Structural requirements for recognition of vasopressin by antibody; Thermodynamic and kinetic characteristics of the interaction

hfolecular Immunology, Vol. 18, No. 5, pp. 439-446, Printed in Great Brirain.

0161-5890/81/050439-08 $02.00/O Pergamon Press Ltd.

1981.

STRUCTURAL REQUIREMENTS VASOPRESSIN BY ANTIBODY; KINETIC CHARACTERISTICS J. BARBET, Centre

G. ROUGON-RAPUZZI,

d’Immunologie

FOR RECOGNITION OF THERMODYNAMIC AND OF THE INTERACTION A. CUP0

INSERM-CNRS de Marseille-Luminy, CEDEX 2, France

and M. A. DELAAGE Case 906, 13288, Marseille

(Received 16 September 1980; accepted 19 November 1980)

AbstractThe thermodynamic and kinetic properties of two conventional antivasopressin antisera were studied. Values for binding affinity were determined by equilibrium measurements to be Kd = 1.4 and 2.7 x lO-Lz M. The kinetic parameters were independently determined. The association rate constants (kas) were calculated by a pseudo-first-order analysis of binding kinetics (ka = 3.1 and 1.7 x 10’ M-’ set-I). The dissociation rate constants (kds) were measured by dissociating antibody-labelled antigen complexes with large excess of unlabelled antigen (kd = 1.6 and 1.7 x 10e5 set- I). A fairly close agreement was achieved between equilibrium and kinetic evaluation of the affinity. The heterogeneity cannot be assessed through equilibrium experiments because of the very low concentrations of reagents to be handled. However, kinetic studies strongly suggested that the molecular heterogeneity with respect to affinity of the antisera is restricted to a narrow range (5 x lo-l3 M to 7 x lo-‘2 M). Despite their very similar physicochemical properties these two antisera exhibited different fine specificities: the study of cross-reactivities with various analogues of the original hapten showed that one antiserum-5-is clearly directed against the C-terminal moiety of the molecule. The antigenic determinant is a sequential one and composed of the last four aminoacids Cys-Pro-Arg-GlyNH,, while the other antiserum is not so sensitive to modifications of the last residue GlyNH,.

INTRODUCTION

While there is a considerable body of knowledge related to immunoglobulin structures and their amino acid sequences, less is known about antibody-hapten interaction. Affinity constants reported as Kd (dissociation constant) can vary over limits as wide as 10m6 to lo-l3 M. The heterogeneity of these antisera in terms of affinity as well as in terms of molecular species (clones) is still giving rise to controversy: from an apparent homogeneity (Montgomery er al., 1972) to a very large heterogeneity. With respect to kinetics, if one generally agrees that the association step is fast, the dissociation rate constants vary broadly and are supposed to reflect the affinity distribution (Smith & Skubitz, 1975). However, considerable studies were done on very small haptens (e.g. DNP) (Day et al., 1963; Kelly et al., 1971; Barisas et al., 1975) but the results obtained are clearly misleading. It is well established that the antibody combining site ‘is relatively large (Segal & Hurwitz, 1976) and that very small molecules like DNP-lysine must not fill completely this combining site. Thus, studies on antisera raised against large haptens may give more relevant information. 439

The development in our laboratory of a vasopressin radioimmunoassay gave us the opportunity to study, from the immunochemical point of\view, the antibody response against a well defined peptidic hapten: I

I

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-GlyNH,.

Several rabbits were hyperimmunized with arginine vasopressin (AVP)* coupled via its amino terminal group to a macromolecular carrier (human myeloma IgA protein). Each animal gave high titered antisera after three boosts (Rougon-Rapuzzi et al., 1977). This paper deals with the determination of the specificities and of the equilibrium and kinetic characteristics of vasopressin binding to two of these antisera.

