Specific Defenses of the Host: The Immune Response

1. Specific Defenses of the Host:The Immune Response Immunogen: A substance that induces a specific immune response • Antigen (Ag): A substance that reacts with the products of a specific immune response

2. Antigenic Determinants • Antibodies recognize and react with antigenic determinants or epitopes. Figure 17.3

3. Haptens Figure 17.4

4. Hapten: A substance that is non-immunogenic but which can react with the products of a specific immune response. Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule .

5. Chemical Nature of Immunogens Proteins Polysaccharides Nucleic Acids Lipids Some glycolipids and phosopholipids can be immunogenic for T cells and illicit a cell mediated immune response

6. Epitope or Antigenic Determinant: That portion of an antigen that combines with the products of a specific immune response Antibody (Ab) : A specific protein which is produced in response to an immunogen and which reacts with an antigen.

7. Antibody Structure Figure 17.5a-c

8. Human Immunoglobulin Classes • IgG - Gamma (γ) heavy chains • IgM - Mu (µ) heavy chains • IgA - Alpha (α) heavy chains • IgD - Delta (δ) heavy chains • IgE - Epsilon (ε) heavy chains

9. 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 forces • Van der Waal forces • Hydrophobic forces • Multiple Bonds • Reversible

10. Low Affinity High Affinity Ab Ab Ag Ag Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site Affinity = ∑ attractive and repulsive forces

11. Calculation of Affinity [Ag-Ab] Keq = [Ag] x [Ab] Ag + Ab  Ag-Ab Applying the Law of Mass Action:

12. 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

13. Specificity The ability of an individual antibody combining site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen.

14. 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

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

17. 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.

18. 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

19. HISTORY

20. 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

21. 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.

22. 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)

23. 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)

24. 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

25. 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

26. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

27. 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.

28. 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