Today's topic is Structures and Functions
of Immunoglobulin.
You have been studying for over several lectures
that B cells are the only cells that make
immunoglobulins as cell surface receptors;
and, the same cells start secreting antibodies,
once they are differentiated to plasma cells.
Now, it was already known that there is a
soluble component in the blood, which can
counteract very effectively with the immunogen
or pathogen as it was known earlier.
And, this was known already in beginning of
the twentieth century.
People since then, were trying to look at
or identify what is this molecule or what
are these molecules that participate in the
humoral immune response.
It was around 1939 that Tiselius and Kabat
were the first report that this component
antibody is present in the gamma globulin
fraction of the serum.
Technology was primitive those days.
And, the only method by which scientist could
identify which were the fractions present
in the serum was electrophoresis, simple electrophoresis,
where proteins could be separated based on
the net charge.
Now, Tiselius and Kabat did a very simple
experiment and it took them several months,
perhaps several years to do.
They have made antiserum to ovalbumin.
When they electrophoresis the antiserum as
is known for even just serum from any individual,
there are essentially two sets of proteins
that one can identify: albumin and globulins.
Now, globulins also could be separated as
can be seen on this graph - into alpha, beta
and gamma.
Now, when they have taken the same aliquot
of antiserum from rabbit raised against ovalbumin,
that is, egg albumin to which now, they added
or a certain amount of T antigen ovalbumin.
They already knew that, on addition of antigen
to antiserum, always or most likely, resulted
in precipitation; precipitates that could
be centrifuged down to a pellet.
So, they did that.
Now, they have taken two aliquots like I said:
one, where they have not added the antigen;
and, the other one, where they have added
the antigen.
After what they thought was appropriate antigen-antibody
interaction, they centrifuged down the particulate
material, which was nothing but the immune
precipitate.
And now, they have run electrophoresis both
the samples on their native gels, where proteins
were separated as albumins, and globulins:
alpha, beta and gamma.
Now, what could be seen very clearly that
in the aliquot, which showed precipitate on
adding the antigen, there was a decrease only
in the gamma globulin fraction.
And, it was quite clear; you can see the decrease.
Therefore, it was easy for them to then conclude
that the immune response that one can generate
by way of secreting antibodies in the blood
resided in the gamma globulin fraction, and
therefore, the name immunoglobulin came about.
So, it was Tiselius and Kabat, who showed
for the first time that antibodies were gamma
globulin fraction.
Since then, people started to do more search
on how to find out the structure of this gamma
globulin fraction or the antibody that we
know now.
Now, there were large number of scientists,
who were looking at the structure.
And now, we know the structure has two heavy
chains, which are held together by disulfide
bond, which inturn hold the light chain.
But, it took many years for Porter and Edelman
to come to this, just because the technology
then was not as advanced as it is today.
And, they always needed to work with milligram
amounts of the molecule anyway.
This picture here shows the results of two
of the major experiments.
One, where they have taken the antibody molecule,
immunoglobulin; and now, reduced the disulfide
bonds with mercaptoethanol.
Always, this resulted in identification of
two bands; they called heavy and light.
There are always two bands.
But, when they looked at the size of the two,
they realized immediately that the structure
should be much large.
Now, the size of the heavy chain is only around
55 kilo Dalton and the light chain is around
25.
And, they knew that the whole protein molecule
was around 150000.
Therefore, they could do simple calculation
and surmise that the immunoglobulin molecule
is made up of two heavy chains and two light
chains.
Now, in addition to these experiments, they
also were digesting the proteins with different
enzymes and they were able to come to a good
conclusion with respect to the structure by
digesting the molecule with pepsin or papain.
Interestingly, pepsin digestion attacks the
site on the immunoglobulin molecule, which
is after the disulfide bonds, which holds
the heavy chain together.
Papain on the other hand, cleaves the molecule
at a site, which is above the disulfide bonds.
Therefore, the pattern of the fragments that
they obtained was quite distinct.
What they obtained with pepsin digestion had
a fragment, which could bind to molecules
of antigen; whereas, with the digestion with
papain, they got a fragment, which could bind
only one; it was monovalent; only one antigen
molecule.