*Abbreviations used: AVP, arginine-8 vasopressin; LVP, lysine-8 vasopressin; AVP-COOH, arginine-8 vasopressin free acid; AVP-(Arg-COOH), tryptic peptide from AVP; AVP-(Gly-Gly-NH,), decapeptide bearing one additional glycine residue on the C-terminal moiety; diA-LVP, diS-LVP, diacetyl, disuccinyl lysine vasopressin; AVT, arginine-8 vasotocin; OT, oxytocin; [*251] AVP, lzsI iodo-arginine-8 vasopressin; DNP, 2,4-dinitrophenyl; As, antiserum.

440

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AND METHODS

Antibodies Antibodies against Arg-8 vasopressin (AVP) coupled to a human IgA via its amino terminal group were raised in rabbits as described by Rougon-Rapuzzi et al. (1977). The response was followed for five rabbits. Sera from 2 rabbits (designated 3 and 5) were used for the present study. Chunicds vasopressin (AVP) and Arg-8 Arg-8 vasopressin free acid (AVP-COOH) were purchased from Bachem Feinekemikalien AG, Bubendorf, Switzerland. Lys-8 vasopressin (LVP), oxytocin (OT), Arg-8 vasotocin (AVT) were purchased from Sigma Chemical Company, St. Louis, MO, LJ.S.A. AVP was hydrolysed by trypsin (TPCK 259 TAME units/mg from Worthington) for 60 min at 4C to obtain the octapeptide lacking the C-terminal glycinamide AVP-(Arg-COOH). AVP-(Gly-Gly-NH,) was synthesized by coupling AVP-COOH (Bachem) to glycinamide with l-ethyl-3(3 dimethylaminopropyl) carbodiimide (Sigma). The peptide acetyl-L-Pro-L-Arg-Gly-NH2 was synthesized from L-Pro-L-Arg-naphthylamide (Bachem) by acetylation, tryptic hydrolysis and coupling to glycinamide. Diacetyl and disuccinyl LVP (diA-LVP) (diS-LVP) were obtained by acylation of LVP with acetic and succinic anhydrides respectively. AVP, AVP-COOH and OT were iodinated according to Hunter & Greenwood (1962) with lz51 sodium iodide (NEN Chemicals GmbH, Dreielch, West Germany). The lz51 labelled peptides were purified by gel filtration on a G-25 Sephadex column, as described by Rougon-Rapuzzi et al. (1977). This procedure was shown to produce the mono-iodoTyr.2 derivative separated from unreacted peptide as well as from sodium lzsI iodide and diiododerivatives. Thus the specific radioactivity of labelled AVP can be approximated by the sodium iodide specific radioactivity of 2000 Ci/mmole. The yield of the 7 counter we used was found to be 75’14using a NEN calibration sample (IzaI simulating 1251). Equilibrium

.studies

These experiments were performed using two different techniques: (a) Equilibrium dialysis: constant amounts of

et al.

