Competitive ELISA Test - Animated Video
Summary
TLDRThis 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
🧪 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.
🔍 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.
Mindmap
Keywords
💡Eliza
💡Plate Coating
💡Polystyrene
💡Blocking Solution
💡Conjugated Protein
💡Incubation
💡Wash Buffer
💡Competitive Eliza
💡Substrate
💡Tetramethylbenzidine (TMB)
💡Photometric Detection
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
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