Transcript Monomer - muhadharaty.com

IMMUNOGLOBULINS (Ig)
“Antibodies”
Immunoglobulins are glycoproteins that are
composed of 82-96% protein and 4-18%
carbohydrate. They bind specifically (lock-and
key) to antigens which stimulated their
formation. The Igs are present at different
body sites including serum, blood, extracellular fluid, CSF, saliva, tears, exocrine
secretions (nasal , intestinal & breast milk),
and on surface of lymphocytes. The serum
protein electrophoresis shows that the Igs are
located mainly at the globulin region. The Igs
are produced from B cells that become
plasma cells which synthesize & secrete
thousands of antibody molecules per second.
Clonal Selection Theory:
The antibodies are produced according
to this theory which is not any more a
theory, but almost a fact. This theory
states that a single epitope stimulates a
single clone of B cells that produce a
single antibody specificity.
Clone: A cluster of identical cells since
they are all daughter cells of a single
mother cell.
• Basic Structure of antibody:
• Monomer: This is the basic structure of the
antibody. It is composed of 2 pairs of
identical polypeptide chains (i.e., the
polypeptides in each pair are identical). In
one pair, the polypeptide chains have twice
the M.W. and twice the numbers of A.A. of
the other pair. The first of pair polypeptides
are called Heavy chains (H), while the second
one are called Light chains (L). These chains
are joined together by disulfide bonds (S-S).
These bonds are between the H-L and H-H
chains. The M.W. of H chain is 46 KD, and
that of L chain is 23 KD.
Domains:
The polypeptide chains are like threads folded on
themselves. These folds are called domains and
are held together by S-S bonds (intra-chain). Each
domain is composed of 110 A.A. The sequences
of these A.A. are of two types:
1.Variable (V):
The domains located at the amino-terminal (H3N+)
have variable (different) A.A sequences between
the various antibody molecules. In each
monomer, there is one V domain in the H chain
(VH) and one V domain in the L chain (VL). The V
domains of both the H & L chains determine the
specificity of the antibody molecule.
2. Constant (C):
All domains ,except the V , have
constant A.A. sequences. The L chain
have only one C domain (CL), but the H
chain have 3 or 4 C domains (CH1, CH2,
CH3 & CH4). These C domains are
located toward the carboxy (COO-)
terminal of the monomer. According to
the C domains the H and L chains are
subdivided into different types.
• Types of H and L chains:
• H chains have 5 types which are
gamma, muta , alpha, delta, and
epsilon. According to these chains the
antibodies are divided into 5 classes
which are IgG, IgM, IgA, IgD, and IgE
respectively. One type of H chain is
present in each monomer.
• L chains have 2 types which are kappa
and lambda. Each monomer have either
kappa or lambda chains (never both).
Generally in the body, the kappa:
lambda ratio is 2:1.
• Subtypes of H & L chains:
• According to minor differences in the
constant regions, both H & L chains are
subdivided into different subtypes.
• The subtypes of H chains include:
• Gamma : 1, 2, 3, & 4 (for IgG1, IgG2, IgG3 &
IgG4 respectively).
• Mu: None
• Alpha: 1, & 2 (for IgA1, & IgA2 respectively).
• Delta : None.
• Epsilon : None
• The subtypes of L chains include:
• Lambda : 1, 2, 3, 4, 5 & 6
• Kappa : None
• ISOTYPES = All types & subtypes of Igs.
• Functions of Ig regions:
• Variable (VH & VL) : Are the Antigen binding
sites via hypervariable areas which are more
variable than the rest of the VH & VL regions.
The areas are also called “ hot spots” or “
idiotypes”. In the VH there are 4 idiotypes,
while in the VL 3 only. Only three idiotypes
from each chain are shared in the actual
binding with the AD residues; these
idiotypes are called “paratopes”.
• These hot spots (paratopes) are the areas of
intimate contact with the residues of the
epitopes (Lock-Key according to sequences
or configuration). Each idiotype is composed
of about 10 A.A.
• Q. Idiotypes are AD of the Ig ( F? Or T?).
• Q. Anti-idiotypes can be prepared (F? Or T?).
