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FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 1 of 6
Specific Description of the Topic Being Discussed Today
I. Introduction: [S1 & S2]
Today will be about: antibodies and how they interact with antigens; the structure of antibodies, focusing on IgG
antibodies; classes and subclasses of antibodies and their biological functions; monoclonal and polyclonal antibodies.
II. Antibodies
a. [S3] Proteins that are made by our immune system to bind antigens
b. Unique in several ways—can exist in two different forms
i. On the B cell membrane
ii. As a secreted protein secreted by plasma cells that are the daughter progeny of the B cell
c. [S4] Everyday about 1 million new B cells are generated in the bone marrow.
d. B cells
i. Will have a slightly different antigen specificity—different receptor for antigens expressed on their
membrane.
ii. Will leave bone marrow and recirculate and go different secondary lymphoid organs like the spleen, lymph
nodes, tonsils, mucosal immune system and so forth.
iii. Circulate for a couple of weeks and then most of them die because they never run into their antigen.
iv. if you are making 1 million new B cells every day, you must get rid of some or you would be full of them
v. On the slide, B cells 1, 3, & 4 all die.
vi. B cell 2 interacts with its antigen (a virus) that induces the proliferation of the B cells, their differentiation
into plasma cells that secrete antibody, and also isotype switching
e. [S5- S6] Ig (immunoglobin) antibodies can exist either as an antigen receptor on a B cell or after interaction
with antigen, they can exist as soluble proteins secreted by plasma cells.
f. Membrane bound antibodies give the particular B cell its antigen specificity.
g. Each B cell has one unique Ig on its surface therefore the B cells are monospecific.
h. The secreted antibody can circulate in the blood and migrate into the tissues; particularly IgA can bathe the
mucosal secretions.
i. Antibodies can have various functions such as neutralization of viruses, eliminating pathogens, and preventing
uptake of antigens—antibodies have lots of different functions that are mostly based on what their isotype is.
III. Discovering Antibodies [S7]
a. Discovered by their ability to neutralize toxins back in the 1890’s, but nothing was known about what
antibodies were, just that there was something in the serum of humans or animals that had been immunized.
b. 50 years ago Tiselus and Kabat did an experiment where they hyper immunized rabbits.
i. Kept immunizing rabbit over and over again with the same antigen.
ii. Rabbit made a huge amount of antibodies.
iii. Wanted to figure out what was the nature of the antibody.
iv. Had developed technique of electrophoresis of the serum by this time
v. Put serum in a tube and apply an electric current and the different serum proteins would migrate either to
the positive or negative direction depending on their charge.
vi. The experiment hyper immunized rabbits—the blue line represents the serum from that hyperimmunized
rabbit.
vii. Major protein in serum is albumin—this is the major peak in the graph.
viii. Saw 3 more peaks which were called alpha, beta, and gamma globulins.
ix. Most of the antibody activity in the hyperimmunized rabbits was in the gamma globulin fraction—could tell
this because the serum with the antigen could be incubated and the antibodies could be pulled out—if you
do that you will see you will lose a lot of the gamma globin peak and will also lose antibody activity
x. Conclusion: gamma globin part of serum contained antibody activity—that’s why even today gamma globin
is used in reference to kids with antibody deficiencies because the kids get injected with gamma globin
from other people.
xi. Eventually it was called immunoglobin to indicate the fact that the gamma globins had immune activity.
xii. Gamma globin and immunoglobin are pretty much synonymous.
IV. Antibody Structure [S8]
a. Knew antibody structure had to be strange because you could show that you could make antibodies to just
about anything—so you could immunize a rabbit with any substance you could dig off the shelf—if done the
right way, you could make an antibody.
b. This immediately raised questions about how you could make so many different kinds of antibodies.
c. Were looking in particular at IgG antibodies because these are the main antibodies found in the serum,
especially if you hyper immunize an animal.
d. Edelmen and Porter eventually got the Nobel Prize for looking at antibody structure—they used a variety of
techniques to find out molecular weight of IgG was about 150,000.
FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 2 of 6
e. Biochemists were using lots of different enzymes at the time to cleave different proteins in order to see how
the enzyme cleavage affected the size of the protein.
f. Papain, an enzyme from papayas, was used to digest IgG antibodies—got two identical fragments of 45,000
called Fab= fragment antigen binding and one fragment called the Fc fragment= fragment crystalizable.
g. See pictures at the bottom of the slide to see what the digestion does and on [S9]
i. Papain digestion gives you 2 Fab fragments which could still bind antigen which is why they are called
fragment antigen binding & the Fc fragment is called this because if you let Fc sit in a tube, it will
crystallize—reason for this is because Fc is so homogeneous. This is a very homogeneous preparation of
proteins.
ii. Pepsin, from the stomach, cuts below the disulfide bond so now there is 2 antigen binding fragments still
held together by disulfide bonds—the rest of the molecule is chewed up because pepsin is so powerful.
iii. Did other experiments to show that there were 2 heavy chains and 2 light chains.
h. Eventually the structure of IgG was discovered.
i. [S9] If you digest with Papain, you cut the antigen binding arms off above the disulfide bond so they become
single antigen binding fragments.
j. If you do pepsin digestion, you get the dimer of antigen binding fragments.
V. Heavy and Light Chains [S10]
a. The next important thing was to discover was how you could combine so many different antigens.
b. Amino acid sequencing of these proteins was done.
c. This was done by looking at the protein’s immunoglobin secreted by myeloma cells—myeloma is a tumor of
plasma cells, a tumor of the antibody secreting cells. People with multiple myeloma have huge numbers of
plasma cells in their bone marrow and all are derived from single plasma cell and all secrete the same
antibody; antibody can be isolated and will pretty much be a monoclonal protein, and all the proteins will be
identical
d. The light and the heavy chains were sequenced.
i. The amino terminal of the light chain was variable and the constant region of the light chain was constant,
except for the fact that there are two different light chains, kappa and lambda.
ii. Light chain can be either kappa or lambda and will have a constant and a variable region.
iii. Heavy chains were much bigger and took longer to sequence.
iv. One end of the heavy chain was highly variable and the rest was constant expect for the fact that there are
different antibody subclasses—mu, gamma, alpha, delta, and epsilon.
v. The constant regions of the heavy chains correspond to the isotypes.
vi. The variable regions or “ends” of the molecules are what would be involved in binding antigen because
each different antibody has a different variable region and each different antibody could bind to different
antigens.
e. [S11] In this IgG molecule, there is a gamma heavy chain that would have either a kappa or a lambda light
chain.
f. Remember in each antibody molecule the light chains are identical and the heavy chains are identical and
each B cell only makes one kind of immunoglobin.
g. This defines the monospecificity of the immune system.
h. [S12] Noticed that after sequencing and gathering up hundreds of the myeloma proteins that the amino acid
sequences of the variable regions of the heavy chains could be aligned with the variable regions of the light
chains.
i. Align all the sequences and look at each amino acid position and say “How much variability is there there?”—
that’s what these plots represent.
j. The y axis is the extent of variability and the x axis is each residue number.
k. Even though it’s called a variable region, there are certain “hot spots” of variability.
l. Some regions are pretty unvariable and other regions are highly variable.
m. The highly variable regions are called “hypervariable regions” (HV).
n. HV are the regions that are actually involved in directly binding to antigen.
o. The HV regions are all spread out on the molecule, how can they be involved in antigen binding? – remember
that when a protein folds up, the HV regions are actually going to come together and make up the antigen
binding site.
p. The HV regions are what binds antigen.
q. [S13] 3-D picture of a crystallographic structure of part of an antibody molecule.
i. Variable regions of the heavy and light chains are shown
ii. CH1 and CL domains are shown.
iii. The antigen is a particular antigen called lysozyme which is a small protein about 12000 MW.
iv. The antibody is binding to a particular sort of “bump” on the surface of the lysozyme.
FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 3 of 6
v. This is what is called the antigenic determinant or epitope.
vi. There are other bumps and grooves on the surface of the protein—one would imagine that you could make
other antibodies if you kicked that one off to recognize other parts of the molecule.
vii. Each individual antigen, particularly protein antigens are going to have multiple epitopes that can be
recognized by different antibodies.
VI. Immunoglobins [S14]
a. The variable region is the “business end” in terms of binding antigens.
b. Many antibody classes have a hinge region—on the previous slide [S13], the antibody molecule folds up into
these variable and constant domains—in between the different domains you can have an extended region
which is called the hinge region and tends to be enriched in proline amino acids.
c. The hinge region allows flexibility in the arms of the antibody molecule so the arms of the antibody molecule
can actually reach out and grab different epitopes—not stiff and rigid structures—can reach out and grab
things.
d. Hinge region is exposed and can also make that region of the molecule susceptible to proteolytic enzymes—in
the old experiments shown earlier the lecture, the enzymes (pepsin and Papain) would cut within the hinge
region, either above or below the disulfide bonds.
e. [S15] Immunoglobin Classes or Isotypes
i. 5 classes in humans
ii. Distinguished by having unique sequences in the heavy chain constant region—each individual antibody
class has a particular heavy chain and can use either a kappa or a lambda light chain.
iii. Important thing about the different classes—they have different functions due to different heavy chain
constant regions.
