Immunofluorescence Lab. 6 - PowerPoint Presentation

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Immunofluorescence

Lab. 6

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Immunofluorescence

It is a technique that uses a fluorescent compound (

fluorophore

or fluorochrome) to indicate a specific antigen-antibody reactionIf antibody molecules are tagged with a fluorescent dye and then binds to an antigen, this immune fluorescently labeled complex can be detected by colored light emission when excited by light of the appropriate wavelength

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UV

Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized

The presence of a specific antigen is determined by the appearance of localized color against a dark background

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Applications of IF

This method is used for:

Rapid identification of microorganisms in cell culture or infected tissue

Antigens on neoplastic tissue & inside cellsand CD antigens on T and B cells through the use of cell flow cytometryDetection of different proteins inside cells3

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Anti-HBV PreS2

(envelope proteins)

Cells infected with adenovirus

infected

cells in green and

nuclei stained with DAPI in blue.

Tubulin (green) M

M

itochondria (red)

Nonspecific stain for

nuclei (blue)

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Examples

Slide5

Fluorophores

Fluorophores

are typically organic molecules with a ring structure

They absorb light energy over a range of wavelengths that is characteristic for that compoundThis absorption of light causes an electron in the fluorescent compound to be raised to a higher energy levelThe excited electron quickly decays to its ground state, emitting the excess energy as a photon of light, which has a longer wavelength and lower energy This transition of energy is called fluorescence

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Fluorescence



Ex

Absorption

Relaxation: Measured as the

Fluorescence Lifetime

(~ 1 – 25 ns)

Em

Fluorescence: Always at a higher

wavelength

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Absorption & Emission Spectrums

The range over which a fluorescent compound can be excited is termed its absorption spectrum (excitation)

The range of emitted wavelengths for a particular compound is termed its emission spectrum

The time interval between absorption of energy and emission of fluorescence is very short and can be measured in nanoseconds7

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Factors Affecting

Fluorescence

Increase fluorescence

StructureAromatic groupsRigidityrigid

structures have lower probability of collisions

Decrease fluorescenceTemperature increaseHeavy atoms in solvent ( Intersystem crossing)Dissolved O2 ( Intersystem crossing)

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Factors Affecting Fluorescence

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Types of Immunofluorescence

Fluorescent staining can be categorized as direct or indirect, depending on whether the original antibody has

a fluorescent

tag attachedDirect IFIndirect IF

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Direct

Immunofluorescence

The antibody to the tissue antigen is conjugated with the fluorochrome and applied directlyThe antibody used is usually monoclonal antibodyFor example, to show the presence of virus antigens in tissue, fluorescence labeled antibodies are applied directly to the tissue

When viewed with

the fluorescence microscope, the tissue will be brightly stained11

Ab to tissue Ag is labeled with

fluorochrome

Ag

Fluorochrome

Labeled Ab

Tissue Section

Ag

Ag

Y

Y

Y

Ex

Ex

Ex

Em

Em

Em

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Indirect

Immunofluorescence

In this double-layer technique,

the unlabeled antibody (primary Ab) is applied directly to the tissue and visualized by treatment with a fluorochrome-conjugated to anti-antibody (secondary antibody)

The secondary antibody is anti-species antibody which is raised against the species where the primary antibody was produced

It is a polyclonal antibody12

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Indirect Immunofluorescence

Ab to tissue Ag is unlabeled

Fluorochrome-labeled anti-Ab is used to detect binding of the first Ab

Fluorochrome

Labeled

Anti-Ab

Tissue Section

Unlabeled

Ab

Ag

Ag

Ag

Y

Y

Y

Y

Y

Y

Ex

Ex

Ex

Em

Em

Em

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Direct & Indirect IF

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Direct

Indirect

Fix specimen on slide

Add labeled antibody specific for the desired antigen

Look for fluorescence

Fix specimen on slide

Add primary antibody, specific for the desired antigen

Add secondary labeled antibody

Look for fluorescence

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Advantages of Indirect IF

The

fluorescence is brighter than with the direct test

since several fluorescent anti-immunoglobulins bind on to each of the antibody molecules present in the first layerEven when many sera have to be screened for specific antibodies it is only necessary to purchase a single labeled reagent