[“‘I]AVP derivative (40,000 cpm,:ml corresponding to a final dilution of 6 x IO-~ I7 M) were incubated with antisera (dilution l/50.000 and l/100,000 for antisera 3 and 5 respectively) and variable quantities of unlabelled AVP or analogues. Concentrations tested were varied from 10-l” A4 to 10mx M. Incubations and separation of free from bound antigen were performed using a dialysis apparatus previously described (Cailla et al., 1973). The incubation medium consisted of 10 mM sodium phosphate, pH 7.2,O. 15 M sodium chloride containing 1 g/l. sodium azide and I g/l. human serum albumin. The incubation time was 48 hr at 4 C. Reference curves obtained with the homologous hapten AVP allowed us to calculate antibody binding site concentrations as described by Cailla rt ul. (1973). Cross-reactive factors for analogues were reported as the ratio of concentrations giving a displacement of 50”,, of the [‘251]AVP antibody binding. (b) In order to determine dissociation constants (K&s), we used another kind of permitting us to test lower incubation, concentrations of antibody and hapten. as suggested by Steensgaard rt d. (1980). Samples were prepared by mixing in 3 ml plastic tubes antisera at dilutions 1O> and 10” ( I .2 x IO ’ z M for As 3 and 5 respectively), 3200 cpm of [I r-il IAVP derivative (final concentration 8 x IO- I.3 A4) and unlabelled AVP at final concentrations varying from 10 ‘A ,21 to lo-” M. The final incubation volume was I ml. The tubes were shaken for 96 hr at 4’C in order to ensure equilibrium completion. Free from bound ’ 2zIantigen separation was performed by filtration through cellulose acetate filters as described for kinetic studies (vi& infiu). In all calculations of Kd values, the background occurring when labelled antigen alone was tiltered ( 1 IO”,,) was substracted. Kinetic .studies The kinetics of association and dissociation were followed by taking advantage of the capability of cellulose acetate filters (Millipore, GSWP, 0.22 ,um, 13 mm) to retain diluted antibodies whether free or complexed with their hapten (Weinryb et ul., 1972). Association rate constants (kas) were determined from binding measurements using dilutions ranging from l/2000 to l/20,000 (6 x IO-” M to6 x IO-‘? M antibody binding sites) for antiserum 3. and from l/10,000 to l/50,000 (I.1 x IO-” M to 2.2 x 10-l l A4 antibodv bindine sites) for

Thermodynamic

and Kinetic

Characteristics

of Anti-vasopressin

441

Antibodies

A

I IO-‘2

IO.13

Concentration of bound antigen,

M

Fig. 1. Scatchard analysis of equilibrium data obtained by Millipore filtration. (A) Scatchard plot for antibody 3 obtained with antiserum dilution of l/lOO,OOO; [12SI]AVP at a concentration of 8 x lo-l3 M in the test and AVP dilutions ranging from IO-” to lo- I3 M. Incubation time was 96 hr at 4°C. (B) Scatchard plot for antibody 5 obtained with antiserum dilution of 1/106, other parameters were similar to those mentioned above. Each point is the mean for three different assays. The closed circle (0) on each plot represents the antibody site concentration calculated from equilibrium dialysis.

antiserum 5. The radiolabelled antigen was diluted to 40,000 cpm/ml in the reaction medium (about lO_” A4). At selected times, 200 ~1 of the incubation mixture were filtered through a Millipore filter and washed with 10 ml buffer. Each sampling was done in duplicate, and the reaction mixture was kept at 4°C. The filter was then directly counted in a y-counter for 2 min. The reaction was followed to completion and the data were plotted as log (a/a-x) (where a is maximum binding and x the binding at time t) versus time (t), for each antiserum dilution. The reaction rates are then calculated by linear regression and used for pseudo-first-order analysis.

Table

1. Thermodynamic

and kinetic constants

Dissociation rates (Us) were determined, at different concentrations of antibody. Antisera were preequilibrated for at least 24 hr with the radio-labelled antigen, then unlabelled antigen (5 x 10e5 A4 in buffer) was added to a final concentration of 1O-6 A4 and at successive times 200 ~1 of the incubation mixture were filtered, washed and counted. The data are plotted as log (x/a) versus time (t) and the dissociation rate constants (kd) are calculated by linear regression. In all calculations the binding occurring when labelled and unlabelled antigens were premixed ( rr_10%) was substracted. Filtration of antibody alone and subsequent incubation of the filtrate with [‘251]AVP

for the interaction

Association rate constant ka (M-l set- ‘1

Dissociation rate constant kd (set- I)

Antiserum

3

3.1 x 10’

1.6 x 1O-5 2 x 10-4

Antiserum

5

1.7 x 10’

1.7 x 10-s

between

Dissociation constant calculated

AVP and anti-AVP

antibodies

Dissociation constant measured (M,

Concentration of binding sites (M,

0.5 x IO_‘2 7 x lo-‘2

1.4 x IO_‘2

1.2 x 10-7

1 x 10-12

2.7 x lO-12

1.1 x lo-6

$n

442

(21al

J. BARBET

showed that essentially all the antibody bound to the Millipore filters in this concentration range (lO-‘2 M to lO_‘” M).

