Immunological Methods

Co.

1

The Quality of Antibodies and Cellular Receptors

S. Fazekas de St. Groth

Publisher Summary

This chapter discusses the theory and practice of estimating equilibrium constants in immune systems. The simplest overall measure of the forces holding epitope and paratope together is the equilibrium constant. For practical measurement, at least one of the three components making up the equilibrium mixture (free epitopes, free paratopes, or epitope–paratope complexes) must be separated from the other two. If there are reasonable differences in size, the separation presents no problems. The simple treatment of equilibria assumes independence of epitopes and paratopes. The immunoglobulin molecules found in sera are multivalent and so are most natural antigenic molecules and particles. The most common interaction between antibodies landing on neighboring sites is electrical. If such interactions are present, an additional term for electrostatic free energy change arises and the observed K will not remain constant but appear as a function of the density of epitope–paratope complexes.

I Introduction

II Simple Equilibria

A Theory

B Practical Restrictions

C Complications

D Practice

1. Equilibrium Dialysis

2. Equilibrium Filtration

3. Equilibrium Sedimentation

III Competitive Equilibria

A Theory

B Practice

1. Compartmental Assays

2. Plaque Assays

References

I INTRODUCTION

Conventional titrations define an end point which depends on both the quantity and quality of reactants. The reaction can be made of practically zero order by working in great excess of one component, and the quantity of the other may then be estimated independently. Quality cannot be assayed in such systems because either it appears confounded with quantity or its effects are eliminated altogether. This holds equally for binary reactions (such as precipitin tests in solution or in gels, active or passive agglutination, complement fixation, etc.) and for ternary systems where an acceptor and an indicator compete for the third component (such as inhibition of any binary reaction, neutralization of toxins or pathogens, etc.).

The simplest overall measure of the forces holding epitope and paratope together, that is, of quality in the immunological sense, is the equilibrium constant. When not only the extent but also the nature of these forces is to be investigated, thermodynamic parameters (such as changes in enthalpy, entropy, heat capacity, etc.) must be determined. All this, however, amounts to no more than measuring equilibrium constants under different environmental conditions (commonly at different temperatures, less frequently by varying pressure or volume experimentally). This chapter deals, therefore, with the theory and practice of estimating equilibrium constants in immune systems.

II SIMPLE EQUILIBRIA

A Theory

1 Monovalent Reactants

Consider the reaction between epitopes at concentration [E] and paratopes at concentration [P]. Both reagents are in thermal motion and have a calculable chance of colliding with each other. Epitope-paratope complexes, x, will be formed at a rate proportional to the concentration of free reagents ([E − x] and [P − x] and to the rate of effective collisions, k1. Formally, the concentration of complexes will increase with time as

(1)

The formed complexes dissociate at a rate of k2, and hence their concentration decreases with time as

(2)

The overall rate is obtained by combining Eqs. (1) and (2):

(3)

Equilibrium is defined as the state where d[x]/dt = 0, that is, where association and dissociation exactly balance each other (Note 1). Thus, at equilibrium we have

(4)

NOTE 1.

The approach to equilibrium is obtained by integrating Eq. (3). Thus, the concentration of epitope-paratope complexes at time t is

or, conversely, the time required to reach a particular concentration of complexes, xt, is

where x0 is the concentration of complexes at zero time and x1,x2 are the roots of Eq. (5),X1, X2 = 1/2{E + P + K + [(E + P + K)²− 4EP]¹/²}. The equilibrium concentration, x, reached at is obviouslyX2, the smaller root of Eq. (5).

The equilibrium constant, K, is simply the ratio of k2 and k1, as seen by rearranging Eq. (4):

(5)

The equilibrium constant has the dimensions of concentration and is conventionally expressed in terms of molarity (moles per liter). With biological material, such as cells, it is more meaningful to use cgs units, that is, to speak of topes per cubic centimeter (cf. Jerne et al., 1974). Since there are 6.023 × 10²³ molecules per mole, and thus 6.023 × 10²⁰ in 1 cm³ of a molar solution, the two notations are readily interconvertible: Kcgs = 6.023 X 10²⁰Kmol.