Upon papain digestion and pepsin digestion,
they were able to get fragments, which they
called FC.
And, we know that this is the constant domain
of immunoglobulin.
Now, putting all these observations together,
they came to the conclusion that the antibody
binds two antigens from the amino terminus
and that antibody molecule is at least bivalent.
But, this was quite remarkable considering
the time at which they were working in trying
to elucidate the structure of immunoglobulin.
Now, already in the 1930s, sequencing of protein,
the methodology at least, came to be established.
So, scientists started to sequence immunoglobulins.
How were the immunoglobulins purified?
By the method of either sorting out with ammonium
sulphate or by electrophoresis as I have shown
in the first slide; but, this was always from
the immunoglobulins from serum.
Now, they could not come to any conclusion
with respect to the sequence.
Now, it is very easy for us to see why, because
there is tremendous variability at the N terminus,
and therefore, the N terminal sequencing was
possible due to tremendous variability 
at the N terminus.
So, how did they then get the sequence of
immunoglobulins?
Now, you know that in the database, we have
large number of sequences of various immunoglobulins.
So, it was after several years that scientists
found, in fact, immunoglobulins could be sequence,
because of the discovery of myeloma proteins.
Now, what are myeloma proteins?
Myeloma proteins are products of cancer of
plasma cells.
Myeloma proteins are also monoclonal; why,
because cancer is usually monoclonal; almost
any cancer one can think about whether it
is epithelial or let us say from hematopoietic
lineage; cancer is usually monoclonal.
If a plasma cell gets transformed - plasma
cell remember, the cell that secrets antibodies,
becomes immortal now or becomes cancerous,
then this cell and the progeny of this cell
would make antibodies of only one class, only
one type.
And, those proteins were known as myeloma
proteins.
At that time when myeloma proteins were discovered,
it was not known what these molecules are;
they came to be known later.
So, what are myeloma proteins?
They are secreted immunoglobulins from the
cancer of plasma cells.
Now, at this junction, those not really relevant,
I would also like to say that people were
also able to identify Bence Jones proteins,
which were not the whole molecule of immunoglobulin,
but light chains.
Now, myeloma patients secrete in their circulation
not only myeloma that is the whole molecule
immunoglobulin, but also some of them express
large amounts of the light chains.
And, those were called Bence Jones proteins.
Finally, anyway they realized, is a whole
molecule myeloma or Bence Jones like chains
alone.
Now, soon after that came to be known, almost
every myeloma protein that could be identified
was sequenced.
The amino acid sequences then were analyzed
and the following observations were made that
the amino terminii of the immunoglobulins,
that is, one third, were very variable.
So, when they looked at all the sequences
of the myeloma protein that they had now a
bank of, they found that the amino terminii
always were very variable.
And therefore, it was logical to conclude
that this region would constitute the antigen
binding region.
Two thirds of the immunoglobulin sequence
towards the C-terminus comprised of common
sequences.
When they looked at those common sequences,
they could see that these myeloma proteins
then could be categorized into five main classes.
And, we know them as IgM, IgD, IgG, IgE and
IgA.
Now, once the sequences came to be known on
closer look of the variable domains...
There are now two graphs here before me: one
shows the variable domain of the heavy chain;
and the other one, variable domain of the
light chain.
Now, when people analyze these variable sequences
closely and they try to see how variable these
variable regions are, they did a very simple
analysis and which is shown here.
Now, the variable domain, first of all, they
could see was around 110 amino acids from
any and every myeloma protein; both heavy
as well as the light; about approximately
110.
Now, they made these graphs by putting on
the x-axis the amino acid number, and then,
they tried to look at how many times how different
is that particular amino acid at that particular
residue, that number.
So, y-axis shows the variability.
The peaks that you can see here would mean,
in this particular case, where it is maximum,
let us say, in the 94th position or 95th position,
the amino acid varied almost more than a 100
percent; which would mean that this would
be the most variable region and residue, and
definitely, this would contribute tremendously
to the antigen binding.