The cross-reactivities for antisera 3 and 5 of related hormones and chemically or enzymatitally modified haptens were evaluated by competition with [ lz51] AVP. The cross-reactive factors Cjs) were expressed as

RESULTS

moles investigated

Equilibrium The dissociation constants (Kds) were determined using the second technique of incubation (see Materials and Methods) which allows us to test much lower concentrations of reagents (lz51 antigen: 8 x 10-i” M and As: 1 x lo- l2 M). Because of the very low concentrations used the experimental values obtained were slightly scattered. Nevertheless, Scatchard plots (Fig. 1) have been fitted by the least square regression straightline and the dissociation constants have been calculated (Table 1). The heterogeneity of these antisera cannot be assessed; however the fact that comparable antibody combining site concentrations were found at different dilutions (1 /SO,000 and l/100,000 for As 3; 1/SO,OOO, 1/lOO,OOO, l/l ,OOO,OOOfor As 5) indicates that there is no significant contribution from lower affinity antibody populations.

Table 2. Cross-reactive

factors

compound

moles AVP the amounts of AVP and investigated compounds were read from the displacement curves at 50”/ inhibition of binding of [1251]AVP (Table 2). Dissociation

rate constants

A typical experiment using antiserum 5 and [’ 2 51] AVP is shown in Fig. 2. The experimental curves can be fitted by single exponentials and the dissociation rate constants (kds) obtained (1.7 x 1O-5 set -i for antiserum 5 and 1.6 x 10 5 sect l for antiserum 3) are quite close and do not depend on the antibody concentrations. In order to ascertain whether lower affinity antibody populations are present in these antisera and are able to bind antigen in the dilution range used in these kinetic measurements we designed two experiments: (i)

for vasopressin

analogue-anti-vasopressin

antibody

complexes Cross-reactive factors us) antiserum 3 5

Peptide

I

2

3

4

5

6

I

Cys

AVP LVP

7

-8.

9

I

-

Tyr

-

Phe

-

AVT

-

Ile

-

OT

-

Ile

-

Gln

-

Asn

-

Cys

-

Pro

-

Arg

-

Lys

-

Gly-NH,

I

I

ND”

I

9.2

diS-LVP

-

Leu

-

-

Lys

-

8.9

2.3 x IO4 3.9 x IO+ ND

1 x 10’

I

NH<-(CH,),COOH Ii 0 -Arg-Gly-Gly-NH,

AVP-(Gly-Gly-NH,) Reduced alkylated

and AVP

cys-

1 S-CH,COOH

-cys-

= not determined

ND

1.2 x IO’

3.6

4.4 x I02

57

I.3 x 103

ND

2.5 x IO’

S-CH,COOH -

Gly-COOH

-Arg-COOH

AVP-(Are-COOH)

“ND

2.9 x 102

I

AVP-COOH

Tripeptide

Y.5

Acetyl

-

Pro

-

Arg

-

Gly-NH,

Thermodynamic

and Kinetic

Characteristics

of Anti-vasopressin

I 1/20,co0

443

Antibodies

I 1/5,cal

I i/3,500

I 1/2,ccm

AntIserum dilution

Fig. 3. Pseudo-first-order analysis of the binding of [izS1]AVP to antiserum 3. The slope of the straight line is 3.1 x 10’ M-i set-i taking 1.2 x IO-’ M as the concentration of antibody binding sites.

