Equine antihapten antibody II. The γG(7Sγ) components and their specific interaction

Int.J. Immunochem. Pergamon Press 1965. Vol. 2, pp. 51-60. Printed in Great Britain EQUINE

ANTIHAPTEN

COMPONENTS

AND

ANTIBODY* THEIR

SPECIFIC

II.

THE

rG(7Sr)

INTERACTION

NORMAN R. KLINMAN,t JOHN H. ROCKEYand FRED KARUSH~ Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

(Received 15 August 1964) Abstract--The ~,G(7S~,) components of antibody prepared against the haptenic group p-azophenyl-fl-lactoside (Lac) were isolated from equine antiserum. The average association constants (KA) for the specific interaction of this antibody with the hapten, p-(p-dimethylaminobenzeneazo)-phenyl-fi-lactoside (Lac dye), were determined at 25 ° and 37"2° by equilibrium dialysis. Thermodynamic parameters have been calculated and shown to be very similar to those previously obtained for rabbit anti-Lac antibody. A physical-chemical and immunochemical characterization of the purified eG equine anti-Lac antibody has demonstrated chromatographic, electrophoretic and antigenic heterogeneity. Three antigenically distinct immunoglobulins have been identified in the purified ~,G anti-Lac antibody each having antibody activity. The antigenic individuality has been related to the heavy chains when compared by immunodiffusion to one another or to ~,A (flzA) antibody from the same serum. A comparison of the binding by the ~,G antibody with the binding previously described for the ~,A antibody shows that the affinity of the latter for the Lac dye is much greater. This difference in the affinity for hapten between different immunoglobulins present in the same serum may partially explain the heterogeneity of hapten binding in purified antibody. Furthermore, variation in the relative amounts of these immunoglobulins with time after immunization could in part account for the temporal changes in the affinity of the antiserum for hapten. INTRODUCTION OUR present knowledge of the thermodynamics of the antigen-antibody reaction is due mainly to studies of the antibody-hapten interaction involving, for the most part, antihapten antibodies prepared with pooled sera from rabbits and guineapigs. T h e present investigation was undertaken with a view to extending the range of species studied and to avoiding the use of pooled antisera. T h e horse was selected both because of its size and because of the extensive fund of knowledge available concerning the immune response of this species. T h e haptezic group used was the p-azophenyl-fl-lactoside (Lac) group which has been employed for studies with the rabbit.(1) Previous studies with equine antisera have been concerned primarily with antibodies directed against protein and polysaccharide antigens. Because of the nature of the antigens, thermodynamic binding studies have been limited, although some information about the affinity of equine antibody was obtained by Jerne(2) using a rabbit skin reaction to assay for uncombined diphtheria toxin in the presence of antitoxin. With the assumption that the antigen-antibody reaction mixture was a reversible system in equilibrium, he was able to infer from the shape of the * This investigation was supported by Public Health Service Research Grants AI-05305 from the National Institute of Allergy and Infectious Diseases and HE-06293 from the National Health Institute. ? Helen Hay Whitney Foundation Fellow. Recipient of a Public Health Research Career Award (S-K6-AI-14,012) from the National Institute of Allergy and Infectious Diseases. 51

