requires a good understanding of the antigen and the design of two antibodies specific to different epitopes of the antigen.