Research Techniques Made Simple: Enzyme Immunoassay and ELISA

1
Research Techniques Made SimpleEnzyme
Immunoassay and ELISA

• Stephanie D. Gan1 and Kruti R. Patel2
• 1Department of Dermatology and 2 Program in
  Molecular and Translational Medicine, Department
  of Medicine, Boston University and Boston Medical
  Center

2
What is EIA/ELISA?

• Enzyme immunosorbant (EIA) and enzyme-linked
  immunosorbant assay (ELISA) are commonly used
  biomedical techniques employed to detect and
  quantify specific antigens or antibodies in a
  given sample.
• EIA and ELISA share similar basic principles.
• Both are modified from the radioimmuno assay, in
  which the radioisotope is replaced with an
  enzyme.

3
What is EIA/ELISA?

• EIA/ELISA is based on the immunologic concept of
  antigen binding to its specific antibody.
• Allows detection of very small quantities of
  antigen, such as proteins, peptides, or hormones,
  or antibody in a sample.
• Detection is qualitatively or quantitatively
  measured using an enzyme-labeled antigen or
  antibody to detect the sample antibody or antigen.

4
Primary antibody
1. Antigens coated onto the ELISA plate
2. Serum/Sample containing primary antibodies is
added
96-well microtiter plate
Secondary antibody
E
E
E
E
E
E
E
4. Secondary antibody-conjugated with an enzyme
is added
5. Excess secondary antibody is washed off the
plate.
3. Non-antigen binding antibodies are washed off
the plate
Steps for performing ELISA
E
E
E
E
E
E
Chromogen
7. Enzyme reacts with the substrate, producing
color. color. Intensity of the color correlates
with the level of antigen.
6. Substrate for the enzyme (Chromogen) is added
5
Types of ELISA

• Indirect
• Sandwich
• Competitive
• Multiple and portable

6
Indirect ELISA

• Antibody or antigen is adhered to the microtiter
  wells.
• Sample containing antibody/antigen is added
• Secondary enzyme-conjugated antibody is added.
• A substrate for the enzyme is introduced to
  quantify the primary antibody through color
  change.
• Disadvantage Method of antigen/antibody
  immobilization is non-specific.

7
Sandwich ELISA

• Used to identify a sample antigen.
• A known quantity of bound antibody is adhered to
  the mictotiter wells to capture antigen .
• Antigen-containing sample is added and a specific
  antibody sandwiches the antigen.
• Enzyme-linked secondary antibody is applied
  followed by a substrate to induce a color change.
• Advantage Eliminates the need to purify the
  antigen from a mixture of other antigens,
  simplifying the assay and increasing sensitivity
  and specificity.

8
Competitive ELISA

• Central to this technique is the competitive
  binding between the sample antigen and antigen
  bound to the microtiter well with the antibody.
• Primary unlabeled antibody is incubated with the
  sample antigen.
• These antibody-antigen complexes are added to the
  well plates that are coated with the same
  antigen.
• Any unbound antibody is washed off.

9
Competitive ELISA (cont.)

• The more antigen in the sample, the more primary
  unlabeled antibody will be bound and thus less
  available to bind to the antigen in the well
  plate.
• Secondary enzyme-linked antibody is added
  followed by substrate.
• Absence of color indicates a positive sample.
• Advantage High sensitivity to compositional
  differences in complex antigen mixtures, even
  when the specific detecting antibody is present
  in relatively small amounts.

10
Multiple and Portable ELISA

• Uses a multicatcher device with 8 or 12
  immunosorbant protruding pins on a central stick
  that is immersed in a sample.
• Washings and incubation with antigens/antibodies
  and chromogen are performed by dipping the pins
  into pre-filled microwells with reagents.
• Advantage Ready-to-use kits, cheap, useful for
  large population screening, does not require
  skilled personnel or laboratory equipment. Ideal
  for low-resource settings.

11
Advantages/Limitations
ADVANTAGES
ELISA is a biochemical assay that uses antibodies and an enzyme-mediated color change to detect the presence of small quantities of a substrate, either antigen or antibody, in a given sample.
Both indirect and sandwich methods are used to quantify the amount of antibody or antigen, respectively.
The competitive method detects compositional differences in complex antigen mixtures with high sensitivity, even when the specific detecting antibody is present in relatively small amounts.
Multiple and portable ELISA is a ready-to-use low-cost lab kit that is ideal for large population screenings in low-resource settings.
LIMITATIONS
The enzyme-mediated color change may yield false-positive results if a sufficiently long period of time has elapsed.
To detect a given antibody or antigen, a known reciprocal antigen or antibody must be generated.
Non-specific binding of the antibody or antigen to the plate would lead to a falsely high positive result