Antigen-Antibody Interactions

For a long time immunologists focused on understanding the ways in which antibodies

and antigens interact, in part because it was feasible to study that process using the techniques

available during the first half of the last century, and in part because it was fairly easy to obtain

large quantities of antibodies from the serum of immunized animals. For that reason, a great deal

is known about antigen-antibody interactions, perhaps more than any other aspect of immune

system function. What I want to do briefly is to talk about the current understanding of how

antigens and antibodies interact, and to discuss several useful applications of that process in

medicine and biology.

First the binding of antigens to antibodies is mediated by non-covalent bonds, and is thus

a reversible reaction. That is, one can write an equilibrium equation of the type

[Ag] + [Ab] [ AgAb], where [AgAb] represents the concentration of the antigen-

antibody complex. The kinds of bonds that hold the antigen and antibody together in this

complex are the same ones that are generally involved in producing the tertiary and quaternary

structures of proteins, namely, ionic bonds, hydrogen bonds, hydrophobic interactions, and van

der Waals forces (see Fig. 6-1). Mostly these are bonds formed between the side chains of

amino acids that make up the antigen-binding site of the antibody (that is, the amino acids in the

three complementarity-determining regions of the heavy and light chains) and the antigen

molecule. If the antigen is a protein, then these are protein-protein interactions with which

you’re familiar, but even for other kinds of antigen molecules, their binding to the antibody is

quite similar in principle to the binding of a substrate to an enzyme or of a hormone to a receptor,

and can be described in similar thermodynamic terms. For example, the binding of antigen to

antibody can be described by an equilibrium constant that indicates the strength of the

interaction. In general antibodies bind with very high affinity to antigens, often several orders of

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magnitude higher affinity than is typical of interaction of enzymes with substrates or of receptor

with their ligands. As shown in Table 6-1 even a relatively small molecule like fluorescein can

bind to its antibody with extremely high affinity, and Ka (association constant) values in the

range of 109 or greater are fairly common. An important measure of the stability of the antigen-

antibody complex is the reverse rate constant k-1, the rate constant that describes the unbinding

reaction. Again as shown in Table 6-1, the forward rate constant for most antigen-antibody

reactions is quite fast, often limited only by the rate of diffusion of the two molecules. Once they

bump into each other, they typically stick (except for some large, complex antigens which must

encounter Ig molecules in a particular orientation). What distinguishes the reactions is typically

how long they stay attached before dissociating, which is indicated by the reverse rate constant.

This can range over several orders of magnitude, and is mostly influenced by the number and

types of bonds that are formed between the antigen and antibody.

Antigen-antibody interactions have been extensively studied and used for three related

but distinct purposes. One is simply to learn more about the nature of the interaction between the

antigen and antibody. A second purpose is to identify the presence or amount of antibodies to a

particular antigen present in an animal (including a human). For example antibodies to the Rh

antigen that are sometimes produced by a pregnant woman to her fetus can be lethal to the fetus,

so it is essential to know whether an Rh negative mother is carrying an Rh positive fetus and if

she has antibodies against the Rh antigen. This can be determined by an immunological assay

designed to detect the presence of such antibodies in the mother’s blood. Tests for HIV exposure

also generally depend on detecting anti-HIV antibodies in a person’s serum. The third

application of antigen-antibody interactions uses antibodies as very specific reagents to

characterize or purify a particular antigen. The common pregnancy test uses antibodies specific

for the hormone human chorionic gonadotropin (produced by women during pregnancy); a high

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level of HCG indicates that a woman is pregnant and a low level indicates that she is not.

Antibodies are also widely used as specific reagents in basic biological research. I want to

discuss each of these types of uses of antigen-antibody interactions in turn.

In order to determine the Ka, k1, or k-1 for a reaction, one must have a way to let the

reaction come to equilibrium and then to measure the concentrations of free (not bound) antigen

and antibody, as well as the concentration of the antigen antibody complex. One way to do so is

equilibrium dialysis (although other methods are now more commonly used). This technique

illustrates the principle well, however, so I want to describe it (see Fig. 6-2). It takes advantage

of the fact that a dialysis membrane is able to allow small molecules to pass through it freely, but

it impedes the movement of large molecules (like immunoglobulins). If one adds a known

amount of antibody to one side of the membrane and a known amount of a small, radioactive

antigen on the other, the antigen will diffuse through the membrane and bind to the antibody. At

equilibrium, the concentration of radioactivity (and hence, of antigen) will be higher on the side

of the membrane with the antibody than it will be on the other side of the membrane (because

some of the antigen binds the antibody and is trapped there, while unbound antigen distributes

itself equally on both sides of the membrane). The concentration of the unbound antigen will be

determined by the concentration on the side of the membrane without the antibody. The amount

of radioactivity on the side with antibody will represent sum of the unbound antigen and the

antigen complexed with antibody. But the concentration of unbound antigen is the same on both

sides of the membrane, so the concentration of the Ag-Ab complex is the difference between the

total concentration of antigen and free concentration of antigen on that side of the membrane.