Now, another observation - in fact, you can
also make looking at this, that both in the
heavy and the light chain variable region,
there is a hyper variable or there are hyper
variable regions.
Look at the residues from around 35 to 38.
Similarly, let us just say, 50 to about 55;
and similarly, around 90 to 94.
Now, these varied tremendously all the myeloma.
Now, these three regions are flanked by more
or less constant domain.
There is a region here, which is more variable
than the others.
So, they coined the terms that these were
the CDR1, 2 and 3; CDR stands for complementarity
determining regions, because by then, they
already knew that these were the regions by
which antibodies of B cell receptors bound
their cognate antigen.
Now, flanking these are the framework regions,
which remain more or less constant.
Now, in fact, today, it is possible to design
primers corresponding to the framework region
and amplify the hypervariable domain, because
one cannot determine or design primers to
get only the hypervariable domains.
Again now, let us look at the heavy and the
light variable domains and you can see immediately,
that in both cases, the CDR3 is most variable.
And overall, the heavy chain domain is much
more variable than the light chain domain.
The CDR2, for example, of the light, contributes
minimal to the antigen binding pocket.
So, the H chain contributes more to the antigen
binding pocket than the L chain.
Now, people have done various experiments
even at the protein level trying to see when
you separate the heavy and the light chains,
whether they are still capable of binding
the antigen.
And, almost with about 95 percent cases, when
H (heavy) chain is separated from the light
chain, a simple reduction of the disulfide
bond, the heavy chain still can bind the antigen;
whereas, light chain almost cannot.
I would like to mention here that the heavy
chain separated from the light chain still
exhibits binding to the antigen, but the affinity
with which the heavy chain still binds could
be 100 and sometime may be a 1000 fold lower.
Now, here after all the information that was
gathered with respect to the amino acid sequence
after reduction of the heavy and light chain,
and the bindability, and all the data, now,
could be put together to come to the final
structure of the monomeric immunoglobulin.
Why I say monomeric, is because there are
classes of immunoglobulin that show oligomeric
structure.
This is a basic structure of an antibody molecule;
or, you can say immunoglobulin molecule.
What does this structure?
We know very well now that every immunoglobulin
is made up of two heavy chains, which are
held together by disulfide bonds.
Now, the heavy chains themselves are again
held to or hold the light chain through disulfide
bond.
Both heavy chains of an immunoglobulin molecule
are identical; so, also the light chains.
An antibody molecule is at least bivalent.
Now, in comparison to those, which have more
than one monomer, where the valency can go
as high as 10; and, we will see that in a
while.
The heavy chains could be either mu gamma,
alpha, delta or eta.
And, in the antibody molecule, the class is
known by the heavy chain; so, M, G and so
on.
The light chain can either be kappa or lambda.
It is important to know that apart from the
heavy chain being identical to each other,
even the kappa would be identical not only
with respect to the variable domain, but also
with respect to the constant domain.
Remember, in my last lecture, I did mention
that always, the recombination of the kappa
light chain allele 1 starts first and if that
fails, the second allele takes over.
If the recombination of the second kappa allele
also is disaster, then the lambda allele 1
starts the recombination process.
Therefore, we know that an antibody molecule
always has only one type of heavy chain, one
type of light chain.
I should have mentioned something about the
constant domains here, which I forgot and
let me go back to the basic structure again;
that the variable domain and the constant
domains are almost similar with respect to
the number of amino acids.
The heavy chain after the variable domain
has three constant domains in three out of
the five classes of immunoglobulin that we
just talked about.
The constant domain of the light chain is
only one.
Immunoglobulins are glycoproteins; they are
glycosylated.
The amino acid that get glycosylated are on
the constant domain 2 of the immunoglobulin
molecule.
Both the heavy chains are glycosylated.
This is a structure, which shows the fold
of the molecules.
It is the same immunoglobulin molecules, but
this elaborates the function of the intra
disulfide bonds.
And, let us look at this a little bit closely.
You can see very clearly from here that the
heavy chain has 1 2 3 and 4 folds.
These are called immunoglobulin folds.
