Serology: Fluorescent antibody tests and other tests employing conjugated antibodies Authors: Adapted by Prof M van Vuuren. Originally compiled by Dr RW Worthington. (Retired) Licensed under a Creative Commons Attribution license. IMMUNOFLUORESCENCE ASSAYS Direct and indirect immunofluorescence procedures provide means whereby the site of an antigenantibody reaction can be observed by microscopic examination. Specific antibody molecules are chemically linked to a fluorescent dye, the most common of which is fluorescein isothiocyanate (FITC). These labelled antibodies retain their specificity for their respective antigens, and after a short period, the reaction site can be visually detected with the aid of a fluorescence microscope. Fluorescent antibody and immunohistochemical tests for the demonstration of antigen Direct fluorescent antibody tests The most elementary form of the FA test is the direct test where specific antibodies (polyclonal or monoclonal) are marked with (conjugated to) a fluorescent dye and then placed directly onto the target or capture antigens. After an appropriate incubation period of 30-45 minutes, the excess unbound antibodies are washed away with phosphate buffered saline (PBS). The PBS is then washed off with distilled water and the preparation covered with a suitable mounting fluid such as buffered glycerol and examined with a fluorescence microscope. The direct FA test can only be used for the detection of specific antigens and not for the detection of antibodies. Fluorescent antibody tests (FAT) are frequently used for the demonstration of antigens in tissue and tissue culture preparations. An example is the demonstration of classical swine fever virus in tissues of infected pigs. In a typical case tonsil from suspected infected pigs would be used as follows: Sections are cut from frozen tissue and acetone fixed to microscope slides using standard histological techniques. The sections are washed in PBS, flooded with a classical swine fever antibody conjugated to fluorescein and incubated in a humid chamber for 30-minutes at 37 0 C. The conjugate is used at a dilution previously determined by titration. 1 P a g e
Known positive and negative control sections are similarly tested The slides are washed in PBS to remove all unbound conjugate, blotted dry and covered with mounting buffer and a coverslip and examined under an UV microscope. The presence of brilliant green fluorescing cells indicates the presence of virus in the tissues. In the case of classical swine fever, cross-reactions can occur with other pestiviruses when polyclonal sera are used as the conjugate. For this reason highly specific monoclonal antibodies that are specific for swine fever and for other pestiviruses can be used on additional sections to differentiate the viruses. In principle the specificity and sensitivity of tests depends on the antibody used. Where high specificity is a prime requirement of the test, high quality MAbs are generally preferred if they are available. Polyclonal antibodies may give results of similar or greater sensitivity but are less specific than MAbs. Similar tests can be done using antibody conjugated to horseradish peroxidase instead of fluorescein. In this case after washing to remove unattached conjugate the slide must be incubated with substrate and then examined by conventional light microscopy to identify stained antigen. Substrates that deposit as a coloured product in the tissues when oxidised by peroxidase must be used. Some of the substrates suitable for use with horseradish peroxidase are: 3-amino-9 ethyl carbazole; N,N- dimethyl-formamide Diaminobenzidine tetrahydrochloride 4-chloro-naphthol FAT and immunohistochemical methods using enzyme conjugated antibodies are used to identify virus in tissues and in tissue culture including tissue culture used in virus neutralisation tests. Use of these methods in tissue culture is useful for identifying virus, when it grows without an easily identifiable cytopathic effect on the tissue cells. The methods are also used to identify bacterial infections such as venereal campylobacteriosis and clostridial infections. A major advantage of these methods over tissue culture methods is that the results are obtained more rapidly. Indirect fluorescent antibody tests Indirect fluorescent antibody tests (IFA) are used for detecting antibody in serum. An antigen preparation such as a smear that contains identifiable antigen particles must be available for the test. IFA is widely used for protozoal diseases where bloodsmears or parasite smears can be used as antigen. The test is also commonly used in a wide variety of viral diseases where tissue culture preparations are used as capture antigens. The indirect test is more versatile in that it can be employed for the detection of both antibodies and antigens. It can for certain applications be more sensitive than the direct test in that its 2 P a g e
fluorescent signal is amplified as a result of stacking of antibodies on top of each other as a result of more binding sites being available, thus leading to more FITC molecules being detected with an UV microscope. To be able to use the indirect FA test for the detection of antigens, one needs good quality known antiserum against the specific antigen in question and an anti-species IgG-FITC conjugate. In other words, if we want to detect the presence of antigens in smears or tissues, we have to react our specific antibodies with the target or capture antigens in the same way as described for the direct test. However, after this step, the antigen-antibody reaction will not be visible microscopically as a result of the absence of fluorescing molecules. To achieve that objective, one must add an anti-species antibody/fitc conjugate prepared in a different species as the one under investigation. This secondary antibody will attach to the primary antibody and give rise to the fluorescent signal. To enhance the contrast between negative and positive organisms or infected and non-infected