Sandwich ELISA Test - Animated Video

Summary

This video explains the sandwich ELISA technique used to detect and quantify thyroid-stimulating hormone (TSH). It covers the step-by-step process, including plate coating with primary antibodies, blocking unoccupied sites, adding samples and standards, and using secondary antibodies for detection. The video highlights washing steps to remove unbound substances, the use of a substrate for signal generation, and concludes with photometric detection to quantify TSH levels. A calibration curve is constructed from the absorbance values to determine TSH concentration in the samples.

Takeaways

• 🧪 ELISA is a plate-based assay used for detecting and quantifying soluble substances such as antibodies, antigens, proteins, peptides, and hormones.
• 🔬 The video focuses on sandwich ELISA, particularly its application in detecting and quantifying thyroid-stimulating hormone (TSH).
• 📋 The first step in sandwich ELISA is plate coating, where a specific primary antibody binds to TSH and is immobilized on the polystyrene surface of a well strip.
• 🔄 The wells are incubated and washed several times to remove unbound antibodies, ensuring only bound antibodies remain on the plate surface.
• 🚫 A blocking step is crucial, where a blocking solution (e.g., BSA or serum) fills unoccupied sites, preventing unwanted binding during further steps.
• 📊 Standard solutions with known concentrations of TSH are added to the wells to create a quantification curve, followed by samples in their respective wells.
• 🔗 TSH in the samples binds to the immobilized antibodies during an incubation step, which is followed by more washes to remove unbound TSH.
• 🧫 A secondary antibody, conjugated for detection, is added to bind specifically to the captured TSH, and the plate undergoes further incubation and washing.
• 🎨 A substrate solution (e.g., pNPP) is introduced, and in the presence of TSH, an enzymatic reaction produces a yellow color in the wells.
• 📈 A spectrometer measures absorbance values from the wells, allowing for the creation of a calibration curve to determine TSH concentrations in the samples.

Q & A

What is the purpose of the ELISA technique described in the video?

The ELISA technique is used to detect and quantify soluble substances such as antibodies, antigens, proteins, peptides, and hormones. In this case, it is used to detect and quantify thyroid-stimulating hormone (TSH) in a sample.

What type of ELISA is discussed in the video, and how does it work?

The video focuses on sandwich ELISA, which works by using a primary antibody to bind the target molecule (TSH), followed by the addition of a secondary antibody for detection and quantification of the TSH present in the sample.

What is the first critical step in the sandwich ELISA process?

The first critical step is the coating of the plate with a primary antibody that binds to the target molecule (TSH). This step ensures that the TSH in the sample will be captured by the immobilized antibodies.

Why are polystyrene plates commonly used in ELISA assays?

Polystyrene plates are preferred because they allow antibodies to immobilize effectively on the solid phase, facilitating the binding of target molecules such as TSH during the assay.

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

The blocking step is crucial for preventing non-specific binding. A blocking solution containing proteins like BSA, serum, or casein is applied to block unoccupied sites on the solid phase, ensuring that only specific interactions occur during the subsequent steps.

How are TSH levels quantified using standard solutions?

Standard solutions with known concentrations of TSH are added to the wells to create a quantification curve. This curve serves as a reference for determining the TSH levels in the test samples based on absorbance values.

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

The secondary antibody is conjugated with an enzyme and specifically binds to the TSH-antibody complex. This antibody enables detection and quantification of TSH by producing a measurable signal in subsequent steps.

How is the signal generated and detected in sandwich ELISA?

A substrate, such as p-nitrophenylphosphate (pNPP), is added to the wells, which is cleaved by an enzyme (e.g., alkaline phosphatase) conjugated to the secondary antibody. This reaction produces a color change, and the intensity of the color is measured using a spectrometer to quantify TSH levels.

What happens during the washing steps of the sandwich ELISA?

The washing steps are critical for removing unbound substances and unbound antibodies. These steps ensure that only the specific antibody-target complexes remain in the wells, which are necessary for accurate detection and quantification.

How is the calibration curve used to determine TSH concentrations in samples?

The calibration curve is constructed using the absorbance values of standard solutions with known TSH concentrations. By comparing the absorbance of the test samples to this curve, the exact concentration of TSH in the samples can be determined.

Outlines

00:00

🔬 Introduction to ELISA and the Plate Coating Process

The video introduces ELISA, a plate-based assay used to detect and quantify soluble substances such as antibodies, antigens, and hormones. The focus is on the sandwich ELISA technique, particularly its application in detecting thyroid-stimulating hormone (TSH). The process begins with plate coating, where a primary antibody designed to bind to TSH is added to wells in 96-well plates or 8-well strips. The wells are coated with the antibody, covered with adhesive plastic for incubation, and then washed to remove unbound antibodies. The polystyrene surface is commonly used for antibody immobilization, and the washing process ensures that any excess unbound antibodies are cleared from the wells.

