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.

Outlines

00:00

🧪 Introduction to Competitive ELISA

This paragraph introduces the Eliza (Enzyme-Linked Immunosorbent Assay) technique, specifically focusing on the competitive ELISA method used to detect and measure soluble substances such as antibodies, antigens, proteins, peptides, and hormones. The process begins with plate coating, where samples are placed in wells and coated with specific antibodies. After incubation, the wells are washed, and a blocking solution is applied to prevent non-specific binding. The paragraph explains the use of a 96-well plate or an 8-well strip as common formats for the assay, with polystyrene as the preferred solid phase material for immobilizing antibodies.

05:00

🔍 Detection and Quantification in Competitive ELISA

This paragraph details the steps following the blocking phase in competitive ELISA. It describes the preparation and addition of the sample mixture, which includes a conjugated protein with an enzyme that allows for detection and quantification. During incubation, a competition occurs between the target protein in the sample and the conjugated protein for binding to the immobilized antibodies. After incubation, unbound substances are washed away, and a substrate is added to initiate a reaction that produces a detectable signal. The intensity of this signal correlates with the concentration of the target protein in the sample. The paragraph also mentions the use of tetramethylbenzidine (TMB) as a chromogenic substrate and the use of a spectrometer to measure absorbance, which is then used to determine the target protein concentration through a calibration curve.

Keywords

💡Eliza

Eliza, short for Enzyme-Linked Immunosorbent Assay, is a laboratory technique used primarily for the detection and measurement of substances such as antibodies, antigens, proteins, peptides, and hormones. In the context of the video, Eliza is the central theme, with a focus on the competitive Eliza method. The script explains how this technique is used to detect and measure a specific protein of interest in a sample.

💡Plate Coating

Plate coating is the initial stage in the Eliza process where a specific antibody is applied to coat the wells of a plate or strip. This step is crucial as it provides a solid phase for the subsequent binding reactions. The script mentions the use of 96-well plates or 8-well strips for this process, highlighting the importance of a controlled environment for effective antibody immobilization.

💡Polystyrene

Polystyrene is a widely preferred material for the solid phase in Eliza assays due to its properties that facilitate antibody immobilization. The script refers to polystyrene as the material onto which antibodies are immobilized, forming the foundation for the binding of target proteins and conjugated proteins.

💡Blocking Solution

A blocking solution is used to occupy any unoccupied sites on the solid phase after antibody immobilization. This step prevents non-specific binding in subsequent steps. The script describes the use of proteins like BSA, serum, non-fat dry milk, or casein in the blocking solution to create a barrier on the plate.

💡Conjugated Protein

In competitive Eliza, a conjugated protein is used, which shares the same affinity for the immobilized antibodies as the target protein in the samples. The protein is conjugated to an enzyme that facilitates detection and quantification. The script explains that the conjugated protein competes with the target protein for binding to the immobilized antibodies, which is a key aspect of the competitive Eliza process.

💡Incubation

Incubation is a critical step in Eliza where the sample mixture, containing the conjugated protein, is added to the wells and allowed to interact with the immobilized antibodies. The script describes the incubation process as a time when competition for antibody binding sites occurs between the target protein and the conjugated protein.

💡Wash Buffer

A wash buffer is a specially formulated solution used to rinse the wells and remove any unbound substances during the Eliza process. The script mentions the use of a wash buffer after incubation to eliminate unbound antibodies and after the blocking step to remove any remaining blocking protein.

💡Competitive Eliza

Competitive Eliza is a type of Eliza assay where the target protein in the sample competes with a conjugated protein for binding to immobilized antibodies. The script focuses on this method, explaining how the concentration of the target protein affects the binding of the conjugated protein and thus the subsequent detection and quantification.

💡Substrate

A substrate is a substance that initiates a reaction in the Eliza process, leading to a detectable signal. The script describes the use of a substrate in conjunction with an enzyme, such as horseradish peroxidase (HRP), to generate a color change that can be measured to determine the concentration of the target protein.

💡Tetramethylbenzidine (TMB)

Tetramethylbenzidine (TMB) is a chromogenic substrate widely used in Eliza assays for detection. The script explains how the HRP enzyme catalyzes the oxidation of TMB in the presence of hydrogen peroxide, resulting in a color change that indicates the presence and concentration of the target protein.

💡Photometric Detection

Photometric detection is the method used to measure the absorbance in each well after the substrate reaction, which correlates to the concentration of the target protein. The script describes the use of a spectrometer to measure the absorbance, which is then used to determine the protein concentration through a calibration curve.

Highlights

Eliza is a plate-based assay technique for detecting and measuring soluble substances like antibodies, antigens, proteins, peptides, and hormones.

This video focuses on competitive Eliza and its use in detecting and measuring specific proteins in a sample.

Plate coating is the initial stage of competitive Eliza, using 96-well plates or 8-well strips.

Antibodies are applied to coat the well strip to bind to the protein of interest.

Polystyrene is the preferred solid phase material for Eliza assays.

Unbound substances are removed through a thorough washing process with a wash buffer.

Blocking solution is applied to prevent non-specific binding in subsequent steps.

Conjugated protein with an enzyme is used to facilitate detection and quantification of the target protein.

A competition occurs during incubation where the protein of interest competes with the conjugated protein for antibody binding.

The concentration of the protein of interest affects its binding competition with the conjugated protein.

Unbound substances are removed after incubation with a thorough wash.