Similarly, the amount of unbound antibody is the total added at the beginning of the experiment,

minus whatever is now tied up in Ag-Ab complexes. In this way one can determine the values of

all the concentrations, [Ag], [Ab], and [Ag-Ab], needed to calculate the equilibrium constant. If

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you take time samples during the equilibration complex, you can also compute the forward rate

constant. And since Ka is the ratio of the forward to reverse rate constants, k1/k-1, you can also

calculate the reverse rate constant for the reaction.

A second way to characterize antigen-antibody interactions is through a Scatchard plot

(see Fig. 6-3), which is similar in concept to the Lineweaver-Burk approach to studying enzyme-

substrate interactions. For immunological reactions one usually keeps the concentration of

antibody constant, and varies the concentration of antigen, determining the equilibrium

concentrations of [Ag-Ab], [Ab], and [Ag] for each initial concentration of [Ag]. Then one plots

the determines the ratio of bound antigen concentration (that is, [Ag-Ab])to total antibody

concentration, a ratio called r, and the concentration of unbound antigen, called c. You then plot

r/c vs. r to obtain the Scatchard plot. If all the antibody molecules have the same affinity

constant for antigen, then the curve is a straight line with a slope of –Ka and an intercept that

corresponds to the number of antigen-binding sites per antibody molecule (called n; n is 2 for

IgG and IgE molecules, 4 for IgA, and 10 (or sometimes less) for IgM). If the “antibody” is a

mixture of different kinds of antibody molecules with varying affinity for antigen, then the curve

has a varying slope. The intercept still shows the number of antigen binding sites per molecule

(assuming that all the antibodies are of the same class). A rough estimate of the average affinity

constant of the antibody mixture for antigen (K0) can be obtained from the r/c value of the curve

at r = 1/2 r intercept. In the example shown in Fig. 6-3b in the book serum #3 has a mixture of

antibodies with a higher average affinity for antigen that serum #4.

Detection of specific antibodies (and a quantitative assessment of their concentration) can

be achieved in a variety of ways. The approach that has been in use for the longest time takes

advantage of the fact that antibodies can form large, extensive complexes with antigens, and

these complexes become insoluble—that is, they precipitate out of solution. The precipitate can

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then be detected in solutions or in gels, which allows for characterization of the binding reaction.

All immunoprecipitation assays rely on the fact that antibody molecules are multivalent; that is,

they can bind to two or more antigen molecules. (See Fig. 6-4a). If the antigen has two or more

epitopes to which the antibody binds (or antibodies, if one is dealing with a polyclonal

serum—which contains a mixture of different antibodies that may recognize the antigen), then a

lattice forms in solution that eventually becomes so large as to be insoluble (see Fig. 6-4a and b).

As shown in 6-4b the maximum precipitate forms when antigen and antibody concentrations are

roughly equivalent, and this there provides a reasonably accurate way to judge the antibody

concentration present in a serum. Similar principles underlie a variety of other assays including

immunodiffusion assays (also know as Ouchterlony assays) and immunoelectrophoresis.

The disadvantages of precipitation assays are that they are relatively insensitive (that is,

you need a lot of antibody and antigen to get a result) and they aren’t very quantitative. For that

reason methods have been developed that amplify the “signal” generated by formation of an

antigen-antibody complex, which allows for both greater sensitivity and accuracy. One

commonly used method is to employ a radioactively labeled antigen to assay either the

concentration of antibody or the concentration of an antigen in an unknown solution. The

principle is similar to one we discussed previously and makes use of the fact that radioactive and

non-radioactive antigen molecules bind equally well to antibody. Thus addition of non-

radioactive antigen to a mixture of antibody and radioactive antigen causes a “competition”

reaction which reduces the amount of radioactive antigen that can bind to a fixed amount of

antibody (see Fig. 6-9). Conversely if one coats plastic wells with a fixed amount of antibody

and then varies the amount of radioactive antigen added, one can measure the quantity of

antibody present by finding the plateau of the binding curve.

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 A very similar assay uses an enzymatic assay instead of radioactivity to measure the

binding of antigen to antibody. This is called an enzyme-linked immunosorbent assay or ELISA.