05:05

🧪 Blocking and Sample Incubation

In this stage, any unoccupied sites on the solid phase are blocked using a solution, typically containing proteins such as BSA, non-fat dry milk, or serum, to prevent non-specific binding in later steps. The blocking solution is added to each well, and after incubation, it is washed out. Standards with known TSH concentrations are then introduced to create a calibration curve, followed by the addition of the test samples. Incubation facilitates the binding of TSH to the immobilized antibodies. The wells are then washed again to remove any unbound substances, ensuring that only the specific antibody-hormone complexes remain.

🧫 Detection Using Secondary Antibodies and Color Change

The secondary antibody, which is conjugated to enable detection, is added to each well. After incubation, the wells are washed to remove any unbound secondary antibodies. Detection involves the addition of a substrate that reacts with the enzyme-labeled secondary antibody, producing a detectable signal, typically a color change. In the case of detecting TSH, the enzyme alkaline phosphatase catalyzes a reaction with pNPP substrate, generating a yellow color. This color change indicates the presence of TSH in the samples.

📊 Quantification of TSH Levels via Spectrometry

Once the color change is visible, a strong base like NaOH is added to stop the reaction, raising the pH of the mixture. The absorbance of each well is then measured using a spectrometer, with the absorbance values of the standards used to construct a calibration curve. This curve allows for the precise quantification of TSH concentrations in the test samples based on their absorbance readings.

Keywords

💡ELISA

ELISA, or Enzyme-Linked Immunosorbent Assay, is a plate-based laboratory technique used to detect and quantify soluble substances such as antibodies, antigens, proteins, peptides, and hormones. In the context of the video, it refers to the method being used to measure thyroid-stimulating hormone (TSH) levels in samples, demonstrating its application in diagnostic tests.

💡Sandwich ELISA

Sandwich ELISA is a subtype of the ELISA assay that involves the use of two antibodies: one to capture the antigen and another to detect it. The primary antibody is first bound to the well, and the antigen (TSH in this case) is sandwiched between this and a secondary detection antibody. The video focuses on how this technique is used to quantify TSH, showing the specific steps involved.

💡Thyroid-stimulating hormone (TSH)

Thyroid-stimulating hormone (TSH) is a hormone that regulates the function of the thyroid gland. In the video, TSH is the target molecule being measured using Sandwich ELISA, emphasizing the importance of accurate detection of this hormone in diagnostic tests related to thyroid function.

💡Polystyrene

Polystyrene is a common material used for the solid phase of the ELISA plate. In the video, it is noted that antibodies are immobilized onto the polystyrene surface of the wells, making it a critical component in ensuring the assay functions properly by capturing the targeted TSH.

💡Primary antibody

A primary antibody is the first antibody used in an ELISA assay that binds specifically to the antigen being measured. In the context of the video, the primary antibody is used to coat the wells and specifically bind to TSH, forming the basis for further steps in the detection process.

💡Blocking solution

A blocking solution contains proteins that block non-specific binding sites on the solid phase of the ELISA plate. This prevents unwanted interactions during the assay. In the video, proteins like BSA or serum are used to coat the wells after antibody immobilization to ensure only the antigen of interest (TSH) is detected.

💡Secondary antibody

The secondary antibody is an antibody conjugated with a detection enzyme, used to bind to the antigen captured by the primary antibody. In the Sandwich ELISA described in the video, the secondary antibody enables the detection of TSH by producing a measurable signal when bound to the antigen-antibody complex.

💡Substrate

A substrate in ELISA is a chemical that reacts with the enzyme linked to the secondary antibody to produce a detectable signal, usually a color change. In the video, p-nitrophenylphosphate (pNPP) is used as the substrate, which turns yellow upon reacting with alkaline phosphatase, indicating the presence of TSH in the samples.

💡Calibration curve

A calibration curve is a graph used in assays to quantify unknown concentrations by comparing them with known standards. In the video, TSH standards are added to create a calibration curve that helps in determining the concentration of TSH in test samples based on their absorbance values.

💡Absorbance

Absorbance refers to the amount of light absorbed by a sample in an assay. In the context of the video, absorbance is measured using a spectrometer to determine how much of the substrate’s reaction product (yellow color) is present, which directly correlates with the concentration of TSH in the sample.

Highlights

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

Sandwich ELISA is applied to detect and quantify thyroid-stimulating hormone (TSH) in a sample.

The first crucial step of Sandwich ELISA is the plate coating, where a primary antibody specific to TSH is used to coat a polystyrene well strip.