Detection and quantification are achieved using a substrate that reacts to produce a detectable signal.

The enzyme HRP and substrate TMBB are used to generate a color change for detection.

The intensity of the color change correlates with the concentration of the protein of interest.

The reaction is stopped using a strong acid like sulfuric acid before photometric detection.

A spectrometer is used to measure the absorbance in each well to determine the target protein concentration.

The target protein concentration is determined using a calibration curve.

Transcripts

Eliza is a plate-based assay technique

designed to detect and measure soluble

substances like antibodies antigens

proteins peptides and hormones the Liza

assays come in various types in this

video we'll focus on competitive

Eliza and explore how it's used to

detect and measure a specific protein of

interest in a

sample the initial stage of competitive

Eliza involves plate coating

to start samples are positioned in a

designated

area and for this coating process 96

well plates or eight well strips are

commonly

employed the well strip is placed into a

support

frame following that a specific antibody

designed to bind to our protein of

interest is applied to coat the well

strip the antibody solution is added

into the wells in ensuring that each

well receives the appropriate

volume next the well strip is covered

with adhesive plastic to establish a

controlled environment for the

incubation

process in Eliza polystyrene is widely

preferred as the solid phase material

antibodies are then immobilized onto

this polystyrene

Surface after immobilizing antibodies

onto the solid phase the adhesive

plastic is removed the well strip is

then overturned and tapped to eliminate

antibody Solutions and an absorbent

paper towel is used to ensure thorough

removal next the wells undergo a

thorough washing with a specially

formulated wash

buffer this solution effectively rinses

the wells removing any Unbound

substances using a wash buffer helps

eliminate any Unbound

antibodies after discarding the wash

buffer an absorbent paper towel is used

to remove any remaining

liquid the next step in competitive

Eliza is to block any unoccupied sites

on the solid

phase during this step a blocking

solution is applied usually containing

proteins like BSA serum non-fat dry milk

or

casine the blocking solution is added

into each well containing immobilized

antibodies subsequently the well strip

is covered and incubated the proteins in

the blocking solution create a barrier

on the plate preventing substances from

binding to these sites in subsequent

steps after completing the blocking step

the adhesive plastic is removed followed

by the discarding of The Blocking

Solutions an absorbent paper towel is

then used to thoroughly absorb any

remaining

solution next the wells are are

thoroughly washed with the wash

buffer this step helps to ensure that

any remaining blocking protein is

Thoroughly removed from the

wells after discarding the wash buffer

any residual liquid is removed using an

absorbent paper towel now moving on to

the next crucial step in competitive

Eliza preparing and adding the sample

mixture

in this stage a conjugated protein is

utilized sharing the same affinity for

the immobilized antibodies as our Target

protein found in the samples the protein

is conjugated to specific enzyme

facilitating both detection and

quantification the solution containing

the conjugated protein is added to each

sample once the samples are mixed with

the conjugated protein it's time to add

each sample into its corresponding

well next the well strip is covered

initiating the incubation

process during the incubation phase a

competition takes place our protein of

Interest competes with the conjugated

protein both seeking binding

opportunities with the immobilized

antibodies when the concentration of

conjugated proteins is higher they

outcompete and bind more effectively to

antibodies than our protein of

Interest conversely when the

concentration of our protein of interest

is higher it outcompetes and binds more

effectively to antibodies than the

conjugated protein if our protein of

interest is absent in the sample only

the conjugated protein will bind to the

antibodies after incubation with the

samples the solution is removed from

each

well subsequently a paper towel is used

to eliminate any remaining

liquid the next critical step is to

perform a thorough wash to remove any

Unbound

substances the washing step effectively

eliminates Unbound substances leaving

behind only the specific antibody

protein

complexes after discarding the wash

buffer an absorbent paper towel is used

to eliminate any remaining

liquid following the final wash the next

critical step involves detection and

quantification this is achieved by using

a substrate that initiates a reaction

resulting in a detectable

signal the substrate solution is

carefully added into each

well then the well strip is covered and

incubated the protein can be labeled

with enzymes such as alkaline phosphat a

beta galact Tod a and horseradish

peroxid a

hrp for hrp various detection reagents

have been developed with

tetramethylbenzidine tmbb standing out

as one of the most widely used

chromogenic substrates in the presence

of hydrogen peroxide hrp enzyme

catalyzes the oxidation of tmbb

resulting in the formation of two

intermediate oxidation state products

one product is a colorless tmbb cat

radical which is in equilibrium with a

blue green colored charge transfer

complex

CTC with an increased presence of

conjugated proteins more substrates will

be catalized generating significant

intensity and a visible blue

color on the other hand when our

proteins of Interest are more prevalent

fewer substrates will be catalyzed

resulting in less intensity and a

lighter blue color when only labeled

proteins are present a substantial

amount of substrate will be catalyzed

leading to significant intensity and a

deep blue

color next prior to photometric

detection the reaction is commonly

stopped by lowering the pH of the

reaction mixture using a strong acid

such as sulfuric

acid following a second one electron

oxidation event facilitated by hrp the

blue colored tmbb product is transformed

into a yellow colored Diamond oxidation

product

finally a spectrometer instrument is

utilized to measure the absorbance in

each

well after measuring the absorbance the

target protein concentration in the

samples is determined using a

calibration

curve thank you for watching if you're

interested in other Eliza tests you can

check out this

channel