immunology

1. Immunoflourescent assey

2. • Immunofluorescence is a serological test where the labeling of antibodies
or antigens is done with fluorescent dyes ( fluorochromes).
• Fluorochromes are dyes which have the ability to absorb the short
wavelength UV radiation and emit light of longer wavelength fluorescence (
visible green light).
• Examples : FITC, Rhodamine , Acridine orange
• First this technique was discovered by albert coons in 1942

3. • There are two ways of doing IF staining
• Direct immunofluorescence
• Indirect immunofluorescence
• Direct immunofluorescence
Use: Direct detection of Pathogens or their Ag’s in tissues or in
pathological samples
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over it
• Slide is washed to remove unattached Ab’s
• Examined under UV light in an fluorescent microscope
• The site where the Ab attaches to its specific Ag will show apple green
fluorescence

4. • DIRECT IMMUNOFLUORESCENE.
• Direct immunofluorescence (DIF) is a technique used in the laboratory to diagnose diseases of
the skin, kidney, and other organ systems. It is also called the direct immune fluorescent test
or primary immunofluorescence.
• Principle of the test
• DIF involves the application of antibody–fluorophore conjugate molecules to samples of patient
tissue obtained from biopsies.These antibody–fluorophore conjugates target
abnormal depositions of proteins in the patient’s tissue.When exposed to light,
the fluorophore emits its own frequency of light, seen with a microscope.The particular staining
pattern and type of abnormal protein deposition seen in the tissue sample help diagnose the
disease.

7. • What happens to the specimen in the laboratory?
1. DIF may be carried out in an automated machine or manually.The process involves first making frozen sections then
carrying out immunofluorescence.
2. Preparing frozen sections
3. A punch biopsy is transported to the lab on saline-soaked gauze.
4. The specimen is placed in a gel.
5. Liquid nitrogen is used to freeze the specimen and gel.
6. The frozen specimen is contained within the frozen gel.
7. The specimen can be stored in liquid nitrogen for about one week.
8. 4–6 micron thick slices are cut

8. • Carrying out direct immunofluorescence
1. Five or six slides are made; each for a different reagent.One slide will be used for a normal H&E stain.
2. A special pen is used to draw a perimeter to keep reagents within the slides.
3. The slides are washed.
4. The reagents are made up.
5. The reagents (antibody–fluorophore conjugates to IgG, IgM, IgA, complement protein C3, and when
required fibrinogen) are dropped onto the slides and the slides are left for some time in the dark.
6. The slides are washed again in solution.
7. Glass covers are placed over the slides.

9. • Interpretation of direct immunofluorescence.
• The prepared immunofluorescence slides are examined by a pathologist to determine
the primary sites of immune deposition (if any), the classes of immunoglobulin or other immune
deposits, and the patterns of deposition. Staining patterns can be classed into five groups:
1. Intercellular surface staining (ICS) pattern
2. Linear basement membrane zone (BMZ) pattern
3. Granular BMZ pattern
4. Shaggy BMZ pattern
5. Vascular and other patterns.

10. INDIRECT IMMUNOFLUORENCETECHNIQUES.
• A laboratory test used to detect antibodies in serum or other body fluid.The
specific antibodies are labeled with a compound that makes them glow an
apple-green color when observed microscopically under ultraviolet light.

12. • Principle of the test
• For the determination of autoantibodies or antibodies against infectious agents,
cells, tissue sections or purified, biochemically characterized substances 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.
• Positive samples can be titrated in steps.The most suitable titration interval is
provided by the dilution factor 3.162 (square root of 10). In this way, every
second step represents in its denominator an integral power of 10 (1:10, 1:32,
1:100, 1 : 320, 1 : 1000, 1 : 3200, 1 : 10000 etc.).

13. • INDIRECT IMMUNOFLUORESCENCETEST (IIFT)
• INTRODUCTION
• Immunofluorescence is a method of using the specific reactivity of antibodies
with antigen to reveal the presence of these antibodies in sera and other body
fluids or to identify antigens in tissues in the presence of fluorescent dyes
(=fluorochromes). Immunofluorescence technique combines the sensitivity and
specificity of immunology with the precision of microscopy.
• Direct immunofluoresecence: Specific antibodies are conjugated with
fluorescent compounds.The conjugated antiserum is added to tissues and thus
fixed to the antigens. Unbound antibodies and non-antibody proteins are
removed by washing and the preparation is observed in a fluorescence
microscope.

14. • Indirect immunofluorescence: Indirect fluorescence is a double antibody
technique. the unlabelled antibodies which have bound to the antigens are
visualized by a fluorescent antiglobulin reagent directed at the unlabelled
antibodies.
1. Criteria for judging a fluorochrome as a suitable dye are:
2. The fluorochrome should posses chemical groups which will form covalent
bonds with protein molecules.
3. Easy removal of unreacted fluorescent material is also important
4. Fluorescent color of the conjugate should be different from that of the
background.
5. Conjugate should be stable under storage conditions.

