Immunofluorescence Lab 6 2 Immunofluorescence It is a

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

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

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 Pre. S 2 (envelope proteins) Cells infected with adenovirus infected cells in green and nuclei stained with DAPI in blue.

6 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 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

Increase fluorescence ØStructure § Aromatic groups § Rigidity rigid structures have lower probability of collisions Decrease fluorescence ØTemperature increase ØHeavy atoms in solvent ( Intersystem crossing) ØDissolved O 2 ( Intersystem crossing)

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

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

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 Ex Em Fluorochrome-labeled anti-Ab is used to detect binding of the first Ab Fluorochrome Labeled Anti-Ab Unlabeled Ab Ag Ag Tissue Section Ag

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

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

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23 Fluorescence Instrument for detection of fluorescence consists of: Xenon Arc Lamp Light source Xenon Arc Lamp or mercury vapor lamp Laser Wavelength selector Excitation filter Emission filter Detector Signal processor 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

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26 Fluorescence Microscope

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

Quenching & Bleaching

29 Indirect Fluorescence Assay For Mumps Virus Ig. G 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 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

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

32 Mumps virus IFA is an indirect immunofluorescence assay for the qualitative and semi quantitative determination of Ig. G 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 Ig. G conjugated to fluoresceinisothiocyanat(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 Ig. G Positive Control: Each vial contains 2 ml mumps virus Ig. G antibody positive human control This component is a ready for use liquid at a 1: 10 working dilution Mumps Virus Ig. G Negative Control: Each vial contains 2 ml mumps virus Ig. G antibody negative human control This component is a ready for use liquid at a 1: 10 working dilution

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: 10 ul sample + 90 ul PBS. Semi-quantitative titration: prepare a 4 fold serial dilution based on the 1: 10 (v/v) dilution using PBS buffer solution, e. g, 100 ul sample dilution + 300 ul PBS, resulting the following dilutions: 1: 10, 1: 40, 1: 160, etc.

37 procedure 1. Bring all reagents to RT before use. Mix gently, remove slides from pouch immediately before use. 2. 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. 3. Incubate 30 min at RT in a moist chamber. 4. Rinse gently with PBS solution using a squeeze wash bottle. don’t focus the PBS stream directly onto the wells. Avoid cross contamination. 5. Wash 2 X 5 min in changing PBS solution in coplin jar, agitate gently at entry and prior to removal. 6. 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. 7. Apply one drop of conjugate to each well of the slides, making sure each well is completely covered. 8. Incubate 30 min at RT in a moist chamber, protected from direct light. 9. Repeat step 4 and 5. 10. 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. 11. 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 semiquantitative following the guidelines established by the CDC, Atlanta, USA: +4 = maximal fluorescence, brilliant yellowgreen +3 = less brilliant yellow-green fluorescence Positive +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 Negative

40 Negative result: A serum dilution is considered negative for mumps virus Ig. G 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 Ig. G 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: Mumps virus Ig. G Titer Negative < 10 positive > or = 10 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: Ø Negative control: the cells should exhibit less than +1 fluorescence and appear reddish orange due counterstain. Ø Positive control: fluorescence intensity of +3 to +4.

43 t. Thank You. .