In fact, there is a super family, where immunoglobulin
is the prototype molecule; and this family,
I can give you a few examples, the alpha 1,
beta 1, the co receptor for the immunoglobulin,
the B cell receptor or the CD4 or the CD8
or the TCR receptor and the co-receptor.
All of these belong to the immunoglobulin
super family.
And, all of them would have perhaps different
sequences at these different regions and the
structure may be very different.
But, all of them are characterized by the
presence of these immunoglobulin folds.
These immunoglobulin folds are held together
or stabilized by intra disulfide bonds.
So, you can see the complexity of the molecule
here.
Now, why is it necessary that there should
be these immunoglobulin folds, is because...
Now, where do you find these immunoglobulin
molecules either as cell surface receptors
or in circulation as the antibodies?
Now, there is a lot of...
Even these cells are built in the blood plasma
or in body fluids; all of which contain large
number of proteases.
To enable these molecules to survive the proteases,
nature has allowed the evolution of such molecules,
which are very stable to proteases.
Now again, one fourth of the molecule is the
variable region and the rest of the molecule
is more or less constant.
And, this definitely then should have common
functions biological activity.
Let us look at that.
This is one more structure, which is based
on crystal structures that were obtained for
all the myeloma proteins and it was seen...
Now, this is very characteristic and I would
like to spend a little time on this.
Now, the immunoglobulin fold - each one of
this is a fold and this is the structure of
the light chain; you can see constant light
and variable light.
Now, very clear here are the presence of these
antiparallel beta pleats.
These are held together as a barrel.
And, if one can think in terms of roof of
the barrel and the flow, one can see that
the roof and the flow are again held together
by the presence of that disulfide bond, which
I had pointed out in the previous picture.
Now, these antiparallel beta sheets are joined
together by these loops: beta bends or loops.
And, as one can imagine that it is the loops,
which constitute the antigen binding pocket.
This is the variable domain and these are
the antigen binding pockets.
What is very interesting to see, that the
immunoglobulin molecule does not have any
helices; total absence of helices.
There are five classes of immunoglobulins
or antibodies.
Are these similar; are they identical?
Obviously, not; so, where are they different
from each other?
Before we come to the molecules level, let
us just look at the domain organization.
IgG, IgD and the monomer of 
IgA are very similar; they have the heavy
chain, which has variable one domain and then
constant three domains; you can see.
However, the remaining two classes: IgE and
IgM; both have four domains.
And, what you might see very clearly here,
they do not have the hinge region, which the
other immunoglobulins have; there is lack
of the hinge region.
In fact, the hinge region in IgE and IgM has
been modified to yet another constant domain.
Therefore, now, IgE monomer and IgM monomer
are longer; the heavy chain is longer than
the heavy chains found in the other three.
Is there any similarity between IgA and IgM?
Yes, both of them can oligomerize.
And, the oligomerization - you can see two
molecules of IgA forming a dimer; IgA can
also find form a trimer.
The dimer and the pentamer seen in the IgM
are held together by the J chain, which is
a 15 kilo Dalton, 16-rich molecule, which
is also synthesized by the B cells.
You can see the J chain here and the J chain
here.
So, you have similarities between these two
molecules because of the presence of J chain.
Let us see if there are any other similarities.
Now, let me come to the subclasses of IgG.
I have already discussed this with you earlier
that IgG is present in the serum as four distinct
subclasses: IgG1, 2, 3 and 4; in human, IgG1,
2a, 2b, 3.
And, these are also present on memory cells
as the antigen receptor.
Remember that there are constant domain regions
in the immunoglobulin gene itself.
And, these not in the sequence, but these
are found soon after the gene for IgD.
So, you have IgM, IgD and then you have the
IgGs, the different domains of the four types
of IgG: IgG1, 2, 3 and 4.
Now, if you look at these four, immediately
you can see, IgG3 is different, because it
has a very long hinge region.
Hinge region is that region, which joins the
constant domain 1 with the constant domain
2.
Now, there can be as many as 13 disulfide
bonds in the extended hinge region of IgG3.
IgG2 and 4 are similar in their hinge region
disulfide bonds.
