宿主的特异性防御-免疫反应Specific Defenses of the Host-The Immune Response
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Immunogen: A substance that induces a specific immune response Antigen (Ag): A substance that reacts with the products of a specific immune response
• Lock and Key Concept
• Non-covalent Bonds
– Hydrogen bonds – Electrostatic forces – Van der Waal forces – Hydrophobic forces
:85/chime2/lyso-abfr.htm
Keq =
104 Affinity
106 Avidity
1010 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.
ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification)
Ab titre reached in Serum Spleen removed
(source of cells)
PRODUCTION OF MONOCLONAL ANTIBODY
• Multiple Bonds • Reversible
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
Affinity
• Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Low Affinity Ab
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.
Immortal Tumor Of Lymphocytes
Myeloma Cells
+8 -
Azaguanine
Myeloma Cells
HGPRTHigh Viability & Rapid Growth
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells
Ab
Ag
Ag
Affinity = ∑ attractive and repulsive forces
Ag + Ab Ag-Ab Applying the Law of Mass Action:
Keq =
[Ag-Ab] [Ag] x [Ab]
Avidity
• The overall strength of binding between an Ag with many determinants and multivalent Abs
Polyclonal antibodies Produced by: Bind to: Many B cell clones Multiple epitopes of all antigens used in the immunization A mixture of different Ab classes (isotypes)
Ab excess
Ag excess
• Ag:Ab ratio
• Physical form of Ag
Equivalence – Lattice formation
Definition:
. Mono: one . Clone: a strain of cells descended form single cell.
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
Antibodies recognize and react with antigenic determinants or epitopes.
Figure 17.3
Figure 17.4
• Hapten: A substance that is nonimmunogenic 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
Titre too low BOOST (PurST (Pure antigen)
2 weeks
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells
High
All Abs have the same antigen binding site
Potential for cross-reactivity:
Low
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
•
• • • • •
1) Immunize animal (mouse or rabbit)
PEG FUSION
SPLEEN CELLS
MYELOMA CELLS
Feeder Cells Growth Medium
1. Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas
HYBRIDOMA CELLS ELISA PLATE HAT Medium
• • •
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells
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
Cross reactions
Anti-A Ab Anti-A Ab Anti-A Ab
Ag A
Ag B
Ag C
Shared epitope
Similar epitope
Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production
After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery)
. 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
2) Isolate spleen cells (containing antibodyproducing 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.
Monoclonal Antibodies A single B cell clone A single epitope of a single antigen
Antibody class:
All of a single Ab class
Ag-binding sites:
A mixture of Abs with different antigen-binding sites
• Lock and Key Concept
• Non-covalent Bonds
– Hydrogen bonds – Electrostatic forces – Van der Waal forces – Hydrophobic forces
:85/chime2/lyso-abfr.htm
Keq =
104 Affinity
106 Avidity
1010 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.
ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification)
Ab titre reached in Serum Spleen removed
(source of cells)
PRODUCTION OF MONOCLONAL ANTIBODY
• Multiple Bonds • Reversible
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
Affinity
• Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Low Affinity Ab
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.
Immortal Tumor Of Lymphocytes
Myeloma Cells
+8 -
Azaguanine
Myeloma Cells
HGPRTHigh Viability & Rapid Growth
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells
Ab
Ag
Ag
Affinity = ∑ attractive and repulsive forces
Ag + Ab Ag-Ab Applying the Law of Mass Action:
Keq =
[Ag-Ab] [Ag] x [Ab]
Avidity
• The overall strength of binding between an Ag with many determinants and multivalent Abs
Polyclonal antibodies Produced by: Bind to: Many B cell clones Multiple epitopes of all antigens used in the immunization A mixture of different Ab classes (isotypes)
Ab excess
Ag excess
• Ag:Ab ratio
• Physical form of Ag
Equivalence – Lattice formation
Definition:
. Mono: one . Clone: a strain of cells descended form single cell.
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
Antibodies recognize and react with antigenic determinants or epitopes.
Figure 17.3
Figure 17.4
• Hapten: A substance that is nonimmunogenic 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
Titre too low BOOST (PurST (Pure antigen)
2 weeks
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells
High
All Abs have the same antigen binding site
Potential for cross-reactivity:
Low
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
•
• • • • •
1) Immunize animal (mouse or rabbit)
PEG FUSION
SPLEEN CELLS
MYELOMA CELLS
Feeder Cells Growth Medium
1. Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas
HYBRIDOMA CELLS ELISA PLATE HAT Medium
• • •
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells
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
Cross reactions
Anti-A Ab Anti-A Ab Anti-A Ab
Ag A
Ag B
Ag C
Shared epitope
Similar epitope
Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production
After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery)
. 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
2) Isolate spleen cells (containing antibodyproducing 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.
Monoclonal Antibodies A single B cell clone A single epitope of a single antigen
Antibody class:
All of a single Ab class
Ag-binding sites:
A mixture of Abs with different antigen-binding sites