(Some immunochemists define K as

a formally unobjectionable alternative which, however, imparts to K the quaint dimension of liters per mole. This may stun those accustomed to thinking in concentrations and has to be inverted in any case for conventional thermodynamic calculations.)

2 Multivalent Reactants

Equation (5) holds for monovalent reagents, such as small haptens and Fab fragments. Considering the valency of whole antibody molecules, that is, a number n of paratopes/antibody (making P = nA), the equilibrium equation becomes

(5a)

For large multivalent antigens (such as cells, bacteria, viruses) of concentration [C], each carrying n epitopes, the corresponding equation is

(5b)

(The valency of antibodies can usually be ignored in this situation. To check the correctness of this practice, the behavior of whole Ig molecules may be compared with Fab fragments derived from them.)

3 Microscopic Equilibria

Equation (5b) does not take into account the fact that epitopes are found on a large particle and thus occur in parcels of n. It can be shown, however, that in the absence of interaction between epitopes their behavior is the same, irrespective of whether they occur singly or as part of the surface of large carriers (Note 2).

NOTE 2.

Consider the microscopic equilibrium between the (i -1)th and the ith epitope on, say, a red cell:

(6)

Here xi classes in which i out of n sites can be occupied. Note that

(7)

If all sites are identical and equivalent, a single equilibrium constant, K, characterizes all associations and Ki = K[n - (i − 1)]/i, since there are n - (i − 1) ways of adding an antibody molecule to make xi out of xi−1 and i ways in which a molecule can dissociate from xi to make xi−1. Equation (7) may be rewritten accordingly as

(7a)

The average number of antibody molecules bound to a cell is

(8)

Substituting the function (7a) and simplifying by the zero term, we have

(9)

The denominator here is the binomial expansion of [1 + K/(P − x)]n and the numerator is its derivative with respect to K/(P − x). Thus,

(10)

which is directly rearranged to give

a result identical with Eq. (5b).

B Practical Restrictions

Equation (5) can be solved numerically for any set of observations. The quadratic form (cf. Note 1), however, is not convenient for plotting and especially not for statistical estimation of parameters. The parabola will have to be transformed into a straight line as soon as it is decided what we wish to estimate.

1 Separation of Terms

For practical measurement, at least one of the three components making up the equilibrium mixture (free epitopes, free paratopes, or epitope-paratope complexes) must be separated from the other two. If there are reasonable differences in size, the separation presents no problems. Thus, haptens will diffuse through membranes which retain antibody, yielding a compartment representing [E − x]. With particulate antigens, [P − x], the antibody remaining free can be separated either in the form of a supernatant after centrifuging or as a filtrate after passing the equilibrium mixture through a sieve retaining the antigen and, hence, a fortiori, antigen-antibody complexes. When the two reactants are of comparable size, for example, in idiotope-antiidiotope reactions, simple mechanical separation will not work, but there are alternative techniques (labeling one or the other reactant and using a precipitating agent directed against one of them; following the formation of complexes by some physical, usually optical, change in the system; or attaching one of the reactants to a solid or particulate carrier and thus reducing the problem to mechanical separation).

In either case, the separated compartment, that is, [E − x] or [P - x], will have to be estimated and compared to the input concentration [E] or [P]. It is customary therefore to express x as a fraction of the input. In most cases this practice becomes mandatory since only titers (unknown multiples of absolute concentrations) can be determined, with [E] or [P] themselves as parameters to be estimated.

2 Linear Transforms

Since the equilibrium equation is symmetric in E and P, the formal treatment is the same for both. Here we shall take as an example a cell with n epitopes on it, that is, an input concentration of [nC] epitopes reacting with [P] paratopes. In this system, the separated compartment will be free antibody, and we define therefore x = αP (i.e., a fraction 0 < α < 1 of antibody is bound in epitope-paratope complexes) and rewrite Eq. (5b) as

(11)

Equation (11) has six possible linear transforms, obtained by multiplying both sides by a factor and rearranging to the general form y = a + bx (Note 3).

