Competitive ELISA Test - Animated Video

Summary

This video delves into the competitive ELISA technique, a method for detecting and quantifying specific proteins in samples. It begins with plate coating using antibodies, followed by incubation and washing. A blocking solution prevents non-specific binding. The key competitive phase involves the sample's protein of interest competing with a conjugated protein for antibody binding. After incubation and washing, a substrate reaction indicates protein presence, with color intensity correlating to concentration. The video concludes with photometric detection and calibration curve analysis to determine protein levels.

Takeaways

• 🧪 Eliza is a plate-based assay technique used to detect and measure soluble substances like antibodies, antigens, proteins, peptides, and hormones.
• 🔍 The video focuses on competitive Eliza, which is used to detect and measure a specific protein of interest in a sample.
• 🏺 Plate coating is the initial stage, where a specific antibody is applied to coat the wells of a 96-well plate or an 8-well strip.
• 🔬 Polystryene is widely used as the solid phase material in Eliza, where antibodies are immobilized.
• 🚿 After immobilizing antibodies, wells are washed with a specially formulated wash buffer to remove unbound substances.
• 🛡 A blocking solution is used to block any unoccupied sites on the solid phase, preventing non-specific binding.
• 🧬 The sample mixture contains a conjugated protein that competes with the target protein in the sample for binding to the immobilized antibodies.
• 🧬 During incubation, a competition occurs between the target protein and the conjugated protein for binding to the antibodies.
• 🔑 If the target protein is absent, only the conjugated protein will bind to the antibodies, which can then be detected and quantified.
• 🔍 A substrate is used to initiate a reaction that results in a detectable signal, allowing for the detection and quantification of the target protein.
• 📊 The target protein concentration in the samples is determined using a calibration curve based on the measured absorbance.

Q & A

What is the primary purpose of ELISA?

ELISA is designed to detect and measure soluble substances such as antibodies, antigens, proteins, peptides, and hormones.

What types of ELISA assays are mentioned in the script?

The script focuses on competitive ELISA, which is used to detect and measure a specific protein of interest in a sample.

What is the first step in the competitive ELISA process described in the script?

The first step is plate coating, where samples are positioned in designated areas and a specific antibody is applied to coat the well strip.

What type of plates are commonly used for ELISA?

96-well plates or 8-well strips are commonly used for ELISA.

Why is polystyrene widely preferred as the solid phase material in ELISA?

Polystyrene is widely preferred because it allows for the immobilization of antibodies onto its surface.

What is the purpose of the blocking solution in ELISA?

The blocking solution is used to block any unoccupied sites on the solid phase to prevent non-specific binding in subsequent steps.

What role does the conjugated protein play in competitive ELISA?

The conjugated protein, which shares the same affinity for the immobilized antibodies as the target protein, is used to facilitate detection and quantification.

How does the competition between the protein of interest and the conjugated protein occur during incubation?

During incubation, the protein of interest competes with the conjugated protein for binding opportunities with the immobilized antibodies.

What is the purpose of the washing step after incubation with the samples?

The washing step is performed to remove any unbound substances, leaving behind only the specific antibody-protein complexes.

How is the detection and quantification of the target protein achieved in ELISA?

Detection and quantification are achieved by using a substrate that initiates a reaction, resulting in a detectable signal, which is then measured using a spectrometer.

What is the role of the chromogenic substrate TMB in the ELISA process?

TMB is a chromogenic substrate that, in the presence of the HRP enzyme and hydrogen peroxide, produces a color change that can be measured to determine the concentration of the target protein.