52

NORMANR. KLINMAN,JOHNH. ROCKErand FREDKARUSH

neutralization curves for a variety of equine antisera that the association constants ranged from 5 × 108 to 10n 1./mole. Much more information has been accumulated concerning the molecular characteristics of equine antibodies. Such antibodies have been characterized as 7S v-globulins (vG),* macroglobulins (vM), and a so-called T component which is not ordinarily detectable in normal serum. The relative concentrations of each of these classes of antibodies has been found to depend on the nature of the antigen, the method of immunization and the duration of immunization.(S-sJ In the studies described herein a thermodynamic evaluation has been made of the interaction of equine antihapten antibody with the hapten p-(p-dimethylaminobenzeneazo)-phenyl-/~-lactoside (Lac dye). A comparison of these results with those obtained with rabbit anti-Lac antibody demonstrates a great similarity in the binding of hapten by antibodies from the two species. In addition, the characterization of the equine antihapten antibody has also led to observations pertinent to two other properties of antihapten antibodies: the heterogeneity of such antibodies with respect both to molecular properties and affinity for hapten(61 and the increase in this affinity during the course of immunizationAV) The results of this investigation together with those obtained previouslyCS) demonstrate that equine antihapten antibody contains at least six antigenicaUy distinct immunoglobulins. These components also have different physical-chemical properties and three of them can be described as 7S y-globulins (vG). The affinity for Lac dye has been shown to be much higher in antibody identified as vA than it is in the 7G antibody components isolated from the same serum. It is therefore possible that the increase in affinity of antihapten antibody with a prolonged course of immunization may be related in part to shifts in the distribution of antibody among the various immunoglobulin classes during the period of immunization and to changes in the distribution of antibody among subclasses within a class. MATERIALS AND M E T H O D S Preparation of antigens. The preparation of p-aminophenyl-fl-lactoside and p(p-dimethylaminobenzeneazo)-phenyl-/~-lactoside (Lac dye) has been previously described.~l~ The diazotization of the amino compound to yield p-azophenyl-fllactoside (Lac group) and its coupling to human serum albumin (Cutter Laboratories, Berkeley, Calif.) and bovine gamma globulin (Armour Pharm. Co., Kankakee, Ill,) to give Lac-HSA and Lac-BvG respectively were carried out as before.a~ For the coupling reaction to hemocyanin similar conditions were used. Hemocyanin from the horseshoe crab, Limulus polyphemus, at a concentration of 4.5-5 mg N/ml as determined by micro-Kjeldahl analysis,~9~ was brought to pH 9 and maintained at this pH during the coupling reaction by the dropwise addition of 1 N NaOH with a pH titrator. The reaction mixture was held at 0-5 ° C while the diazotized paminophenyl-/~-lactoside (0.1-0.5 mg/mg protein) dissolved in 1 N HCI (10 ml/g p-aminophenyl-fl-lactoside) was slowly added. After the addition was completed, the solution was maintained at pH 8.9-9.0 at 0-4 ° C for 24 hr and then dialyzed * We shall use in this paper the new nomenclature for immunoglobulins proposed recently by a committee organized by the World Health Organization. In the report of this committee~zS~the following replacements were recommended: ~,G for 7Sy, ~,A for fl2A(~qA) and vM for fi~M(ylM).