NOTE 3

In equilibrium dialysis tests [where Eq. (5a) is used], the concentration of free hapten is called c (c = [E - X], in our notation), and the ratio of bound to total antibody r (r = x/A, in our notation). The equilibrium constant is defined as k1/k2. The unknown parameters are usually estimated by one of two linear transforms. The first,

or

in our notation, is clearly Eq. (11a), scaled by the factor 1/A. The second equation,

becomes

in our notation and is seen to be equivalent to Eq. (11c), scaled by A/E. These linear forms are occasionally referred to by special names (Lineweaver-Burk plot, Scatchard plot, etc.). Perpetuation of proprietary names for trivial transforms is a tiresome conceit.

3 The Informative Range

Each of these equations can be used to estimate the parameters from the y intercept a and the slope b. (In case of emergency, the x intercept c = −a/b may also be used.) We wish then to choose the plots which provide the greatest precision, that is, estimate intercepts with minimal extrapolation and have a slope close to unity. Each equation will be examined therefore from this aspect once the optimal range of concentrations for the reactants has been set. We have here two considerations to guide us. First, the system should be far from saturation, to avoid interactions between sites. In the example, this means that most epitopes should remain free. This sets [nC] ≥ 10[αP]. Second, the separated reactant should be a small fraction of the input, to make the difference highly significant. This sets α ≥ 0.9. These two conditions define the relations between the parameters over the most informative range of equilibrium tests. Thus, P ˜ K and nC ˜ 10K.

Figure 1 illustrates the course of the six linear transforms on varying the concentration of antigen over a tenfold range. For the plots, nC was taken as constant and its dilution factor d (1 ≤ d ≤ 10) was incorporated into the appropriate variables.

Fig. 1 Linear transforms of the mass equation.

4 Problems of Estimation

Equations (11a) and (11f) have, statistically speaking, the most to recommend them. Both have slopes that can be estimated with precision, while the y intercept of Eq. (11a) and the x intercept of Eq. (11f) are reached by minimal extrapolation and differ greatly from zero. The information extracted from either of these is the same, as they happen to be inverse functions of each other. Their common shortcoming is that P cannot be estimated independently and will always carry the error of K, too.

The next pair, Eqs. (11d) and (11e), gives satisfactory estimates of K(contained in their y and x intercepts, respectively) but are poor estimators of P, even though the slope is independent of K.

The last pair, (11b) and (11c), have both their x and y intercepts close to the origin and will therefore give reliable estimates of P only if the experimental points are not scattered widely about the theoretical line. Of the two, Eq. (11c) is preferable because it estimates the parameters P and K independently.

C Complications

1 Heterogeneity

Since even the simplest antigen may interact with and stimulate a large variety of cells, the observed immune response is the sum of a number, usually an unknown number, of clonal responses. Equation (5) does not take this into account, and thus the observed equilibrium constant, K0, is in fact the central measure of an unknown distribution of particular equilibrium constants, Ki (1 ≤ i ≤ n), where the number n itself is unknown.

As is usual in such situations, a normal distribution was assumed for the free energies of association (Pauling et al., 1944), that is, log-normal for K (Note 4).

NOTE 4.

While this assumption is certainly wrong in its limits, it also seems intuitively inappropriate as there are more ways of obtaining poor than perfect fits. Indeed, each of the few reports analyzing subpopulations of antisera (Fazekas de St. Groth, 1967; Werblin and Siskind, 1972; Werblin et al., 1973; Kim et al., 1974) demonstrated distributions heavily skewed toward antibodies of low affinity. The true form of the distribution, however, is still not known and, what is worse, it is known to change with time even in the same individual, as the response matures. Under the circumstances, then, the normal assumption is retained as the most convenient compromise; it is found also to work in practice, provided the tests do not cover extreme ranges.

The measure of heterogeneity, σ, was defined as the square root of the estimated variance. Since the value of σ is obtained by cumbersome empirical fitting, the usual treatment fits a distribution which is similar to the normal, but more manageable. Of the many distributions which approximate the normal, the choice of immunochemists (Nisonoff and Pressman, 1958) fell on one of the distributions proposed by Sips (1948) (Note 5).