All of them again would be similar with respect
to the domains.
They have only three constant domains and
the heavy chain.
Why do these subclasses differ from each other?
The difference lies mostly in the amino acid
sequence, which is in the hinge region and
a little bit in the FC region.
Therefore, one can have reagents or antibodies,
which can recognize IgG1, but not the others
and so on.
So, one can generate isotype-specific antibodies.
These are isotypes of IgG.
Something very interesting about the IgA molecule;
I will be coming to that a little later, but
IgA molecules are present mostly the dimeric
form, is present mostly in the body fluids.
Therefore, the molecule has to transcytose
travel across the epithelial barrier to go
on the other side.
How is this achieved?
Now, the transport of the IgA across epithelial
cells was discovered a little later; if I
remember correctly, it was sometime in the
early 90s.
Now, how does this happen, is what I will
tell you that now you have the plasma cell,
which synthesizes IgA.
IgA dimerises by the J chain.
Now, this allows accessibility to a region
on the FC portion of the IgA, one of the monomers,
to recognize or bind to a receptor, which
is also a member of the immunoglobulin super
family, which is known as poly-Ig receptor,
which is present on the basolateral surface
of the epithelial cells.
So, this is IgA synthesize.
The dimer now binds to the poly-immunoglobulin
receptor triggering receptor mediated endocytosis
of the entire complex in the vesicle now;
this is a vesicle, where there is cleavage
of the immunoglobulin receptor.
This is the site, where cleavage takes place.
And now, the lose end can wrap itself around
the entire molecule finding a binding site
on the other monomer of the dimer.
Now, this complex through receptor mediated
exocytosis is thrown out.
And, you have now, this molecule of the dimer
of IgA, which has the J chain and the secretory
component now.
The same poly immunoglobulin receptor on wrapping
itself around the dimer is known as secretory
IgA.
What does the secretory component do?
As the receptor helps the transcytosis and
after it is secreted, you have the wrapping
of this secretory chain, which is around quite
large, is about 7000 Dalton protein.
And, this can further add to the stability
of the dimer stabilizing the protein against
proteolytic degradation.
Like you to look more closely at the immunoglobulin
that is secreted; the IgA that is secreted.
You have the dimer formed by J chain and then
the secretory component.
Now, this is something quite interesting though
we know that in the serum is the IgG, which
constitutes major part of the immunoglobulin.
But, it is the IgA, which is secreted at the
rate of something like 5 to 15 grams in humans;
but, most of it is secreted or transcytosed
across epithelial barriers into body fluids.
Now, IgA constitutes only 10 to 15 percent
of the serum.
And, in the serum or blood, the protein is
present as a monomer; as an oligomer either
dimer or timer.
IgA is present in milk, saliva, tears, mucus
of the gastrointestinal tract as well as urogenital
tract as well as digestive tract.
Therefore, this in fact, protects the body
surfaces from pathogens.
Let us talk now about IgE.
When we think about IgE, we always think in
terms of the hypersensitivity reaction that
IgE mediates.
Now, that is a separate lecture.
But let us look at IgE, which is secreted
into the blood.
Average serum concentration of IgE is only
0.3 micro grams per ml, because as soon as
IgE, as soon as a memory cell now, upon activation
by the antigen, undergoes class switching.
And, if it is an allergen and the class switching
is to IgE, IgE synthesizes immediately captured
by specific high affinity receptors present
on mast cells; and, this is the picture of
the mast cell, basophils as well as eosinophils.
And, IgE may not have a very long half-life
in circulation.
But, present as bound to the FC receptors,
IgE molecules can be stable for weeks.
Cross linking of such molecules by the allergen
will induce cross linking of the FC receptors,
which are present on mast cells.
And, this leads to degranulation and release
of the preformed granules, stored granules
components of which are histamine and several
mediators, which we will again deal with later.
So, this is what IgE does, You might remember
that both IgE and IgM have four constant domains.
Now, this is a graph just to tell you that
the concentrations, the circulatory levels
of human Igs.
Now, there has been a little bit of a shift
of the writing here.
The first one is IgG; the x-axis is all the
immunoglobulins.