The Specific Interaction of 7G Antibody Components

53

extensively in the cold room against a solvent containing 0.02 M sodium phosphate, pH 7.8 and 0.15 M NaC1. It was then passed through a column of Sephadex G-25 (Pharmacia, Uppsala, Sweden), equilibrated with the above buffer, to remove the uncoupled dye. The final solution contained 2.5-3 mg N/ml and 20-25 moles of the Lac group per 100,000 g of protein as determined by the use of a molar extinction coefficient for the haptenic group of 2.48 × 104 at its wavelength of maximum absorption, 365 m/~.o) The ratio of the optical density at 280 m/~/365 m# in 0.02 M phosphate, pH 7.2, or 287 m~/400 m/~ in 0.1 N NaOH varied from 0.7 to 1.0 depending on the number of groups coupled to the protein. This material, Lachemocyanin (Lac-Hy), was used for both immunization and specific precipitation. Lac-HSA was used only for specific precipitation and Lac-ByG only for immunization. The Lac-HSA had 15-20 moles of the Lac group per 65,000 g of protein and the Lac-ByG had 14-20 moles of the Lac group per 160,000 g of protein. Preparation of antisera. A 500 kg horse underwent two series of intramuscular injections with solutions of Lac-coupled proteins dispersed in incomplete Freund's adjuvant. The first series extended over a 5 month period with monthly injections of 40-50 mg of Lac-BTG. The anti-Lac antibody titer during this series never exceeded 0.25 mg of protein/ml of serum as determined by quantitative precipitin tests.t 9) Lac-Hy was used in the second injection series which began two months after the first series ceased. The dosage of Lac-Hy was 60-80 mg per injection and injections were given on a biweekly basis. Serum was collected one week after each injection and a maximum anti-Lac antibody titer of 2.14 mg of protein/ml of serum was reached after six weeks. Purification of antibody. Quantitative precipitin tests were used to determine the optimum antigen concentration for precipitation of antibody from each serum. The precipitating antigens used for these determinations were Lac-Hy and LacHSA. As Lac-Hy was also used for immunization it was necessary, after the third bleeding, to absorb the serum with hemocyanin prior to the quantitative precipitin assay. The amounts of precipitate were measured both by optical density in 0.1 N NaOH with correction for the contribution of antigen, and by micro-Kjeldahl. All optical density measurements were carried out with a Zeiss spectrophotometer. Prior to specific precipitation of the anti-Lac antibody, the serum was treated at 37 ° with hemocyanin and bovine gamma globulin (the carrier protein in earlier immunizations) to remove complement components and other possible coprecipitating components as well as antiprotein antibody. Over 2 mg of antiprotein antibody per ml of serum was routinely precipitated by this procedure. Specific precipitation of anti-Lac antibody was carried out in slight antigen excess, usually with Lac-Hy as the precipitating antigen. The specific precipitate was washed extensively in cold 0.02 M phosphate buffer, pH 7.2 containing 0.15 M NaC1 and then in phosphate buffer alone. Following the method of Utsumi and Karush,¢101 the specific precipitate was then dissolved in 0.02 M sodium phosphate buffer, pH 7.2 containing 0.5 M lactose (Baker Chem. Co., Phillipsburg, N.J.) at 37 °. The solution was placed on a column (5 × 40 cm) containing DEAE-cellulose (0.9 m-equiv./g, Bio-Rad Laboratories, Richmond, Calif.) which was equilibrated with a solution of 0.02 M phosphate, pH 7.2 containing 0.5 M lactose. Elution was carried out at room temperature with a flow rate of 1-2 ml per min and the effluent was collected in 5 ml fractions. After elution with 1-2 1. of this initial buffer, NaC1

