INDIRECT IMMUNOFLUORESCENCE TEST (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.

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.

Criteria for judging a fluorochrome as a suitable dye are:

- The fluorochrome should posses chemical groups which will form covalent bonds with protein molecules.
- Easy removal of unreacted fluorescent material is also important
- Fluorescent color of the conjugate should be different from that of the background.
- Conjugate should be stable under storage conditions.

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.

MATERIALS

- Slides with antigen
- Positive control serum
- A positive patient's serum with a high IFT titre (at least 1:1,280) is preserved with merthiolate (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

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.

- Incubation chamber
- 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.
- Staining jar
- Glycerin-PBS
- Mix 9 parts glycerin (water-free, distilled) and 1 part PBS pH 7.4
- Coverslips
- Fluorescence microscope

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 with Teflon 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 with Teflon, apart from the spots where the glycerin drops were put.

Drop about 10 ul of a leptospira culture on the Teflon 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.

PERFORMANCE OF THE TEST

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.

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.

READING OF THE TEST

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.

REFERENCES

Nairn R.C. Fluorescent protein tracing. E.&.S. Livingstone Ltd., Edinburgh and London, 1969