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Indirect ELISA - Protocol

ELISA (enzyme-linked immunoSorbent assay) is an immunoassay that detects antigens via antibody binding that are linked to enzymes to allow for a diverse choice of detection methods. The indirect ELISA detects antigens that have been immobilized onto solid surfaces, commonly in the form of microplates. Once non-specific sites are blocked, primary antibodies and then enzyme conjugated secondary antibodies are incubated to build a complex for detection. Detection methods can include colorimetric, fluorometric, or luminometric depending on the type of secondary antibody conjugation.

Materials:

  • Coating Solution: Antigen or antibody is diluted in coating solution for immobilization onto the microplate. Commonly used coating solutions are: 50 mM sodium carbonate, pH 9.6; 20 mM Tris HCL, pH 8.5; or 10 mM pBS, pH 7.2. A protein concentration of 1-10 ug/mL is usually sufficient.
  • Blocking Solution: Commonly used blocking agents are: BSA, nonfat dry milk, casein and gelatin. Different assay systems may require different blocking agents.
  • Primary/Secondary Antibody Solution: Primary/secondary antibody should be diluted in 1x Blocking solution to prevent non specific binding. It is recommended to dilute antibodies between 1:100 and 1:500. Follow the manufacturer’s advice for secondary antibodies.
  • Wash Buffer Solution: Typically 0.1 M phosphate buffered saline or Tris buffered saline (pH 7.4) containing a detergent such as Tween 20 (0.02% 0.05% v/v).

Protocol:

  • 1. Dilute the antigen to 1-2 ug/ml in coating solution
  • 2. Add 100 ul of diluted antigen to appropriate wells. Incubate 2 hours at room temperature or 4 °C overnight.
  • 3. Empty plate and tap out residual liquid.
  • 4. Wash twice with 300 ul Wash solution.
  • 5. Add 300 ul Blocking solution to each well. Incubate 1 hour.
  • 6. Empty plate and tap out residual liquid.
  • 7. Wash twice with 300 ul Wash solution.
  • 8. Add 100 ul diluted primary antibody to each well. Incubate 1 hour at 37 °C or 3 hours at room temperature.
  • 9. Empty plate, tap out residual liquid.
  • 10. Fill each well with Wash solution. Invert plate to empty, tap out residual liquid. Repeat 3 times.
  • 11. Add 100 ul diluted secondary antibody to each well. Incubate 1 hour at room temperature.
  • 12. Empty plate, tap out residual liquid and wash as described in step 10.
  • 13. Give final 5 minutes soak with Wash solution. Tap residual liquid from plate. This washing step is critical to reduce signal background.
  • 14. Fill each well with Wash solution. Invert plate to empty, tap out residual liquid. Repeat 5 times.
  • 15. Dispense 100 ul of substrate (e.g. pNPP, TMB) into each well. Develop the color for 30 minutes.
  • 16. Stop reaction if necessary and read plate with plate reader at the correct wavelength.