54-

NORMANR. KLINMAN,Jom~H. ROCKEYand FREDI~USH

was added to the buffer by a continuous gradient increasing to 1 M within 2 1. The optical density of the fractions was read on a Zeiss spectrophotometer at 280 m/~ and 365 m~. Selected samples were analyzed by agar double diffusion and immunoelectrophoresis to ascertain the pattern of elution of the various immunoglobulins. Fractions were then pooled in 100-300 ml portions so as to minimize the mixing of the various immunoglobulins. These were dialyzed for 3 days against 3 changes of 14 1. of 0.002 M phosphate, pH 7.2 and concentrated by lyophilization or by ultrafiltration. The concentrated samples were brought to 5-10 ml in 0.002 M phosphate and 0"1 M galactose (Fisher Scientific Co., Fair Lawn, N.J.) and dialyzed for 3 days against 3 changes of 500 ml of the same solution. The galactose was then removed by dialysis against 0.001 M phosphate, pH 7.2. These fractions were then analyzed, as before, for their immunoglobulin content. Determination of protein concentrations. The protein concentration of the pooled fractions was determined by the micro-Kjeldahl method assuming 16 per cent N content and by optical density at 279 m/~ and 251 m/~. Protein concentrations of fractions from zone eletrophoresis were determined by the modified method of Folin-Ciocalteu. (ii) Zone electrophoresis. Zone electrophoresis was carried out on a starch supporting medium(12) in barbital buffer 1"/2 0.05, pH 8.6 at 4 °. Fractions were eluted with 0.15 M NaC1. Preparation of antisera against equine serum and y-globulin components. Rabbits were immunized with normal equine serum and gamma globulin by injection of these materials, 1 mg per rabbit, in complete Freund's adjuvant into the foot pad followed three weeks later by an intravenous booster dose of 1 mg of the protein. Bleedings were taken one week later. The y-globulin used for immunization was prepared by repeated ammonium sulfate precipitation at 30 per cent saturation using sera taken prior to immunization of the horse. For the preparation of the heavy and light chains of horse y-globulin for immunization, the same y-globulin preparation was placed on a DEAE-cellulose column and eluted with 0.02 M sodium phosphate, pH 7.2. This material, containing 7G and yA globulins, was then separated into the subunits by the method of Utsumi and Karush. (1°) These preparations were injected intramuscularly, in complete Freund's adjuvant, into pairs of female goats, at the level of 20 mg of protein per animal with a 5 mg intramuscular booster injection after 2 weeks. Bleedings were taken 10 days following the booster injections. The preparation of antisera specific for 7A globulin was accomplished by absorbing rabbit anti-equine y-globulin antisera with purified 7S y-globulin (7G). The latter was prepared by zone electrophoresis of normal equine serum and purification of the protein fraction of lowest mobility by DEAE-cellulose chromatography in 0.015 M sodium phosphate, pH 7.85. Goat and rabbit anti-equine whole serum and rabbit anti-equine y-globulin were obtained from Baltimore Biological Laboratories Incorporated, Baltimore, Md. Agar diffusion studies. Immunoelectrophoresis was carried out by the micro technique of Scheidegger. (18) Mixtures of antigen and antibody in the same well, for identification of antibody components, were prepared either by mixing the antibody with excess antigen and allowing the mixture to incubate at 37 ° for 1 hr or by adding the antigen to the well before and after the antibody was added.

The Specific Interaction of yG Antibody Components

55

Double diffusion studies were carried out on glass microscope slides layered with 1 per cent agar (Special Agar-Noble, Difco Laboratories, Detroit, Mich.). Ultracentrifugation. Analytical ultracentrifugation was carried out in a Spinco model E centrifuge at 52,640 revs/min. To determine values of s°~o,w, protein solutions at four concentrations, ranging from 0.5 to 3.2 mg/ml, were examined at 20 ° in a solvent containing 0.2 M NaC1 and 0.01 M sodium phosphate, pH 7.8. The values of s2o,w were extrapolated to zero concentration of protein. A partial specific volume of 0.74 was used for the protein. A combination of Schlieren and absorption optics was achieved by placing a blue Klett filter (maximum transmission 440 mt~) over the mercury light source. The transmitted light was thus limited to the wavelength range corresponding to the absorption region of the Lac dye so that dye sedimenting with the protein could be observed. Measurement of hapten binding. Hapten binding was measured by equilibrium dialysis as previously described.(1) Protein concentrations were varied from 1.48 × 10-5 to 2.07 × 10-5 M and dye concentrations from 1-12 × 10-5 M. The experiments were carried out at 25.0 ° and 37.2 ° in a constant temperature water bath. RESULTS

