Affigen ELISA

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Affigen ELISA detect and quantifies the presence of specific proteins, antibodies, or antigens. ELISA is based on the specific binding of an antigen (or antibody) to a corresponding antibody (or antigen) immobilized on a solid surface. The interpretation of results in both types involves evaluating optical density or color development, which correlates with the concentration of the target substance in the sample.There are different types of ELISA, including direct, indirect, sandwich, and competitive ELISA.

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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 present, the primary antibody binds to the immobilized antigen.
  4. Secondary Antibody Incubation: An enzyme-conjugated secondary antibody is added, which binds to the primary antibody.
  5. Substrate Addition: Addition of a substrate for the enzyme leads to a color change.
  6. Measurement: Optical density (OD) or color intensity is measured, correlating with the concentration of the target substance.

Interpretation:

  • Positive Result: Higher OD values indicate higher concentrations of the target substance.
  • 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: Addition of a substrate for the enzyme leads to a color change.
  5. Measurement: Optical density (OD) or color intensity is measured, correlating with the concentration of the target substance.

Interpretation:

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

Tips for Interpretation:

  1. Standard Curve: Use a standard curve with known concentrations of the target substance to correlate OD values with concentrations.
  2. Controls: Include positive and negative controls to validate assay 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.

 

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