بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

3. IMMUNOLOGY SEROLOGY Over View Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

4. Clinical Immunology & Serology Practice (Code: MLIS 201) Teaching Objectives: 1- To know the elements of serological reactions 2- To define serological reactions 3- To describe the basics of primary and secondary serological reactions

7. A N T I G E N S Definition: Any molecular structure that when introduced is capable of Antibody production. Parts of Antigen Carrier portion - responsible for the molecular weight of antigen Epitope or Determinant - determines specificity of antigen

8. Two Properties of Antigens 1. IMMUNOGENICITY - inherent ability of a substance to induce the specific immune response resulting in the formation of immune lymphocytes or antibodies

9. Factors influencing Immunogenicity (Summary) 1-Foreigness : Foreign substances are immunogenic 2- Molecular size: High molecular weight increase immunogenicity 3- Chemical structure complexity: High complexity increase immunogenicity 4- Route of administration: Parenteral routes are more immunogenic to oral route SC>IP>IV>Intragastric

10. Factors influencing Immunogenicity (Summary) (cont.) 5- Degradability of the immunogen 6-Genotype of the recipient 7- immunogendose: Appropriate dose optimum antigenicity Low dose low- zone tolerance High dose high-zone tolerance 8- Adjuvant: Substance when injected with an immunogen enhance immunogenicity

11. 2. ANTIGENICITY/SPECIFICITY - the ability to react specifically with the antibody or cell that caused it to be produced

12. A N T I B O D I E S Definition: Spec. glycoproteins, produced by plasma cells in response to antigenic stimulation of B lymphocytes. Immunoglobulins.

13. Structure of Immunoglobulin 1.Four (4) polypeptide chains: 2 identical LIGHT chains and 2 identical HEAVY chains. 2.Both light and heavy chains are held together by DISULFIDE BONDS. 3.Heavy chains are interconnected by DISULFIDE bonds in the HINGE region. • 4.Ig has 2 terminal regions: • Carboxyterminal - with constant • amino acid sequence • (constant region). • Aminoterminal - with varying • antibody specificity (variable • region)

14. Classes of Immunoglobulins

17. Complement System

18. COMPLEMENT SYSTEM - A set of serum proteins that play a role in cytolytic destruction of cellular antigen by specific antibody. - reaction is nonspecific to the target cell - destroyed at 56ºC for 30 minutes

20. - the in vitro study of antigen-antibody reaction - laboratory study of the activities of the components of blood serum that contributes to immunity

21. Immunologic Reactions: • Primary- combination of Ag-Ab; non-visible reaction • Secondary - demonstrable Ag-Abrxn (e.g. precipitation, agglutination)

22. PRIMARY IMMUNOLOGIC TESTS. IMMUNOASSAYS Ligand - any substance that will complex to another substance; (the substance to be measured.

23. FLUORESCENCE IMMUNOASSAY • Fluorescent Probes used: • a. FITC (Fluorescein isothiocyanate) -emits green light***mostly used • b. Phycocyanin -emits red light • c. Texas red -emits red light • d.Tetramethyl rhodamine -emits red orange light Techniques: 1. Direct/Single Layer Immunofluorescent Assay 2. Indirect/Double Layer Immunofluorescent Assay

24. positive FA test for rabies (Image: Centers for Disease Control) negative FA test for rabies (Image: Centers for Disease Control)

25. C.ENZYME IMMUNOASSAY • colorimetric reaction • Enzymes Used: • a. ALP • b. Horseradish Peroxidase • c. Glucose oxidase • d. B-galactosidase • Techniques: • 1. Direct • 2. Indirect • 3. Sandwich/Double Ab • 4. Competitive Binding • 5. Enzyme Inhibition

27. ELISA: DOUBLE ANTIBODY TECHNIQUE

29. Secondary tests • PRECIPITATION • AGGLUTINATION • COMPLEMENT FIXATION • NEUTRALIZATION

30. I.PRECIPITATION RXNS - Antigens involved are soluble antigens. Types of Precipitation reaction: 1. Single diffusion, Single Dimension Px serum w/ soluble Ag (+) rxn - formation of pptn line Gel/Agar impregnated w/ known Ab

31. 2. (RADIAL IMMUNODIFFUSION) - uses a plate containing agar with known antibody - Px serum is placed on the wells - Diameter of the pptn line is directly proportional to the concentration of the target antigen

34. 5. Immunoelectrophoresis (IEP) - useful procedure for the ID of monoclonal proteins (Bence Jones Protein) - utilizes both double diffusion and electrophoresis a. Ags migrate under an electric current (-) Ag (+) b. Rgt. Ab Added. Diffusion through gel (-) (+) Antibody c. (+) rxn - Pptn arcs formed (-) (+)

35. STEP 1. STEP 2.

36. II. AGGLUTINATION REACTIONS - Ags involved are particulate antigens. Types of Agglutination Rxn 1.Direct Agglutination (e.g. Blood Typing)

37. 2. Antiglobulin Technique (CoombsTest)/Indirect Agglutination • anti-human IgG is added to bridge the gap between the cells • to demonstrate incomplete antibodies

38. 3.Passive Agglutination - A soluble Ag is artificially attached to a particulate carrier (e.g. cells, latex, bentonite, celloidin, or charcoal

39. 6. Hemagglutination - Agglutination of rbc due to antibody, viruses, bacteria, or other biologic substance. - It is not the antigen of RBC but the artificially attached Ag (after undergoing tx) that are made to react with the Ab - ex. TPHA

40. Study Questions: Compare between: Different Classes of Immunoglobulins

41. Assignment Write notes on A- Primary Ag-Abreactions شروق ابو الحسن – شروق كمال – غادة عز الدين – فاطمة على – مروة اشرف B- Secondary Ag-Abreactions صلاح ميلود – محمد زغلول – محمد فوزى – محمود محمد رمضان

42. Thanks