This module will help you

• learn antibody structure.
• see how antibody structure and function are related.
• test your knowledge and immunology problem-solving skills.

Molecular Structure
Biological Functions

Molecular structure

Antibodies are plasma glycoproteins, called gamma globulins because of their mobility in an electric field and immunoglobulin (Ig) because of their role in immunity. Antibodies were initially characterized using myeloma proteins, homogenous antibodies produced by cancerous plasma cells in individuals with multiple myeloma. Antibodies which are identical with each other at every amino acid (because they have all been produced by the descendants of a single B cell) are called monoclonal antibodies (mAb). Myeloma proteins are naturally occurring monoclonal antibodies, because the myeloma develops from a single cancerous plasma cell (a clonal tumor). Monoclonal antibodies can also be produced in the lab (see ToolBox Designer Antibodies). Serum antibodies are polyclonal antibodies, because they are produced by the descendants of several B cells that recognize different epitopes on the same antigen.

All antibodies share a basic structure; IgG molecules closely resemble this Ig prototype. Each antibody "monomer" has a molecular weight of approximately 150,000 Daltons (150 KD) and is composed of two identical heavy (H) polypeptide chains and two identical light (L) chains, covalently bonded via interchain disulfide (S-S) linkages between cysteine residues. Each H chain is about 440 amino acids long; each L chain is about 220 amino acids long. H and L chains each contain intrachain disulfide bonds which stabilize their folding into 110-amino acid domains. Immunoglobulin domains are a common feature of many soluble molecules and membrane-bound receptors of the immune system, comprising the Ig superfamily.

Sequence analysis of many myeloma proteins showed that all antibodies have one of two kinds of L chain, kappa (k) or lambda (l); each antibody has two identical k chains or two identical l chains. Five different H chains have been found: alpha (a), gamma (g) , delta (d), epsilon (e), and mu (m). Antibody isotypes (classes) are named IgA, IgG, IgD, IgE, and IgM to correspond to their H chain types, which influence the effector functions of the antibody molecules.

The amino acid sequence of the H and L amino terminal domains vary considerably from one Ig to the next and are responsible for the antigen-binding diversity of antibodies; these make up the variable regions, V_H and V_L. Each light chain has one V_L domain and each heavy chain has one V_H domain. One V_H and one V_L fold together to form an antigen-binding site, so each Ig molecule has two identical antigen-binding sites. The amino acid sequence of the carboxyl half of the L chain and three-fourths of the H chain show relatively limited variability, and make up the constant regions (C_H and C_L). Each light chain has one C_L domain, and each heavy chain has three (a, g, and d chains) or four (e and m chains) C_H domains. The hinge region is a more extended region (not folded into a domain) between H chain C_H1 and C_H2 that is present in a, g, and d chains. It allows the two antigen-binding regions of each antibody molecule to move independently to bind antigen.

Limited proteolytic digestion with papain cleaves the Ig prototype into three fragments. Two identical amino terminal fragments, each containing one entire L chain and about half an H chain, are the antigen binding fragments (Fab). The third fragment, similar in size but containing the carboxyl terminal half of both H chains with their interchain disulfide bond, is the crystalizable fragment (Fc). The Fc contains carbohydrates, complement-binding, and FcR-binding sites. Limited pepsin digestion yields a single F(ab')₂ fragment containing both Fab pieces and the hinge region, including the H-H interchain disulfide bond. F(ab')₂ is divalent for antigen binding. Pepsin digests the carboxyl halves of the H chains.

Within V_H and V_L there are hypervariable regions which show the most sequence variability from one antibody to another and framework regions which are less variable. Folding brings the hypervariable regions together to form the antigen-binding pockets. These sites of closest contact between antibody and antigen are the complementarity determining regions (CDR) of the antibody.

The antigen-binding site of a typical antibody is a cleft formed by folded VH and VL regions. It can accommodate approximately four to seven amino acids or sugar residues. Contact between large antigens and antibodies probably extends along the surface of the antibody outside as well as inside the antigen-binding pocket. Antigen-binding specificity of antibody resembles that of enzyme binding substrate. Antibodies bind their specific antigens using hydrogen bonds, ionic bonds, hydrophobic interactions, and Van der Waals interactions. Covalent bonds are not formed between antigen and antibody, so binding is reversible.

An epitope is the portion of an antigen bound by an antibody. Viral capsid proteins and bacterial cell wall components usually have multiple epitopes. Antibody is produced to each epitope in relation to its immunogenicity; epitopes to which the most antibody is produced are called immunodominant epitopes. Antibodies distinguish antigenic differences (serotypes) between members of the same bacterial or viral species. Some epitopes are shared by different antigens, so that antibody made to one also binds the other (is cross-reactive). Cross-reactive antibodies are one mechanism by which autoimmunity is induced. For example, antibodies made by some people to Streptococcus pyogenes bind a cross-reactive antigen on their heart valves and cause rheumatic heart disease.

The strength of the interaction between a single antigen-binding site on the antibody and its specific antigen epitope is called the binding affinity of the antibody. The higher the affinity, the tighter the association between antigen and antibody, and the more likely the antigen is to remain in the binding site. Antibody affinity generally increases with repeated exposure to antigen because B cells with higher affinity antigen receptors are selected to produce larger clones of antibody-secreting plasma cells.

The affinity constant Ka is the ratio between the rate constants for binding and dissociation of antibody and antigen. Typical affinities for IgG antibodies are 10⁵-10⁹ L/mole. Antibody affinity is measured by equilibrium dialysis.  The relationship between bound and free antigen and antibody affinity is expressed by the Scatchard equation, r/c = Kn - Kr, where r = the ratio of [bound antigen] to [total antibody], c = [free antigen], K = affinity, and n = number of binding sites per antibody molecule (valence). If all the antibodies have the same affinity for antigen (monoclonal antibody), a plot of r/c versus r will yield a straight line with a slope of -K and an r intercept approaching n. If the antibody is heterogeneous (polyclonal), the plot of r/c versus r will yield a curved line; the average affinity can be determined by the slope of the curve when half the binding sites are full (r=1).

IgG, IgD, IgE, and "monomeric" IgA have two identical antigen-binding sites (valence = 2). Dimeric IgA has four. Serum IgM has ten, although the observed valence of IgM is five because all the binding sites cannot make contact with antigen simultaneously due to steric hindrance. Avidity is the functional affinity of multivalent antigen binding to multivalent antibody molecules. Avidity strengthens binding to antigens with repeating identical epitopes. The more antigen-binding sites an individual antibody molecule has, the higher its avidity for antigen. Cross-linking antibody by binding two different Ig molecules to the same antigen, common with pathogens which have many copies of the same epitopes on their surface, is crucial for activating both complement and B cells.

Antibodies are named by the species from which they were obtained and the antigen to which they were produced. For example, human anti-cholera O129 is antibody from a human who produced it against Vibrio cholerae O antigen (LPS) serotype 129. Mouse anti-human g chain is mouse antibody specific for g chain of human IgG. Antibodies are assumed to be polyclonal unless otherwise specified. Mouse monoclonal anti-human CD4 would be a monoclonal antibody specific for the CD4 antigen on human helper T cells.

Immunoglobulins can be used as antigens to generate antibodies that distinguish several Ig epitopes: isotypes, allotypes, and idiotypes. Anti-isotype sera differentiate epitopes in the constant regions of H and L chains. All members of a species share the same isotypes. For example, anti-isotype made against one cloned human m chain would bind to all human m chains. Humans have four subisotypes of g chain ( g₁- g₄) and two subisotypes of a chain. Subisotypes differ in amino acid sequence and biological functions but are more closely related to each other than to other isotypes.

Within a species there is some variation in amino acid sequence within an isotype or subisotype; these differentiate Ig allotypes. Allotypic epitopes are in C_H and C_L. They have been identified in all four human subisotypes of g, in a₂, and in k chain and are called Gm, Am, and Km. Each individual B cell or plasma cell produces antibody of a single allotype (allotypic exclusion), and both H chains or both L chains of an individual antibody molecule have the same allotype. Each person has antibodies with one (homozygous) or two (heterozygous) allotypes, depending on whether they inherited the same or different allotypes from each parent. For example, a person who inherited GM1 from one parent and GM3 from the other would have B cells making either GM1 or GM3 H chains, but their serum would contain roughly equal numbers of IgG molecules with each allotype.

Idiotypic epitopes are due to sequence differences within V_H and V_L, so each individual makes antibodies with many idiotypes. Monoclonal antibody molecules share the same idiotype. Polyclonal antibodies, even those made against the same epitope, may have different idiotypes. Anti-idiotype antibody and antigen usually compete for the antigen-binding region of the Ig.

Biological Functions

Antibody is synthesized on membrane-bound polyribosomes (rough endoplasmic reticulum, RER) in the cytoplasm of the B cell or plasma cell. A signal recognition protein attached to the H and L chain leader sequences sends the chains into the endoplasmic reticulum (ER). H and L chains assemble into H₂L₂ monomers with formation of the interchain disulfide bonds; carbohydrate is added to the C_H regions. The vesicle containing antibody moves via the Golgi apparatus to the plasma membrane and exocytosis releases secreted antibody from the plasma cell. Membrane-bound antibody has an additional transmembrane sequence on its carboxyl terminal C_H region which anchors the molecule to the lipid bilayer.

Pick the one BEST answer for each question by clicking on the letter of the correct answer.

1. An antibody Fab contains

a. complementarity determining regions.
b. H and L chain variable regions.
c. one antigen binding region.
d. one H-L interchain disulfide bond.
e. all of the above.

2. Myeloma proteins are

a. abnormally formed antibodies secreted from cancerous plasma cells.
b. cancerous plasma cells that divide without requiring antigen activation.
c. cell lines that secrete specific antibodies for a short time, then die.
d. homogeneous antibody molecules secreted by plasma cell tumors.
e. protein signaling molecules that make a plasma cell become a multiple myeloma.

3. The regions of the antibody molecule which contribute MOST to the affinity of the antibody for antigen are the

a. CDR.
b. Fab regions.
c. Fc regions.
d. framework regions.
e. hinge regions.

4. Antibody Fc fragments contain

a. antigen-binding sites.
b. CDR.
c. complement-binding sites.
d. framework residues.
e. light chain variable domains.

5. The immunoglobulin isotype is determined by the

a. antigen specificity.
b. H chain constant region.
c. L chain variable region.
d. number of antigen-binding sites.
e. number of V_H domains.

6. Which statement about antigen epitopes is FALSE?

a. An epitope may be shared by two different antigens.
b. A protein molecule usually contains multiple epitopes.
c. B cells bind only processed antigen epitopes.
d. Epitopes may be linear (composed of sequential amino acids) or assembled by protein folding from amino acids far apart in the protein primary amino acid sequence.
e. Some epitopes are more immunogenic than others.

7. An example of an antigen epitope from an infectious organism would be

a. a bacterial endotoxin (LPS) molecule.
b. a fungal cell wall protein.
c. a peptide on the surface of a virus capsid protein.
d. a whole virus.
e. All of the above are antigen epitopes.

8. Antibody affinity for antigen depends on

a. the antibody isotype.
b. the complementary shape and charge of each antibody V region for its antigen epitope.
c. the number of Fab regions in each antibody molecule.
d. whether the antibody is in the serum or on the cell surface.
e. whether the light chains are kappa or lambda.

9. Avidity

a. is a pathogenic agent, causing a very serious disease.
b. occurs when the ratio of antibody to antigen is optimal.
c. refers to the strength of interactions between a multivalent antibody and a multivalent antigen.
d. results in a loss of antibody reactivity.
e. results in cross-reactivity when antibody binds two different antigens.

10. A colleague sends you an antibody to polio virus capsid protein. You perform equilibrium dialysis on the antibody to measure its affinity. Plotting r/c versus r gives you a curved line with K= 2.5 X 10⁸ L/mole and an r intercept of 4. From these results, you conclude that the antibody is probably

a. a cross-reactive antibody.
b. a monoclonal anti-polio virus antibody.
c. a polyclonal IgG antibody.
d. IgA anti-polio virus.
e. not specific for polio virus.

11. Allotypic determinants are

a. constant region determinants that distinguish each Ig class and subclass within a species.
b. expressed only from the paternal chromosome.
c. generated by the conformation of antigen-specific V_H and V_L sequences.
d. Not immunogenic in individuals who do not have that allotype.
e. amino acid differences encoded by different alleles for the same H or L chain locus.

12. Which of the following is NOT a characteristic of IgG?

a. It contains 2 g and 2 L chains
b. It crosses the placenta.
c. It is the predominant immunoglobulin in blood, lymph, and peritoneal fluid.
d. It is the largest of all the Igs.
e. Its L chains are either k or l.

13. Human serum IgA is isolated and injected into a rabbit. The rabbit anti-IgA antibodies will react against all of the following EXCEPT human

a. a chain.
b. IgG.
c. k chain.
d. l chain.
e. secretory component.

14. You have purified some Fab from an IgG myeloma protein. Under appropriate conditions, you could use this Fab to generate antibodies to

a. both k and l chain.
b. g chain hinge region.
c. J chain.
d. g chain allotypic determinants.
e. the idiotype of this myeloma.

15. The Ig isotype which would be most important for neutralizing polio virus before it could infect intestinal cells would be

a. secretory IgA.
b. serum IgA.
c. serum IgD.
d. serum IgG.
e. membrane IgM.

16. Which of the following changes to a serum IgM antibody molecule would definitely DECREASE its avidity?

a. Increase noncovalent antigen-antibody interactions in the CDR.
b. Remove the secretory component.
c. Replace the Fc portion of the mu chains with the Fc portion of alpha chains.
d. Replace V_H and V_L framework regions with those from a different antibody.
e. Use limited enzyme digestion to make Fab fragments.

17. IgA can be secreted from the body because it

a. binds poly-Ig receptor on mucosal epithelial cells.
b. has a specialized H chain called secretory chain.
c. has a special secretory idiotype.
d. is small enough to pass between mucosal epithelial cells and leave the body.
e. is synthesized by mucosal epithelial cells and secreted directly into the intestinal lumen.

18. The ability to make antibody with the same antigen specificity but different Fc regions

a. causes allelic exclusion of Ig molecules.
b. does not occur against bacterial antigens.
c. improves the antigen binding specificity of an Ig molecule.
d. increases the effector functions of Ig molecules.
e. requires clonal elimination.

19. Allergy symptoms are produced when antigen binds to IgE on FcR on

a. A cells.
b. macrophages.
c. mast cells.
d. neutrophils.
e. Th1 cells.

20. One amino acid difference in the Fc region of different human g chains is the epitope recognized by anti-

a. allotype.
b. idiotype.
c. isotype.
d. IgG.
e. g chain.

Problems

1. Decide if the antibodies listed on the left will bind to the immunoglobulins (antigens) listed on the right. If they do bind, think about what parts of the immunoglobulins are bound (V_H, V_L, C_H, C_L).

2. You inject a rabbit on several occasions with pure human serum albumin (HSA), collect some blood from the rabbit and allow it to clot. The yellowish liquid remaining is your antiserum.

a. Why did you give several injections of antigen? How long would you wait after the last one before collecting blood?

b. Are all the molecules in the antiserum antibodies? Are all the antibody molecules in the serum specific for HSA? How can you check your answer?

c. How can you separate the anti-HSA from the other molecules (purify it)?

After purification, you measure the binding of your antiserum to human serum albumin (HSA), human immunoglobulin (HIg), rabbit serum albumin (RSA), and chimpanzee serum albumin (CSA). The results are shown in the table below:

d. Why should an antibody to HSA bind CSA? What can you deduce about the structures of these four molecules from the binding data?

e. From amino acid sequencing, HSA and CSA are about 90% homologous (have the same amino acids at about 90% of the sequence) and HSA and RSA are about 45% homologous. Why does the antiserum show so little binding to RSA? Are all epitopes of an antigen equally immunogenic? Predict the results if the antiserum had been made in a chimp.

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