1. Applied Immunology
Aftab Jasir, European Centre for Disease Prevention and Control (ECDC)
European Public Health Microbiology training program (EUPHEM)

2. Define basic components of immune system
Objectives
Define basic components of immune system
Define important terms in immunology
Explain major applications of immunology

3. What is immunology?
Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all living organisms.
It deals with the physiological functioning of the immune system in states of both health and disease
The specificity of the bond between antibody and antigen has made it an excellent tool in the detection of substances in a variety of diagnostic techniques. Antibodies specific for a desired antigen can be conjugated with a radiolabel, fluorescent label, or color- forming enzyme and are used as a "probe" to detect it. However, the similarity between some antigens can lead to false positives and other errors in such tests by antibodies cross-reacting with antigens that aren't exact matches

4. The immune system is the ministry of defence of the human/animal body
What is the immune system?
The immune system is the ministry of defence of the human/animal body

5. Major defence components of the human immune system
Cells
Immunoglobulins

6. Definitions/terminology
Antigens (Ag)
Large molecules, is anything that obtain the formation of a specific immune response (Anomy)
Ag determinants (epitopes) are the particular chemical groups on a molecule that are antigenic
Antibody(Ab)/immunoglobulin (Ig).
A special group of soluble proteins that are produced in response to foreign antigens (substances)
Anomy and forgion

7. Antigen and antibody

8. Haptens

9. a. IgG (secondary exposure, small, passing placenta)
5 classes of IGs
a. IgG (secondary exposure, small, passing placenta)
b. IgM (first exposure, large, not passing placenta, huge amont)
c. IgA (mucosal immunity, respiratory tract)
d. IgE (Allergy and parasites)
e. IgD (proteins in the plasma membranes of mature B-lymphocytes, same time as IgM)
IgG is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids, comprising 75% of serum immunoglobulins in humans, IgG molecules are synthesized and secreted by plasma B cells.
Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. More IgA is produced in mucosal linings than all other types of antibody combined
In biology, Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin "isotype") that has only been found in mammals. IgE is a monomeric antibody with 4 Ig-like domains (CH1->CH4). [1] It plays an important role in allergy, and is especially associated with type 1 hypersensitivity.[2] IgE has also been implicated in immune system responses to most parasitic worms[3
Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in the plasma membranes of mature B-lymphocytes where it is usually coexpressed with another cell surface antibody called IgM. IgD is also produced in a secreted form that is found in very small amounts in blood serum.

10. Ministry of defence of the human body

12. Factors influencing immunogenicity
Contribution of immunogen
Contribution of biological system
Method of administration

13. Immunogenicity: contribution of biological system
Genetics
Species
Individual
Responders vs Non-responders
Age

14. Major practical applications of immunology
Use of antiserum and vaccination to provide protection against disease.
Diagnostic tool to detect disease.
Epidemiological investigation of vaccine preventable diseases 14

15. My face is my fortune
Where are you going, my pretty maid? I’m going a-milking, sir, she said
May I go with you, my pretty maid? You’re kindly welcome, sir, she said
What is your father, my pretty maid? My father is a farmer, sir, she said
What is your fortune, my pretty maid? My face is my fortune, sir, she said
Illustration of the famous first vaccination experiment. In 1796 Edward Jenner infected eight-year-old James Phipps with fluid from a lesion on the hand of Sarah Nelmes, who had caught cowpox. The word ‘vaccination’ comes from the Latin word ‘vaccinia’ for cowpox. This was a successful experiment, because the boy was subsequently protected against a human smallpox infection. Years later Louis Pasteur proposed that all inoculations intended to protect against infectious diseases should be called vaccinations, in honour of Jenner.

17. Variolation
The word ‘variolation’ comes from the Latin word ‘variola’ for human smallpox.
Figure 1. Ancient Oriental print illustrating the technique of variolation. Dried material from scabs of human smallpox is blown into the nose of an unaffected person with a small blowpipe. The word ‘variolation’ comes from the Latin word ‘variola’ for human smallpox.
source: Claire JP Boog

18. Discovery of small pox vaccine
In 1798, Jenner introduced 1st vaccination (vacca: cow) following his experimentation with isolates of cow pox virus from ‘Blossom’.
Cartoon on smallpox vaccination from 1802 entitled “The Cow Pock or the Wonderful Effects of the New Inoculation”. It shows Edward Jenner and persons who are developing cow-like projections as a result of allowing themselves to be inoculated with an ‘animal disease
Edward Jenner 1780AD
Blossom 18 18

20. Edward Jenner Among patients awaiting small pox vaccination
The mass vaccination against became a standard practice. 20 20

21. Types of acquired immunity

22. Passive – receive Abs made by another 1. natural
2. artificial - γ globulin, hyperimmune serum
Natural
Artificial

23. Mode of delivery

24. Advantages and Disadvantages of Active Immunization
Not immediate
Immune suppressed/deficiency
Long term immunity
Herd immunity
Risk of infection
Risk of contamination
Animal ???
Attenuated can revert to their pathogenic form 24