Purification of 7G antihapten antibody. The specific precipitate formed with LacHy was found to be soluble to the extent of 90-95 per cent in the lactose-phosphate solvent. Elution with this solvent from the DEAE-cellulose column yielded an early protein peak, consisting of 20-30 per cent of the non-antigen protein placed on the column, foUowed by a long trailing shoulder which occasionally showed smaller peaks and contained 15-25 per cent of the non-antigen protein (Fig. 1). After 1500 ml of the initial eluate was collected, the antigen was still restricted to the top third of the column. A sodium chloride gradient in the initial buffer was used to elute more non-antigen protein from the column. The elution of this material and its characteristics have been described in a previous publication.iS) The first peak and its tail were collected in fractions of 100-300 ml, concentrated and freed of lactose as described above. The types of immunoglobulins in each of these fractions were characterized by immunoelectrophoresis with anti-equine y-globulin antisera. The results of this analysis are illustrated in Fig. 1. The ascending limb of the first peak shows two slow-moving bands and a faint trace of a third, faster-migrating, component. The remainder of the peak shows these three components with the emergence of a fourth, faster-moving, antigenically distinct component. Fractions from the tail of this peak show the gradual decline in the amount of the slower-migrating components and the relative enhancement of the faster two components, especially the fastest component. This latter component has been found to be antigenically identical to a component eluted early in the gradient of NaC1 and characterized as a 7S fl2A(yA) globulin.iS) Analysis by agar double diffusion of fractions taken from the first peak, with antisera absorbed with 7S y-globulins, demonstrated that 9,A immunoglobulin is absent in the ascending limb of the peak and present in the remainder of the peak and its tail (see Fig. 1). In order to obtain additional 7G globulin free of 7A globulin, fractions containing a mixture of the two classes of immunoglobulins were subjected to zone electrophoresis. The protein elution pattern obtained is illustrated in Fig. 2 and the immunoelectrophoretic patterns of the designated fractions is shown in the insert.

56

NORMAN R. KLINHAM, JOHN H. ROCKEYand FRED KARUSH

Peak A appears to be free of yA globulin. A pool of yG globulins consisting of fraction 1 (Fig. 1) and peak A (Fig. 2) of zone electrophoresis was used for all subsequent studies. Purity of yG antihapten antibody. The above agar diffusion studies demonstrated that the yG antibody prepared by this method was essentially free of any other antibody components. Analysis by immunoelectrophoresis with antiserum directed against whole equine serum demonstrated that the antibody preparation contained no other equine serum proteins. The absence of contaminating Lac-Hy was demonstrated by the lack o f any detectable absorption peak at 365 m/~ and the extrapolation to two binding sites per molecule of antibody by equilibrium dialysis. A demonstration that all of the protein components observed contain antibody activity was accomplished by a modification of immunoelectrophoresis.(14) Fig. 3 shows photographs of an immunoelectrophoretic slide in which the bottom well contained an antibody preparation consisting of 7G,,b,c, and 7A, and the top well '500 . . . . . O'02M PO4 pNT.2 0"25" ACETIC ACIn "4OO

~

~

PROTEIN CONCENTRATION = •

~Z' 300

~'200

"100

240

260

280 300 320 340 WAVELENGTH (mJJ)

360

380

FIc. 4. Absorption spectra of purified equine anti-Lac ~,G antibody in 0"02 M sodium phosphate buffer, pH 7.2 and 0-25 M acetic acid. contained the same antibody preparation at the same concentration but mixed with Lac-HSA. As the amount of Lac-HSA was in large excess, any antibody protein should have been in a soluble complex with the antigen. Electrophoresis of this material should be expected, then, to show antibody molecules migrating more rapidly than would be expected in the absence of the antigen. It can be seen therefore that all of the components present in the samples tested had anti-Lac antibody activity. Physical and chemicalproperties of equine ~,G antihapten antibody. Optical density measurements of the purified ~,G antihapten antibody showed an absorption maximum at 279 mtz and a minimum at 251 mt~ both in 0.02 M sodium phosphate buffer, pH 7.2 and in 0.25 N acetic acid (see Fig. 4). The ratio of absorption at

