FLORESCENT ANTIBODY TECHNIQUE
(FAT)
• Ag/Immunogen-in vivo
• Antibody-specific
• Ag-antibody/primary
• Primary Ab (protein) human----enzymes/proteolytic---Fab (2)& Fc----centrifuge----Fc
(protein) injected in rabbit/goat/mice ….Anti Fc Abs /secondary Abs
• Test System-----Ag and Ab-----invisible
• Indicator System---+ve/-ve…..Florescent dyes
• Epitope/antigenic determinant
• In vivo-----to defend
• In vitro ----diagnose
• Florescent dye labelled Abs---conjugate-----Fc
• Fab ----antigen
• Antigen ---in vitro / Immunogen-in vivo
• Primary Ab & Secondary Ab
• In vitro---diagnose
• Ag-----Abs ([Link]) Human…..Protein….Proteolytic enzyme ---Fab(2) & Fc----centrifuge----
Fc….Rabbit/goat/mice…..Anti Fc Abs ([Link])
• Florescent dyes…Rhodamine, Auramine
• Conjugate….enzymes/ Radioisotopes/ [Link] labelled Abs…Fc
• Fab/
• Epitope /antigenic determinant…..on surface….
• Test System Ag& Abs----invisible
• Indicator System----
• Ag ----Ab ([Link])
• FAT, RIA, ELISA
• Test System-----Ag &Ab
• Indicator System-----+ve/-ve
• Florescent dyes (green, yellow , red), Radioisotopes (radiations), Enzyme (colored
product)
• Antigen (in vitro)/immunogen (in vivo)
• Epitope/antigenic determinant
• Antibodies (human) …..[Link] (Antigen)……. Protein …..Proteolytic enzyme…
Fab(2)…..Fc…….centrifuge….Fc ------Rabbit/Goat/Mice….[Link] (Anti Fc Abs)
• Abs linked/labelled with [Link]/Radioisotope/enzyme……Conjugate
• UV light-----excitation of photon----visible……Florescent microscope
• IgM, IgG, IgD, IgA, IgE
• 25 students ----serum…..IgG……YYYYYYYY…Fc Part of a class of a specie
• 1 Ab…weak…….2/3/4 Ab-labelled-----strong…..+ve sample
What is Florescence
• The property of certain dyes absorbing light rays at one particular wavelength
(ultraviolet light) and emitting them at a different wavelength (visible light) is known
as fluorescence.
• In the immunofluorescence test, a fluorescent dye that illuminates in UV light is used
to detect/show the specific combination of an antigen and antibody.
• The dye usually used is fluorescein isothiocyanate, which gives yellow-green
fluorescence.
• Immunofluorescence tests are also termed fluorescent antibody tests (FAT)Commonly
used Fluorochromes are:
• Fluorescein
• Rhodamine
• Phycoerythrin
• Auramine
❖The florescence can be visualized by florescent microscopy
Radioimmunoassay (RIA)
Radioimmunoassay (RIA)
• Radioimmunoassay is one of the sensitive immunoassay techniques which helps in the
determination of antigens or antibodies in a sample with the use of radioisotopes.
• It is an in vitro type of antigen-antibody interaction.
• When radioisotopes (I-125) instead of enzymes are used as labels to be conjugated with
antigens or antibodies, the technique of detection of the antigen-antibody complex is
called radioimmunoassay (RIA).
➢Application;
• It was first used for the detection of peptide hormones.
• Detection of different viral antigens
• Detection of many hormones and drugs
• Detection of Hepatitis B surface antigens
• Detection of mycotoxins
• Detection of the early stage of cancer