cells, a counter-stain such as Evan s blue is added in conjunction with the FITC conjugate. The indirect FA test can similarly be employed to detect antibodies. In this approach however, the unknown element is the serum specimen and the known entity is the target cell or organism. The target or capture antigens are prepared in the laboratory in the form of tissue culture-infected cells (in the case of intracellular microorganisms), or smears of larger microorganisms such as protozoa. These targets are fixed onto microscope slides, and combined/reacted with the known primary antibodies. The antigen-antibody complexes are then again made visible by adding an anti-species IgG/FITC conjugate diluted in Evan s blue counter-stain. The example given below describes an IFA test for detection of anti-viral antibodies. Protocol for the indirect fluorescent antibody test 1. Remove slides to be used from the -20 C freezer (chest freezer) or -80 C freezer to thaw 2. Make two-fold serial dilutions of the animal serum sample starting at 1:10 and ending at 1:5 120 3. Place 10µl of each serum dilution on wells on the slide. To be able to use only one pipette tip, start with the highest dilution (1:5 120) and work backwards to fill the spots on the slide. The first well on top left is reserved for the positive control serum, and the first well bottom left for the negative control serum 4. Incubate at 37 C for 30 minutes in a moist chamber 5. Wash slide for 5 minutes in PBS (preferably sterile but not compulsory) with a magnetic stirrer. Rinse slide in distilled water and blow dry 6. Add 10µl of an anti-species IgG or IgM conjugate (e.g. anti-cat IgG conjugated with fluorescein isothiocyanate (FITC) diluted in 0.05% Evans Blue solution The freeze-dried commercial conjugate is reconstituted in distilled water as recommended by the manufacturer (usually 1 ml) and aliquoted into 0.25µl volumes in Nunc tubes and stored at -20 C 3 P a g e
Evans Blue is prepared from 0.5% stock that is stored in a dark bottle at room temperature. Dilute 1:10 in PBS to obtain a 0.05% solution To use in the test, take a vial with 25µl conjugate and add 2 ml of the 0.05% Evans Blue to obtain a 1:80 dilution. If some remains after the test, it can be left in the fridge at 4 C for up to 10 days maximum 7. Incubate at 37 C for 30 minutes in a moist chamber 8. Wash slide for 5 minutes with a magnetic stirrer using PBS (preferably sterile but not compulsory). Rinse slide in distilled water and blow dry 9. Place 2-3 drops of glycerol-containing mounting fluid on slide and cover with a coverslip to allow mounting fluid to fill all wells 10. Examine slide with an UV microscope Anti-IgM/FITC conjugates Detection of pathogen-specific IgM is used to diagnose recent infections with a single serum sample. Different antibody classes behave differently during the course of an infectious disease. Antibodies of the primary response belong to the IgM class, and fall below detectable levels after 2-3 months. Already in the first two weeks following infection, they are starting to be replaced with antibodies of the IgG class, the latter which may persist for years, but often drop to undetectable levels after 14-18 months. Detection of pathogen-specific IgM antibodies therefore usually indicates a recent infection. Monoclonal antibodies for us in FA tests Monoclonal antibodies result from the fusion of normal antibody-producing plasma cells and nonantibody-producing myeloma cells. The hybrid cells are permanently adapted to grow in cell cultures and are capable of producing antibodies of desired specificity. In contrast, polyclonal antibodies have traditionally been limited by difficulty in generating reagents of high quality and specificity. Purified antigens used to induce polyclonal antibody formation often are contaminated with small amounts of macromolecules that induce large amounts of unwanted antibodies. Even high titre specific antiserum is composed of antibodies heterogenous in their affinity, biological activity and cross-reactivity. Procedure for fluorescent antibody staining of virus-infected cell cultures in plastic plates Plastic multi-well plates are useful when testing a large number of samples. The effect of acetone on plastic plates however, must be considered when using acetone to fix cells in the plates. Acetone alone or as an 80% solution in PBS will etch the plate, making it impossible to read the results. To overcome this problem, the PBS must be added to the wells before adding the acetone. PBS is added in 0.2 ml volumes to each well of a 24-well plated to be stained. Acetone is then gently added to the PBS using a Pasteur pipette to fill the wells about three-quarters full. Trouble shooting Non-specific staining 4 P a g e
The IFA is a subjective test method which does not always allow an easy interpretation. Ideally one should include three positive sera (high, medium and low) and a negative serum to serve as controls. Serum dilutions less than 1:20 (or 1:40 for certain preparations) may lead to nonspecific staining. Look critically at positive controls to determine: The ratio of negative to positive cells/organisms The nature of the fluorescence Trapping of conjugate among the cells or organisms The presence of anti-nuclear or anti-cytoplasmic fluorescence Control wells may give an incorrect result Check capture/target antigens by reacting it with antigen-specific conjugates with the aid of a direct FA test Check anti-species conjugates with known antigens and known primary antibody. Once reconstituted, it can only be kept for up to 10 days at 4 C. Check positive control sera with known antigens and known secondary antibody. Serum can deteriorate following several freeze and thaw cycles. Use at a dilution of four-fold less than the antibody titre. Check ph of wash buffer Check for presence of mounting fluid under coverslip Check that the fluorescence objective of the microscope is working Check the life-expectancy (in working hours) of the fluorescent light source (bulb) 5 P a g e