The Specific Interaction of 7G Antibody Components

57

279 m# to that at 251 m# was 2.36 in 0.02 M phosphate and 2.39 in 0.25 M acetic acid. At 279 m~ E 17° was 14.66 in the former solvent and 15.07 in the latter solvent when calculated with protein concentrations determined by micro-Kjeldahl analysis assuming 16 per cent N. Ultracentrifugation of this material showed a single symmetrical peak with an s020,w value of 6.8 S. The dye binding of this protein was also demonstrated by ultracentrifugation (Fig. 5). Immunoelectrophoresis developed with anti-equine y-globulin demonstrated that while the material showed a migration pattern typical for yG globulin, it contained three antigenically distinct bands (cf. Fig. 6). These were distinguishable from other globulin components such as yA by their electrophoretic mobility and their antigenic individuality. Similar heterogeneity of human and mouse ~,G has been reported recently. ~1~-19) To ascertain whether the heterogeneity demonstrated with 7G antihapten antibody could be attributed to differences in the heavy (A) chains, the material was analyzed by immunoelectrophoresis with antisera specific for equine 7G globulin heavy (A) chains and equine ~,G globulin light (B) chains. The results in Fig. 6 show that the antisera directed against the heavy chains (A) distinguished three components in the 7G preparations while antiserum against light (B) chains gave rise to a double band. These results were unchanged even if the anti-A and anti-B antisera were previously absorbed with excess B chains and A chains respectively. The finding of multiple heavy chains is similar to that reported for the ~,G immunoglobulins of other species.(16 19) The presence of these three yG components in normal equine serum was shown by the immunoelectrophoretic technique (Fig. 7). This figure also demonstrates that the heavy chains of the three yG components are antigenically distinct from ~,A heavy chains. The upper electrophoretogram shows normal equine serum developed against rabbit anti-equine serum and goat antiserum specific for the A chains of equine y-globulin. The existence of the four components yGa,b,c and yA in normal serum was demonstrated by the reactions with these antisera. In the lower electrophoretogram the technique of Osserman~20) was used to demonstrate that the three components of yG antibody are immunologically identical to components of normal serum and distinct from vA in the normal equine serum. Here equine serum was electrophoresed and developed against anti-A chain antiserum while yGa,b, and c, diffused from the lower well. The presence of yGa,b and c in normal serum is shown by the fusion of three of its lines with those of the yGa,b,c while the ~,A line of normal serum is distinct. Binding of Lac dye by purified yG antibody. The binding experiments were carried out with three preparations of yG anti-Lac antibody. Preparation #3 contained antibody purified from serum taken 6 weeks after the second course of immunization was initiated. Preparations # 4 and #5 contained antibody from sera taken at 8 and 10 weeks respectively. The binding curves of these preparations at 25 ° and 37.2 ° are shown in Fig. 8. The data are plotted with r, the average number of dye molecules bound per antibody molecule, as the ordinate and r/c as the abscissa where c is the free dye concentration at equilibrium. The data plotted in this form gives the average association constant as the value of r/c at half saturation of antibody, i.e. r = 1. A value of two binding sites per molecule, based on a molecular weight of 150,000, was obtained by additional binding measurements

58

NORMAN R. KLINMAN, JOHN H. ROCKE¥ and FRED KARUSH

with these and other equine antibody preparations in the region of higher free dye concentrations and extrapolation of the values of r to infinite free dye concentration. T h e thermodynamic parameters were calculated using the association constants obtained for the various antibody solutions at the two temperatures and are shown in Table 1. T h e results are expressed as unitary free energy and entropy to eliminate the entropy of mixing for reasons discussed elsewhere. (51) 70

25"C

372"C

i.5

60

5O

T

o x

40

~4~ #5 30

20 I0

d8

i'z

1:6 oYs

1'.2

,'.6

F

FIG. 8. H a p t e n - b i n d i n g curves c o n s t r u c t e d w i t h t h r e e e q u i n e ~,G a n t i - L a c antib o d y p r e p a r a t i o n s at 25 o a n d 37.2 °. r is t h e n u m b e r of moles of L a c dye b o u n d p e r 150,000 g of a n t i b o d y at t h e e q u i l i b r i u m free dye c o n c e n t r a t i o n c.

