IMMUNOFLUORESCENCE

Dr Rania Abo-ShadyASS.Prof. of Clinical Pathology

Ain Shams University

Immunofluorescence assay

• Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluoresceinisothiocyanate (FITC).

• The specific antibodies are labeled with a compound (FITC) that makes them glow an apple-green color when observed microscopically under ultraviolet light.

• Fluorescence is the property of certain molecules or fluorophores to absorb light at one wave length and emit light at longer wave length (emission wavelength) when it is illuminated by light of a different wavelength (excitation wavelength).

• The incident light excites the molecule to a higher level of vibrational energy. As the molecules return to the ground state, the excited fluorophore emits a photon(= fluorescence emission ).

Principle of the Test

There are two major types of immunofluorescence staining methods:

• 1) direct immunofluorescence: staining in which the primary antibody is labeled with fluorescence dye,

• 2) indirect immunofluorescence: staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody.

Advantages of indirect:(1). Gives an amplification effect -- more tag or label ('signal') per molecule of target protein.

(2). Requires only one labeled antibody to identify many proteins. Same labeled secondary antibody can be used to bind to ("light up") many different proteins. (Preparation of labeled antibody is difficult and expensive.)(a). A different primary antibody is used for each target protein. (Not labeled -- no tag.) Variable part of primary antibody binds to specific part of target protein. (b). The secondary antibody binds to the constant part of the primary antibody. Therefore a sample of the same (labeled or tagged) batch of secondary antibody can bind to many different (unlabeled) primary antibodies

• Indirect immunofluorescence uses two antibodies; the first (the primary antibody) recognises the target molecule and binds to it, and the second (the secondary antibody), which carries the fluorophore, recognises the primary antibody and binds to it.

• For the determation of autoantibodies, tissue sections are used as antigen substrates.

• If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to a solid phase.

• In a second step, the attached antibodies are stained with fluorescein-labelled anti-human antibodies and visualized with the fluorescence microscope.

Indirect immunofluorescence assay:A laboratory test used to detect antibodies in serum

or other body fluid.Examples of autoantibodies:– Anti-dsDNA Abs.– ANA .– APA.– ASMA.– AMA.– Anti LKM.– ANCA.– Antithyroid Abs.

Indirect immunofluorescence is considered the standard technique for detection of autoantibodies. It offers unique advantages:

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

• For every antibody there is a characteristic fluorescence pattern.

• High specificity through visual discrimination: Antibodies are localized morphologically in exactly the same spots as their corresponding antigens.

• The combination of different substrates in one test field is highly suitable for determining autoantibody profiles (mouse –stomach –kidney substrate CT3 )

Autoantibodies are detected on specific substrates

– Anti-dsDNA Ab Crithedia Lucilae substrate

– ANA on Hep-2 substateon mouse stomach kidney substrate

– APA.– ASMA. on mouse stomach kidney substrate(CT2)

(CT2)– AMA.

– Anti LKM on mouse liver stomach kidney (CT3)

– ANCA on neutrophil substrate– Antithyroid Abs on Thyroid tissue

Advantage of Hep2 cells over rodent tissue

i. Higher sensitivity (greater Ag expression)ii. Human origin ensure better specificity iii. Cell division rates are higher so cell cycle

dependent Ab are easily identifiediv. Nucleus are much larger ,visible & complex

nucleolar detail can be seenv. Ags distribution are uniform not obscuring

intercellular matrix

Antinuclear antibodies (ANA)

• Are autoantibodies directed against various nuclear antigens, and are used to report the titer of the ANA and the pattern of

nuclear staining of the ANA.

• Comment on :

-Type of substrate

-Autoantibody (positive/negative)

-Pattern

• STAINING PATTERNS• Diffuse / homogeneous: antibodies to histone• Rim: antibodies to nuclear envelope proteins and to double-stranded (ds) DNA • Speckled: antibodies to Sm, RNP, Ro/SS-A, La/SS-B, and other antigens

• Nucleolar: associated with diffuse scleroderma• Centromeric: highly specific for the CREST

syndrome

Patterns of ANA

Negative ANA on Hep2

ANA (Homogenous pattern)

ANA( Speckled)

ANA (Nucleolar)

ANA(Rim pattern)

ANTIBODIES TO DOUBLE-STRANDED DNA (Positive anti dsDNA)

Negative Anti-ds DNA

Anti-neutrophil cytoplasmic Abs (ANCA)

P-ANCA

Antithyroid Abs (anti-microsomal)

Antithyroid Abs (anti-thyroglobulin)

Mouse stomach-kidney substrate

AMA onMouse stomach-kidney substrate

AMA (renal tubules)

ASMA on Mouse stomach-kidney substrate

ASMA (blood vessel )

APCA

Anti-LKM on mouse liver stomach kidney substrate(CT3)

• The pattern was consistently found to be that of a typical LKM antibody without any evidence of a mitochondrial antibody pattern is as follows;

Liver – strong positive cytoplasmic stain

Kidney – strong positive cytoplasmic stain in inner proximal tubules, negative distal tubules.

Stomach – negative

LKM bright liver, negative stomach

LKM close up of proximal renal tubule staining

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