• Constant region:
• 1. Opsonization : Is the binging of IgG (IgG1
& IgG3 that are called Opsonin Igs) to an
antigen which is then followed by
phogocytosis via CH3. Also, binding of IgG
to macrophages via CH3 results in arming of
these cells.
• 2. NK cells: Bind to antigens (e.g. Cells or
m.o.) via CH3 of IgG (CD16); part of ADCC
(Antibody-Dependent Cell Mediated Cytoto.).
• 3. Complement fixation: Starts via C1 (C1q).
IgM is good complement fixing antibody
(+++), while IgG (+ - ++). All Ig subclaases
can fix the C except IgG4.
• 4. Placenta cross: IgG is the only Ig that can
cross the placental barrier; all subclassis can
do this efficiently except IgG2 (not efficient).
Immunochemistry of Ig (enzymatic
digestion by proteolytic enzymes):
• 1. Papain:
• Digest the IgG monomer at a flexible area
between CH1 & CH2 in front (toward H3N+
terminal) of the enter-H chains disulfide
bonds. This area is called “Hinge Region”.
The result is 3 fragments; two fragments are
called Fab (Fragment of Antigen Binding)
and the third one is named Fc ( Crystallizable
Fragment).
Fab =
VH+VL+CH1+CL;
Fc = CH2 + CH3 + Hinge R.
•
• 2. Pepsin:
• Digest the IgG monomer behind the
disulfide bond (toward COO- terminal)
of the hinge region. The result is 2
fragments; one is F(ab)2 and the
second is small negligible portion. The
F(ab)2 = 2 Fabs + Hinge Region.
• F(ab)2 has clinical & laboratory
applications. Example: Therapeutic use
when I.R. to Fc portion is unwanted.
• Polymers: Are composed of more than
one monomer; 2 monomers= Dimer, 3=
Trimer, 4= Teramer, 5= Pentamer, 6=
Hexamer. Examples: IgM in the serum
is usually pentamer and rarely
hexamer, while on lymphocyte surface
is monomer. Also, secretory IgA (IgA2
in exocrine secretions) is usually dimer
and some times trimer, while in the
serum is monomer.
IgM pentamer structure:
It is composed of 5 IgM
monomers linked covalently by
disulfide bonds and only one J
(joining) chain of 15 KD M.W.
Also, the J-chain is linked by –
S-S- to the main structure.
Secretory IgA (IgA2):
It is composed of 2 IgA monomers that are
linked covalently by J-chain (15 KD) with
non-covalent binding of Sc (Secretory)
component of 70 KD. Its light chains are
present in opposite position to the usual one
, and linked together by –S-S- bonds (L to L), but
not to the H chains (as usual). The Sc
component is produced by the epithelial
lining of the mucosal surfaces and its
function is to assist in the transfer (crossing)
of secretory IgA across the epithelial barrier.
Items
IgG
IgA
IgM
IgD
IgE
H chain class
gamma
alpha
mu
delta
epsilone
H chain subclasses
1, 2, 3, 4
1, 2
none
none
none
M.W.
150,000
160,000
or
400,000
900,000
180,000
190,000
Sedimentation coefficient
(S)
6-7
7
Percentage (%) in serum
75%
15 %
10-15%
0.2%
0.004%
120
3
0.05 or 10-15
IU / ml
Serum Concentration
(mg/dl)
1000
200
Serum half life (days)
23
6
7-8
19
5
3
8
2
Items
IgG
IgA
Complement fixation
+
0
++++
0
0
Placental transfer
+
0
0
0
0
IgG4 only
0
0
0
++++
Bacterial lysis
+
+
+++
?
?
Antiviral Activity
+
+++
+
?
?
Immune Response
Secondary
(mainly)
Secondary
(uncommon)
Primary
(mainly)
Secondary
(rare)
Secondary)
uncommon)
Heavy cain M.W.