The first one is IgG; the green is IgM.
This is IgA 1, 2 - the monomer, the dimer;
this is IgD; and, this is IgE.
Now, the y axis says immunoglobulin levels
milligram per ml.
But, I would like you to look at the numbers;
we start with the highest number, which is
10 mg per ml, which goes down to 1, 0.1, 0.01.
So, this is increments of 10 fold.
So, you can see the concentration of IgE,
which is this as being not more than 3 micrograms
per ml; whereas, IgG in circulation is about
13 milligrams per ml.
So, IgG is maximum in the serum.
What are the properties of the different classes
of immunoglobulin?
I will just point out a few.
Now, this is a table that talks about the
molecular weight and the other properties
of the subclasses of immunoglobulin G, IgG1,
2, 3 and 4.
And, you can see that all of them are around
the same molecular size.
I should have in fact said around 150000.
The normal serum levels are IgG1 is maximum
9, 3, 1 and IgG4 is very low - 0.5 milligram
per ml.
The half life of IgG in circulation is 23
days.
This says days; I missed that.
Half life in vivo days - 23 except for IgG3;
now, you can immediately link this to the
presence of that very long hinge region, which
allows proteolytic degradation.
So, IgG3 has a stability of only 8 days.
Can all of them activate complement?
That is going to be my next lecture.
IgG3 of the classes can, but you will see
that IgM does that much better.
But, you can see IgG1 and 3 do; IgG3 does
that better; and, IgG4 does not activate the
complement.
Do all of them cross placenta to confer immunity
to the fetus - IgG scan, but IgG2 has minimal
ability to cross the placenta.
IgG1 and 3 can bind to the FC receptors on
macrophages.
This allows antibody-dependent - the antibody
recruiting the cells of the innate immune
response.
You may have studied thus in some of your
earlier classes.
None of them are present in body secretions;
none of them can induce mast cell degranulation,
which IgE can.
Now, properties of different classes of immunoglobulins
other than IgG, that is, IgA1, IgA2; this
is a monomer; this would be a dimer, trimer;
IgM, E and D. You can see that IgM being a
pentamer along with the J chain, has a molecular
size, which is around 900000; IgE because
of an extra constant domain has a molecular
size larger than IgA1 and IgD, which is 190000.
IgA1 is present in 3 milligrams per ml; IgM
half of that, 1.5; and, IgD as I have told
you several time, that IgD is mostly present
as cells of this receptor and almost no antibodies
in circulation, which represent the IgD class.
Half life in vivo - all of them have much
shorter half life than IgG, which I have told
you is 23 days.
These are only maximum 6 days and IgE is only
about 2.5 days in circulation.
But, remember on the mast cells, it is few
weeks.
Activation of complement - only IgM can activate
complement from this group; IgG can as I have
told you while referring to the earlier slide.
None of them can cross the placenta; only
IgG can.
Only IgM can bind to the FC receptors of the
macrophages or monocytes.
IgA and M can be present in body fluids; definitely,
the dimer form of IgA2.
And, except IgE, none of them can induce mast
cell granulation.
And, we have talked about the structure of
the immunoglobulins what are their functions.
Since we have been discussing so much with
respect to antigen binding and specificity,
obviously, that would be the first on the
list.
And, let us begin with the molecule as it
is made as the cells of its receptor.
Now, immunoglobulins are antigen receptors.
They are present on naive and memory cells;
and, they are associated with the immunoglobulin
alpha-immunoglobulin beta for signaling.
This signaling can happen only because the
antigen receptor can bind very specifically
to the cognate epitope on any antigen.
In the plasma cells, secrete antibodies; the
secreted forms of immunoglobulin can form
complexes with antigens.
And, these are very nicely cleared by phagocytes.
The secreted forms can activate complement
cascade.
And, like I said, my next lecture is going
to be that.
And, the complement activation is a very important
part of the immune system.
Again, one part of that complement cascade
recruits proteins, which otherwise would have
been proteins of the innate immune system,
which is now recruited to the acquired immune
system.