The primary antibody does not need to be conjugated with a fluorochromeBecause

the supply of primary antibody is often a limiting factor, indirect methods avoid the loss of antibody that usually occurs during the conjugation reaction 16

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Controls

Reagent and tissue controls are necessary for the validation of immunofluorescence staining

results

Without their use, interpretation of staining would be haphazard and the results of doubtful valueMore specifically, controls determine if the staining protocols were:

followed correctly

whether day-to-day and worker-to-worker variations have occurredand that reagents remain in good working order17

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Controls

In carrying out an immunofluorescence experiment one has to be confident

that:

the reaction is specific and that the Ab is in fact binding selectively to the target Ag and not to other components of the cell or other closely related AgsIn addition if no fluorescence is observed with the probe does this mean that:the

Ag is not present or it mean that there may be a problem with preparation or with the tissue

itselfIf the correct controls are included in the experiment we can, with high certainty, answer these questions18

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Positive and Negative Controls

Negative Tissue Controls:

Specimens

serving as negative controls must be processed (fixed, embedded) identically to the unknown, but do not contain the target antigenIf a

signal is detected then this suggests

that a problem exists within your technique or protocolPositive Tissue Controls: Again, these controls must be processed identically to the specimen but contain the target antigenIf a signal is not detected

then this suggests the problem exists within your technique, protocol or reagent

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Detection of signal

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Fluorescence Instrument

Instrument for detection of fluorescence consists of:

Light source

Xenon Arc Lamp or mercury vapor lampLaserWavelength selectorExcitation filterEmission filterDetector Signal processor

Emission filter

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Xenon Arc Lamp

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Fluorescence Microscope

It is a microscope that uses fluorescence to generate an image

The combination of exciter filter, dichroic mirror and emission filter should be selected according to the

fluorochrome

label

The 3 components are usually built into a single module called the filter block22

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Quenching &

Bleaching

Quenching

is when excited molecules relax to ground states via nonradiative pathways avoiding fluorescence emission (vibration, collision, intersystem crossing)Molecular oxygen quenches by increasing the probability of intersystem

crossingPhotobleaching is defined

as the irreversible destruction of an excited fluorophore.

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Indirect Fluorescence Assay For Mumps Virus IgG Antibody

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Introduction and Summary

of

Test Procedures

Mumps, an acute, contagious disease, is generally characterized clinically by parotitis.Invasion of the central nervous system, testes, ovaries, and other visceral organs can accompany the infection

The introduction of a mumps virus vaccine in 1967 has resulted in a decline in the incidence of mumps

But because of the vaccine's restricted use, mumps will remain a common worldwide problem.Laboratory confirmation of mumps infection is usually not required in those patients with characteristic parotitisHowever, the two most common complications, meningoencephalitis and orchitis, can occur without the classic parotitisIn these cases, laboratory detection is necessary to confirm mumps infection

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Principle of the Test

Fluorescent

antibody assays use the indirect method of

antibody detection and titer determinationPatient serum or plasma samples are applied to cultured cells containing inactivated viral antigens provided on wells on

glass microscope slidesDuring a 30 minute incubation, antibody

specific for mumps virus antigens forms an antigen/antibody complex with the mumps virus antigens in the infected cells26

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Principle of the Test

In a

brief

washing step, nonspecific antibody and other unreacted serum proteins are eliminatedFluorescein-conjugated goat antihuman IgG is then applied to the wells of the glass slideThe anti-IgG conjugate combines with human IgG, if present, during a

30 minute incubationAfter a brief wash to remove unreacted

conjugate, the slides are viewed by fluorescence microscopyA positive antibody reaction is denoted by bright green fluorescence at the antigen sites27

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Controls

Mumps Virus IgG Positive Control:

Each

vial contains 0.5 ml mumps virus IgG antibody positive human controlThis component is a ready for use liquid at a 1:10 working dilutionMumps Virus IgG Negative Control: Each vial contains 0.5 ml mumps

virus IgG antibody negative human controlThis component is a ready for use liquid at a 1:10 working dilution

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Interpretation of Results

Bright

green fluorescent staining of the infected cells

denotes a mumps virus IgG antibody positive reactionAbsence of specific fluorescent staining of the infected cells denotes a mumps virus IgG antibody negative reactionFluorescence found in both infected and uninfected

cells, test sample is exhibiting a nonspecific reaction

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Positive

Negative