NOTE 5.

The Sips function,

stated here in immunochemical terms, amounts to a late rediscovery of the logistic distribution, whose metametric transformation is P = eY/(1 + eY). In the Sips formulation, P defines the chance of a single paratope being occupied, while Y (the logit of P) is equated to a In K0c.

This is by far not the best approximation (it deviates seriously from the normal around ±σ and becomes increasingly inappropriate with the distance from the mean beyond ±2σ) and is also unsuited to routine statistical estimation of precision. But it has the redeeming virtue of being stated in the same terms as have been historically used in the treatment of equilibrium data (cf. Note 3). Thus,

(12)

The heterogeneity index, a, can be read off the slope of the straight line and K0 worked out from the y intercept or, better, obtained directly from the x intercept. The value of a lies between 0 and 1, homogeneity being indicated by a = 1.

The equilibrium equation underlying Eq. (12) would read, in our notation,

and the linear transform suitable for estimating a

(12a)

Equations (12) and (12a) use all the information for the estimation of a and K0 and can be solved only if all other parameters ([E], [P], and [x]) are known.

2 Incestuous Combinations

The simple treatment of equilibria assumes independence of epitopes and paratopes. In fact, immunoglobulin molecules found in sera are multivalent and so are most natural antigenic molecules and particles. It should be considered, therefore, that some of the n-valent antibody molecules already attached by one of their paratopes may form a second, third, … nth epitope-paratope bond with the same antigenic particle. That such complexes exist has been well known since the 1960s (Lafferty, 1963; Lafferty and Oertelis, 1963; Almeida et al., 1963; Greenbury et al., 1965; Klinman and Karush, 1967; Feinstein and Munn, 1969), but their rate of formation and the proportion present in a particular equilibrium mixture are not known. The problem is difficult as not only the number and flexibility of paratope-bearing limbs on the globulin molecule but also the topography of epitopes on the antigen will influence the outcome. Experimental tests on one system (Klinman et al., 1967; Hornick and Karush, 1969, 1972; Gopalakrishnan and Karush, 1974) showed that incestuous bonds occurred with high probability and thus the observed K0 for IgG was close to the square of K0 measured for single attachments. In another system, van Regenmortel and Hardie (1976) found that, depending on the concentration of the reactants, the frequency of incestuous combinations could be varied to involve from practically none to practically all IgG molecules.

The danger of this phenomenon lies in its insidious effect on estimates of heterogeneity which, by their very nature, are estimates at different concentrations of reactants. The way out is simple: Fab fragments of a particular antibody population have the same affinity and heterogeneity as the parent population, but cannot form incestuous complexes. Thus, the difference in K0 and a of an antiserum and the Fab preparation obtained from it is the measure of incestuous combinations.

3 Interaction between Sites

The most common interaction between antibodies landing on neighboring sites is electrical. If such interactions are present, an additional term for electrostatic free energy change arises and the observed K will not remain constant but appear as a function of the density of epitope-paratope complexes (Note 6).

NOTE 6.

If all the epitopes on an antigenic particle are equivalent and there is no interaction between them, the equilibrium constant characterizing the binding of the ith paratope is Ki = (n - i + 1)K/i [cf. Eqs. (6) and (7a)]. The corresponding change in standard free energy is

As may be derived from the Debye-Hückel theory, electrostatic interaction will change the free energy by

where N is Avogadro’s number, ∈ the electronic charge, z the charge on the antibody molecule, D the dielectric constant, ρ the radius of nearest approach of opposed ions, and 1/K the thickness of the ionic atmosphere as defined in the Debye-Hückel theory. Thus,

if we write w for the constant term

Katchalsky and Spitnik (1947), as well as Scatchard (1949), have proposed a very simple approximation which, when written in a form analogous to Eq. (5b), reads

(13)

It can be shown to carry a maximal error of less than ±1.2% when n = 3, less than ±0.3% when n = 4, and one that is entirely negligible with the number of epitopes on large carriers such as cells.

