This document discusses immunofluorescence techniques. It begins by defining immunoassays and fluorescence. It then explains the principles and types of immunofluorescence, including direct and indirect immunofluorescence. Direct immunofluorescence uses an antibody directly labeled with a fluorophore, while indirect uses an unlabeled primary antibody and a secondary antibody labeled with the fluorophore. The document outlines the advantages and applications of immunofluorescence, such as for diagnosing autoimmune disorders and evaluating cells. It concludes by noting some limitations, such as antibody quality and fluorophore photo bleaching.

1. SUBMITTED BY- SK AZIZ UDDIN.
SUBMITTED TO- DR. RIKESHWAR PRASAD DEWANGAN.
COURSE- M.PHARM.
SUBJECT- PHARMACEUTICAL ANALYSIS.
BATCH-2020-2022.
COLLEGE NAME- JAMIA HAMDARD.
FLUORO IMMUNOASSAY

2. Introduction
Principle of fluro immunoassay
Types of immuno fluorescence
Advantages & Disadvantages
Applications
Limitation
CONTENTS

3. IMMUNITY
 DEFINITION- Immunity is the capability of multi
cellular organisms to resist harmful microorganisms.
 Immunity involves both specific and non specific
components.

4. Introduction
 Immunoassay-: Immunoassay is a test that uses
antibodies and antigen complexes as a means of
generating a measurable result.
 An antibody : antigen complex is also known as an
immune complex.
 An immunoassay is a test that utilizes immune
complex when antibodies and antigens are brought
together.
 Immunoassays are used to quantify molecules of
biological interest based on the specificity and
selectivity of antibody reagents generated.

5. Continue;-
Immunoassay are categorised into different category,
these are
Competitive
immunoassay
Non-competitive
immunoassay
Homogenous immunoassay
Heterogeneous immunoassay

6. Competitive immunoassay
 In a competitive format, unable analyte (usually the
antigen) in the test sample is measured by its ability to
compete with the labelled antigen in the immunoassay.
 It is less label measured in the means more of the
unlabelled (test antigen) antigen is present.

7. NON COMPETITIVE
IMMUNOASSAY
 In non competitive assay , the measurement of the
labelled analyte (usually the antibody) is directly
proportional to the amount of antigen present in
the sample.
 Non competitive assay formats can use either one
step or two step methods.
 In two step assays format, there are wash steps in
which the sandwich binding complex is isolate and
wash to remove excess unbound labelled antigen.

8. DIFFERENCE
COMPETITIVE VERSUS NON COMPETITIVE IMMUNOASSAY

9. HOMOGENEOUS IMMUNOASSAY
 Homogenous methods have been generally applied to
the measurement of small analytes such as abused and
therapeutics drugs.
 Since homogenous methods do not require the
separation of the bound Ab- Ag* from the free
Ag*,there are generally much easier and faster to
perform.

10. HETEROGENOUS IMMUNOASSAY
In the
heterogeneous
immunoassays
the labelled,
unbound analyte
is separated or
washed away,
and the
remaining
labelled , bound
analyte is
measured.

11. TECHNIQUES OF IMMUNOASSAY
Radio immunoassay(RIA)
ELISA
Enzyme immunoassay
Optical immunoassay
Fluoro immunoassay
Luminescence immunoassay

12. IMMUNO FLUORESCENCE
 Immuno fluorescence is a technique allowing the
visualization of a specific antigen by binding a specific
antibody chemically conjugated with a fluorescent dye
such as fluorescein isothiocyanate(FITC).
 The specific antibodies are labelled with a compound
(FITC) that makes them glow an apple-green colour
when observed microscopically under ultraviolet light.

13. FLUORESCENCE
 Fluorescence is the property of certain molecules
to absorb light at one wave length and emit light at
longer wave length when it is illuminated by light
of a different wavelength.
 The fluorescence can be visualized using
fluorescence microscopy. The IF technique allows
for a visualization of the presence as well as the
distribution of target molecules in a sample.

14. PRINCIPLE

15. CONTINUE;-
 An immunofluorescence assay is based on following
principles steps;-
1. Specific antibodies bind to the protein of interest
2. Fluorescent dyes are coupled to these immune complexes
in order to visualize the protein of interest using
microscopy
 Immunofluorescence is a powerful approach for getting
insight into cellular structures and processes using
microscopy. specific proteins can be assessed for their
expression and location, making immunofluorescence
indispensable for scientist to solve many cell biological
questions.

16. Immunofluorescence staining
1. Direct immunofluorescence;- staining in which
primary antibody is labelled with fluorescence dye.
2. Indirect immunofluorescence-; Staining in which a
secondary antibody labelled with fluorochrome is
used to recognize a primary antibody.

17. Direct immunofluorescence
 Direct immunofluorescence 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 microscopy

18. Advantages of direct
immunofluorescence
 This technique has several advantages over
indirect immunofluorescence because of the direct
conjugation of the antibody to the fluorophore.
This residues the number of steps in the staining
procedure making the process faster and can
reduce background signal by avoiding some issues
with antibody cross-reactivity.

19. Indirect immunofluorescence
 Indirect immunofluorescence uses two antibodies; the
unlabelled primary antibody specifically binds the
target molecule, and the secondary antibody, which
carries the fluorophore, recognises the primary
antibody and binds to it.

20. Advantages of indirect
immunofluorescence
 Gives an amplification effect- more label per molecule of
target protein.
 Requires only one labelled antibody to identify many
proteins- same labelled secondary antibody can be used to
bind to many different proteins.
 A different primary antibody is used for each target protein.
variable part of primary antibody binds to specific part of
target protein.
 The secondary antibody binds to the constant part of the
primary antibody. Therefore a sample of the same batch of
secondary antibody can bind to many different primary
antibodies.

21. Application
 Immunofluorescence can be used on tissue sections,
cultured cell lines, or individual cells, and may be used
to analyse the distribution of proteins and small
biological and non-biological molecules.
 Immunofluorescence can be used in combination with
other , non-antibody methods of fluorescent staining,
for example, use of DAPI,( 4’,6-diamidino-2-
phenylindole) is a fluorescent stain that binds strongly
to A-T rich regions in DNA, to label DNA.
 It also play a key role in the diagnosis of autoimmune
disorder.
 This technique has a number of different biological
applications including evaluation of cells in
suspension,cultured cells,tissue, beads.

22. Limitation
 Quality and concentration of the antibody.
 Proper handling of the specimen.
 Choice of secondary antibodies.
 Fluorophores undergoes photo bleaching as
they are exposed to light.

23. THANK YOU