Polystyrene is the widely preferred material for the solid phase in ELISA due to its ability to immobilize antibodies.

Washing the wells with a specially formulated wash buffer is essential to remove unbound antibodies after each step.

A blocking solution containing proteins like BSA, serum, or casein is applied to prevent non-specific binding by covering unoccupied sites on the plate.

Samples and standard solutions with various known concentrations of TSH are added to the wells to create a quantification curve for TSH measurement.

The next step is incubation, where TSH from the samples and standards binds to the immobilized antibodies in the wells.

After incubation, washing is performed again to remove any unbound TSH or other substances.

A secondary antibody, conjugated for detection, is added to the wells, which binds specifically to TSH present in the sample.

Following incubation with the secondary antibody, another wash is performed to remove unbound antibodies, leaving only the specific sandwich complexes.

Detection and quantification are achieved using a substrate solution that initiates a color change in the presence of TSH, producing a yellow color.

The enzyme alkaline phosphatase catalyzes the reaction, transforming pNPP into p-nitrophenol, which generates the yellow color used for detection.

The reaction is stopped by increasing the pH with a strong base, such as NaOH, to prepare for photometric detection.

Absorbance is measured using a spectrometer, and a calibration curve based on standard points enables the quantification of TSH levels in the sample.

Transcripts

ELISA is a plate-based assay technique designed for detecting and quantifying soluble substances

such as antibodies, antigens, proteins, peptides, and hormones.

ELISA assays are categorized into various types. In this video,

we will focus on sandwich ELISA and explore how it can be applied

to detect and quantify thyroid-stimulating hormone present in a sample.

The crucial first step of Sandwich ELISA is plate coating.

To begin, samples are placed in a designated area, and for the coating step

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

The well strip is positioned in a support frame.

Next, a specific primary antibody designed to bind to TSH is used to coat the well strip.

The primary antibody solution is added into the wells, ensuring that each well receives the appropriate volume

Next, the well strip is covered with adhesive plastic

to create a controlled environment for an incubation process.

In ELISA, polystyrene stands as the widely preferred material for the solid phase.

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.

After eliminating excess solutions

the wells undergo thorough washing with a specially formulated wash buffer.

This solution 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 sandwich ELISA 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 casein.

the blocking solution is added to each well.

Subsequently, the well strip is covered and incubated

The proteins in the blocking solution form 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 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.

After the blocking step, the subsequent stage involves incubation with the samples.

Prior to loading the samples, standard solutions with various known concentrations of TSH

are added into the wells.

These standards serve as reference points to establish a quantification curve

for measuring the TSH levels in the samples.

Following the addition of standard solutions, each sample is carefully placed into its corresponding well.

Next, the well strip is covered and incubated.

Incubation facilitates the binding of TSH, present in both the samples and standards,

to the immobilized antibodies

Next, the solutions are removed from the wells, ensuring the elimination of any remaining liquid.

The next critical step is to perform a thorough wash to remove any unbound substances.

The washing step effectively removes both unbound substances and unbound TSH

leaving only the specific antibody-hormone complexes.

The next step involves incubation with a secondary antibody

which is a conjugated antibody designed to enable the detection and quantification of targeted TSH.

The secondary antibody solution is added to each well, and then the well strip is covered and incubated.

The secondary antibody is specifically designed to bind to the targeted

thyroid-stimulating hormone present in the wells.

Next, the secondary antibody solution is removed from each well.

Following the use of the conjugated antibody, the next crucial step involves performing a final wash

to remove any excess secondary antibody

The wash buffer effectively removes any unbound antibody, leaving only the specific sandwich complexes

Following the final wash, the next critical step involves detection and quantification.

This is achieved using a substrate that initiates a reaction, producing a detectable signal.

the substrate solution is carefully added to each well.

then the well strip is covered and incubated.

the detection antibody is typically labeled with an enzyme, such as alkaline phosphatase.

The most common chromogenic substrate for this enzyme is p-nitrophenylphosphat pNPP

the alkaline phosphatase enzyme catalyzes the cleavage of phosphate groups from pNPP molecules.

This enzymatic reaction transforms pNPP into p-nitrophenol

which generates a yellow color upon deprotonation in an alkaline medium.

in the presence of TSH, a color change takes place, causing the wells to display a yellow color.

Next, Prior to photometric detection, the reaction is commonly stopped by increasing the pH

of the reaction mixture using a strong base such as NaOH.

Finally, a spectrometer instrument is utilized to measure the absorbance in each well,

allowing for the quantification of TSH levels.

Using the absorbance values of standard points, a calibration curve is constructed.

This curve enables the determination of TSH concentration in samples.