TABLE 1

Serum Equine ~:3 Equine ~4 Equine @5 Rabbit

KA ×10 -5 KA ×10 -5 25'0 ° 37"2° 1./m 1./m 3"5 4"24 6'2 1' 57

1.92 2"08 3.23 --

AH ° Kcal/m

AF,, Kcal/m

AS= eu/m

-9"06 -10"07 -9'94 - 9"7

-9"81 -10'08 -10.29 - 9"47

+2"52 +0.04 +1"18 - 0-8

DISCUSSION T h e thermodynamic parameters of the antibody-hapten interaction of three preparations of equine anti-Lac antibody and one of rabbit anti-Lac antibody(t, 21) are presented in Table 1. A comparison of the results obtained with the anti-Lac antibody preparations from the two species shows almost identical values for the free energy and enthalpy of the reaction with Lac dye. In both cases the enthalpy is almost entirely responsible for the specific binding activity while the unitary entropy of the reaction is either negligible or slightly favorable.

F ++

÷

÷

+ +, .......... % - -

2i0 i

8 E

t '~6

o

~- t:2 Z

0:8

0"4

o 0:2 0

200

400

600

800

1000

1200

1400

ml

FIG. 1. Chromatographic pattern of anti-Lac antibody from the solubilized specific precipitate eluted from DEAE-ceUulose with 0"02 M sodium phosphate buffer pH 7'2 and 0-5 M lactose. /~2A (TA) content of selected samples is given above the elution diagram. Insert shows the immunoelectrophoresis pattern of the indicated fractions developed against rabbit anti-equine serum antiserum. On the basis of the designations employed by Rockey et al.(s) fraction I shows 7G(a), ~,G(b) and a trace of 7G(c), and fraction II shows 7G(a), (b) and (c) and a trace of 7A. All four components are present in fractions I I I and IV with 7G(c) and 7A increasing relative to ~,G(a) and 7G(b).

0"5

F--A--t

F----

B ----,

o

~0-3 Z W

o 0-2

\

U

ORIGIN

g J .....

5

I0

--..~v

15

FRACTION

20

25

30

35

NUMBER

FIG. 2. Starch block zone electrophoresis of F11 (Fig. 1) in barbital buffer, p H 8"6 /'[2 0"05 at 4 °. Insert shows immunoelectrophoresis pattern of protein from the two peaks developed against rabbit anti-equine serum antiserum. Peak A shows only 7G components while Peak B contains ~,G and 7A. (Facing p. 58)

FIG. 3. Immunoelectrophoresis of a preparation of yG(a, b, c) and z,A anti-Lac antibody developed against rabbit anti-equine serum antiserum in the absence (below) and presence (above) of excess Lac-HSA. The changed migration of all four components in the presence of L a c - H S A demonstrates that they all have anti°Lac antibody activity.

FIG. 5. Sedimentation diagram of purified equine anti-Lac ~,G antibody equilibrated with Lac dye. Picture taken at 45 ° schlieren phase plate angle 52 rain after reaching speed of 52,640 revs/min. Sedimentation of the dye with the antibody is demonstrated by absorption of filtered light by the dye.

FIG. 6. Immunoelectrophoresis of equine 7G anti-Lac antibody developed tgainst goat anti-equine 7-globulin A chains (upper) and goat anti-equine 7,~lobulin B chains (lower). T h e antiserum against heavy chains shows three distinct bands denoted a, b and c. The antiserum against light chains shows only two bands.

FIG. 7. (Upper). Immunoelectrophoresis of normal equine serum developed against rabbit anti-equine serum antiserum (above) and goat anti-equine vglobulin A chain antiserum (below). T h e presence of yGa,b,c and yA are demonstrated in normal equine serum. (Lower). Immunoelectrophoresis of normal equine serum developed against goat anti-equine y-globulin A chain antiserum (above) with equine yG anti-Lac antibody in the lower trough. T h e presence of yGa,b, and c heavy chains identical to those present in the antibody preparation is demonstrated by t h e complete fusion of the bands, while vA appears absent in the antibody preparation and is antigenieally distinct.

