Presentation on theme: "Applied Immunology Aftab Jasir, European Centre for Disease Prevention and Control (ECDC) European Public Health Microbiology training program (EUPHEM)"— Presentation transcript:
1 Applied ImmunologyAftab Jasir, European Centre for Disease Prevention and Control (ECDC)European Public Health Microbiology training program (EUPHEM)
2 Define basic components of immune system ObjectivesDefine basic components of immune systemDefine important terms in immunologyExplain 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 diseaseThe 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 CellsImmunoglobulins
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 antigenicAntibody(Ab)/immunoglobulin (Ig).A special group of soluble proteins that are produced in response to foreign antigens (substances)Anomy and forgion
9 a. IgG (secondary exposure, small, passing placenta) 5 classes of IGsa. 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 combinedIn 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).  It plays an important role in allergy, and is especially associated with type 1 hypersensitivity. IgE has also been implicated in immune system responses to most parasitic worms[3Immunoglobulin 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.
12 Factors influencing immunogenicity Contribution of immunogenContribution of biological systemMethod of administration
13 Immunogenicity: contribution of biological system GeneticsSpeciesIndividualResponders vs Non-respondersAge
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 diseases14
15 My face is my fortuneWhere are you going, my pretty maid? I’m going a-milking, sir, she saidMay I go with you, my pretty maid? You’re kindly welcome, sir, she saidWhat is your father, my pretty maid? My father is a farmer, sir, she saidWhat is your fortune, my pretty maid? My face is my fortune, sir, she saidIllustration 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 VariolationThe 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 diseaseEdward Jenner 1780ADBlossom1818
24 Advantages and Disadvantages of Active Immunization Not immediateImmune suppressed/deficiencyLong term immunityHerd immunityRisk of infectionRisk of contaminationAnimal ???Attenuated can revert to their pathogenic form24
25 Advantages and Disadvantages of Passive Immunization no long term protectionserum sicknessimmediate protectionrisk of hepatitis and AIDSgraft vs. host disease25
26 Serological tests based on Abs specifically binding to Ag SerologyA science that attempts to detect signs of infection in a patient’s serum such as Ab for a specific microbeSerological tests based on Abs specifically binding to AgAg of known identity will react with Ab in an unknown serum sample.Known Ab can be used to detect Ag in serumAg-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 microbes 27
28 Specificity, sensitivity, and cross reactivity a) SpecificityAb attaches with great exact- ness to only one type of Ag.b) SensitivityAb can locate Ag, even when it is greatly diluted.c) Cross reactivitythe 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 testsPrecipitation testsImmunoelectrophoresisWestern blot testsComplement fixation testsImmunofluorescence testingImmunoassays29
30 ELISAEnzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA),is a biochemical techniqueused 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 antigenantibody is linked to an enzymea substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substratePrimary 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 testsAb cross-links whole cell Ag, forming complexes that settle out and from visible clumps in the test chamberPurpose of agglutination testing:Qualitative testingblood typing, some bacterial & viral diseases.Quantitative testingUsed to detect titer (maxium dilution that will still give visible agglutination)Difference between agglutination and precipitation?Agglutination => clumping together of insoluble moleculesPrecipitation => aggregation of soluble molecules32
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 Ex21999, 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 phaseamount of virusantigen/genomedaysamount of antibodieslate phase of infectionmonthsyearsdetection limitvirus detectionPCR, capture-ELISAvirus isolation, electron-microscopy, hybridisationserology testsimmunofluorescence, NTELISA, immunoblot, HIAProf. 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 outbreakAny unusual or unexpected results should be confirmed by genetic testIf possible use other methods than serology in an outbreak situation or combine with other methods