VII. Immunoglobin G (IgG) [S16]
a. 4 subclasses of IgG, but don’t worry about them, just IgG in general
b. Most abundant Ig (immunoglobin) in serum---in humans it is about 10 mg/ml, which is a lot of protein
c. Monomer with 2 gamma heavy chains and either a kappa or a lambda light chain.
d. From the cartoons, the major differences between the subclasses are the length of the hinge region—ex. IgG
3 has a very long hinge region.
e. Don’t need to know the different details of the subclasses, just that they have functional properties—ex. Some
of them can cross placenta so mother has receptors for these Igs and can cross placenta and transfer into
circulation of the fetus—fetus will have same antibodies as the mother at least in the subclasses that can cross
the placenta.
f. Some of them are more effective at activating complement, which is a system of proteins and some bind better
to Fc receptors.
g. Again, IgG is the most abundant in serum and we will later talk about mucosal surfaces in which IgA is the
most abundant antibody and they have different functions.
VIII. Immunoglobin M (IgM) [S17]
a. Huge molecule of about 1,000,000 MW because it’s a pentamer of the individual subunits. (IgG = 160,000
MW)
b. In serum, it’s about 1 mg/ml, about 10% of IgG is.
c. The five monomers give rise to 10 antigen binding sites; IgG only has 2.
d. Unique feature: polypeptide J chain which is involved in holding the Ig together in the proper way to make a
pentamer rather than hexamers; J chain is required for correct polymerization.
e. If you immunize somebody with tetanus and look with time that the antibodies that are being made, the 1 st
antibody in the so-called primary immune response will be IgM and then later on you get isotype switching and
you make IgG antibodies.
f. IgM is so big that it pretty much has to stay in circulation; IgG, when it gets into the capillaries, can leak out
into the tissues and protect you there from pathogens. IgM has a problem with that because it’s so large
g. Primary immunodeficiency in humans called hyper IgM immunodeficiency—how can you be immunodeficient if
you make IgM?
i. It’s hyper IgM—more IgM than normal is made. Because of this, IgG can’t be made and people will this
condition get lots of infections in their skin for example because the IgM antibodies can’t get out of the
circulation to neutralize those bacteria—leads to significant problems
IX. Immunoglobin A (IgA) [S18,19,20]
a. Weird class of antibody for many reasons.
b. It is a dimer, held together by J-chains just like IgM.
c. It is 1mg/mL (serum level) and it is most important in mucosal secretions like in intestines, eyes, and the oral
cavity.
d. In the oral cavity, the predominant Ig class is IgA.
FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 4 of 6
e. In humans, if you look in serum, it is not a dimer, it is actually a monomer.
f. In secretions, it is a dimer. If you look at the amount of Ig made every day, IgA is the made the most. If you
look in serum, IgG has the highest concentration.
i. Reason for this is because you are dumping much of your IgA into your intestine. It does not go into
serum; it goes into intestine.
g. IgA is strange because of how it enters your intestine.
h. Cartoon, image [S19] shows the lumen of the intestine where all the bacteria are and where all the pathogens
can get in.
i. Structure here in the intestine is called a Peyer’s patch. If you get this nasty bug coming across your
intestine (referring to image), it will get into this Peyer's patch and that is where you will generate an
immune response.
ii. You will have B cells in there that are specific for these bacteria and they start to divide and make
antibodies.
iii. In here is where the immune response is taking place in these Peyer’s patches. The cells divide,
differentiate into plasma cells, and these plasma cells migrate underneath the epithelial cells in the intestine
or in your oral cavity.
iv. These cells then secrete IgA and it gets into the lumen.
v. Cartoon from the book just shows an arrow of the antibodies going from under here to over there, but in
fact there are cells in the way here. You can’t just squirt right through the cell; it is complicated mechanism
to get the IgA made by the plasma cell out to where it will do its job.
i. States about [S20]: don’t read all this text but here is this IgA secreting plasma cell.
j. It is sitting underneath the epithelial cells that line the intestine and it is secreting IgA.