L

I IO

I 20

I 30

Time,

I

I

40

50

h

Fig. 2. Dissociation of the complex [iz51]AVP-anti-AVP antibody from serum 5. Antiserum 5 at dilutions l/lO,OOO (0) and l/50,000 (+) with radiolabelled antigen IO- *1 M for 24 hr at 4°C. AVP (50 PM) was added at time zero to a final concentration of 10e6 M. Similar curves were obtained with antiserum 3 at dilutions l/2000, l/3000, l/SO00 and l/20,000. The solid line represents the exponential derived from experimental data as described in Materials and Methods.

the dissociation of a fully saturated antiserum (antiserum 3 diluted l/3500 and incubated with an excess of radiolabelled antigen: 8 x lo- 11 A4 final concentration) was followed by chasing with the cold hormone; (ii) the dissociation was initiated after a short time of incubation (30 min) of the antibody (antiserum 3, dilution l/3500) and the radiolabelled antigen (1.5 x lo-” M’). In both cases the dissociation curves cannot be fitted by single exponentials and, as expected, faster dissociating complexes are observed. Nevertheless, in both cases the amplitude of the first exponential represents only about 2.5% of the total binding. The associated dissociation constant was found to be -2 x low4 set-‘, while the remaining 75% dissociated more slowly with a rate constant very close to the one determined under the normal conditions. Similar results were obtained for antiserum 5. Association kinetics In these experiments the concentration of radiolabelled antigen used (about 1.5 x 10-l l

n/n is relatively high as compared to the of antibody binding sites concentrations calculated from equilibrium data. However, these values may be slightly underestimated because of the contribution to the binding of lower affinity antibody populations which can be of some influence in experiments with such concentrations. In the dilution range l/20,000 to l/2000 requirements for pseudo-firs t-order approximation are fulfilled. Figure 3 shows this analysis for antiserum 3. Using the concentration of antibody binding sites derived from Scatchard plots the association rate constant (ka) was determined to be about 3.1 x 10’ M- ’ set- I for antiserum 3. The same analysis for antiserum 5 was done using dilutions of antiserum from l/50,000 to l/10,000 (not shown). The association rate constant was then found to be 1.7 x 10’ M-l set-l. The comparison between kinetic and equilibrium is straightforward: the ratio of the dissociation rate constant over the association rate constant (kd/ka) gives the equilibrium dissociation constant (Kd). For both antisera the agreement between these two determinations of Kd is satisfactory (Table 1). Binding of vasopressin analogues No significant binding to antisera 3 (dilution l/2000) and 5 (dilution 1/lO,OOO) was found with either [1251]OT or [lZSI]diacetyl-LVP by equilibrium dialysis or by kinetic measurements, in agreement with competition measurements which give for these compounds dissociation constants at least 30 times higher than the

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444

concentrations of antibody binding sites used in these experiments. By contrast, the iz51 free acid AVP exhibited strong binding to antiserum 3 (dilution l/2000) as evidenced by its association kinetics. The time constant under these conditions is 9 x 10m5 set I. This binding may be reversed by chasing with cold AVP and the dissociation rate constant (kri) is 6.7 x lo- s set- ‘. In equilibrium dialysis the competition of cold AVP-COOH with either [izSI]AVP or [12sI]AVP-COOH gives the same displacement curves (K& thus AVP and AVP-COOH compete for the same antibody binding sites. So we can calculate from kinetic experiments a dissociation constant of 4.5 x 10mi2 A4 (ka = 1.5 x IO7 M-l set-I) which favourably with the equilibrium compares dialysis measurements cf = 3.6) For antiserum 5 the cross-reaction between AVP and AVP-COOH is much lower cf’ = 440) as evidenced by equilibrium dialysis and no significant binding was observed when [1251]AVP-COOH is added to antiserum 5 at dilution 1/10,000. DISCUSSION