Interactions become manifest only near saturation (i.e., when x/nC approaches unity). The effect is an apparent gradual increase in K and hence a curve concave toward the ordinate when plotting one of the transforms where K appears as a multiplier of the independent variable, such as Eq. (11f). Since the slope at low values of x/nC still gives a valid estimate of K0, its exponential term [(cf. Eq. (13)] is best evaluated by plotting 2αP/C (twice the ratio of complexes to cells) against ln{[(1 − α)/α] (nC − αP)} (the logarithm of the concentration of free epitopes multiplied by the ratio of free to bound paratopes). The slope gives directly the value of w and the line is constrained to cut the ordinate at ln K, as derived from the plot of Eq. (11f). The functional relationship is

(14)

For interactions of unknown mechanism and undefined magnitude (such as steric inhibition or allosteric changes in the neighborhood of an occupied epitope), an apparent equilibrium constant may be found according to Wyman (1948) by plotting ln[(nC/αP) − 1] against ln[P(1 − α)]. The x intercept (where nC = 2αP, i.e., the point of 50% saturation) represents this value. A slope greater than unity at the intercept reveals positive interactions between sites; slopes less than unity suggest negative interactions, although the existence of some positive interactions is not excluded here. The magnitude of the interaction at particular levels of saturation may be gauged by comparing the apparent equilibrium constant with K0 obtained from plots of Eqs. (11a)–(11f) far from saturation.

D Practice

1 Equilibrium Dialysis

a Principle

Equilibrium dialysis relies on the retention of antibodies by semipermeable membranes that freely pass the antigen. Dialyzable antigens (i.e., molecules of molecular weight less than 3000, preferably less than 1000) are, generally, not immunogenic. They are haptens, capable of combining with antibodies but incapable of inducing the production of antibodies. The method of equilibrium dialysis thus implies the nonidentity of immunogen and test antigen. It is well to keep this fact in mind as it restricts equilibrium dialysis to the study of heterologous reactions, nonidentiy of immunogen and test antigen being the formal definition of a cross reaction.

Apart from this restriction, the method yields unequivocal answers: Free hapten appears on one side of the membrane and, knowing the input concentrations of hapten and antibody, the average equilibrium constant may be calculated from a set of such data by substituting into any of Eqs. (11a)–(11f). This holds for the ideal case where (1) equilibrium is attained, (2) the equilibrium is not distorted by osmotic effects, and (3) the membrane or, generally, the equipment does not interact with any of the reactants. None of these conditions is automatically fulfilled in practical systems; the ways of recognizing and correcting potential systematic errors will be given below.

b Equipment

Some of the classical equilibrium measurements have been performed in dialysis bags tied at both ends and dangled in a solution of hapten. Nowadays the technique is both more economical in materials and less accident prone.

Diffusion chambers of 1.5-ml capacity are commercially available¹or can be homemade by cutting off the top third of 5-ml antibiotic vials and grinding their cut surfaces flat. Before putting them into service, the chambers are cleaned in hot nitric acid or KOH-ethanol, thoroughly rinsed in distilled water, and siliconed to cut down adsorption. Washing under the tap followed by a distilled-water rinse is usually sufficient between runs. The chambers are dried and stored in a dust-free container, such as a large Petri dish. A pair of diffusion chambers may be clamped together (Quickfit JC-19 clamps) or mounted in an appropriate brass frame (Drummond Scientific Co., Broomall, Pennsylvania).

Dialyzing membranes (20/32–32/32, Visking Corp., Chicago, Illinois) are cut to convenient size, say 10 × 10 cm², and soaked in distilled water. After an hour, the water is brought to boiling and allowed to cool over the next hour. Then the membranes are rinsed in cold distilled water, three changes of 30 min each, and finally stored in 0.01 M NaN3 (0.06% in H2O), ready for use.

