October 5, 2004

IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL

IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL ANTIBODIES Structure Sequence diversity Nature of reaction with Ag Different functional properties

If bacteria are injected into blood specific substances are found that exhibit different biologic properties Agglutinin - specifically clump only the bacteria used for immunization Opsonin - facilitate engulfment Antitoxin - neutralize toxin associated with the immunizing bacteria Cytolysis - lyse bacteria Precipitins - form flocculate precipitates when added to supernatant from the bacteria

If bacteria are injected into blood specific substances are found that exhibit different biologic properties Agglutinin - specifically clump only the bacteria used for immunization Opsonin - facilitate engulfment Antitoxin - neutralize toxin associated with the immunizing bacteria Cytolysis - lyse bacteria Precipitins - form flocculate precipitates when added to supernatant from the bacteria

Antibodies are found both as cell-associated receptors (BCR) and as circulating effector molecules

We will now consider the experiments that were done to determine antibody atructure

Immune serum from rabbits before and after the antibodies were removed by precipitation with Ag (ovalbumin)

The antibodies migrated as a  -globulin Showed they w

When purified antibodies (produced by dissolving immunoprecipiates) were analyzed in an ultracentrifuge they ran with a 7S sedimentation co-efficient indicating a m.w. of approx. 150,000

Two molecules of Ag were precipitated by each Ab

Digestion of antibodies with the enzyme papain yielded two fragments, the Fab (fragment antigen binding), and the Fc (fragment crystallizable) Ratio: 2 Fab and 1 Fc

If break disulfide bonds and separate on the basis of size you get two products

This is the structure of the fundamental building block of an Ab molecule. Some antibodies (such as IgG) are made of one of these building blocks. Others (IgA and IgM) are made of multiple copies of this basic building block.

One problem with further analysis was the heterogeneity of antibodies: note the narrow peak of the homogenous protein albumin compared to the broad peak of the Ig

It was a major breakthrough when it was realized that multiple myeloma is a tumor of plasma cells generally synthesizing a single species of Ig and that the myeloma proteins are homogeneous Abs produced by the plasma cells

Bence-Jones proteins are monoclonal (homogeneous) L chains in patients with multiple myeloma

Take two kappa (or lambda) lights chains from two different patients with multiple myeloma and produce proteolytic fragments

Take two kappa (or lambda) lights chains from two different patients with multiple myeloma, produce proteolytic fragments. Analyze the products by 2-D peptide map (chromatography in one dimension, electrophoresis in the other).

You find that about half of the cleavage products are identical between the two chains while the other half differ. The shaded spots indicate peptides shared by the two  or chains. Note the difference between  and. The conclusion is that the lights chains have a portion that is variable and a portion that is constant.

Amino acid sequence analysis shows that the variable portions is at one end of the light (and heavy) chains

More extensive analysis of the sequences of variable regions shows that they in fact contain relatively conserved regions and other regions which are hypervariable

More extensive analysis of the sequences of variable regions shows that they in fact contain relatively conserved regions and other regions which are hypervariable. There are 20 different amino acids. If all are found with equal frequency variability is 20/.05 = 400. If only one amino acid is found the variability is 1/1 =1/

There are 20 different amino acids. If all are found with equal frequency variability is 20/.05 = 400. If only one amino acid is found the variability is 1/1 =1/ Hypervariable regions are also called complementarity determining regions (CDRs). The prediction is that the CDRs contact antigen.

Hypervariable regions are also called complementarity determining regions (CDRs). The prediction is that the CDRs contact antigen. X-crystallography shows that the Ab molecule folds into compact domains. The CDRs are loops extending from the variable regions so that they are easily accessible for interaction with Ag. The other amino acids in the variable region are the “framework” amino acids and provide a scaffold to maintain the CDRs in the proper orientation.

Light chains have two domains: one variable and one constant Heavy chains have four or five domains, one of which is variable with the remains constant All heavy chains have at least one carbohydrate moiety attached

The forces are weak and operate at short distances Therefore the closer the approach (the better the fit) the stronger the interaction

Another characteristic of the antibody molecule is a flexible hinge

When a divalent hapten reacts with Abs, trimers, tetramers and other large complexes form. Because of the flexibility of the hinge region the angle between the arms of the Abs vary.

Digestion with papain removes the Fc

There are several different possible structure of the constant regions of the heavy chains

IgM H chain has four constant region domains and no hinge In addition to H and L also contains J chain (H 2 L 2 ) 5 J or (H 2 L 2 ) 6 Usually the first Ig made in response to antigen Increased valence leads to increased avidity

IgG Most abundant serum immunoglobulin

IgA Present in secretions where it provides protection at epithelial surfaces Frequently found as a polymer with J chain In secretions it also has a fourth chain, secretory component, a product of the epithelial cells

IgE Present at low concentrations Responsible for allergy Binds to receptors on mast cells

IgD Principle role seems to be as a membrane Ig

All isotypes can exist as both secreted and membrane bound antibodies