The immunization of rabbits against AVP resulted in the production of high titered antisera (Rougon-Rapuzzi cr al., 1977) and in the occurrence of an autoimmune disease in immunized animals (Cau & Rougon-Rapuzzi, 1979). These phenomenons led us to investigate further the fine specificity and the thermodynamic properties of these antisera. The study of cross-reactivities of AVP analogues allowed us to define more precisely the site of fixation of antibody on the antigenic molecule and to determine the immunodominant residues; this was done essentially for antiserum 5, which is used in radioimmunological measurements of AVP in biological fluids and tissues (Rougon-Rapuzzi ~>tal., 1978): (1) The conformation of the peptide plays only a minor role in the interaction. AVT is only 9 times less immunoreactive than AVP though it was demonstrated by Deslauriers & Smith (1970) that replacing Phe 3 by Ile causes conformational changes in the cyclic moiety. Furthermore, the reduced and alkylated peptide which must differ strongly from the original peptide in terms of conformation but also in terms of charge is nevertheless strongly immunoreactive (f = 120) (2) A positive charge on the 8th amino acid is essential for recognition. While antiserum 5 possesses the same affinity for AVP or I-VP,

e/ L/I

acetylation or succinylation of I-VP result in it considerable loss of immunoreactivity. OT. which lacks the positive amino acid in position 8 and presents a ‘tocin’ confortnation binds only weakly to antiserum 5 (f = 3.9 x lOJ). (3) Modifications of the C’-terminal tnoiety reduce the immunoreactivity dramatically. Cross-reactive factors for AVP-(Gly-Gly-NH2), AVP-COOH and AVP-(Arg-COOH) are respectively 290. 440 and 1300. The small peptide Pro-Arg-GlyNH,. which represents the C‘terminal sequence of AVP. possesses a low but measureable immunoreactivity by itself cf’ = 2.5 x 10s). These considerations lead us to propose that the major antigenic determinant recognized by antiserum 5 is the C-terminal moiety of AVP: Cys-Pro-Arg-GlyNH,. As the tracer is a hormone modihed on its cyclic moiety. this eliminates the possibility of interaction with some antibody populations specific for this cyclic moiety. The same kind of results were obtained by Czernichow et ul. (1974) for some antisera elicited against AVP coupled to serum albumin. Other specificities are possible, as evidenced with antiserum 3 which strongly binds AVP-(Gly-Gly-NH,), AVP-COOH and to a lesser extent AVP-(Arg-COOH). Thus, the major antigenic determinant for this antiserum is somewhat displaced toward the cyclic moiety of AVP. However, neither of these antisera were able to recognize the ‘pressin’ ring of I-VP from AVT as did those studied by Czernichow ct ul. (1974). These results afford an opportunity to discuss the possible size and nature of antigenic determinants recognized by our antibodies. The equilibrium binding data clearly indicate that the antigen determinant, for antiserum 5, is composed of the last four sequential amino signifcant influence of the acids, without conformation. This is in agreement with the proposal of 4 to 5 residues for the size of a sequential determinant on a polypeptide (Schwartz et al., 1978; Janski & Graves. 1979). In the case of conformational determinant. it is clear that all the amino acids responsible for the conformation are needed for antibody recognition (Schechter er ul., 1971). Such LiS opposed to anti-sequence antibodies. the classical ‘anti-conformational’ antibodies generally described for proteins, could be considered as ideal probes for studying precursor-product relationships in proteins or for detecting homologies even when evolution has not maintained the confortnational de-

