Presentation on Immunofluorescence

Presented by:Farhan ali

Naveen bhattMsc Biotechnology II sem

IMMUNOFLUORESCENCE

Immunofluorescence is an antigen-antibody reaction where the antibodies are tagged (labelled)

with a fluorescent

dye and the antigen-antibody complex is visualized using ultra-violet (fluorescent) microscope.

Anti-alpha smooth muscle Actin antibody.(left)

A fluorescent stain for actin in the smooth muscle of the skin.(right)

History

• Immunofluorescence studies are considered the ‘gold standard’ for the diagnosis of autoimmune blistering diseases.

• Coons et al. (1941) developed the immunofluorescence techniques for the first time, a discovery which made possible to observe microscopically antigens, antibodies and their related substances on tissue sections or on cell smears

PRINCIPLE

• Fluorescence and phosphorescence are both

types of luminescence.

• In fluorescence the emission of light occurs

extremely rapidly after the absorption of

excitation light.

• phosphorescence emission continues for

milliseconds to minutes after the energy source

has been removed.

Fluorochromes are dyes that absorb ultra-

violet rays and emit visible light. This

process is called fluorescence.

The fluorochromes commonly used

in immunofluorescence are

fluorescein isothiocyanate (green)

and and tetramethyl rhodamine

isothiocyanate (red).

Types of immunofluorescence:

Direct immunofluorescence

Indirect immunofluorescence

Direct immunofluorescence:

• This is the one step histological staining process for identifying in vivo antibodies that are bound to tissues antigen.

• This technique is used to detect antigen in clinical specimens using specific fluorochrome labeled antibody. The steps involved are:

• Fixation of smear on the slide, treating with labeled antibody, incubation, washing to remove unbound excess labeled antibody and visualization under fluorescent microscope. When viewed under fluorescent microscope, the field is dark and areas with bound antibody fluorensce green.

Uses of Direct immunoflourescence

• This technique can be used to detect viral, parasitic,

tumor antigens from patient specimens or monolayer

of cells.

• Another application is identification of anatomic

distribution of an antigen within a tissue or within

compartments of a cell.

Advantages of direct immunofluorescence

• Shorter sample staining times and simpler dual

and triple labeling procedures.

• In cases where one has multiple antibodies

raised in the same species, for example two

mouse monoclonals, a direct labeling may be

necessary.

Disadvantages of direct immunofluorescence

• lower signal, generally higher cost, less

flexibility and difficulties with the

• labeling procedure when commercially labeled

direct conjugates are unavailable.

Indirect Immunofluorescence

• It is considered as the Standard technique for the Detection of the

Autoantibodies.

• Indirect immunofluorescence is employed to detect antibodies in patient

serum. The antigen on smear are made to react with specific unlabeled

antibody (raised in mouse) and washed. The unbound antibody gets washed

off.

• The presence of specific mouse antibody bound to the antigen on smear is

detected by adding another antibody.

• The second antibody is labeled anti-gamma globulin (rabbit antibody

against mouse antibody) antibodies. This antibody binds to Fc portion of

first antibody and persists despite washing.

• The presence of the second antibody is detecting by observing under

fluorescent microscope.

Uses of Indirect immunoflourescence

• It is often used to detect autoantibodies.

• Commonly used in the detection of anti-nuclear

antibodies (ANA) found in the serum of patients with

SLE (Systematic lupus Erythematosus)

Advantages of indirect immunofluorescence

• A negative result excudes the presence of all these antibodies.

• For every Antibody there is a characteristic fluorecence

pattern.

• Greater sensitivity than direct immunofluorescence.

• There is amplification of the signal in indirect

immunofluorescence because more than one secondary

antibody can attach to each primary.

• Commercially produced secondary antibodies are relatively

inexpensive, available in an array of colors, and quality

controlled.

Disadvantages of indirect immunofluorescence

• The potential for cross-reactivity and the need to find

primary antibodies that are not raised in the same

species or of different isotypes.

• When performing multiple-labeling experiments.

Samples with endogenous immunoglobulin may

exhibit a high background.