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SEROLOGY

SEROLOGY. IMMUNOLOGY AND THE IMMUNE SYSTEM. Immunology Study of the components and function of the immune system. Immune System Molecules, cells, tissues and organs which provide non-specific and specific protection against: Microorganisms. Microbial toxins. Tumor cells. Serology.

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SEROLOGY

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  1. SEROLOGY

  2. IMMUNOLOGY AND THE IMMUNE SYSTEM • Immunology • Study of the components and function of the immune system. • Immune System • Molecules, cells, tissues and organs which provide non-specific and specific protection against: • Microorganisms. • Microbial toxins. • Tumor cells.

  3. Serology • Is the branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibodyreactions in vitro. In practice, the term usually refers to the diagnostic identification of antibodies in the serum; but we can also detect antigens.

  4. Plasma and Serum • Serum: cell-free liquid, minus the clotting factors • Plasma: cell-free liquid with clotting factors in solution (must use an anticoagulant)

  5. Antigen & Antibody Antigen is the substance that the body is trying to “fight off” (eliminate or reduce) by mounting an immune response. Antibody is a protein that is produced by the body in response to an “invading” (foreign) substance. Antibodies possess high a) specificity and b) affinity for a specific antigen.

  6. Antigen • Is any substance that causes your immune system to prompts the generation of antibodies • Antigens can be proteins, polysaccharides, conjugates of lipids with : proteins (lipoproteins) and polysaccharides (glycolipids) • An antigen may be a foreign substance from the environment such as chemicals, bacteria, viruses, or pollen.  • An antigen may also be formed within the body, as with bacterial toxins or tissue cells.

  7. Antibody Each antibody consists of four polypeptides - two heavy chains and two light chains connected by disulfide bonds, joined to form a "Y" shaped molecule.

  8. Agglutination

  9. Agglutination Reactions • Principle Agglutiogen +Agglutinin Agglutination (Particulate Ag) + (Ab) Ag-Ab Complex -It is a serological reaction in which antibodies interact with particulate/cellular antigen. -Agglutinins are antibodies that aggultinate (adhere together) bacterial cells, RBCs, WBCs, or anything that is cellular.

  10. Agglutination When antibodies are mixed with their corresponding antigens on the surface of large, easily sedimented particles such as animal cells, erythrocytes, or bacteria, the antibodies cross-link the particles, forming visible clumps. This reaction is termed agglutination.

  11. (A) Direct Agglutination Tests • Used to determine antibody titer against large cellular antigens(Bacteria, Fungi or RBCs) • Particulate antigen reacts directly with antibodies. • Antigen found naturally on particle (cell). • Easy to perform • Applications • Blood group typing. • 2.The identification of viruses.

  12. ABO blood grouping system According to the ABO blood typing system there are four different kinds of blood types: A, B, AB or O (null).

  13. Blood group AIf you belong to the blood group A, you have A antigens on the surface of your RBCs and B antibodies in your blood plasma. Blood group BIf you belong to the blood group B, you have B antigens on the surface of your RBCs and A antibodies in your blood plasma. AB0 blood grouping system

  14. Blood group O If you belong to the blood group O (null), you have neither A or B antigens on the surface of your RBCs but you have both A and B antibodies in your blood plasma. Blood Groups, Blood Typing and Blood Transfusions The discovery of blood groups Experiments with blood transfusions, the transfer of blood or blood components into a person's blood stream, have been carried out for hundreds of years. Many patients have died and it was not until 1901, when the Austrian Karl Landsteiner discovered human blood groups, that blood transfusions became safer. Mixing blood from two individuals can lead to blood clumping or agglutination. The clumped red cells can crack and cause toxic reactions. This can have fatal consequences. Karl Landsteiner discovered that blood clumping was an immunological reaction which occurs when the receiver of a blood transfusion has antibodies against the donor blood cells.Karl Landsteiner's work made it possible to determine blood types and thus paved the way for blood transfusions to be carried out safely. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1930. What is blood made up of? An adult human has about 4–6 liters of blood circulating in the body. Among other things, blood transports oxygen to various parts of the body. Blood consists of several types of cells floating around in a fluid called plasma.The red blood cells contain hemoglobin, a protein that binds oxygen. Red blood cells transport oxygen to, and remove carbon dioxide from, the body tissues.The white blood cells fight infection.The platelets help the blood to clot, if you get a wound for example.The plasma contains salts and various kinds of proteins. What are the different blood groups? The differences in human blood are due to the presence or absence of certain protein molecules called antigens and antibodies. The antigens are located on the surface of the red blood cells and the antibodies are in the blood plasma. Individuals have different types and combinations of these molecules. The blood group you belong to depends on what you have inherited from your parents. There are more than 20 genetically determined blood group systems known today, but the AB0 and Rh systems are the most important ones used for blood transfusions. Not all blood groups are compatible with each other. Mixing incompatible blood groups leads to blood clumping or agglutination, which is dangerous or individuals. Nobel Laureate Karl Landsteiner was involved in the discovery of both the AB0 and Rh blood groups. AB0 blood grouping system Blood group AB If you belong to the blood group AB, you have both A and B antigens on the surface of your RBCs and no A or B antibodies at all in your blood plasma.

