Seminar topic

on
Immunofluorescence

Submitted to: Submitted by:

Dr. Salma Khan Saurabh Maoanker

Professor Msc 2 nd semester
Rd university biochemistry
jabalpur
Contents
 History
 Definition
 What is fluorescent compound?
 Types of immunofluorescence
 Direct immunofluorescence
 Indirect immunofluorescence
 Special points
 History

 First proposed in 1944 by Albert Coons.

 He showed that antibody could be labeled with the molecule that have
the property fluorescence.
Introduction
 Immunofluorescence is a technique used with a fluorescent
microscope and is used primarily on biological sample.
 This technique uses the specificity of antibody molecule to their
antigen, and also uses the fluorescent dye, which can be covalently
attached to antibody molecule without impairing its biological
activity.
 Fluorescent compound For example fluorescein isothiocynate form
covalent bond with the free amino group on antibody molecule.
 Such labelled antibody retains its ability to bind with specific antigen
molecule.
 It makes antibody molecule highly fluorescent so that they can
easily visualized under a fluorescent microscope.
What is fluorescence compound?

 These are the compounds which absorb light of one wavelength

and emit light of another wavelength.
 The emitted light can be viewed by a fluorescence microscope.
 Examples:
 Fluorescein: it is a organic dye and absorb light of 490nm, and
emit an intense yellow green fluorescence.
 Rhodamine: absorb yellow green light (515 nm) and emits deep
red fluorescence of 546 nm.
Types of immunofluorescence

 Mainly there are two types :

 Direct immunofluorescence
 Indirect immunofluorescence

 Direct immunofluorescence: In this technique there is a uses

a single antibody that is chemically linked to a fluorophore. The
antibody recognizes the target molecule and binds to it, and the
fluorophore it carries can be detected via microscope.
Direct immunofluorescence technique for the detection of
Rabies virus infection:

Smear of brain tissue Fluorescent labelled Ag-Ab complex

antibody against
rabies virus

If the antigen present in the smear than the antigen antibody complex
will be form and it will seen under the fluorescence microscope.
Fig: Visualization of Ag-Ab complex under the fluorescence microscope.
Indirect immunofluorescence

 In this technique two types of antibody used:

 Primary antibody
 Secondary antibody

 Primary antibody recognizes the target molecule and binds to it, and
the secondary antibody which carries the fluorophore, recognizes
the primary antibody and binds to it.
 Indirect Immunofluorescence technique for detection of Syphilis

Smear of T. pallidium drop of test serum Ag-Ab complex

Ag-Ab complex fluorescent labelled examined under

antiserum against uv illumination
human gamma globulin
Fig: Ag-Ab complex with the secondary in indirect immunofluorescence
technique.
Special points:

 This technique suitable for identify bacterial species, by detecting

Ag-Ab complex.

 This technique involve a small amount of sample to detect Ag

molecule.

 The test gives the result in very less time as compare to Elisa
technique.
References
 Immunology by kuby

 Methods in cell biology by Zbigniew Darzynkiewicz, J. Paul

Robinson, Harry A. Crissman

 Immunology/Immunofluorescence Protocol at Protocal-online.org