50,000
55,000
70,000
62,000
70,000
15
18
Mast cells/ basophils
degranulation
Carbohydrate %
4
10
IgM
IgD
IgE
18
items
IgG
IgA
IgM
IgD
IgE
Common
Molecular form
Monomer
Dimer or
Monomer
Pentamer
or
Monomer
Monomer
Monomer
Antigen receptor
on B cells
-
-
+
+
-
J chain
-
+
+
-
-
Secretory piece
(component)
-
+
-
-
-
IgG Functions:
1. Opsonin: It enhances phagocytosis (opsonization) by coating
bacteria and attaching by its Fc portion to Fc receptors on
phagocytic cells.
2. Precipitin: It precipitates soluble antigens.
3. Incomplete-Haemolysin: Does not cause haemolysis by
complement fixation and activation as Anti-Rh.
4. Complement Fixing Antibody.
5. NK Fc receptor (CD16) which functions as ADCC.
6. Neutralizing antibody as anti-toxin,& anti-viruses.
7. Present in C.S.F. and extracellular fluids.
•8. Can Cross the Placents .
•9. IgG4 reaginic antibody in allergy (mainly ingestants).
•10. Present in active diseases as Brucellosis.
•11. Generally against protein antigens except IgG2 which is antiCHO.
•12. The major antibody in secondary immune response
(anamnestic I.R.).
IgA Functions:
1.Local immunity at mucous surfaces (coat m.o.,
prevent adherance & entery). It is synthesized locally by
submucosal plasma cells (80% of plasma cells in MALT
synthesize IgA). It protects 400 sq.m. of mucous
membranes.
2. Present in mucous secretions (RT, GIT,GUT) and
in tears,sweat,saliva, colostrum& milk.
3. IgA1 (monomeric) is mainly present in the serum,
while IgA2 (di- or trimeric) is present as secretory
antibody.
4. IgA1 can be inactivated by an IgA protease produced
by Gonococci, Pneumococci, & H. Influenzae, while IgA2
can not be inactivated.
IgM Functions:
1. Agglutinin antibody that agglutinates particle
antigens.
2. Haeagglutnin as Natural isohaemagglutinin
(Anti-ABO).
3. Complete haemolysin.
4. Good complement fixingِ ِ
5. Antigen Receptor on mature lymphocytes (B
cells) together with IgD.
6. Main antibody of primary immune response.
*
• IgD Functions:
• 1. It is co-expressed with IgM on the suface
of mature B lymphocytes. Like IgM functions
as an antigen receptor.
• 2. It may act as antibody against certain
antigens as insulin receptor...etc.
• IgE Functions:
• 1. It plays a pathogenic role in type I (immediate)
hypersensitivity. It has Fc receptor (Fc-epsilon) on
mast / basophil cells.
• 2. High levels are also detected during parasitic
infestations mainly helminthic (e.g. Ascariasis).
• 3. It is monmeric, does not fix complement, and does
not cross the placenta.
• Immunoglobulin Diversity
• There are about 10 trillions different Ig specificities
in the human body which is called “ repertoire” )i.e.,
equipped with all the possibilities of antigens
present in nature). This is achieved by different
mechanisms by the immune system and controlled
genetically.
• The B cells differentiate and mature to plasma cells
that synthesize and secrete Igs. The mechanism of Ig
formation consists of 2 parts:
• 1. Synthesis of light chains (L).
• 2. Synthesis of heavy chains (H).
• The combination of L & H chains result in the
formation of complete Ig. Each of the L & H chains
are formed from two portions (Variable & constant
regions) which are joined together by joining (J)
region or portion. The regions are all formed under
the control of genes.
• L : Leader
• Vk : Genes controlling the peptides that
form the Kappa chain.
There are 30
different types of variable region of Kappa
chain ( VK1,2,3,...30).
• C : Constant
• K : Kappa
• J
: Joining chain of 5 types (J1,2,3,4,5).
• IVS : Intervening segment (intron).
• Kappa Chain Formation:
• The first process in the formation of Kappa
chain is the rearrangement which is the
binding of one J chain with one VK (brought
together). Then the C region will bind. This
process occurs at the DNA. Then there is
transcription process on the RNA and the
formation of mRNA which will be transfered
to the ribosome for protein synthesis (Ig
synthesis). Also, there is a process of
removal of all the IVS segments which is
called RNA splicing (result in L + VK + J +
CK). The last process is the translation of
these information by removal of the L
segment and the completion of the Kappa
chain formation (VK + J + CK). Probabilities =
30 V X 5 J X 1 C = 150
• Antibody Diversity:
• This process depends on:
• 1. Multiple gene segments.