15. • The fluorescence emission of FITC (Fluorescein isothiocyanate) conjugates
is green with a maximum wavelength at 529 nm.
• Fading of fluorescence:The fluorescence of microscopical preparations is
subject to fading during illumination and there may be a color change.There
should be minimum exposure to illumination during the microscopic
examination.

16. • MATERIALS
• Slides with antigen
• Positive control serum
• A positive patient's serum with a high IFT titer (at least 1:1,280) is preserved with me
thiolate (final concentration 1:10,000), divided into aliquots of 0.2 ml and kept frozen at
-20 C.
• Negative control serum
• The negative control serum should not give any fluorescence. Handle and store in the
same way as the positive control.
• PBS pH 7.4 (Check pH before use and adjust if necessary with 1N NaOH or 1N HCl)
• Conjugated anti-human globulin

17. • Use polyvalent anti-human conjugate e.g. anti-human immuno globulin (sheep) fluorescein-labeled. The working dilution of every batch
should be determined by making a series of dilutions of the dissolved conjugate.The optimal working dilution is considered to be that
dilution which is one double dilution lower than the highest dilution giving maximal fluorescence. A dilution of 1:100 is usually suitable.
1. Incubation chamber
2. A large Petri dish can be used as incubation chamber. The filter paper is placed on the bottom to keep the atmosphere humid. The slides
can be placed on glass rods.
3. Staining jar
4. Glycerin-PBS
5. Mix 9 parts glycerin (water-free, distilled) and 1 part PBS pH 7.4
6. Coverslips
7. Fluorescence microscope

18. • PREPARATION OF ANTIGEN SLIDES
• Clean the microscope slides thoroughly in hot water with a detergent or leave overnight in a
dichromate-sulfuric acid mixture.
• Rinse the slides 4 to 5 times with hot tapwater and twice with distilled water, then rub dry with a
towel.
• Put 10 drops of glycerol on the slides in two rows of 5 drops each.
• Spray the slides withTeflon in a fume cupboard to avoid inhalation.
• After drying at room temperature, rinse the slides thoroughly with lukewarm tap water and
distilled water, dry on filter paper.The slides have now been coated withTeflon, apart from the
spots where the glycerin drops were put

19. • Drop about 10 ul of a leptospira culture on theTeflon free spots. Leptospira copenhageni strain Wijnberg is
used, grown 2-3 days in EMJH medium.
• The density of the culture is considered optimal when the leptospira are lying separately in the dried drops.
If the culture is too dense, dilute the antigen with culture medium.
• Let the slides with antigen dry at room temperature on the bench. Fix the antigen on the slides by plunging
them for 10 minutes in cold acetone and dry again at room temperature.
• Now the slides are ready for use.The antigen is stable for at least 1 year at 4 C.To store the antigen, wrap
the slides in soft tissue (or soft toilet paper) and seal air tight in plastic bags.
• Before use allow the slides to come to room temperature before opening the plastic bag, to avoid
condensation of water vapor from the air on the antigen spots, which deteriorates the antigen.

20. • PERFORMANCE OFTHETEST
• Make doubling dilutions of the patients' serum in PBS, pH 7.4, starting with 1:10
up to 1:5,120.The positive serum is also diluted from 1:10 to 1:5,120.The
negative control serum is diluted from 1:10 to 1:160.
• Put 10 ul of the dilutions of patient's and control sera on the antigen spots,
starting with the highest dilution. As the negative control serum uses only half a
slide (5 antigen spots), use the other half of the slide for antigen control by
dropping 10 ul of PBS on the antigen spots.
• Incubate the slides for 30 minutes at room temperature in a humid closed Petri
dish.
• Put the slides in a staining jar with PBS and discard the PBS carefully. Add fresh
PBS. Keep for 10 minutes in PBS.
• Discard the PBS. Dry the slides around the antigen spots with soft tissue paper
and place the slides in the humid Petri dish.
• Drop 10 ul of the diluted conjugate on every antigen spot.

21. • Incubate in the dark for 30 minutes at room temperature.
• Repeat step 4 but put the staining jar in the dark, during the washing procedure.
• Repeat step 5 but place the slides in a slide-holder.
• Put a few drops of glycerin-PBS on the slide.
• Cover the slide with a coverslip, remove air bubbles by pressing softly. Wipe off
the superfluous glycerin-PBS.
• When washing the slides do not forget first to fill the jar with PBS. Avoid the
lower serum dilutions flowing over the stops with the higher dilutions.This is to
prevent false positive reactions in the higher dilutions.
• It is important to squeeze out the superfluous glycerin-PBS. A too thick
preparation gives difficulties in focussing.

22. • READING OFTHETEST
• Examine the slides under the fluorescence microscope. For fluorescence
microscopy filter combinations are dependent on the light source and the
fluorescent labels.
• The intensity of the fluorescence is read as follows:
• ++++ brilliant fluorescence
• +++ bright fluorescence
• ++ clearly visible fluorescence
• +/- weak fluorescence
• The titre of the serum is the highest dilution giving 1+ fluorescence. It is
characteristic for a positive serum that the intensity of the fluorescence
diminishes gradually in the higher dilutions