The Enzyme Linked Immunosorbent Assay (ELISA)

Introduction to ELISA

  • ELISA (Enzyme-Linked Immunosorbent Assay) is a technique used to detect and quantify proteins, antibodies, and antigens in a sample.

  • Essential components needed for the experiment include:

    • Microtiter plate

    • Phosphate-buffered saline (PBS)

    • Antibody and antigen samples

    • Color-changing substrate solution

    • 37-degree incubator

    • Adjustable micropipette and tips

    • Transfer pipettes

Step-by-Step Procedure

Step 1: Preparation

  • Label the microtiter plate and transfer pipettes according to the instructions in the product literature.

Step 2: Antigen Addition

  • Add 100 microliters of the antigen solution to all wells of the microtiter plate.

Step 3: Incubation of Antigen

  • Incubate the plate for five minutes at room temperature.

    • During this time, antigens will adhere to the plate through hydrophobic and electrostatic interactions.

Step 4: Liquid Removal

  • Use a transfer pipette to remove all liquid from the wells.

Step 5: Washing Procedure

  • Wash the wells with approximately eight drops of PBS buffer.

  • Carefully remove all PBS from each well to avoid spillage into adjacent wells.

  • Traditional protocols involve blocking wells to prevent nonspecific antibody interactions, but this protocol optimizes this step by eliminating it.

Step 6: Reagent Addition

  • Add reagents as specified in the product literature.

  • Ensure the use of a clean micropipette tip for each reagent to avoid contamination.

Step 7: Incubation of Reagents

  • Incubate the plate for 15 minutes at 37 degrees Celsius.

Step 8: Liquid Removal

  • Remove the liquid from the wells using the designated transfer pipette.

Step 9: Washing with PBS

  • Wash each well with fresh PBS and remove the liquid with the specific transfer pipette for each sample.

Step 10: Secondary Antibody Addition

  • Add 100 microliters of the secondary antibody to each well.

Step 11: Secondary Antibody Incubation

  • Incubate for an additional 15 minutes at 37 degrees Celsius.

Step 12: Substrate Preparation

  • Prepare the detection substrate as outlined in the product literature while the samples are incubating.

Step 13: Secondary Antibody Removal and Washing

  • Remove the secondary antibody solution using the transfer pipette designated for each sample.

  • Wash each well once with fresh PBS buffer, ensuring to remove the liquid appropriately.

Step 14: Substrate Addition

  • Add 100 microliters of the substrate solution to each well.

Step 15: Final Incubation

  • Incubate at 37 degrees Celsius for five minutes.

Step 16: Result Examination

  • Analyze the results after incubation:

    • Differences between negative and positive samples should be noticeable, with positive samples typically developing a brown color.

    • If coloration is insufficient, extend the incubation time at 37 degrees Celsius.