  15. ABO Blood Typing

  16. Illustration of the reaction patterns of the ABO groups using a blood group tile. http://www.bh.rmit.edu.au/mls/subjects/abo/resources/genetics1.htm

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  18. Immunofluorescence (IF)

  19. Immunofluorescence (IF) • Principle: • Immunofluorescence is the labeling of antibodies with fluorescent dyes. Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.

  20. Immunofluorescence (IF) • Principle: • Antigen+ flourescent antibody= visualization of the reaction by ultraviolet light or flourescent microscope.

  21. Immunofluorescence (IF) • There are two ways of doing IF staining: • Direct immunofluorescence. • Indirect immunofluorescence.

  22. 1.Direct immunofluorescence: It’s called direct flourescent antibody test because it directly tests the presence of antigen with a tagged antibody.

  23. Immunofluorescence (IF) 1.Direct immunofluorescence: • Ag is fixed on the slide. • Fluorescein labeled Ab’s are layered over it. • Slide is washed to remove unattached Ab’s. • Examined under UV light in an fluorescent microscope

  24. Direct immunofluorescence

  25. Uses: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples e.g. bacteria or viruses (e.g. diagnosis of rabies in animals). A drop of antirabies-flourescein labled antibody is added to sections of brain tissue from a suspected infected animals.

  26. Immunofluorescence (IF) 2. Indirect immunofluorescence: Indirect test is a double-layer technique. The unlabelled antibody is applied directly to the tissue substrate. Treated with a fluorochrome-conjugated anti-immunoglobulin serum.

  27. 2. Indirect immunofluorescence: Uses: Used to detect the presence of antibody e.g. in serodiagnosis of syphilis.

  28. A positive immunoflouresecnce of cardiomyocytes infected with adenovirus.

  29. A positive immunofluorescence test of Haemophilus influenza bacteria.

  30. Enzyme Linked Immunoassay (ELISA) • ELISA test A type of immunoassay aimed to detect the Ag or Ab present in an inflicted individual for proper diagnosis and further treatment.

  31. Enzyme Linked Immunoassay (ELISA)

  32. Enzyme Linked Immunoassay (ELISA) • Procedures: • Bring all components to room temperature before use. • Add +ve control, -ve control, and sample to their specific wells in the microtire plate. • Incubate at 37 °C for 40 minutes. • Wash each well using the washing buffer. • Add enzyme and mix gently. • Incubate at 37 °C for 40 minutes. • Wash each well using the washing buffer. • Add substrate and mix well. • Incubate at 37 °C for 20 minutes. • Add stopping solution to each well to stop the color reaction. • Visually compare the development of sample color against +ve and –ve controls, then measure the color intensity using spectrophotometer.

  33. Enzyme Linked Immunoassay (ELISA) • Functions of Components Microtitre Plate

  34. Enzyme Linked Immunoassay (ELISA) Micropipette

  35. Enzyme Linked Immunoassay (ELISA) Micropipette

  36. Enzyme Linked Immunoassay (ELISA) • Washing Buffer Removes unbound material.

  37. Enzyme Linked Immunoassay (ELISA) • Stop Solution Stops the enzymatic reaction.

  38. Enzyme Linked Immunoassay (ELISA) • Enzyme Acts as an amplifier, it conjugates with Ag-Ab complex.

  39. Enzyme Linked Immunoassay (ELISA) • Substrate

  40. Staining of Bacteria Simple Staining Type of staining:-Simple Stain Name of stain:-Methylene blue Size:medium Shape of cells:-bacilli Arrangement of cells:-chain Color:-Blue Name of M.O.:-Bacillus spp.

  41. Staining of Bacteria Simple Staining Name of stain:-Crystal violet. Type of staining:-Simple Stain Shape of cells:-cocci Arrangement of cells:clusters Color:-violet Size: small Name of M.O.:- Staphylococcus aureus

  42. Staining of BacteriaSimple Stain Type of stain: Simple stain Name of stain: Crystal violet Shape of cells: oval Size: large Colour: Violet Size: large Name of microorganism: Candida albicans 44

  43. Staining of Bacteria Simple Staining Type of stain: Simple Stain Name of stain: Saffranin Shape of cells: rods Size: Small Colour: Red Size: large Name of microorganism: E.coli

  44. Staining of Bacteria Simple Staining Results

  45. Staining of BacteriaGram Stain Type of stain: Gram stain Shape: Cocci Arrangement:clusters Size: small Color: Violet Gram’s reaction: Gram’s+ve Name of microorganism: Staphylococcus aureus

  46. Staining of BacteriaGram Stain Type of stain: Gram stain Shape: Oval Arrangement: Single Size: large Color: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Candida albicans

  47. Staining of BacteriaGram Stain Type of stain: Gram stain Shape: Bacilli Arrangement: Chains Size: medium Color: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Bacillus spp.

  48. Staining of Bacteria Gram Stain Type of stain: Gram stain Shape: Rods Arrangement: Single Color: red Gram’s reaction: Gram’s –ve Name of microorganism: E.coli

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