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Practical Applications of Immunology

Practical Applications of Immunology. Serological Tests. Agglutination: Particulate antigens Hemagglutination: Agglutination of RBCs Precipitation: Soluble antigens Fluorescent-antibody technique: Antibodies linked to fluorescent dye Complement fixation: RBCs are indicator

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Practical Applications of Immunology

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  1. Practical Applications of Immunology

  2. Serological Tests • Agglutination: Particulate antigens • Hemagglutination: Agglutination of RBCs • Precipitation: Soluble antigens • Fluorescent-antibody technique: Antibodies linked to fluorescent dye • Complement fixation: RBCs are indicator • Neutralization: Inactivates toxin or virus • ELISA: Peroxidase enzyme is the indicator

  3. Nature of Ag/Ab Reactions http://www.med.sc.edu:85/chime2/lyso-abfr.htm Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 • Lock and Key Concept • Non-covalent Bonds • Hydrogen bonds • Electrostatic bonds • Van der Waal forces • Hydrophobic bonds • Multiple Bonds • Reversible

  4. Y Y Y Y Y Y Y 104 106 1010 Keq = Avidity Affinity Avidity Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs

  5. Cross reactions Anti-A Ab Anti-A Ab Anti-A Ab Ag B Ag C Shared epitope Similar epitope Ag A Cross Reactivity • The ability of an individual Ab combining site to react with more than one antigenic determinant. • The ability of a population of Ab molecules to react with more than one Ag

  6. Ab excess Ag excess Equivalence – Lattice formation Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity • Ag:Ab ratio • Physical form of Ag

  7. Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

  8. Monoclonal antibody Definition: . Mono: one .Clone: a strain of cells descended form single cell. .Antibody: a molecule of animal origin that has immunological activity only against the antigen to which it was made.

  9. Monoclonal antibodies • MAbs produced from a single clone of B cells • Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity • Mostly produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture

  10. HISTORY

  11. ANTIBODIES Polyclonal antibodies Monoclonal Antibodies Produced by: Many B cell clones A single B cell clone Bind to: Multiple epitopes of all A single epitope of a single antigens used in the antigen immunization Antibody class: A mixture of different All of a single Ab class Ab classes (isotypes) Ag-binding sites: A mixture of Abs with All Abs have the same antigen different antigen-binding binding site sites Potential for cross-reactivity: High Low

  12. Fusions generated by Sendai virus or PEG Hybridoma1975 Kohler and Milstein

  13. Antibodies Polyclonal Monoclonal Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media Antibodies that are collected from sera of exposed animal recognize multiple antigenic sites of injected biochemical. recognize ONE antigenic site of injected biochemical

  14. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY • 1) Immunize animal (mouse or rabbit) • 2) Isolate spleen cells (containing antibody-producing B cells) • 3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol) • 4) Allow unfused B cells to die • 5) Add HAT culture to kill unfused myeloma cells • 6) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells) • 7) Screen supernatant of each clone for presence of the desired antibody (ELISA) • 8) Grow the chosen clone of cells in tissue culture indefinitely. • 9) Harvest antibody from the culture supernatant.

  15. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)

  16. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre High Titre too low 2 weeks BOOST(Pure antigen) BOOST(Pure antigen)

  17. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells + 8 - Azaguanine Myeloma Cells Immortal Tumor Of Lymphocytes Myeloma Cells HGPRT- High Viability & Rapid Growth hypothanthin-aminopetrin Medium HGPRT = Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

  18. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells PEG FUSION MYELOMA CELLS SPLEEN CELLS Feeder Cells Growth Medium • Plating of Cells in HAT selective Medium • Scanning of Viable Hybridomas HYBRIDOMA CELLS ELISA PLATE HAT Medium

  19. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

  20. Disadvantages • Need high experience qualification • High risk of contamination • Some mcABs are very liable so activity may be lost on freezing and thawing or long term storage • The frequent need to purify a mcAB from monoclonal culture medium Advantages -useful for the production of a specific antibodies to impure or mixed immunogenes.

  21. Uses Measuring protein and drug levels in serum Typing tissue and blood Identifying infectious agents Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas Identifying tumor metastasis Identifying and quantifying hormones Immunoaffinity Purification

  22. Serological Tests Direct Indirect Detect Ag from patient sample . Detect Ab from patient sample

  23. Precipitation : ( Continued ) Precipitation curve :

  24. Precipitation Reactions • Involve soluble antigens with antibodies

  25. Precipitation : ( Continued ) - Precipitation reactions

  26. Precipitation : ( Continued ) Ouchterlony (double diffusion)

  27. Agglutination Reactions • Involve particulate antigens and antibodies • Antigens may be: • On a cell (direct agglutination) • Attached to latex spheres (indirect or passive agglutination)

  28. Antibody Titer • Is the concentration of antibodies against a particular antigen Figure 18.5

  29. Agglutination : ( continued ) - Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens. - Cellular/molecular view of agglutination and Precipitation reactions that produce visible Ag-Ab complexes.

  30. Hemagglutination • Hemagglutination involves agglutination of RBCs. • Viral hemagglutination inhibition tests for antibodies by the antibodies' ability to prevent viruses from agglutinating RBCs. Figure 18.7

  31. Neutralization Reactions • Eliminate the harmful effect of a virus or exotoxin

  32. Agglutination Tests Lattice Formation

  33. Qualitative agglutination test • Ag or Ab Y +  Y Y Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

  34. Agglutination/Hemagglutination Quantitative agglutination test Titer Prozone 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8

  35. Agglutination/Hemagglutination Definition Qualitative test Quantitative test 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 • Applications • Blood typing • Bacterial infections • Fourfold rise in titer • Practical considerations • Easy • Semi-quantitative

  36. Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y Y +  Y • Applications • Measurement of antibodies to soluble antigens

  37. Coombs (Antiglobulin)Tests Y Y + Y Y Y Y  Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) • Incomplete Ab • Direct Coombs Test • Detects antibodies on erythrocytes

  38. Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum anti-rhesus factor (Rh) antibodies Step 1 Y +  Y Y Y Y Target RBCs Patient’s Serum Y Y Y Step 2 Y Y Y Y Y Y Y Y Y Y Y Y +  Y Y Y Y Coombs Reagent (Antiglobulin)

  39. Complement Fixation Ag mixed with test serum (Preheated) to be assayed for Ab No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y • Methodology • Standard amount of complement is added • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag

  40. Complement Fixation

  41. Complement Fixation

  42. Complement fixation test

  43. Fluorescent Antibody Techniques (Direct) Figure 18.10a

  44. Fluorescent Antibody Techniques (Indirect)

  45. Enzyme-Linked Immunosorbent Assay(Direct ELISA)

  46. Enzyme-Linked Immunosorbent Assay (Indirect ELISA)

  47. ELISAEnzyme-LinkedImmunosorbent Assay

  48. Test antigen Test antiserum

  49. Visualization of reaction

  50. +ve clone +ve control -ve control ELISA to test MAb production itself The dark blue spot represents a positive clone. The light blue spots are positive controls, the next two wells to the right are negative controls

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