ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique used to detect antibodies, antigens, or other proteins. It works by binding an antigen or antibody to a well, adding an enzyme-linked antibody or antigen, and detecting the reaction using a chromogenic substrate. There are three main types: direct ELISA detects antigens, indirect ELISA detects antibodies, and competitive ELISA detects antigens by competition for antibody binding sites. The intensity of color developed is proportional to the concentration of the target being detected.

1. 1
Enzyme-Linked Immunosorbent Assay (ELISA)
Definition of ELISA
ELISA, or enzyme-linked immunosorbent assay, is an immunoassay
technique involving the reaction of antigen and antibody in vitro.
 EL = Enzyme-linked
 IS = Immunosorbent
 An antibody or antigen (“immuno”) is adsorbed (“sorbent”)
onto the polystyrene wells in which we conduct the test.
 One antibody is already adsorbed added to the wells and a
second antibody labeled with enzyme.
 Possible enzymes used in the ELISA system are horseradish
peroxidase (HRP) and alkaline phosphatase (AP). Each has a
rapid conversion of chromogenic substrate to coloured product
resulting in high sensitivity.
Application:
• Hormone measurement.
• Detection of small amounts of antibodies or antigens in tissues or
blood.
Types of ELISA
 Direct or Sandwich ELISA
 INDIRECT ELISA
 COMPETETIVE ELISA

2. 2
Direct or Sandwich ELISA
Principle:
- Suppose we want to detect an antigen in the serum of a patient.
- A specific antibody to this antigen is fixed to the well of a microtitre plate.
- The patient’s serum is added in the well and incubated for 30 minutes at 37°C.
- If the serum contains the antigen, it is fixed on the antibody.
- After washing, another antibody against the same antigen, tagged with the
enzyme horseradish peroxidase (HRP), is added.
- If the antigen is fixed then the antibody-HRP conjugate is fixed in the well.
- A color reagent is added, containing hydrogen peroxide and diamino benzidine
(DAB). The following reaction occurs:
H2O2 H2O + Nascent oxygen (O)
Diamino benzidine Oxidized DAB
(Colorless) (Brown color)
- Development of a brown color indicates the presence of the antigen in the
patient’s serum. The color developed is proportional to the antigen
concentration in the serum. So, the intensity of the color is measured, from
which the antigen level is calculated.
Enzyme
e
HRP

3. 3
Indirect ELISA
Principle:
- Suppose we want to detect an antibody in the serum of a patient.
- A good example is the test for detection of HIV antibody. In patients with
AIDS, the human immunodeficiency virus (HIV) enters the body and produces
specific antibody, to detect it the following method is used.
- The antigen from HIV is coated in the well of a microtitre plate.
- The patient’s serum is added, and incubated. If it contains the antibody, it is
fixed.
- A second antibody (antibody against human immunoglobulin) conjugated with
HRP is added.
- Then the color reagent (same as described above) is added. The development
of a brown color indicates that the antibody was originally present in the
patient’s serum.
- Here, also the color developed is proportional to the antibody concentration
originally present in the patient’s serum. Therefore from the color intensity, the
concentration of the antibody can be calculated.

4. 4
COMPETITIVE ELISA
Principle:
 Primary antibody (unlabeled) is incubated with sample antigen.
 Antibody-antigen complexes are then added to 96-well plates which are pre-
coated with the same antigen.
 Unbound antibody is removed by washing the plate. (The more antigen in the
sample, the less antibody will be able to bind to the antigen in the well, hence
"competition.")
 The secondary antibody that is specific to the primary antibody and conjugated
with an enzyme is added.
 A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent
signal.
ELISA Reader