Thermodynamic

and Kinetic

Characteristics

terminants. In the case we studied, in high molecular forms (precursor) an elongation of the AVP hormonal sequence on the C-terminal side prevents binding to antiserum. A tryptic digestion released peptides with a C-terminal sequence similar to that of AVP-(Arg-COOH) which is still not well recognized by antiserum 5. But a reconstitution of the antigenic determinant by covalent coupling of glycinamide allowed the quantification of the precursor forms by the same radioimmunoassay as AVP itself. The application of these concepts will be presented elsewhere (Cup0 et al., 1981). By contrast with most of the published data we are dealing with a relatively large hapten specifically bound to the carrier via its amino terminal group, which is the unique reactive function. Besides, the reactivity of these antisera was monitored by a radiolabelled derivative which bears the iodine atom on the Tyr-2 residue. Thus, structural modifications that always occur in the chemical coupling to a carrier or in the radiolabelling with iodine are restricted to the same moiety of the AVP molecule. So, one can expect that the antigenic determinants presented on the carrier and born by the hapten, labelled or not, are quite similar. This could be one explanation for the strong interaction between the hapten (AVP) and the antibodies. Indeed, the classical technique, i.e. equilibrium dialysis does not permit a correct evaluation of affinity. In fact, with the concentrations used for the RIA, the binding of the radiolabelled peptide was almost stoichiometric and the competition with unlabelled antigen only allows estimation of the total binding site concentration. So, in order to give reasonably precise numerical estimates of dissociation constants (Kds), we were obliged to choose a more convenient method allowing to test lower concentrations of all reagents. Because of their slight dispersion, the values obtained by Millipore filtration do not permit a precise analysis of the Scatchard plots (Fig. 1) and thus the heterogeneity of the antibody binding affinities cannot be estimated. However, we noted that analysis of experiments performed with different dilutions of antisera (from l/100,000 to l/10,000 for As 3 and l/lo6 to I/50,000 for As 5) always gave very similar concentrations of antibody binding sites. These findings rule out the possibility of a broad distribution of affinity in the range lo- l2 M to 10 - l O M expressed as Kd. When affinities are too large with respect to the sensitivity of the methods used to follow

of Anti-vasopressin

Antibodies

445

the interaction between the two molecules, make possible an measurements kinetic independent determination of these thermodynamic parameters (Capelle et al., 1980). For the reaction considered, the ratio of the dissociation rate constant (k4) to the association rate constant (ka) must be equal, if no major rearrangement of the complex occurred, to the equilibrium dissociation constant (Kd). Association rate constants (kas) cannot exceed the diffusion limit which can be estimated for such molecular weights to be about lo8 M- * set-l. For a low affinity system the association constants were found to be around lo7 M-l set-’ (Day et ul., 1963; Froese et al., 1962). Similar values were also mentioned for other systems such as fluorescein (Levison et al., 1971) and ouabain (Skubitz et al., 1977) (ka = 5 x lo7 M-’ set-‘; Kd = 6 x lo-lo M and 8 x lo6 M-l set-l; Kd = 3.5 x lo-” M respectively). So, in all these examples, dissociation rate constants showed a greater degree of variability than did association rate constants, in keeping with the conclusions of Smith & Skubitz (1975) that differences in antibody affinity arise mainly through variations in dissociation values. This was also supported by our conclusion measurements which showed that the high affinities of our antisera are reached through slow dissociation rates (kd = 1.6 x lo- 5 set- i and 1.7 x lop5 see-‘) and by those of Kriise (1979) for antithyroxine antibodies. However, one exception has been reported by Denizot et al. (1979) for anticyclic AMP which exhibited lower association rateconstants(ka = 1.2 x lo5 M-l set-’ and 6.6 x lo5 M-’ set-‘j. These kinetic measurements bring some additional data with respect to the heterogeneity of the antisera. On one hand, no detectable amount of very slowly dissociating antibodies was observed, so we can exclude an appreciable contribution of a higher affinity antibody population. On the other hand, several experiments support the conclusion that important low affinity populations do not exist: (1) pseudo-first-order analysis of association kinetics were conducted at fairly hiah c concentrations of antibody, where low affinity populations were likely to have an influence; (2) dissociation kinetics performed after incubation of antibody with an excess of hapten showed only a minor (25%) population dissociating more rapidly, with an affinity constricted to 7 x IO- l* M, (3) the pulse binding (30 min) also showed only a minor contribution of the lower affinity complexes.