Since concentration gradients during dialysis are to be minimized, the assembled cells must be agitated. A simple device for this purpose is a vertical disk rotating at 5–10 rpm, as used in tissue culture laboratories. It will accommodate 20–60 assembled dialysis units and can be mounted on a bench top. For critical thermodynamic work, more accurate temperature control is required than can be achieved in incubators or constant-temperature rooms. Commercially available agitators have either insufficient capacity or improper action, but a simple and effective rotating device has been designed by Karush and Karush (1971), holding 36 dialysis units and fitting readily available refrigerated constant-temperature baths.

c Performance of Test

The dialysis cells are assembled the night before the test. First the ground-glass flanges are given a thin coating of silicone

grease (e.g., Merck, Darmstadt, Art. 7921) and placed, face up, on the bench. Then a sheet of dialyzing membrane is blotted between two sheets of lint-free absorbent paper (e.g., Photowipes, Sorg Paper Co., Middletown, Ohio) and disks of a size exactly fitting the dialysis cell are punched out with a sharp cork borer. (A sheet of 10 × 10 cm² is meant to give 16 disks of 22.5-mm diameter.) A membrane free of visible imperfections is placed on top of each half-cell, smoothed out, covered with the other half-cell, and the assembly clamped together. As a check for invisible imperfections and imperfect sealing at the edges, the top halves are filled with 1 ml of water and connected to a source of compressed air or nitrogen through a rubber stopper fitting the mouth of the cells. The pressure is set at 0.1 atm (1.5 psi) and any significant leak through the membrane or between the membrane and the top compartment should show up within 5 min. After that the pressure is released, the dialyzing unit inverted, and the procedure repeated to test the seal between the membrane and the bottom compartment. If the experiment is to be performed in a water bath, the joining perimeter of the two half-cells is painted with molten paraffin wax (melting point, 62°–65°C; heated to ca. 100°C) to prevent lateral seepage during dialysis. The assembled cells, lying on their sides, are stored overnight at room temperature. The small residue of water, usually less than 0.1 ml, evaporates during this period and the cells are ready for use

Before setting up an equilibrium test the dialysis units are labeled. Then the various hapten solutions are measured in with volumetric pipettes and the top half-cell is sealed. The commercial cells have screw caps and these we found unsatisfactory on two accounts. First, tightening the caps occasionally twists the cell and thereby distorts or tears the membrane; even if great care is taken to avoid such damage, the sealing itself puts the cell under positive pressure. Second, the caps collect water between the threads and, unless they are sealed with wax (i.e., by an extra operation), this water will dilute the samples to be assayed after completion of the run. We use, therefore, the rubber stoppers employed in sealing antibiotic vials. The stopper is first pierced through its center by a 15-mm (5/8th-in.) 22-gauge needle (to allow equalization of pressure during insertion) and then pressed into the cell to give a flush fit. After all solutions have been delivered, the needles are withdrawn and the units inverted. Measuring in the antibody solutions and sealing their compartments are done similarly. The dialysis units are then mounted on the rotating device and diffusion is allowed to proceed for a predetermined time.

After the equilibration period the neck of the cells is blotted, a needle inserted into the stopper of the hapten compartment, and the stopper removed. The sample is withdrawn with a Pasteur pipette by slightly tilting the cell and touching its side (not the membrane!) with the tip of the pipette. By this technique usually not more than 0.05 ml of fluid is left in the cell. While the exact contents are difficult to assess, the error will be only of the order of ±2% by using a correction of +0.05 ml for the capillary layer of fluid left behind. Even though in most tests only the concentration of hapten is to be determined, the antibody compartment is drained in a similar fashion and the yield of fluid recorded.

d Experimental Design and Controls

Since one of the reactants, the hapten, is usually monovalent, the precautions aimed at avoiding interaction between sites (cf. Section II,C,3) may be relaxed for equilibrium dialysis. There are, however, other considerations that limit the scope of informative tests. First, the concentration of epitope-paratope complexes is obtained as a difference, by subtracting free from total hapten. This calls for special care in determining the input concentrations, since their imprecision will enter as systematic error into the estimate of complexes. It also demands that the difference between free and bound hapten be made as large as possible, their error variances being additive. In practice, this amounts to setting the antibody concentration above the equilibrium constant, to ensure better than 50% binding (Note