Research Techniques Made Simple:Enzyme 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

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.

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.

96-well microtiter plate

1. Antigens coated onto the ELISA plate

2. Serum/Sample containing primary antibodies is added

3. Non-antigen binding antibodies are washed

off the plate

4. Secondary antibody-conjugated with an enzyme is

added

E

5. Excess secondary antibody is washed off the

plate.

6. Substrate for the enzyme (Chromogen) is added

7. Enzyme reacts with the substrate, producing color. color. Intensity of the

color correlates with the level of antigen.

E E E E E E

E E EE E E Chromogen

Secondary antibody

Primary antibody

Steps for performing

ELISA

Types of ELISA

• Indirect• Sandwich• Competitive• Multiple and portable

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.

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.

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.

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.

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.

Advantages/LimitationsADVANTAGES

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