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 .

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

• 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, 2000

http://www.med.sc.edu:85/chime2/lyso-abfr.htm

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

Ag + Ab Ag-Ab

Keq = [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

Keq = 104

Affinity106

Avidity1010

Avidity

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 clones A single B cell clone

Bind to: Multiple epitopes of all A single epitope of a singleantigens used in the antigenimmunization

Antibody class: A mixture of different All of a single Ab classAb classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same antigendifferent antigen-binding binding sitesites

Potential for cross-reactivity: High Low

PRODUCTION OF MONOCLONAL ANTIBODYHYBRIDOMA

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 immunizati

onSerum 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

+8 - 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 CELLSELISA PLATE

Feeder CellsGrowth 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.

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 hormonesImmunoaffinity Purification

Uses

Uses