The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. The technique has revolutionized immunology and is commonly used in medical research laboratories and commercial applications. Although there are many variants of ELISA, they all depend on the same basic elements: coating and capture, plate blocking, probing and detection, and signal measurement.

For a quick look at ELISA, watch this video about direct and indirect ELISA

Tech Tip #65: ELISA Technical Guide and Protocols is a 14-page pdf document that covers the key elements of ELISA assays as well as general methods for optimization.

Topics covered in this downloadable guide include:

  • Introduction
  • Variations between ELISA protocols
    • Antigen immobilization for ELISA
    • Direct vs. indirect ELISA detection of antigen
    • Biotin signal amplification in ELISA
    • Fluorescence ELISA
    • Enzymatic detection in ELISA
    • Substrates for ELISA
  • Factors affecting assembly of the immune complex in ELISA
    • Types of plate and coupling options
    • Antibodies
    • Blocking buffer
    • Target antigen
    • Enzyme conjugate
    • Washing the plate
    • Substrates and signal detection
  • Common ELISA occurrences and explanations
    • Low signal-to-noise ratio
    • Signal fades quickly (observed with chemiluminescence)
    • Substrate forms a precipitate or plate visibly glows in the dark
    • Fluorescent signal is low
    • Poor standard curve linearity and dynamic range
  • General capture and sandwich ELISA protocol
    • Materials required for ELISA
    • Preparation of standards for ELISA
    • Preparation of samples for ELISA
    • ELISA procedure
  • Modifications to the ELISA protocol
    • Performing direct antigen immobilization
    • Using an enzyme labeled secondary antibody
    • Using an alkaline phosphatase system
    • Using fluorescence
    • Using chemifluorescence
    • Using chemiluminescence
    • Competitive ELISAs – Watch video below
  • ELISA optimization
    • Optimizing capture antibody concentration
    • Optimizing the blocking buffer
    • Optimizing the standard diluent
    • Optimizing sample concentration
    • Optimizing the detection antibody concentration
    • Optimizing the enzyme conjugate concentration
    • Optimizing signal detection

Video about competitive ELISA