Question

Immunoassay : Enzyme linked immunosorbent assay

(ELISA) -> Ag Detection Method and Ab Detection Method with the way

Answer 1

ELISA ( ENZYME LINKED IMMUNOSORBENT ASSAY) :

ELISA is a immunoassay technique used to detect the Antibodies in the blood. It is widely used immunodiagnostic format in health care, industrial and bioanalytical settings.

Direct ELISA or Antigen Detection Method:

The simplest direct enzyme linked immunosorbent assay has been employed for the Immuno Assay of highly defined antigens. The first step involves

• Passive antigen adsorption onto the microtiter plate (MTP) wells by incubation for few hours to overnight. (Incubation at 37 degree celsius)
• Intensive washing to be done to remove unbound excess antigen.
• Non specific protein binding sites are blocked by blocking buffer such as Bovine Serum albumin.
• The adsorbed antigens are detected by incubating enzyme labeled antibody specific to antigen forming immunocomplex.
• After the unbound Antibody is washed away by repeated washing a specific substrate solution is provided for the enzyme labelled to Antibody.
• The enzyme catalyzes the conversion of substrate molecules into the colorimetric product and the enzyme substrate reaction is stopped by addition of stop solution
• The absorbance of resulting colorimetric product solution is measured at specific wavelength.

Incubate with Ag Microtiter plate Incubate with blocking buffer Incubate with enzyme-labeled Ab PRODUCT (Colorimetric/ chemiluminescent) SUBSTRATE I. Incubate with enzyme substrate

Indirect ELISA or Antibody Detection Method:

In indirect ELISA the unlabeled detection Antibody is used for specific binding to the coated antigen molecules. It has been widely used by the invitro diagnosis industry to examine large number of antisera for binding to target antigen using specific antispecies antibodies.

• The antigens are to bound onto the microtiter plate which is solid phase and non specific protein binding sites on the MTP are covered by treatment with a blocking buffer.
• The unlabeled detection antibody is used for specific binding to the coated antigen molecules.
• Incubation with enzyme labeled antispecies antibodies against the immunoglobulins of the species in which the detection antibodies are produced
• The enzyme catalyzes the conversion of substrate molecules into the colorimetric product and the enzyme substrate reaction is stopped by addition of stop solution.
• The absorbance of resulting colorimetric product solution is measured at specific wavelength

Incubate with Ag Microtiter plate Incubate with blocking buffer Incubate with Ab Incubate with enzyme-labeled anti-Ab PRODUCT (Colorimetric/ chemiluminescent) SUBSTRATE Incubate with enzyme substrate