An Enzyme-Linked Immunosorbent Assay, also called ELISA or EIA.  This is quantitative immunological assay / test commonly used to detects/measure antibodies, antigens & proteins in biological samples. ( Blood, Urine, CSF etc.) Use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The most commonly used conjugate enzyme labels are Horseradish Peroxidase (HRP) and Alkaline phosphatase (AP). Other enzymes have been used as well; these include β-galactosidase, catalase and acetylcholinesterase.

1. ELISA – Enzyme Linked
ImmunoSorbent Assay
Amjad Khan Afridi
Chapter # 05

2. An Enzyme-Linked Immunosorbent Assay, also called ELISA or
EIA.
• This is quantitative immunological assay / test commonly used to
detects/measure antibodies, antigens & proteins in biological
samples. ( Blood, Urine, CSF etc.)
• Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).
• The most commonly used conjugate enzyme labels are Horseradish
Peroxidase (HRP) and Alkaline phosphatase (AP).
• Other enzymes have been used as well; these include β-
galactosidase, catalase and acetylcholinesterase.
ELISA – Enzyme Linked ImmunoSorbent Assay:

3.  The enzyme converts a colorless substrate (chromogen) to a colored
product, indicating the presence of Ag : Ab binding.
 Very sensitive & powerful technique to detect Ag & Ab.
 ELISA assays are generally carried out in 96 microtiter plate (well plates),
allowing multiple samples to be measured in a single experiment.
 Each ELISA measures a specific antigen, and kits for a variety of antigens
are widely available.
ELISA – Enzyme Linked ImmunoSorbent Assay:

4. Some examples include:
 Diagnosis of HIV infection,
 Pregnancy tests,
 Measurement of cytokines in the serum, etc.
ELISA – Enzyme Linked ImmunoSorbent Assay:

5. ELISA is of 3 types.
 (1) Indirect ELISA
 This technique is used for the detection of an Antibodies from the
clinical sample.
 The microtire plates are coated with an antigen
 Suspects serum is added, host antibody will bind with coated an
antigen and unbound proteins are washed off.
 The conjugated Antibody (secondary antibody conjugated to an
enzyme) will added and will bind with host an Antibodies and will
produce color.
Antigen Coated
microtire plates
Clinical Sample
Conjugated
antibody
Color is produced
Antigen
Host Antibody
Conjugated Antibody
with subtract

6. (2) Sandwich ELISA
 Used to detect the presence of Ag in a sample.
 The well is coated with Ab specific to the Ag and
then suspect serum is added allowed to react.
 The wells are washed to remove unbound Ag’s.
Antibody Coated
microtire plates
Clinical Sample Conjugated
antibody
Color is produced
Antibody
Host Antigen
Conjugated Antibody
with subtract (Enzyme)

7. (3) Competitive/ Inhibition ELISA
 Also called blocking ELISA
 This technique is used when we do not know which is the
particular agents (Ag or Ab) which is causing the problem/disease
 To measure the concentration of an antigen or antibody
 To find either Ab-Ag complex is present or not, or only Ab are
present
 In this technique, antibody is first incubated in solution with a
sample containing antigen
 The antigen-antibody mixture is then added to an antigen coated
micro titer well.
The more antigen present in the sample, the less free antibody will
be available to bind to the antigen- coated well. Addition of an
enzyme-conjugated secondary antibody (Ab2) specific for the
isotype of the primary antibody can be used to determine the
amount of primary antibody bound to the well as in an indirect
ELISA.

8. (3) Competitive/ Inhibition ELISA

9. Comparison between Indirect Sandwich & Competitive ELISA
to detect Ab (HIV, HCV)
to detect Ag ( Tumor Markers, Hormones )
to detect Ag ( Free Testosterone)

10. The chief use of antigen-antibody reactions are:
• Determination of blood groups for transfusion.
•Serological ascertainment of exposure to infectious
agents.
•Development of immunoassays for the
quantification of various substances.
•To detect the presence or absence of protein in
serum.
•Determining the characteristics of certain immuno-
deficiency disease.
Application of Antigen – AntibodyReaction: