Structure of antibodies

At a glance

1. An antibody, or immunoglobulin, is the B cell's antigen receptor. A B cell carries it on its surface; a plasma cell, the B cell's final form, secretes it.
2. The basic unit is four chains, two identical heavy and two identical light, held by disulfide bonds into a Y of about 150 kDa. A light chain is κ or λ, never both in one molecule.
3. Every chain is built of domains of about 110 amino acids with the same fold. The first domain of each chain is variable (V), the rest constant (C).
4. Three hypervariable loops of VH and three of VL, the complementarity-determining regions, form one antigen-binding site at the tip of each arm. Each unit has two identical sites.
5. Papain cuts an IgG into two Fab arms and one Fc stem; pepsin leaves the arms joined as F(ab′)2. The hinge between them lets the arms move. The Fc binds complement and Fc receptors.
6. The heavy chain's constant region decides the class: IgG (four subclasses), IgA (dimer in secretions), IgM (pentamer with ten sites), IgE and IgD.

01What an antibody is

Antibodies, also called immunoglobulins (Ig), are the antigen receptors of B lymphocytes. A B cell carries its antibody anchored in its membrane, where it is the B-cell receptor; when the cell has been activated and turned into a plasma cell, it secretes the same antibody in large amounts. Every B cell makes antibodies of a single specificity, and so does the clone it gives rise to.

Antibodies make up about a fifth of the plasma proteins, most of the gamma globulin fraction of a serum electrophoresis. They are not toxic themselves: they bind an antigen and mark it, and the rest of the immune system destroys what they hold.

an IgG, two heavy chains of about 50 kDa and two light chains of about 25 kDa

150kDaan IgG, two heavy chains of about 50 kDa and two light chains of about 25 kDa

in each immunoglobulin domain, about

110amino acidsin each immunoglobulin domain, about

on an IgM pentamer

10binding siteson an IgM pentamer

02Four chains

The basic unit is a heavy chain paired with a light chain, joined to an identical pair. The two heavy chains are held together by disulfide bonds in the hinge and by contact between their last domains; each light chain is joined to its heavy chain by one disulfide bond. The four chains fold into a Y: two arms and a stem.

There are two kinds of light chain, κ (kappa) and λ (lambda). They work the same way, and about 60% of human antibodies carry κ and 40% λ, but a single antibody carries two κ or two λ, never one of each. Heavy chains come in five kinds, γ, α, μ, ε and δ, which make the five classes.

03Domains

Each chain is a row of immunoglobulin domains, about 110 amino acids each, which fold the same way: two β-sheets packed face to face like a sandwich, held by a disulfide bond inside the domain. The first domain of every chain, at its amino end, is the variable domain: its sequence differs from one antibody to the next. The others are constant domains, the same in every antibody of a class.

• A light chain has one variable and one constant domain: VL and CL.
• A heavy chain of IgG, IgA or IgD has one variable and three constant domains, VH, CH1, CH2 and CH3, with a hinge between CH1 and CH2.
• A heavy chain of IgM or IgE has four constant domains and no hinge. Its extra domain, CH2, sits where the hinge would be and lets the arms move instead.

VL lies against VH, and CL against CH1, so each arm holds one light chain and the first half of a heavy chain. The stem is the rest of the two heavy chains.

04The antigen-binding site

Variation in the variable domains is not spread evenly. Most of it falls in three short loops of each domain, the hypervariable regions, also called the complementarity-determining regions (CDRs). The β-strands between them, the framework regions, change little. When VH and VL pair, their six loops come together at the tip of the arm and make one surface, the antigen-binding site (paratope), which fits a small part of the antigen, its epitope. The third loop of the heavy chain, CDR-H3, varies most in length and sequence and usually matters most for binding.

The binding is non-covalent: hydrogen bonds, ionic bonds and van der Waals forces between surfaces that fit, so it can be undone. Its strength at one site is the antibody's affinity. Because each unit has two sites, and an IgM pentamer ten, an antibody can bind several copies of an epitope on one surface at once, and holds far more strongly than one site alone could, its avidity.

The diversity of the CDRs comes from the genes. The variable domain is not encoded as one piece but assembled in each developing B cell by recombining gene segments, V, D and J for the heavy chain and V and J for the light, with nucleotides lost and added at the joins. Later, in an immune response, point mutations in the variable genes (somatic hypermutation) let B cells with higher-affinity antibodies be selected. Together these can make more than 10¹⁴ different antibodies.

05Fab, Fc and the hinge

In the 1950s the enzyme papain was found to cut IgG into three pieces, each about a third of the whole. Two of them still bound antigen, one site each, so they could no longer link it into a precipitate; the third bound none, and was named for the crystals it formed. The names stayed:

• Fab, fragment antigen binding: one arm, a whole light chain with VH and CH1. It binds one antigen.
• Fc, fragment crystallisable: the stem, CH2 and CH3 of both heavy chains.
• F(ab′)2: what pepsin leaves. It cuts below the hinge's disulfide bonds, so the two arms stay joined and can still bind two antigens and cross-link them.

The hinge is a stretch of heavy chain between CH1 and CH2, whose cysteines make the disulfide bonds that join the two heavy chains. It lets each arm swing and turn, so the two sites can reach epitopes at different distances, and lets the Fc move on its own. IgG's four subclasses differ most in their hinges: IgG3's is the longest, up to 62 amino acids with up to 11 disulfide bonds between the heavy chains, against 12 to 15 amino acids and 2 to 4 bonds in the others.

The Fc is where the antibody meets the rest of the immune system. A sugar chain is attached to each CH2 domain (at asparagine 297 in IgG) and fills the space between them; it takes part in binding the Fc receptors. The lower hinge and the top of CH2 hold the binding sites for C1q, which starts the classical complement pathway, and for the Fc-γ receptors of phagocytes and NK cells. Where CH2 meets CH3 the neonatal Fc receptor (FcRn) binds, which carries IgG across the placenta and protects it from breakdown, so it lasts about three weeks in the blood. What each class does with its Fc is the subject of the next topic.

06The five classes

The constant region of the heavy chain decides the class, or isotype, and with it where the antibody goes and what it does.

• IgG makes up about three quarters of plasma antibody. Its four subclasses, named IgG1 to IgG4 in order of falling abundance, are more than 90% identical and differ mostly in the hinge and upper CH2, which changes how well they bind C1q and Fc receptors. IgG4 can swap arms with another IgG4, so most IgG4 in the blood binds two different antigens.
• IgM is the first class a B cell makes, as its receptor and in its first secreted antibody. Plasma cells secrete it as a pentamer of five units joined by disulfide bonds through their tail pieces and clasped by the small J chain, about 900 kDa. A smaller share forms hexamers without a J chain.
• IgA in the serum is mostly a monomer, about 90% of it IgA1. Plasma cells under the mucosae make it as a dimer, two units joined by a J chain. The polymeric immunoglobulin receptor on the epithelial cells binds the dimer, carries it through the cell and is cut off on the far side with it, and the piece of receptor that stays is the secretory component, now part of secretory IgA. IgA1 has a longer hinge than IgA2.
• IgE, like IgM, has an extra constant domain in place of the hinge. Mast cells and basophils hold it by its Fc on a high-affinity receptor, and its level in the serum is tens of thousands of times lower than that of IgG.
• IgD shares its variable region with the IgM on the same naive B cell; little is secreted.

A B cell can change the class it makes, guided by T cells (class switching). The constant region changes and the variable region stays, so the antibody keeps its specificity and gains a new function.

07Isotype, allotype, idiotype

Antibodies are proteins, and so antigens themselves. Their differences are named at three levels:

• Isotype: what every healthy person has, the classes and subclasses of heavy chain and the types of light chain, set by the constant regions.
• Allotype: inherited variants of those constant regions that differ between people, such as the Gm allotypes of IgG.
• Idiotype: what is unique to one antibody, or one clone, the shape of its variable regions and so of its binding site.

Check yourselfAnswer, then say how sure you were: a lucky guess still counts as a weak point.

1An IgG is digested with papain. What is left?

One F(ab′)2 fragment and small peptidesTwo Fab fragments and one Fc fragmentTwo heavy chains and two light chainsSix separate CDR loops

2Which part of an antibody makes contact with the epitope?

The hingeThe CH2 domains and their sugarSix hypervariable loops, three from VH and three from VLThe J chain

3What decides whether an antibody is IgG, IgA, IgM, IgE or IgD?

The type of light chain, κ or λThe constant region of its heavy chainThe sequence of its CDRsWhether it is bound to a membrane

4Why does an IgM pentamer bind a bacterial surface strongly although each of its sites binds weakly?

Its hinge is longer than that of IgGIt has ten antigen-binding sites, which bind togetherIts light chains are always κIt has no constant domains

5A patient's urine contains a large amount of a single kind of free κ light chain. What does it suggest?

A clone of plasma cells, as in myelomaIgA deficiencyA lack of the J chainA deficiency of complement C1q