ELISA (Enzyme-Linked Immunosorbent Assay) kits are designed to detect and quantify specific proteins, hormones, antibodies, or other biomolecules in biological samples. They are widely used in research, diagnostics, and quality control.
Components
- Coated Microplate:
- A 96-well plate pre-coated with capture antibodies specific to the target analyte. This allows the target molecule to bind to the plate.
- Standards:
- Known concentrations of the target analyte used to generate a standard curve. This curve is used to quantify the analyte concentration in unknown samples.
- Detection Antibody:
- A secondary antibody that binds to the target analyte, typically conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). This enables detection.
- Substrate Solution:
- Contains a substrate that reacts with the enzyme linked to the detection antibody, producing a color change proportional to the amount of analyte present.
- Stop Solution:
- An acid solution that halts the enzyme reaction and stabilizes the color, allowing for accurate measurement.
- Wash Buffer:
- Used to wash away unbound substances and reduce background noise, ensuring that only specifically bound analytes are measured.
Procedure
- Sample Preparation:
- Dilute and add samples, as well as standards, to the wells of the microplate.
- Incubation:
- Allow time for the target analyte in the samples to bind to the coated antibodies.
- Detection:
- Add the detection antibody to bind to the captured analyte.
- Substrate Addition:
- Add substrate solution to develop a color proportional to the amount of analyte.
- Stopping Reaction:
- Add stop solution to halt the color development.
- Measurement:
- Measure optical density (OD) using a microplate reader. The OD values are compared to the standard curve to determine the analyte concentration.
Applications
- Research: Quantifying biomolecules in studies related to cellular processes, disease mechanisms, and therapeutic development.
- Clinical Diagnostics: Diagnosing diseases, monitoring health conditions, and assessing biomarker levels for treatment decisions.
- Quality Control: Ensuring the quality and consistency of products in industries such as pharmaceuticals and food production.
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