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 reaction

• If 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

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

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Applications of IF

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

• This method is used for:• Rapid identification of microorganisms in cell

culture or infected tissue• Antigens on neoplastic tissue & inside cells

and• CD antigens on T and B cells through the use

of cell flow cytometry

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Examples

Anti-HBV preS2 (envelope proteins)

Anti- AIF Ab

Tubulin (green) MMitochondria (red)Nonspecific stain for nuclei (blue)

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Fluorophores• Fluorophores are typically organic molecules

with a ring structure• They absorb light energy over a range of

wavelengths that is characteristic for that compound

• This absorption of light causes an electron in the fluorescent compound to be raised to a higher energy level

• The 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

AbsorptionRelaxation: Measured as the Fluorescence Lifetime (~ 1 – 25 ns)

Em

Fluorescence: Always at a higher wavelenth

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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 nanoseconds

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Fluorphores Excitation & Emission Wavelenghts

Factors Affecting Fluorescence

• Increase fluorescence• Structure

• Aromatic groups• Rigidity

• Decrease fluorescence• Temperature increase• Heavy atoms in solvent• Dissolved O2

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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 attached

• Direct IF• Indirect IF

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Direct Immunofluorescence • The antibody to the tissue antigen is conjugated with the fluorochrome and applied directly

• The antibody used is usually monoclonal antibody

• For 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 stained

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Direct Immunofluorescence• Ab to tissue Ag is labeled with fluorochrome

Ag

FluorochromeLabeled Ab

Tissue Section

AgAg

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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 antibody

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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

FluorochromeLabeled Anti-Ab

Tissue Section

UnlabeledAb

Ag AgAg

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Direct & Indirect IF

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 layer

• Even 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 fluorochrome• Because the supply of primary antibody is often a limiting

factor, indirect methods avoid the loss of antibody that usually occurs during the conjugation reaction

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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 value• More specifically, controls determine if the staining

protocols were: • followed correctly• whether day-to-day and worker-to-worker variations

have occurred• and that reagents remain in good working order

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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 Ags

• In 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 itself

• If the correct controls are included in the experiment we can, with high certainty, answer these questions

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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 antigen• If a signal is detected then this suggests that a problem exists

within your technique or protocol

• Positive Tissue Controls: • Again, these controls must be processed identically to the specimen but contain the target antigen• If 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 lamp• Laser

• Wavelength selector• Excitation filter• Emission filter

• Detector • Signal processor Emission filter

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 block

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Fluorescence Microscope

Principles of confocal microscopy

• A focused laser beam serves as a high intensity light source

• Light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane

• A sensitive detector (photomultipler) behind a pinhole to measure the intensity of light

• The laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor

Use of confocal microscope

• Performs optical sectioning of thick samples• Three dimensional image reconstruction• Detects very weak fluorescent signals• Selective photobleaching

Quenching, Bleaching & Saturation

• 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 crossing

• Photobleaching 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

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Introduction and Summary of Test Procedures

• Laboratory confirmation of mumps infection is usually not required in those patients with characteristic parotitis

• However, the two most common complications, meningoencephalitis and orchitis, can occur without the classic parotitis

• In 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 determination

• Patient serum or plasma samples are applied to cultured cells containing inactivated viral antigens provided on wells on glass microscope slides

• During a 30 minute incubation, antibody specific for mumps virus antigens forms an antigen/antibody complex with the mumps virus antigens in the infected cells

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Principle of the Test

• In a brief washing step, nonspecific antibody and other unreacted serum proteins are eliminated

• Fluorescein-conjugated goat antihuman IgG is then applied to the wells of the glass slide

• The anti-IgG conjugate combines with human IgG, if present, during a 30 minute incubation

• After a brief wash to remove unreacted conjugate, the slides are viewed by fluorescence microscopy

• A positive antibody reaction is denoted by bright green fluorescence at the antigen sites

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Controls

• Mumps Virus IgG Positive Control: • Each vial contains 0.5 ml mumps virus IgG antibody

positive human control• This component is a ready for use liquid at a 1:10

working dilution

• Mumps Virus IgG Negative Control: • Each vial contains 0.5 ml mumps virus IgG antibody

negative human control• This component is a ready for use liquid at a 1:10

working dilution

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Procedure1. For qualitative IgG antibody determination, prepare a 1:10

screening dilution of each test sample in PBS• Prepare all dilutions in a minimum volume of 0.10 ml with PBS as the

diluent

2. For quantitative titration of sera, prepare two-fold serial

dilutions of the serum sample in PBS, starting with a 1:10

dilution, and adding equal volumes of diluted serum or plasma

and PBS for each consecutive dilution

3. Remove slides from protective pouch and apply 1 drop

(approximately 20 µl) of the diluted test sample(s) to each well• Add sufficient volume to completely cover each well, but cross-mixing of

contents between wells should not occur

• Note: Each day’s test run requires one well each for positive control,

negative control, and PBS (conjugate control)

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Procedure

4. Incubate the slides in a moist chamber for 30 minutes at 37°C

5. Rinse the slides in a light stream of buffer• Avoid directing the stream at the wells

6. Wash the slides for 10 minutes with a change of PBS solution after 5 minutes

• Agitate the slides by moving the rack up and down in the buffer

7. Blot the paint mask surrounding the test wells with the special blotters

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Procedure

8. Apply one drop of the ready to use conjugate to each test well

9. Repeat steps 4 (incubation), 5 (PBS rinse), 6 (10 minute PBS wash), and 7 (blot)

10. Apply the glycerol mounting media and 22 X 50 mm glass coverslip

11. Observe the reactivity under fluorescence microscopy

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Interpretation of Results

• Bright green fluorescent staining of the infected cells denotes a mumps virus IgG antibody positive reaction

• Absence of specific fluorescent staining of the infected cells denotes a mumps virus IgG antibody negative reaction

• Fluorescence found in both infected and uninfected cells, test sample is exhibiting a nonspecific reaction

Positive

Negative

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• https://www.youtube.com/watch?v=23KXl3wPdPY

• https://www.youtube.com/watch?v=g6Q1eel3YHs