1. Immunofluorescence
Lab. 6

2. 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 UV
phycoerythrin an intensely colored and highly fluorescent pigment obtained from algae, an efficient absorber of light (~30-fold greater than fluorescein), and a brilliant emitter of red fluorescence, stimulating its wide use as a label for immunofluorescence. UV

3. Immunofluorescence 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 Y

4. Applications of IF 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
Detection of different proteins inside cells

5. Examples Tubulin (green) M Mitochondria (red) Nonspecific stain for
nuclei (blue)
Anti-HBV PreS2
(envelope proteins)
The HBV surface protein antigens (HBsAg) are comprised of three carboxyl co terminal HBs proteins termed large (LHBs), middle (MHBs) and small (SHBs, also called major) protein. LHBs and MHBs also share the highly hydrophobic, repetitive, membrane spanning S domain. In addition, MHBs has a 55 amino acid region called preS2.
Cells infected with adenovirus
infected cells in green and
nuclei stained with DAPI in blue.

6. 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

7. Fluorescence Absorption
Relaxation: Measured as the Fluorescence Lifetime (~ 1 – 25 ns) Em
Fluorescence: Always at a higher wavelenth Ex 

8. 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

9. Factors Affecting Fluorescence
Increase fluorescence
Structure
Aromatic groups
Rigidity
rigid structures have lower probability of collisions
Decrease fluorescence
Temperature increase
Heavy atoms in solvent ( Intersystem crossing)
Dissolved O2 ( Intersystem crossing)

10. Factors Affecting Fluorescence

11. 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

12. 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

13. Direct Immunofluorescence
Ab to tissue Ag is labeled with fluorochrome Em Em Em Ex
Fluorochrome
Labeled Ab Y Ag Ag Ag
Tissue Section

14. 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

15. Indirect Immunofluorescence
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ab is used to detect binding of the first Ab Em Em Em Ex Y
Fluorochrome
Labeled Anti-Ab
Unlabeled Ab Y Ag Ag Ag
Tissue Section

17. Direct & Indirect IF Direct Indirect Fix specimen on slide
Add labeled antibody specific for the desired antigen
Look for fluorescence
Add primary antibody, specific for the desired antigen
Add secondary labeled antibody

18. 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

19. 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

20. Controls, cont.
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

21. 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

22. Detection of signal

23. 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
Xenon Arc Lamp
Emission filter

24. 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
A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.

26. Fluorescence Microscope

27. 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 crossing
Photobleaching is defined as the irreversible destruction of an excited fluorophore
Following absorption, molecules can relax via a non-radiative transition to the T1 rather than the S1 state - this is called an intersystem crossing

28. Quenching & Bleaching

29. Indirect Fluorescence Assay For Mumps Virus IgG Antibody

30. 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 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
Parotitis: التهاب الغدة النكفية

31. Introduction and Summary of Test Procedures, cont.
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
Orchitis: inflammation of one or both testes

32. Principle of the Test
Mumps virus IFA is an indirect immunofluorescence assay for the qualitative and semi quantitative determination of IgG antibodies to mumps virus.
The antibodies of the diluted patient samples and controls react specifically with the antigens of the tissue sections immobilized on the slides. After an incubation period of 30 min at room temperature (RT), unbound serum components are removed by a wash step.

33. Principle of the Test, cont.
The bound antibodies react specifically with anti- human IgG conjugated to fluorescein- isothiocyanat(FITC). After an incubation period of 30 min at RT excessive conjugate is separated from the solid phase immune complexes by an additional wash step.
Stained slides are read using a fluorescence microscope (excitation wavelength 490 nm, emission wavelength 520 nm).
A positive antibody reaction is denoted by bright green fluorescence at the antigen sites

34. Kit components Substrate slides
PBS buffer. ( as powder, dissolved in 1 L distelled water)
Conjugate
Mounting medium
Blotting templates
Coverslips
Positive control
Negative control

35. Controls Mumps Virus IgG Positive Control:
Each vial contains 2 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 2 ml mumps virus IgG antibody negative human control

36. Sample preparation:
Allow serum samples to reach RT prior to assay. Take care to agitate serum samples gently in order to insure homogeneity.
Screening: patient samples have to be diluted 1:10 (v/v) prior to the assay, e.g: 10ul sample + 90ul PBS.
Semi-quantitative titration: prepare a 4 fold serial dilution based on the1:10 (v/v) dilution using PBS buffer solution, e.g, 100ul sample dilution + 300ul PBS, resulting the following dilutions: 1:10, 1:40, 1:160, etc.

37. procedure
Bring all reagents to RT before use. Mix gently, remove slides from pouch immediately before use.
Apply 1-2 drops (30–50)ul controls( P, N) and (30-50)ul diluted patient samples onto the respective wells. Completely cover the immobilized tissue section. Don’t touch antigen surface.
Incubate 30 min at RT in a moist chamber.
Rinse gently with PBS solution using a squeeze wash bottle. don’t focus the PBS stream directly onto the wells. Avoid cross contamination.
Wash 2 X 5 min in changing PBS solution in coplin jar, agitate gently at entry and prior to removal.
Remove slides from the wash, shake off excesses PBS by tapping the edge of the slides onto absorbent towel, carefully dry around the wells using blotting templates.

38. Procedure, cont.
Apply one drop of conjugate to each well of the slides, making sure each well is completely covered.
Incubate 30 min at RT in a moist chamber, protected from direct light.
Repeat step 4 and 5.
Apply 2-4 drops of mounting medium across the slide. Rest the edge of a coverslip against the bottom of the slide allowing the mounting medium to form a continuous bead between coverslip and slide. Gently lower the coverslip from the bottom to the top of the slide. Avoid air bubbles, drain excess mounting medium from the edge of the slide with absorbent paper.
Read stained slides using a fluorescence microscope. Avoid longer exposition of one field of vision to minimize bleaching of FITC fluorescence.

39. Interpretation of Results
Fluorescence intensity may be semi-quantitative following the guidelines established by the CDC, Atlanta, USA:
+4 = maximal fluorescence, brilliant yellow-green
+3 = less brilliant yellow-green fluorescence
+2 = definite but dull yellow-green fluorescence
+1 = very dim subdued fluorescence
The degree of intensity is not of clinically relevance and has only limited values as an indicator of titer. Differences in microscope optics, filters and light source may result in differences of +1 or more in intensity.
Positive
Negative

40. Negative result:
A serum dilution is considered negative for mumps virus IgG if the fluorescent intensity is less than +1 and the tissue lacks specific fluorescence pattern.
Positive result:
A serum dilution is considered positive for mumps virus IgG if the fluorescent intensity of +1 or greater with a clearly discernable pattern of fluorescence.

41. Semi-quantitative titration:
If a Semi-quantitative titration is performed. The result should be reported as the reciprocal of the last dilution in which +1 apple-green fluorescent intensity with a clearly discernable pattern is detected
Using the 4 fold serial dilution the endpoint titer can be extrapolated.
1:10 = +1
1:40 = +2
1:160 = +/-
1:640 = -
Then the extrapolated titer is 80.
Fluorescence found in both infected and uninfected cells, test sample is exhibiting a nonspecific reaction

42. Reference values: Test validity:
Controls must be included in each test run and must be examined prior to reading test samples and should demonstrate the following results:
Negative control: the cells should exhibit less than +1 fluorescence and appear reddish orange due counterstain.
Positive control: fluorescence intensity of +3 to +4.
Titer
Mumps virus IgG
< 10
Negative
> or = 10
positive

43. tThank You..