This document discusses immunofluorescence assays. It describes that immunofluorescence uses fluorescent dyes to detect the location of antigens or antibodies in tissue samples. There are two main types of immunofluorescence tests: direct and indirect. Direct tests use fluorescently-labeled antibodies to detect unknown antigens. Indirect tests detect antibodies in samples like serum by using a known antigen on a slide and fluorescently-labeled antibodies that bind to any human antibodies. The indirect test has the advantage of only requiring a single fluorescent conjugate that can detect antibodies to any antigen due to binding to all human antibodies.

1. IMMUNOFLUORESCENSE ASSAY
PRESENTER:
SAMIR MOIRANGTHEM
Msc. MLT, Medical Microbiology
RIPANS

2. IMMUNOFLUORESCENCE
• Fluorescence is the property of certain dyes which
absorb rays of one particular wavelength (
ultraviolet) and emits rays with a different
wavelength( visible light).
• It's is a method of determining the location of
antigen (or antibody) in a tissue section or smear
using a specific antibody (or antigen) labeled with
flurochrome.
• commonly used dyes are fluorescin isothiocyanate ,
lissamine rhodamine.

3. HISTORY
• First described in 1942 and refined by Albert Coons
in 1950.
• Coons and his colleagues showed that fluorescent
dyes can be conjugated to antibodies and these
"labelled" antibodies can be used to detect antigen
in tissues.

5. TYPES
• 1. DIRECT IMMUNOFLUORESCENSE TEST
• 2. INDIRECT IMMUNOFLUORESCENSE TEST

6. • 1. DIRECT IMMUNOFLUORESCENCE TEST
• PRINCIPLE:
• The specific antibodies tagged with fluorescent
dye(i.e.labelled antibodies) are used for detection
of unknown antigen in a specimen.
• If antigen is present, it reacts with labelled
antibodies and fluorescence can be observed
under ultraviolet light of fluorescent microscope.

9. USES

10. DISADVANTAGE
• SEPERATE SPECIFIC FLUORESCENT LABELLED
ANTIBODY HAS TO BE PREPARED AGAINST EACH
ANTIGEN TO BE TESTED.

11. 2. INDIRECT IMMUNOFLUORESCENCE
TEST
• used for detection of antibodies in serum or other body
fluids.
• PRINCIPLE : A known antigen is fixed on a slide. The
unknown antibody(serum) is applied to the slide.
• if antibody (globulin) is present in the serum, it attaches to
known antigen on the slide.
• for detection of this antigen-antibody reaction ,
fluorescein-tagged antibody to human globulin is added.
• in positive test, fluorescence occurs under ultraviolet light.

13. ADVANTAGES
• A single antihuman globulin fluorescent
conjugate can be employed for detection of
antibody to any antigen.
• All antibodies are globulin in nature,
therefore, antihuman globulin attaches to
all antibodies.
• This has overcome the disadvantage of
direct immunofluorescence test.

15. Sandwich' Technique of Immunofluorescence
Antigen being in the middle with labelled and unlabelled antibody
on either side, forms a sandwich. This is used for detection of
antibodies.
Antibody
+
Antigen
+
Labelled antibody

17. REFERENCE
Textbook Of Microbiology by C.P. Baveja
3rd edition

18. THANK YOU