1. Laboratory Techniques in Immunology
Practical Applications of Immunology

2. ANTIGEN-ANTIBODY INTERACTIONS
Many diagnoses in infectious disease and pathology would not be possible without laboratory procedures that identify antibodies or antigens in the patient
Interaction of antigen and antibody occurs in vivo, and in clinical settings it provides the basis for all serologically based tests.
The formation of immune complexes produces a visible reaction that is the basis of precipitation and agglutination tests.

3. Diagnostic Immunology
Many tests based have been developed to determine the presence of antibodies or antigens in a patient to diagnose infectious diseases and pathology
Clinical sensitivity - ability of a test to provide a positive result if the patient has the disease (no false negatives).
Clinical specificity – ability of a test to give a negative result if the patient does not have disease (no false positives).

4. Lattice formation not required
Labeled immunoassay
Lattice formation not required

5. FLUORESCENT ANTIBODY TESTS
The direct fluorescent antibody test (DFA) is used to detect and localize antigen in the patient. The tissue sample to be tested is treated with antibodies against that particular antigen that have been labeled with a fluorescent dye. If the antigen is present in the tissues, the fluorescent-labeled antibodies will bind, and their binding can be detected with a fluorescence microscope.
Variations of this test are used to diagnose respiratory syncytial virus, herpes simplex- 1 and -2, and Pneumocystis infections.

6. Excitation/Emission Spectra

7. Immunofluorescence Qualitative Direct
Ab to tissue Ag is labeled with a fluorochrome. Ag Y
Fluorochrome
Labeled Ab
Tissue Section
Direct Fluorescent-Antibody Tests
Direct fluorescent-antibody tests are used to identify specific microorganisms (antigens).
Antibodies directed against antigens on the surface of a specific microorganism are labeled with fluorescent dye.
Fluorescent antibodies are incubated with the sample and antigen-specific binding allowed to occur
Excess and non-specifically attached antibodies are washed from the sample
The sample is viewed with a fluorescence viewer, whether a fluorescence microscope or plate reader or even fluorescence-activated flow cytometer

8. Direct Immunofluorescence

9. The indirect fluorescent antibody test (IFA) is used to detect pathogen-specific antibodies in the patient. In this case, a laboratory-generated sample of infected tissue is mixed with serum from the patient. A fluorescent dye-labeled anti-immunoglobulin (raised in animals) is then added. If binding of antibodies from the patient to the tissue sample occurs, then the fluorescent antibodies can be bound, and fluorescence can be detected in the tissue by microscopy.
This technique is used to detect antinuclear antibodies, anti-dsDNA antibodies, antithyroid antibodies, antiglomerular basement-membrane antibodies, and anti-Epstein-Barr virus viral-capsid antigen antibodies.

10. Immunofluorescence Indirect Ab to tissue Ag is unlabeled.
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled Ab
Indirect Fluorescent-Antibody Tests
Indirect fluorescent-antibody tests are used to demonstrate the presence of antibodies against a specific antigen in serum.
Antigen or the microorganism itself is incubated with the patient's serum

11. Indirect Immunofluorescence

12. In suspected Graves' disease the first-line test is antibodies against TSH receptors (TRAb), which occur with a prevalence of 90 to 100 % (ELISA). The detection of antibodies against TPO can support the diagnosis (ELISA or IIFT).
In Hashimoto's thyroiditis serological antibodies against TPO are detectable with a prevalence of up to 90 %, while antibodies against TG are occur in 60 to 70 % of cases.

13. RADIOIMMUNOASSAY (RIA) AND ENZVME-LINKED IMMUNOABSORBENT ASSAY (EIA OR ELISA)
RIA and ELISA are extremely sensitive tests (as little as 10-9 g of material can be detected) that are common in medical laboratories.
They can be used to detect the presence of hormones, drugs, antibiotics, serum proteins, infectious disease antigens, and tumor markers.
Both tests are conducted similarly, but the RIA uses the detection of a radiolabeled product and the ELISA detects the presence of enzyme-mediated color changes in a chromogenic substrate.

15. ELISA
In the screening test for HIV infection, the ELISA is used, with the p24 capsid antigen from the virus coated on to microtiter plates. The serum from the patient is then added, followed by addition of an enzyme-labeled antihuman immunoglobulin.
Finally, the enzyme substrate is added, and the production of a color change in the well can be observed if all reagents bind one another in sequence.

17. Double Antibody ELISA(direct)
Ag detection
Immobilize Ab
Incubate with sample
Add labeled antibody
Amount of labeled Ab bound is proportional to the amount of Ag in the sample
Solid
Phase Y Ag
Immobilized
Ag in
Patient’s
sample
Labeled Ab
Quantitative

18. Indirect ELISA Ab detection Immobilize Ag Incubate with sample
Add labeled anti-Ig
Amount of labeled Ab bound is proportional to amount of Ab in the sample
Solid
Phase Y Ag
Immobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig
Quantitative

19. Applications
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen.
It has also found applications in the food industry in detecting potential food allergens such as milk,peanuts,walnuts,almonds, and eggs. 19

20. FLUORESCENCE-ACTIVATED CELL SORTER
This procedure is used to rapidly analyze cell types in a complex mixture and sort them into different populations based on their binding to specific fluorescent dyes.
By using antibodies against specific cell-surface markers conjugated to different fluorescent dyes, it is possible to analyze the relative numbers of cells present in a specific tissue location.

21. As cells pass through the apparatus in a single file, a computer-generated graph is produced, plotting the intensity and color of fluorescence of each cell along the axes.
Each dot on the graph reflects the passage of a cell with a certain level and color of fluorescence, so the darkly dotted areas of the graph reflect the presence of many cells of similar attributes. Cells with high fluorescence from both dyes will therefore be found in the top right quadrant.

23. Flow Cytometry Cells in suspension are labeled with fluorescent tag.
Direct or indirect fluorescence
Cells analyzed on a flow cytometer.

24. Principle of Flow Cytometry
Cell sample labeled with appropriate fluorescent Abs
Cells in suspension are passed through machine in single file in a stream of fluid
Stream is focused through one or more laser beams, measuring light scatter and fluorescence characteristics
Fluorescence detected by photomultiplier tubes (PMTs)
Signals sent to computer for analysis

25. Flow Cytometry Interpretation
One Parameter Histogram
Red Fluorescence Intensity
Green Fluorescence Intensity
Two Parameter Histogram
Unstained cells
FITC-labeled cells
Number of Cells
Green Fluorescence Intensity

26. Myeloma

27. Common Applications of Flow Cytometry in Immunology
Phenotype of cell, surface molecules
Intracellular cytokine staining
Antigen specificity
Cell proliferation (e.g. CFSE, BrdU incorporation)
Cell sorting
Apoptosis analysis
Cytotoxicity assays
Phagocytosis assays
Cell cycle analysis (DNA content analysis)
Cell signalling molecules, Calcium flux assays
Organelle-specific studies (e.g. lysosome)
Cellular transport assays
Transfection efficiencies