Gentaur ELISA tests can be categorized into two main types: indirect ELISA and direct ELISA. The interpretation of results in both types involves assessing the optical density or color development, which is proportional to the amount of the target substance present in the Polyclonal antibody sample.

Indirect Affigen ELISA:

Procedure:

  1. Coating: The target antigen is immobilized on the microplate.
  2. Blocking: Unoccupied binding sites on the microplate are blocked to prevent nonspecific binding.
  3. Primary Antibody Incubation: The sample containing the primary antibody is added. If the primary antibody is present in the sample, it will bind to the immobilized antigen.
  4. Secondary Antibody Incubation: A secondary antibody conjugated with an enzyme is added. This secondary antibody binds to the primary antibody.
  5. Substrate Addition: A substrate for the enzyme is added, leading to a color change.
  6. Measurement: The optical density (OD) or color intensity is measured, and it correlates with the concentration of the target substance.

Interpretation:

  • Positive Result: Higher OD values indicate higher concentrations of the target substance in the sample.
  • Negative Result: Lower or background-level OD values suggest the absence or very low concentration of the target substance.

Direct Affigen ELISA:

Procedure:

  1. Coating: The target antigen is immobilized on the microplate.
  2. Blocking: Unoccupied binding sites on the microplate are blocked to prevent nonspecific binding.
  3. Primary Antibody Incubation: A directly conjugated primary antibody is added to the sample.
  4. Substrate Addition: A substrate for the enzyme is added, leading to a color change.
  5. Measurement: The optical density (OD) or color intensity is measured, and it correlates with the concentration of the target substance.

Interpretation:

  • Positive Result: Higher OD values indicate higher concentrations of the target substance in the sample.
  • Negative Result: Lower or background-level OD values suggest the absence or very low concentration of the target substance.

Tips for Interpretation:

  1. Standard Curve: A standard curve with known concentrations of the target substance is used to correlate OD values with concentrations.
  2. Controls: Include positive and negative controls to validate the assay’s performance.
  3. Background Correction: Subtract background readings (values obtained without the target substance) from sample readings.
  4. Normalization: Normalize results based on dilution factors or sample volumes.
  5. Replicates: Perform experiments in replicates for statistical reliability.