k. IgA is a dimer with a J-chain. On the basal lateral surface of these epithelial cells there is something called the
polymeric Ig receptor (pIgR)
l. As the name implies (pIgR), it binds polymeric immunoglobins which normally is IgA.
m. The J-chain part of the dimeric IgA binds to the pIgR.
i. Where it wants to go is out here where the bugs are (image of [S20]).
ii. What happens is that the receptor gets internalized and transported through the cell and then actually a
little bit of it is cleaved off and you now have secretory IgA.
n. Secretory IgA is made up of dimeric IgA secreted by the plasma cells. Most of the pIgR transported the IgA
across the cells.
o. The IgA in your mucosal secretions is the product of 2 different cells types – the plasma cell that makes the
antibody and the epithelial cell that makes the pIgR.
p. The importance of this is thought to be – remember we said that some of these antibodies are sensitive to
proteases – well, there are a lot of proteases in your GI tract.
q. The secretory component as it is now called is thought to protect the IgA from being degraded.
r. Again, secretory IgA is the main effector antibody in mucosal secretions.
s. There are people with IgA deficiency so they cannot make IgA.
i. What other isotope could you use in that case?
ii. This is called a pIgR, so what else could you use?
iii. Another polymeric immunoglobin – use IgM. The other polymeric immunoglobin is IgM. In that case, some
of these patients actually transport IgM across the intestine rather than IgA.
X. IgE [S21 & S22]
a. IgE- probably heard about it if you have heard about allergies because IgE is the bad immunoglobin that is
involved in things like asthma, hives, hay fever and anaphylactic shock which you can go into if you are
allergic to something like bee venom and you get stung by a bee.
b. In serum it is really low, barely detectable but most of where it does its job is where it binds to mast cells and
basophils.
c. Mast cells and basophils are the cells that give you the symptoms if you have hay fever or some other allergy.
They work by degranulating and squirting histamine, which is why you take antihistamines if you have
allergies.
d. Way this works – you have basophils, mast cells and they have so called “Fc” receptors for IgE antibodies.
e. IgE produced goes immediately to basophils or mast cells and binds there to the Fc receptor. It is binding by
the constant region of the antibody molecule. (Essentially this sits here and nothing happens.)
f. If the allergen or antigen comes in and cross links those IgE molecules that induces all the bad things that are
going to happen.
g. Mast cells have granules that are filled up with histamine and other stuff. When you crosslink these IgE
molecules, it induces degranulation, release of histamine and all the other things that make you sneeze and
feel bad.
FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 5 of 6
h. May wonder why we have IgE if it is doing this kind of thing--supposedly it is important in the elimination of
parasites, particularly from intestine because some of the mediators released by the mast cells induce smooth
muscle contraction.
i. If you imagine the intestine is lined by smooth muscle and if you induce contraction, you squirt out the
parasites.
ii. That is a thought for what the original protective function of IgE--protect against parasite.
iii. In developed countries we do not have very many parasites so our immune system, for lack of anything
else to do, responds to allergens in those people susceptible to getting allergies.
XI. IgD [S23]
a. Still a mysterious antibody class.
b. In serum, it is lower than IgE but if you look on B cells (ex. B cells from blood or spleen), they will have IgM on
the surface and also IgD.
c. Those 2 antibodies will have identical variable regions so they will bind the same antigen.
d. It has been thought that IgD is involved in activating B cells but in mice you can mutate the mouse so he
doesn’t have IgD anymore and there is no obvious problem with the mice in terms of making antibodies.
e. It is still not clear what the function of IgD is. If you immunize people or animals, you cannot find very many
plasma cells making IgD.
i. Not a major antibody for any antigen, but it is a receptor on mature B cells.
f. Question: You said that B cells only express one type of antibody?
g. Answer: I was lying. They do, except for IgM and IgD. They are the same in the sense that they both have
the exact same variable regions. They bind the same antigen. B cells can express two antibody classes.
Plasma cells are totally restricted to one class of antibody.
XII. Properties and biologic activities of Ig isotopes [S24]
a. This next table is not to be memorized.
b. It summarizes the idea that different classes of antibodies have different functions.
c. Just for examples--they are different sizes of course, depending on how many different monomers they are
composed of.
d. Some activate complement better than others, some cross the placenta better than others, some bind Fc
receptors better than others.
e. Don’t be concerned about which particular subclass of IgG binds Fc receptors; it is just to remind you that
these have different biological activities and that is the important point.