25. Advantages and Disadvantages of Passive Immunization
no long term protection
serum sickness
immediate protection
risk of hepatitis and AIDS
graft vs. host disease 25

26. Serological tests based on Abs specifically binding to Ag
Serology
A science that attempts to detect signs of infection in a patient’s serum such as Ab for a specific microbe
Serological tests based on Abs specifically binding to Ag
Ag of known identity will react with Ab in an unknown serum sample.
Known Ab can be used to detect Ag in serum
Ag-Ab reactions are visible by clumps, precipitates, color changes or release of radioactivity.
The most effective tests have high specificity and sensitivity. 26

27. a) The presence of a specific Ab b) Identification of microbes27

28. Specificity, sensitivity, and cross reactivity
a) Specificity
Ab attaches with great exact- ness to only one type of Ag.
b) Sensitivity
Ab can locate Ag, even when it is greatly diluted.
c) Cross reactivity
the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. 28

29. Examples of serological tests
Agglutination tests
Precipitation tests
Immunoelectrophoresis
Western blot tests
Complement fixation tests
Immunofluorescence testing
Immunoassays 29

30. ELISA
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA),
is a biochemical technique
used mainly in immunology to detect the presence of an antibody or an antigen in a sample.
has been used as a diagnostic tool in medicine as well as a quality control check in various industries.
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate.

31. ELISA an unknown amount of antigen is affixed to a surface
a specific antibody is applied over the surface that binds to the antigen
antibody is linked to an enzyme
a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate
Primary and secondary antibodies are two groups of antibodies based on whether they target a target of interest directly or targets another (primary) antibody that, in turn, is bound to a target of interest.
Primary antibodies are antibodies raised against an antigenic target of interest (a protein, peptide, carbohydrate, or other small molecule) and are typically unconjugated (unlabelled). Primary antibodies that recognize and bind with high affinity and specificity to unique epitopes across a broad spectrum of biomolecules are available as high specificity monoclonal antibodies and/or as polyclonal antibodies. These antibodies are useful not only to detect specific biomolecules but also to measure changes in their level and specificity of modification by processes such as phosphorylation, methylation, or glycosylation. A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of ADME and multi-drug resistance (MDR) of therapeutic agents.
A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.
Secondary antibodies may be polyclonal or monoclonal, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions.
Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. Identifying the optimal secondary antibody is normally done through trial and error.
Secondary antibodies are used in many biochemical assays including:
ELISA, including many HIV tests

32. Agglutination tests
Ab cross-links whole cell Ag, forming complexes that settle out and from visible clumps in the test chamber
Purpose of agglutination testing:
Qualitative testing
blood typing, some bacterial & viral diseases.
Quantitative testing
Used to detect titer (maxium dilution that will still give visible agglutination)
Difference between agglutination and precipitation?
Agglutination => clumping together of insoluble molecules
Precipitation => aggregation of soluble molecules 32

33. tination is the clumping of particles
tination is the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue to."
This occurs in biology in three main examples:
The clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex.
The coalescing of small particles that are suspended in a solution; these larger masses are then (usually) precipitated.
An allergic reaction type occurrence where cells become more compacted together to prevent foreign materials entering them. This is usually the result of an antigen in the vicinity of the cells. 33

34. Importance for epidemiologist Ex1
2005, outbreak of Salmonella like illness in Skåne ) Sothern Sweden=, in the same time report from Denmark of Salmonella enterica. Diagnostic of pathogen in Sweden was not successful Many of patients had common sort of relation (eating in the same restaurant, buying meat from same market or meat imported from Denmark) Media reported a new sort of (unknown) infection Speculation of new type of Salmonella among doctors 6 days later Salmonella enterica was detected in the main lab in Skåne What was wrong?

35. Ex2
1999, Outbreak of scarlatina like (Scarlet fever) in 2 daycares in Lund, Sweden 28 Children were diagnosed by symptoms Two teacher, one working in both daycares, one developed STSS No Lab confirmation of Group A streptococci Microscopy showed gram positive chained bacteria One weak later two children were confirmed by lab results having Group A streptococci What was wrong know??

36. Diagnostic of viral infections
early phase
amount of virus
antigen/genome
days
amount of antibodies
late phase of infection
months
years
detection limit
virus detection
PCR, capture-ELISA
virus isolation, electron-
microscopy, hybridisation
serology tests
immunofluorescence, NT
ELISA, immunoblot, HIA
Prof. Matthias Niedrig, RKI

37. What should you have in mind!!!
Some times Ag x Ab based tests can results in wrong alarm of outbreak ( Salmonella)
Antigen variation is always a problem (Chlamydia, grouping of streptococci)
Cross-reactivity can give wrong information of an outbreak
Any unusual or unexpected results should be confirmed by genetic test
If possible use other methods than serology in an outbreak situation or combine with other methods