ELISA Method

Jelian Mae Cinchez

 What is ELISA?

➔ Enzyme-linked Immunosorbent Assay

➔ Used to measure antibodies, antigens, proteins and glycoproteins in

biological samples.
ELISA test may be used to diagnose:
➔ HIV/AIDS
➔ Lyme disease
➔ Pernicious anemia
➔ Rocky Mountain spotted fever
➔ Rotavirus
➔ Squamous cell carcinoma
➔ Syphilis
➔ Toxoplasma
➔ Zika virus
 Procedure

➔ ELISA assays are generally carried out in 96 well plates, allowing multiple
samples to be measured in a single experiment.

◆ A detection enzyme or other tag can be linked directly to the primary antibody or
introduced through a secondary antibody that recognizes the primary antibody.
(horseradish peroxidase (HRP) and alkaline phosphatase (AP).

➔ The plates need to be special absorbent plates to ensure the antibody or antigen
sticks to the surface.

➔ Each ELISA measures a specific antigen, and kits for a variety of antigens are
widely available.
ELISA Method
 Types of Capture Assay

➔ The direct detection method uses a labeled primary antibody that reacts directly
with the antigen. It can be performed with an antigen that is directly immobilized
on the assay plate or with the capture assay format.

➔ The indirect detection method uses a labeled secondary antibody for detection
and is the most popular format for ELISA. The secondary antibody has specificity
for the primary antibody.

➔ In a sandwich ELISA, it is critical that the secondary antibody be specific for the
detection primary antibody only (and not the capture antibody) or the assay will
not be specific for the antigen.
Steps
Measuring Antigens in ELISA
➔ Determination of antigen concentration in a sample requires production of a
standard curve using antigens of a known concentration.

➔ The concentration of antigen in a sample can then be calculated using the

optical density (OD).