XIII. Polyclonal and Monoclonal antibodies [S25,S26]
a. New subject – polyclonal and monoclonal antibodies.
b. This is the picture we looked at earlier–-we have this antigen lysozyme that has, in this case, antibody binding
to this particular epitope.
c. There could be multiple epitomes. If you immunize a mouse or a human with lysozyme, you are going to
induce activation of multiple B cell clones.
d. You are going to induce the activation of the B cell with this receptor on the surface, some other B cell might
recognize that part of the protein and you are going to induce a polyclonal response.
e. That means that is basically a mixture of antibodies.
f. There is a polyclonal response that is heterogeneous – it is good for us because it induces a better protective
immune response.
g. Think about it--if instead of a lysozyme it was some virus and we could only make a particular antibody to a
particular epitope.
i. The virus, after a while, is trying to get away from the immune system so it makes a mutation. It does not
have that epitope anymore. We are in trouble because we can’t make antibodies to this virus.
ii. That is why a heterogeneous polyclonal antibody response is good because even if the virus mutates and
gets rid of that epitope we are going to have other epitopes present to that we can make antibody too.
h. That is good for us, but antibodies are used in a lot of research and diagnostic and even theraputic purposes
and there you want something highly specific so you know exactly what it is recognizing.
i. You don’t have to worry about cross reacting with other antigens and so forth.
j. This would be called a monoclonal antibody response.
k. [S27]If you immunize a rabbit with some sort of antigen and then took its serum out, it is tough to purify
monoclonal antibodies from that serum.
i. Kohler and Milstein developed a method that they got the Nobel prize for by making monoclonal antibodies.
ii. Called making hybridomas.
iii. If you take a normal B cell out of a person or mouse and put it in a tissue culture, within a couple of days it
will die because it is lacking things it needs to survive; can’t really isolate individual plasma cells from an
immunized animal and isolate the secreted antibody.
FUN2: 10:00-11:00
Scribe: Maggie Law
Tuesday, October 21, 2008
Proof: Kallie Law
Dr. Burrows
Antibodies
Page 6 of 6
iv. What they did was take a myeloma cell (a cancerous plasma cell) and they fused that together with normal
B cells.
v. What comes out of there – the B cell gets immortality from the cancerous plasma cell and the plasma cell
gets the antibody secreting capacity of that B cell.
vi. These plasma cells used to make hybridomas don’t make any of their own immunoglobin.
vii. You have an immunoglobin negative plasma cell, a normal B cell, fuse them together and you get a cell out
of that which is immortal.
viii. Now it is going to take and make whatever antibody that B cell is programmed to make.
ix. [S28] This is a cartoon depicting that
x. Here we have a mouse that we are going to immunize with this antigen that has four epitopes.
xi. If we immunize this mouse a couple of times and get a serum later, it is going to be a mixture of antibodies
for each one of these epitopes.
xii. Like I said, if this is a virus, that is good for the mouse because he has polyclonal antibody response and
will get good protection against the virus.
xiii. But if we want to use antibodies in some sort of clinical assay, we would like to get antibodies that
recognize epitope 1,2,3 and 4 and so on.
xiv. What you do is take spleen cells from the immunized mouse and the plasma cells are fused with the
myeloma cells and you get cells that have constituents of both cells – so called hybridoma’s.
xv. Then you can select for the ones that make the antibody you want and pretty soon you have a reagent.
xvi. You have cells that are cranking out a huge amount of a particular antibody to a particular epitope.
xvii. Have to select against the myeloma cells that are in there and haven’t fused because they are otherwise
not going to make any antibody and be of any use.
xviii. End product of all this is the production of monoclonal antibodies.
XIV.
Summary [S29, S30, S31]
a. It is the gamma globin fraction of serum that contains immunoglobins.
b. Immunoglobins have 2 identical heavy and 2 identical light chains. You do not have mixed up antibody
molecules.
c. They have variable regions which are involved in binding antigens and their constant regions which define the
heavy chain isotype.
d. Mu for IgM, Delta for IgD,IgG, IgA, and IgE.
e. They make the antigen binding sites from the hypervariable regions and the isotype is defined by the heavy
chain.
f. Each antibody molecule does not have 2 light chains—it either has a kappa or a lambda light chain.
g. Some of these molecules have hinge regions—they promote flexibility of the protein.
h. IgG is the most abundant immunoglobin.
i. IgM is the primary immune response.
j. IgA is the major isotype in mucosal secretions and it has that complicated method of getting across epithelial
cells using the pIgR.
k. IgE is important in asthma, hay fever, and other hypersensitivity reactions.
l. Also talked about polyclonal and monoclonal antibodies.