Here either antigen or antibody is used to coat a well in a plastic dish, and detection of bound

antibody depends on a secondary antibody which is covalently linked to an enzyme. (A primary

antibody is directed against the antigen of interest. A secondary antibody is typically directed

against the primary antibody. For example if you immunize a rabbit against bovine serum

albumin, then the rabbit makes primary antibodies to BSA. If you also immunize a goat with

immunoglobulin from rabbits, the goat makes antibodies to rabbit Ig. In this case you’d use the

goat antibodies to make an enzyme conjugate). As shown in Figure 6-10a, the enzyme will be

present only if the secondary antibody binds to primary antibody (and therefore only if primary

antibody binds to antigen). The sensitivity of the assay depends on how easily the product of the

enzymatic reaction can be detected, but there are quite sensitive methods for detecting colored or

light-emitting products. Variants of the basic procedure have also been developed, as shown in

Figs. 6-10b and c.

Finally, biologists take advantage of the fact that antibodies have characteristics that

make them very useful for a variety of medical and biological studies. In particular antibodies

typically are exquisitely specific for a particular antigen (especially well selected monoclonal

antibodies) and have extremely high binding affinities for antigens. This makes them quite

valuable for detecting particular antigens and for manipulating or purifying them. Nowadays

many diagnostic tests are based on immunological techniques—a number of which are

mentioned in the book like pregnancy tests for human chorionic gonadotropin, exposure to HIV,

a test for prostate cancer based on prostate-specific antigen (PSA) and many others. By

combining immunological detection with electrophoresis, the Western blotting (or

immunoblotting) technique is a sensitive way to find very small amounts of a protein of interest

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in a diverse mixture of proteins. In this procedure proteins are separated by SDS-

polyacrylamide electrophoresis, and then transferred to (“blotted on”) a support membrane

(usually made of nylon or nitrocellulose) to immobilize the proteins and to allow further

manipulations. The membrane is incubated with primary antibodies to the protein of interest,

and then with secondary antibodies coupled to an enzyme. The substrate of the enzyme is

provided and then either a colored product or a light-emitting product (if the substrate is luminol

and the enzyme is horseradish peroxidase) can be readily detected. (See Fig. 6-12).

Antibodies can also be used to pull a protein out of a mixture of proteins (such as a cell

extract). Because of the high specificity and affinity of the antibodies, even tiny amounts of

protein can be isolated from other proteins in this way. First a primary antibody is incubated

with the extract and forms complexes with the antigen (usually not large enough complexes to

precipitate because there is typically little antigen present). Then a secondary antibody coupled

to a large substance such as an agarose or magnetic bead is added. When the secondary antibody

gloms onto the primary antigen-antibody complex, the antigen is now precipitated out of

solution, leaving behind all the other proteins in the supernatant.

Another very useful techinique is called indirect immunofluorescence, which allows

proteins to be studied in place inside cells. Here the primary antibody is added to the cell and

allowed to bind to the antigen. (If the protein of interest is on the cell membrane, no preparation

of the cell is necessary. If the protein is intracellular, the cell must be treated to make the

membrane permeable to antibodies). A secondary antibody that is covalently labeled with a

fluorescent molecule is then attached to the primary antibody, and the fluorescence can be

observed with a specially equipped light microscope. (A fluorescent molecule is one that

absorbs light in the UV range and emits visible light). It’s possible to fluorescently label the

primary antibody but that is seldom done because the labeling process can damage the antibody

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and make it less effective and because use of a secondary antibody increases, or amplifies, the

fluorescent signal. That is, it’s brighter and easier to see (see Fig. 6-14).

Finally, I mentioned previously that Lloyd Old hypothesized that cells had unique surface

proteins, which he called differentiation antigens, that are related to their functions. He also

showed that these differentiation antigens could be identified by antibodies raised to them. For

example, B lymphocytes express surface Ig (and other cell types don’t) so an antibody against Ig

will bind only to B cells and no other type. Subsequently, a method called fluorescence-

activated cell sorting (FACS, pronounced like fax) was developed. Cells are fluorescently

labeled by antibodies to their unique antigens, passed through a narrow nozzle in solution at low

concentration so that each droplet contains only one cell. The droplets are given an electrical

charge and are scanned by laser, which indicates the type and amount of fluorescence that the

cell carries. A computer “reads” the output of the laser scan and applies a charge to two metal

plates as the droplet passes between them, deflecting the cell into a collection tube, based on the

amount and type of fluorescence it expresses. The tubes thus contain cells sorted by the level of

their differentiation antigen, and thus by function. The computer readout also provides

information on how many cells in the original mixture carried what kinds of differentiation

antigens (or “markers”) (see Fig. 6-15).

In short immunological assays have found widespread applications in immunology, for

the study of the immune system, but also in many other areas of biology and medicine.

Created and copyright by Gary Reiness

Last updated: Feb. 15, 2004