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ELISA protocol

ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used technique in research and diagnostics for detecting the presence of specific proteins, antibodies, antigens, or hormones. Below is a general protocol for performing an ELISA assay:

Plate Selection:

  1. Choose a microtiter plate (96-well format is common) suitable for ELISA assays.
  2. Ensure the plate is made of a material (e.g., polystyrene) that binds proteins well.


  1. Dilute the capture antibody (usually monoclonal) in a coating buffer (e.g., PBS) to the desired concentration.
  2. Add the diluted capture antibody to each well of the microtiter plate.
  3. Incubate the plate overnight at 4°C or for 2 hours at room temperature to allow the capture antibody to adhere to the plate.
  4. After incubation, remove the excess antibody solution by washing the plate with a wash buffer (e.g., PBS with Tween-20).


  1. Add a blocking buffer (e.g., 1% BSA in PBS) to each well to block any remaining protein-binding sites on the plate.
  2. Incubate the plate for 1 hour at room temperature or 4°C.
  3. Wash the plate with wash buffer to remove excess blocking buffer.

Sample Preparation:

  1. Prepare samples (e.g., serum, cell lysates) by diluting them in an appropriate dilution buffer.
  2. Add the prepared samples to the wells of the microtiter plate.
  3. Incubate the plate for a specified period to allow binding of the target antigen to the capture antibody.


  1. Wash the plate multiple times with wash buffer to remove unbound proteins and other contaminants.
  2. Ensure thorough washing to minimize background noise and non-specific binding.

Detector Antibody:

  1. Dilute the detector antibody (usually polyclonal) in a dilution buffer.
  2. Add the diluted detector antibody to each well of the microtiter plate.
  3. Incubate the plate for a specified period to allow the detector antibody to bind to the captured antigen.
  4. Wash the plate to remove unbound detector antibody.


  1. Add the substrate solution appropriate for the enzyme linked to the detector antibody (e.g., TMB, ABTS).
  2. Incubate the plate in the dark for a specified period to allow color development.
  3. Stop the enzyme-substrate reaction by adding a stop solution (e.g., sulfuric acid for TMB).
  4. Measure the absorbance of each well at the appropriate wavelength using a microplate reader.

Data Analysis:

  1. Analyze the absorbance readings to determine the concentration of the target antigen in the samples.
  2. Use appropriate controls (e.g., positive and negative controls) for data interpretation.
  3. Perform statistical analysis if required to assess the significance of the results.


  • Ensure proper handling and storage of reagents to maintain their stability and functionality.
  • Optimize incubation times, concentrations, and other parameters based on the specific assay requirements and conditions.
  • Follow safety precautions when working with chemicals and biological materials.
  • Document all steps, observations, and results carefully for reproducibility and future reference.

This protocol provides a general framework for performing an ELISA assay, but it may require optimization and customization based on the specific requirements of the experiment and the characteristics of the target antigen.