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.