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Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme immunoassays – like ELISA – are diagnostic test that either use antibodies to detect the presence of antigens, or antigens to detect for the presence of antibodies. The assays are generally conducted in microtiter plates. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen to be visualized or quantified.



  •  ELISA are diagnositc assay that test for the presence of antigens or antibodies.
  • There are several different types of ELISA: indirect, direct, and sandwhich amongst the most popular.

What is an ELISA?

Enzyme-Linked Immunosorbent Assays (ELISA) are a very common diagnotistic tool for identifying the presence of antigens or antibodies in an individual. By proxy, these test determine whether an inidividual has been exposed to a particular type of pathogen. There are several types of ELISA, but perhaps the most common is the indirect ELISA.

Indirect ELISA 

In an indirect ELISA, we can quantify an antigen-specific antibody.

    1.  We start with attaching known antigen to the bottom of the microtiter plate wells. The antigen sticks to the wells through electrostatic forces.
    2. We then add blocking agent to prevent any non-specific binding to the plate wells in the following steps.
    3. Patient sample is then added. If the sample contains antibodies to the antigen in the wells, they will bind that antigen.
    4. A wash step prevents any non-bound antigen from our plate wells.
    5. Anti-human antibodies conjugated to an enzyme are added. If, human antibodies are attached to the antigens, this antibody with bind the human antibody.
    6. A wash step prevents any non-bound antigen from our plate wells.
    7. A substrate is added. If the enzyme-linked antibody is present, it will react with the substrate causing a color change.
The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.

What Do You Need for an indirect ELISA?

      • ✔ Patient sample – this is what we will test for antibodies.
      • ✔ Well plate – this is where we will mix our solutions for the experiment. 
      • ✔ Antigen – know antigen that the antibody we are testing for, will recognize.
      • ✔ Anti-human antibodies linked to enzyme – this one part of the indicator mechanism
      • ✔ Substrate – The enzyme will react with the sustrate to cause a color change.
Adapted From: OpenStaxLicense: CC BY 4.0

Direct ELISA

The direct ELISA is less common and has some key differences from the indirect ELISA. Perhaps the biggest difference is that we are testing for the presence of antigen, rather than antibody.
    1. Direct ELISA patient sample is used to adhere antigens onto the well surface.
    2. Enzyme-linked antibodies that recognize the antigen of interest used. 


While this technique s faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).

Adapted From: OpenStaxLicense: CC BY 4.0

Sandwhich ELISA

In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples.

    1. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate. The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away.
    2. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well.
    3. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and,
    4. following a wash,
    5. the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme.
    6. After a final wash,
    7. a colorless substrate  is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10–9 g) per mL range.
Adapted From: OpenStaxLicense: CC BY 4.0

Enzyme-Linked Immunosorbent Assay FAQ & Review?

Key Terms

    • Antigen
    • Antibody
    • Secondary Antibody
    • Enzyme-linked
    • Substrate
  1. Parker N,  Schneegurt M, Thi Tu AH, Lister P, Forster BM. “20.4 EIAs and ELISAs.” Microbiology, Houston, TX, OpenStax, 1 Nov 2016. License: CC BY 4.0 License Terms: Edited & Adapted | Access for free at