446

J. BARBET

Thus all the measurements, kinetic and equilibrium, seem to demonstrate that the heterogeneity of these antisera is fairly restricted in terms of affinity to the range 5 x lo- l3 to 7 x IO-” M. This information on the quality of immunosera may lead to a better understanding of the autoimmune disease induced by antivasopressin immunization in rabbits. The equilibrium and kinetic measurements establish that the diabetes insipidus observed in immunized animals could be satisfactorily explained in terms of hormone trapping by circulating antibodies. But. if the antibody concentrations decrease or if in some animals the on-rate of the raised antibodies is smaller, this trapping might not be efficient enough to lower the free AVP concentration below the regulation level (2.5 x 10p12 M. In conclusion, one can point out a somewhat striking feature of immunochemical analysis of conventional antisera raised against a structurally defined peptide. While the fine specificity may differ significantly from an antiserum to another (that is from one rabbit to another). the physicochemical characteristics of a hapten-antibody interaction appear to be much mot-e constant (our measurements and those of Czernichow et a/., 1974). Keeping in mind that non covalent interaction between protein and small molecules can be much stronger as illustrated by the binding of biotin to avidin (Hofmann & Kiso, 1976) one can wonder if the immune response ij restricted by a functional selection which would eliminate quasi-irreversible complexe;.

et al.

Day IL. A.. Sturtevant J. M. & Sin_eel- S. J. (1963) Kmetics ol the reaction between antibodIes to 2.4,-dinitrophenyl (DNP) group and specific hapten>. ,4nn. ,li.Y Accrd. SC,;. 103, 61 l-625. Denizot F. C.. Hirn M. H. & Delaagc M. A. (1979) Relationship between affinity and kinetics in a hapten-antibody reaction. Studies on anticyclic AMP antibodies. M&c. Imtnunoi. 16, 509-513. Deslauriers R. & Smith I. C. P. t 1970) Evidence from proton magnetic resonance data for the itacking of aromatic amino-acid in lysine va,opl-esain. Comparison with oxytocin derivated and related peptides. Biochrm. Bioplg~. Re.s. (‘ommun. 40, 179-l X5. Froese A., Sehon A. H. & Eigen M. (1962) Kinetic studies of protein-dye and antibody-hapten interactions with the temper-ature-jump method. Can. .I. C’hmt. 40, 1786-1797. Hofmann K. & Kibo Y. (1976) An approach to the targetted attachment of peptides and proteins to solid supports. Proc. nuts. Ac,ad. Sci., U.S.A. 73, 3516-3518. Hunter W. M. & Greenwood F. C. (1962) Preparation of iodide-131 labelled human growth hormone of high specific activity. Nuruw Land. 194, 4951196. Jansky A. M.&Graves D. J. (1979) Useofantibody probe to study regulation of plycogen phosphatase by its NH2 terminal I-don. J. hiol. Chrm. 254. 1644-1652. Kelly K. A., Skhon A. H. & Froese A. (1971) Kinetic studies on antibody-hapten reactlons. I. Reactions with antibodies theit and univalent Fab’ flragmentr. In?munochen?i.~tr~. 8, 6 13-625. Kriise V. (1979) Dissociahon rate constants and fractional binding of tracer estimated for three antibody populations in unstripped and stripped antiserum. Scam/. J. C/in. Luh. In~~.st. 39, 2 15-221 Levison S. A.. Portmann A. J., Kierszenbaum F. & Dandliker W. B. (1971) Kinetic behaviour of anti-hapten antibody of Irestricted heterogeneity by stopped flow fluorescence polarization kinetics. Bioc~lwm. Bioph,tx. Rer. C‘ommun. 43, 258-266. Montgomery P. C.. Rockey J. H. & Williamion A. R. (1972) Homogeneous antibody elicited with dinitrophenylgramicidin-S. Proc. natn. Acad. Sci., U.S.A. 69, 228-232. Rougon-Rapuzzi G., Cau P., Boudier J. A. & Cupo A. (1978) Evolution of vasopressin levels in the hypothalamo-posthypophysial system of the rat during rehydration following water depri\ Revlon. Neuroendocrinology 27, 4642,. Rougon-RaptI// G., Millet Y. A. & Delaage M. A. (1977) Preparation of antivasopressin antibodies using an IgA carrier. Application to radioimmunoassay. Biochimle 59, 939-942.

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