Immuno Flu Re Scence

8/12/2019 Immuno Flu Re Scence

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Benita

Immunofluorescence



fluorescence

•

When molecules with luminescent properties absorbthey emit light of a different wavelength.

• With fluorescence the emission of light occurs extrem

rapidly (nanoseconds) after the absorption of excitati

Fluorochromes• essentially dyes, which accept light energy (e.g. from

at a given wavelength and re-emit it at a longer wave

• These two processes are called excitation and emissio



Stoke’s shift



Maximal absorbance and maximal emi

• The wavelength of excitation is critical to the total photo

light the fluorochrome will absorb.

• FITC (fluorescein isothiocyanate), for example, will absor

within the range 400 –550 nm but the closer the wavelen

490 nm (its peak or maximum), the greater the absorban

• In turn, the more photons absorbed, the more intense th

fluorescence emission will be.

• These optimal conditions are termed maximal absorbanc

maximal emission wavelength



Fluorochromes



Immunofluorescence

•Antigen-antibody reaction where the antibodies are tagg

(labelled) with a fluorescent dye and the antigen-antibod

complex is visualized using ultra-violet (fluorescent) micr

• This technique based on pioneering work by coons and k

and later by mary o sborne.

• Widely used both in research and clinical diagnostics.



• IF techniques can be used on both fresh and fixed samples.

• In if techniques, antibodies are chemically conjugated to fluorescent d

• These labeled antibodies bind (directly or indirectly) to the antigen of i

which allows for antigen detection through fluorescence techniques.

•The fluorescence can then be quantified using a flow cytometer, array s

automated imaging instrument, or visualized using fluorescence or con

microscopy

Immunofluorescence



Types of immunofluorescence:

• ‰ Direct immunofluorescence

• ‰ Indirect immunofluorescence

• ‰ Microimmunofluorescence



Direct immunofluorescence:



Direct immunofluorescence

Advantages

• shorter sample staining times and simpler dual and tr

labeling procedures.

Disadvantages

•lower signal, generally higher cost, less flexibility anddifficulties with the labeling procedure when comme

labeled direct conjugates are unavailable



Indirect immunofluorescence

• Indirect test is a double-layer technique

• The unlabelled antibody is applied directly to the tis

substrate

• Treated with a fluorochrome-conjugated anti-

immunoglobulin serum



Indirect immunofluorescence



Indirect immunofluorescenceAdvantages

•

greater sensitivity than direct immunofluorescence.

• there is amplification of the signal in indirect immunofluorescence because more than one secondary antib

to each primary

• Commercially produced secondary antibodies are relatively inexpensive, available in an array of colors,

controlled.

Disadvantages

• the potential for cross-reactivity

• the need to find primary antibodies that are not raised in the same species or of different isotypes when p

multiple-labeling experiments.

• Samples with endogenous immunoglobulin may exhibit a high background



Microimmunofluorescence

•

This is a serological technique employed to detect antibodies iserum.

• It works on the same principle as that of indirect immunofluore

but is performed on Teflon slides with many wells dotted with

• This technique is used in the serodiagnosis of Q fever, Mediterr

spotted fever, Detection of IgG, IgA and IgM Antibodies to Chla

toxoplasmosis, epidemic typhus etc.



Antinuclear Antibody Testing

• Antinuclear antibody (ANA) testing is commonly used in the assessmen

patients who may have an autoimmune disease.

• Target – nucleic acids and nucleoproteins

• One method is a blood test called the Fluorescent Antinuclear AntibodFANA.



HISTORY

• Hargraves et al., – LE cell in bone marrow in 1948

• Dr. George Friou in 1957, introduced FANA



ANA TYPES

Autoantibodies to DNA and histone

Autoantibodies to extractable nuclear antigens (ENA):

• These nuclear antigens were named ENA as originally they were extracted fro

with saline.

•Autoantibody to Smith antigen (Sm) which is considered to be specific for SLE

first anti-ENA detected in 1966.

• Thereafter further sub-types of ENA were more clearly identified

• i.e. ribonucleoproteins (RNP), SSA/Ro, or SSB/La, Scl-70, Jo-1 and PM1





FANA

• The test detects the presence of ANA in the blood of the patient which adhere to reagent test

(substrate), forming distinct fluorescence patterns.

• different substrates used:

• tissue sections, desquamated cells, chicken erythrocytes , HeLa cells.

• tissue sections using rat liver or a composite multiblock substrate (mouse stomach, rat liver an

became the standard substrate.

• In 1975,HEp-2 cells were introduced which have further increased the sensitivity of the test.

• cultured cells of laryngeal squamous cell carcinoma and are available commercially in the for

on glass slides.





Fluorescence patterns and intensit



Homogenous nuclear pattern



Nuclear rim pattern



Speckled nuclear pattern



• Discrete cytoplasmic and nucleolar pattern of

anti-ribosome abs

• Discrete speckled



Grainy nuclear and nucleolar pattern



• Diffuse nucleolar and sparse

nucleoplasmic pattern

• Punctate nucleolar staini





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