The Specific Interaction of yG Antibody Components

59

The equine antibody used for this comparative study was a selected population of yG immunoglobulin. It closely resembled, therefore, the population of molecules present in the rabbit antibody preparation with which it is compared in Table 1. The selection of such a molecular population was made necessary by the fact that the equine anti-Lac antibody was found to contain other classes of immunoglobulins which bind the hapten.(S) In addition to the above fractionation of the equine antibody, the antibody preparations from the two species differed in other respects. While the general method for antibody purification was similar for both rabbit and equine antibodies the methods for removing precipitating antigen and lactose from the solubilized antibody differed. In the case of the rabbit antibody the precipitating antigen (Lac-human fibrinogen) and the lactose were removed by salt precipitation of the antibody, while DEAE-cellulose chromatography and dialysis were used to remove the precipitating antigen and lactose respectively from the equine antibody. In addition the methods of immunization differed in that the immunizing antigens were not the same and the equine serum was obtained 6 to 10 weeks after the start of the second course of immunization while the rabbit antibody was obtained 5 weeks after the first immunization. In spite of these differences the binding energetics for the equine and rabbit antibodies are virtually identical. The slightly higher affinity of horse antiserum # 5 and of the horse antisera generally, when compared to the rabbit antiserum, may be a reflection of the tendency for higher affinity with increased time after immunization. The yG equine antibody preparation used for this study has been shown by immunodiffusion to contain at least three antigenically distinguishable populations of molecules denoted as yG(a), yG(b), and yG(c). These have also been demonstrated as components of normal equine serum. The yG(c) component may be a new class of immunoglobulin possibly related to the faster-migrating yG component reported by Ovary et al.(22) in guinea-pig antibody. The fact that yG(c) is indeed an antibody is demonstrated both by its presence in specifically purified antibody preparations and by the fact that when it is mixed with antigen its electrophoretic mobility changes. The distinctive antigenic determinants of the three yG immunoglobulins as well as the yA protein present in the antibody preparations have been demonstrated to be on the heavy (A) chains by immunodiffusion studies employing antisera specific for equine A chains. Antisera directed against the light (B) chains show a double band which apparently does not distinguish among the various immunoglobulins. This indicates that the three yG components represent three types of heavy (A) chains. Thus in addition to the yM, 10S y-globulin and yA previously described as components in the anti-Lac antibody population of this animal, t8) three antigenically distinct 7 S y-globulins have been demonstrated to have binding activity to the Lac haptenic group. Therefore, at least six distinct immunoglobulins can be formed in a single animal, all having antibody activity against a single haptenic group. A previous publication(s) has shown that the yA antibody prepared from serum ~4 had an association constant of approximately 107 1./m at 25 °. This value is considerably higher than the association constant of the yG antibody obtained in

60

NORMANR. KLINMAN,JOHN H. ROCKEYand FRED KARUSH

this study from the same serum. T h i s demonstration of differing affinities for the same hapten among the various classes of immunoglobulins from the same serum may partially explain the heterogeneity of hapten binding by purified antibody. If, in addition, there also exist subclasses which have not yet been detected by immunodiffusion, then it is possible that thc affinity of the antibody preparation as a whole, being the average of the affinities of the populations of molecules it contains, would change as the relative amounts of these subclasses changed. Such population shifts could be responsible for the increased affinity observed in antibody with increasing time after immunization.

SUMMARY Purified equine y G anti-Lac antibody contains at least three immunoglobulins with antigenically distinct heavy chains. A comparison of the thermodynamic parameters of its hapten binding show a great similarity to those obtained from a similar rabbit antibody. T h e affinity for hapten, however, is considerably less than that exhibited by ~,A anti-Lac antibody from the same serum. Such a difference in the affinity for hapten may partially explain the heterogeneity of affinity of purified antihapten antibodies as well as the increase in affinity with time after immunization. Acknowledgement~We are grateful to the School of Veterinary Medicine of the University of Pennsylvania and to Dr Lawrence S. Cushing for their generous and invaluable assistance in the research program. We also wish to thank Mrs Sally Karush, Miss Ada C. BeUo and Mr Robert Marks for excellent technical assistance.

REFERENCES

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