So, IgM can; then, IgG3, IgG1 and 2; these
can these molecules can activate the complement
cascade.
IgA2 can confer immunity in the body fluid
by transcytosis itself across the epithelial
membranes.
IgG1, 3 and 4 can travel trans-placentally
to confer maternal human immunity to the fetus.
The FC receptors - FC gamma RIA, which are
present on phagocytes or macrophages, now,
can bind to antibodies, which have inturn
bound to pathogens.
Now, once such a complex is bound to the FC
gamma RIA, this activates the macrophages
to internalize the pathogen.
Therefore, antibodies here by binding FC gamma
RIA, induce phagocytosis macrophages and granulocytes.
Antibodies can all also by similar method,
bind to NK cells.
And, now NK cells here in case of the phagocytes,
it is phagocytosis, which is enhanced in case
of NK cells.
Proxicity is increased by ADCC, which is antibody
dependent cellular cytotoxicity, because these
cells have FC receptors for IgG.
And, again by a similar mechanism, can now
bring the pathogen closer to the cells; the
linking would be through IgG.
I have already told you that IgE brings about
degranulation of mast cells, basophils and
eosinophils; thereby, again contributing to
the immune system or the humoral immunity.
Now, what I talked about with respect to antigen
receptors a little while ago is that antigen
receptors can recognize bind and internalize
antigen.
What I talked about only was recognition.
But, remember that binding of the antigen
triggers the cells to internalize the antigen,
where peptides are generated in the cell in
the lysosomal compartment.
And, these peptides can be presented on MHC
class II for activating T cells.
Thereby, antigen receptors are now allowing
B cells to become antigen presenting cells.
Can immunoglobulins themselves be antigens?
Yes, immunoglobulins are glycoproteins.
They are very complex structures as you have
seen so far.
And therefore, they themselves can evoke an
immune response by way of dictating B cells
to make antibodies to them.
So, what are the regions on the immunoglobulin
molecules, which can be antigenic?
Let us come to the isotypic determinants.
Now, if one takes let us say, IgG from human
and injects into mouse, antibodies will be
made to the isotypic determinants, which are
the determinants, which are present on mostly
the constant domain of the immunoglobulin.
Now, here the example was human immunoglobulin.
Therefore, those antigenic determinants that
are present on the constant domain on the
human immunoglobulin...
So, antibodies would be made by one species
to the immunoglobulins of another and though
would be called isotypic determinants.
And, you all may have used the secondary antibodies;
what we call secondary antibodies as reagents
in a LISA, western blotting, where we use
secondary antibodies are nothing but...
Let us say if you are using the primary antibodies
mouse, then you would be using rapid antibodies
to mouse immunoglobulins.
And therefore, those rapid antibodies would
be binding to such determinants.
What are allotypic determinants then?
Allotypic determinants are those that have
arisen during evolution.
Now, though we say that the constant domain
is exactly the same in all IgGs, let us say,
all IgG1 from the human species would be identical
yet, during evolution, there is every chance
that there could be one amino acid mutation.
Now, if those mutations 1 or 2 lie in antigenic
determinants, that determinant now would become
antigenic, which would be different from the
isotypic determinants.
Now, there are these allotypic determinants
that have been identified.
And, let me elaborate little further.
When would you see generation of an immune
response to allotypic determinants?
In blood transfusions; it is very apparent
that normally, we should not be making antibodies
even if we receive antibodies from other members
of our species.
But, in blood donation, people found that
there was a reaction or antibodies were made
by the recipient or the receiver from the
donors' immunoglobulins.
And, on closer look, these allotypic determinants
were identified.
What are idiotypic determinants?
Idiotypic determinants are those which are
present on the variable region.
Now, as is shown here, the idiotypic determinants
would be those that would bind to that region
or an immune response that is generated to
that variable region.
And in fact, which I have not mentioned earlier
that these idiotypic antibodies, which can
be generated, can also regulate the immune
response; not can too regulate the immune
response.
Idiotypic antibodies can be shown very easily
in an experiment; in fact, you can think of
doing that experiment; a rabbit, which is
injected with one antigen, you will see no
idiotypic antibodies made in the first two
exposure to the same antibody.
