FUN2: 10:00-11:00 Scribe: Maggie Law
Tuesday, October 21, 2008 Proof: Kallie Law
Dr. Burrows Antibodies Page 6 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.
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.
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 1st 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