Immunogen: A substance that induces a specific immune response  Antigen (Ag): A substance that reacts with the products of a specific immune response

 Antibodies recognize and react with antigenic determinants or epitopes. Figure 17.3

Figure 17.4

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.

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

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.

Figure 17.5a-c

 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

Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds – Hydrogen bonds – Electrostatic forces – Van der Waal forces – Hydrophobic forces Reversible Multiple Bonds Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296,

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

Calculation of Affinity Ag + Ab  Ag-Ab K eq = [Ag-Ab] [Ag] x [Ab] Applying the Law of Mass Action:

Avidity The overall strength of binding between an Ag with many determinants and multivalent Abs K eq = 10 4 Affinity 10 6 Avidity 10

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.

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 Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions

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

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.

 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

Polyclonal antibodies Monoclonal Antibodies Produced by:Many B cell clonesA single B cell clone Bind to: Multiple epitopes of allA single epitope of a single antigens used in the antigen immunization Antibody class:A mixture of differentAll of a single Ab class Ab classes (isotypes) Ag-binding sites:A mixture of Abs withAll Abs have the same antigen different antigen-bindingbinding site sites Potential for cross-reactivity:HighLow

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.

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

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

PRODUCTION OF MONOCLONAL ANTIBODY Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Immortal Tumor Of Lymphocytes Azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT - Myeloma Cells

PRODUCTION OF MONOCLONAL ANTIBODY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium 1.Plating of Cells in HAT selective Medium 2.Scanning of Viable Hybridomas

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

 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.

M easuring protein and drug levels in serum T yping tissue and blood I dentifying infectious agents I dentifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas I dentifying tumor metastasis I dentifying and quantifying hormones I mmunoaffinity Purification Uses