antibodies. introduction definition: immunoglobulins represent animal proteins, which possess...
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Antibodies
Introduction
Definition:Immunoglobulins represent animal proteins, which possess antibody activity and other proteins with related chemical structure and antigenic specificity.
(according WHO)
AntibodiesAntibodies
StructureStructure
Immunoglobulin structure
• Heavy chain
IgM, IgG1-4, IgA1-2, IgD, IgE
• Light chain
• Domain
• Ag binding site
Immunoglobulin structure
Immunoglobulin domains
-Variable: VH, VL
-Constant:CH, CL
Complementarity-determining regions [CDR] hypervariable loops
AntibodiesAntibodies
Antigen binding siteAntigen binding site
domains, complementarity determining domains, complementarity determining regions (CDR)- hypervariable regions/loopsregions (CDR)- hypervariable regions/loops
-VVL L : CDR1, CDR2, CDR3: CDR1, CDR2, CDR3
- VVHH : CDR1, CDR2, CDR3: CDR1, CDR2, CDR3
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ImmunoglobulinImmunoglobulin sstructuretructure
IgIg = (H= (H22LL22))nnn=1 for n=1 for IgGIgG, , IgEIgE, , IgDIgDn=1 or 5 for n=1 or 5 for IgMIgMn=1,2,3, or 4 for n=1,2,3, or 4 for IgAIgA
HHeavy eavy chains:chains: IgMIgM, , IgGIgG, , IgAIgA, ,
IgDIgD,,IgEIgE
Light chainsLight chains:: ,,
Hinge regionHinge region:: IgGIgG, , IgDIgD, , IgAIgA
Ag
AntibodiesAntibodies
Ig Class Ig subclass
IgM no
IgG IgG1, IgG2, IgG3, IgG4,
IgA IgA1, IgA2
IgD no
IgE no
Class and subclass (isotypes)
Immunoglobulin structure
• Fragments
Immunoglobulin structure
• Immunoglobulin domain
Immunoglobulin superfamily
• Immunoglobulin
domain
Immunoglobulin structure
• Antigen binding site
AntibodiesAntibodies
Light chain Heavy chain
domains VVLL CCLL VVHH CCHH
IgM 1 1 1 4
IgG 1 1 1 3
IgA 1 1 1 3
IgD 1 1 1 3
IgE 1 1 1 4
Immunoglobulin domainsImmunoglobulin domains
Antigenic Determinants on Immunoglobulins
Name Localization of Ag determinants
Distribution on
cells / molecules
Idiotype VH and VL
(in and out CDR)
•several B cell clones >> public i.
•individual B cell clone
>> private i.
Isotype
-class, subclass CH all members of a species
-type and subtype CL
Allotype
Gm, Am
Km
CH
CL
[ in animals also VH,L]
some, but not all members of a species
Immunoglobulin Isotypes
• Importance
– Ig levels
– B cell tumors
– Immunodeficiencies
IgM (kappa)IgG1 (kappa)
Immunoglobulin Allotypes
• Definition - Antigenic determinants specified by allelic forms of the Ig genes
IgG1 (kappa)
Person 2
IgG1 (kappa)
Person 1
•Importance
-Monitoring bone marrow grafts
-Forensic medicine
-Paternity testing
Immunoglobulin Idiotypes
IgG1 (kappa)
Person 1
anti-B
IgG1 (kappa)
Person 1
anti-A
•Importance
- V-region marker
- Regulation of immune responses
AntibodiesAntibodies
FunctionFunction
Biological activities of immunoglobulins
Property / activity IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Serum level [mg/ml]
9 3 1 0.5 3 0.5 1.5 0.0003 0.03
Half life [days] 23 23 8 23 6 6 5 2.5 3
1-st in primary response
+
Activates classical complement way
++
CH2
+
CH2
++
CH2
+/-
CH2
- - ++
CH4
- -
Crosses placenta + +/- + + - - - - -
Present on mature B cells
- - - - - - + - +
Binds to mph Fc receptors
++ + ++ +/- - - + - -
Present in secretions
- - - - ++ ++ + - -
Induces mast-cell degranulation
- - - - - - - + -
AntibodiesAntibodies
Ig genesIg genes
Susumu Tonegawa
„for his discovery of the genetic principle for generation of antibody diversity“
The Nobel Prize in Physiology or Medicine 1987
AntibodiesAntibodies
Antibody responseAntibody response
Cellular leverCellular lever
B cell activation
Two signals are required for activation of B cell.
The first signal: binding of Ag to BCR(surface Ig)The second signal: binding of Th cellto B cell.
After activation, B cell differentiate to - plasma cell >> secretion of Ab- memory B cells >> secondary response
B cell activation
The second signal represents contact between CD40 receptor on B cell and CD40L ligand expressed on Th cells
B cell activation
Only activated Th cell is able to provide the second signal for B cells.
Th cells are activated by antigen presenting cell (APC).
Activation of Th requires also two signals (steps):1st> APC engulf Ag, process Ag and express Ag fragments in the frame of MHCII on the cell surface for TCR (T cell receptor) on T cells.2nd > APC produce IL-1 which binds to IL-1 receptor on Th cell.
AntibodiesAntibodies
Antibody productionAntibody productionPrimary and Secondary Primary and Secondary
ResponceResponce
Primary and secondary response
Primary response - 1st contact with Ag- IgM
Secondary response - 2nd contact with Ag- based on memory B cells- CLASS SWITCHING Under the influence of cytokines B cells switch from IgM production to IgG and other classes of Ab.
- afinity maturation: Ab have high afinity to Ag compared with the afinity of Ab produced in primary response
- faster onset of Ab production- higher levels (titers) of Ab- longer persistence of Ab (slower drop of Ab titers).
Monoclonal antibodies
Principle:
1) “Immortalization” of Ag specific B cell clone by fusion with myeloma cell.
2) Selection of this clone in HAT medium.
3) Propagation of Ag specific clone in vitro or in mice.
____________________________(Myeloma is a cancer cell “without limitation” of proliferation)