• 2. Their arrangement into different
sequences.
• 3. The combing of different L & H
chains in the assembly of Ig molecules.
• 4. Mutation.
• 5. Junctional diversity (D) ; applies to
the H chain only.
• Isotype switching:
• During the immune response the plasma
cells switch from producing IgM to IgG or
any other class. This could occurs directly or
in a programmed downward process (e.g. Mu
to gamma 1 , then gamma 2 ..etc...).There is
no change of the V region and the L chain,
but the C region is replaced . So, there is no
change in the specificity, but in the class of
Ig. This occurs through the process of DNA
rearrangement and RNA splicing.
• Heavy chain formation:
• This process is controlled by genes on
chromosome No. 14.
• Comparable to that of L. There are L, 50
different VH of gamma, mu, alpha, delta, or
epsilon, 25 Diversity (D), 6 J, and CH. The
formation starts by binding of one D and one
J the to one VH forming VDJ. The
transcription and RNA splicing by the
removal of IVS. Then into the translation
process and the removal of L segment.
• N.B. : Lambda light chain is controlled by
genes on chromosome No. 22. Also is has 4
constant (C) segments.
Monoclonal Antibodies
Discovery:
George Kohler and Cesar Milstein published in
Journal “Nature” in 1975 a new technique for
antibody production. The technique is named
“Hybridization” for the production of
monoclonal antibodies in tissue culture.
Principles of the technique:
1. Immunization of a mouse with an antigen (X).
2. Spleenocytes are taken from the immunized
mouse.
3. Hybridize using poly Ethylene Glycol (PEG) the
spleen cells (which synthesize immunoglobulin,
but can NOT live in tissue culture) with mice
non-secretory myeloma cell lines (which do NOT
synthesize Ig, but can live in tissue culture).
4. Hybrid cells (of tetraploid chromosomes) are
cultured in fluid named RPMI in which Ig are
secreted by these cells, the collected as
supernatants.
Tissue Culture:
1. Microtitre plates of 96 wells are used.
2. In each well ONE hybrid cell will grow and
form ONE clone of cells.
ONE cell
ONE clone
3. This clone secretes ONE type of antibody
of a single specificity to ONE epitope that
is why it is named Monoclonal Antibody.
4. The non-hybridized myeloma cells are
killed by HAT, while the non-hybridized
spleen cells die spontaneously in 5-7 days
Applications of MCA:
I. Diagnostic:
1. Leucocyte identification.
2. Lymphocyte subset determination.
3. HLA detection and typing.
4. Detection of different microbes (bacteria,
viruses, fungi & parasites).
5. Detection of tumour markers or antigens.
6. Determination of concentration of hormones.
7. Typing of lymphomas and leukaemias.
8. Detection of different cardiac or other
tissues markers as cardiac myosin in
myocardial infarction.
9. Tissues immunohistochemical applications.
II. Therapeutic:
1. Anti-Tumour as anti-tumour antigens or antiidiotypes for lymphomas. Antibodies labelled
with toxin as Ricin or radioactive material can be
used against tumours and named ‘Magic Bullet’
or ‘War Head’.
2. Immuno-suppressive therapy in organ
transplantation, autoimmune diseases,
hypersensitivity, and treatment of graft versus
host disease (GvHD).
3. Control and treatment of infertility as using antihCG.
4. Treatment of drug toxicity as digitalis
intoxication.
CD Markers (Clusters of Designation or
Differentiation):
CD markers are a group of special molecules
on the surface of body cells. There are more
than 400 CD markers. They are detected by
specific monoclonal antibodies which share
with them the name All body cells have one
or more of these markers. However, they are
most important or useful for the
identification and differentiation of W.B.C.
and their subsets. Examples: CD4 = Helper Tcells; CD8 = Cytotoxic T-cells; CD56 = NK
cells …etc. MCA as anti-CD4, Anti-CD8, AntiCD56.. etc
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