Now, let us say, the rabbit have received
ovalbumin; there will be antibodies to ovalbumin.
In the first generation, the primary dose,
the secondary immune response also will give
you mostly antibodies to ovalbumin.
But, let us say, from the third immunization
onwards, you may start getting antibodies
to the antibodies to ovalbumin.
And, these in fact are regulators of the immune
response not allowing the immune response
to And, there is a decrease in the amplitude
of the immune response.
So, antibodies to idiotypes can also have
several uses apart from the regular immune
response, which dictates the suppression to
some extent of response to the primary antigen.
We will come to that a little later.
So, just to go over the antigenic determinants
on immunoglobulins, we have isotypic determinants,
which define each heavy chain class and subclass
and each L chain type within a species; within
a species, each normal individual will express
all isotypes.
This is important.
However, allotypic determinants - some alleles
encode subtle amino acid differences in a
few member of a species.
Now, these are the ones that have been well-known;
and therefore, I have written them here.
In case of human, the gamma chain allotype,
there are about 25 such allotypic determinants.
And also on IgA, there are two; and kappa
light chain, there are 3.
Idiotypic determinants - I have already said
- are those determinants that are found because
of the presence of unique amino acid sequences
of the VH and VL.
We should also think about immunoglobulin
molecule as being a glycosylated protein or
a glycoprotein.
So, there has to be some role that glycans
play.
It was thought that since immunoglobulin,
when it is secreted in B cells, it is secreted
as heavy chain and light chain and these have
to come together.
And then, when they held together, disulfide
bonds have to be formed.
Now, these are exported outside either cells
of receptors or secreted as antibodies.
Therefore, the glycosylation function is only
to help the protein folding.
But, we know that the glycans also stabilize
the molecule protected from proteolytic attack.
The importance of the sugars have become known
as glycobiologists have started looking at
the glycans.
And, I would like to just mention here that
there are about 30 different glycoforms of
immunoglobulins; and, some of these glycoforms
are conserved; others are highly variable.
This glycans change, which I have told you
are present on the ch2 region of the FC region.
These molecules, the glycans are very important
for the immunoglobulin to bind to the FC.
I just told you about the importance of these
FC receptors.
And, it is the sugars, which allow this linking.
Decrease in the glycan structure now, does
not allow a tight binding of the immunoglobulin
with the FC receptors.
Also, the sequences of these oligosaccharides,
the sugars that are present have been shown
to inhibit an auto immune response; the auto
immune response, which might result in rheumatoid
arthritis.
Now, let us see how that happens.
There are people who have the condition of
G0 glycoform.
That is, they do not have those enzymes, which
transfer galactose, terminal galactose.
Therefore, in the absence of galactose, there
is exposure of N-acetylglucosamine.
Now, this allows binding to the MBL, which
is mannose binding lectin.
And, we will be discussing what MBL is in
the next class.
And, this MBL is a cascade of the complement
pathway, which can lead to inflammation and
one type of rheumatoid arthritis.
Now, what I would like to do is, stop here
and then begin my next lecture with two immunoglobulins
that I have not discussed with.
But, they are quite important; specially,
one of them, the single chain domain antibody,
which is known as camelid antibodies, because
this has helped molecular biologist to be
able to express only the variable domain of
the immunoglobulin.
And, these are known as single chain variable
fragment.
And, nature has done this in camels, where
30 percent of the antibodies in camels are
made up of only heavy chain.
So, I will stop here.
And, in my next class, I will start with the
introduction on the camelid antibodies as
well as the IgY antibodies.
Now, why IgY antibodies, which are from the
avian system?
Why this is important is, because they can
be used much in diagnostics.
Also, immunoglobulins made by births are deposited
in the egg yolk.
And therefore, it is much easier to be able
to purify antibodies form the egg yolk rather
than from the serum, because of the presence
of large amount of albumin.
More importantly, 3 grams 
of immunoglobulin Y can be got from eggs in
a single month from one hen.
So, I will talk about this as well as introduce
you all to the complement cascade.
Thank you.
