Recognition of imidazole and histamine derivatives by monoclonal antibodies

MolecularImmunology,Vol. 27, No. 10,pp. 995-1000,1990 Printed in Great Britain.

RECOGNITION DERIVATIVES

0161~5890/90 $3.00+ 0.00 Q 1990Pergamon Press plc

OF IMIDAZOLE AND HISTAMINE BY MONOCLONAL ANTIBODIES

A. MOREL,* M. DARMON and M. DELAAGE Immunotech, Luminy, Case 915, 13288 Marseille Cedex 9, France (First received 26 December

1989; accepted in revised form 22 March 1990)

Abstract-The different ways of raising antibodies to histamine are reviewed. High affinity monoclonal antibodies could be raised only against derivatized histamine. Succinyl glycinamide derivatization provided the basis of an efficient radioimmunoassay. In this paper the molecular pattern and the thermodynamical properties of histamine recognition were thoroughly investigated. Only the neutral form and not the cationic form of imidazole was recognized. As expected, the ligand recognition increased, with improved structural homology to the immunogen. However, a detailed analysis revealed a zwitterionic effect whenever a carboxylic group was present on the side chain of the ligand.

INTRODUCTION

The immunoanalysis of histamine has long puzzled immunochemists. The small size of the hapten seemed to preclude the emergence of high affinity antibodies. Actually for small haptens, like DNP, antibody affinity hardly reaches the level required for immunoanalytical purposes. Recently several attempts to solve the problem have been published. They can be classified into two categories. Those aiming at raising antibodies able to directly recognize histamine. Haydik (1983) has obtained antibody to unmodified histamine, merely adsorbed onto proteins. In spite of its rudimentary character, this approach led to antibodies with 30nM affinity, a value which permitted the assay of histamine in histamine release in vitro but not plasma histamine. A second approach was that of Buckler et al. (1987) who synthesized a N-alkyl immunogen which kept intact the cationic charge of histamine. However the affinity for histamine was very low (& 500nM). The second set of studies used the recognition not of intact histamine but of a derivative ressembling an immunogenic conjugate. The coupling of histamine to a carrier can be done in different ways but in all cases the size of the antibody binding site encompasses the histamine residue and includes the chemical linker and the amino-acid residue on the carrier. To achieve maximum affinity one has to carry out a chemical derivatization on the samples to be assayed.

*Author to whom correspondence should be addressed. BSA, Bovine serum albumin; SHIAA, 5 hydroxy indole acetic acid; His-S, Histamine-succinyl; His-SG, Histamine-succinyl-glycine; His-SGA, His. -_ tamine-succinyl-glycinamide; His-SGG, Histaminesuccinvl-nlvcvklvcine: His-SGGA. Histamine-suc. __ - _. cinyl-glycyl-glycinamide; His-SGTA, Histamine-succinyl-glycyl-tyrosinamide; PEG, Polyethylene glycol; tMet-histamine, tele-methyl-histamine.

Abbreuiufions:

This approach stemmed from the work of Cailla et al. (1973) on cyclic AMP. In this case the succinyl group was sufficient to restore full affinity (& 5 x lo-” M for succinyl CAMP, 5 x 10m9M for CAMP). Later on, succinylation was applied to other cyclic nucleotides (Cailla et al., 1976, 1978) and serotonin (Delaage and Puizillout, 1981). Puizillout and Delaage (1981) also applied the derivatization with glycinamide for haptens bearing carboxylic groups, namely S-hydroxy indole acetic acid (SHIAA). In this case the affinity rose by a factor 50 and rose further by 500 for antibodies raised to SHIAA-glycyl albumin after glycinamidation of SHIAA (Puizillout et al., 1982). This led us to the concept of succinyl-glycinamide derivatization, described in detail in a recent paper (Morel and Delaage, 1988). Guesdon et al. (1986) presented another approach to histamine immunoanalysis in which the chemical coupling agent was benzoquinone. Although histarnine-benzoquinone-lysine conjugate exhibited high affinity for the proper monoclonal antibody, the complete chemical derivatization was not possible in crude extracts. Another attempt of the same type was made by Peyret et al. (1986). Using diisocyanate derivatization they could only produce antibodies of 3 nM affinity and the system required a prior purification of samples to give reliable results. The last two approaches dealt with the modification of the imidazole ring. The diazo-coupling used by Mita ef al. (1984) could not be applied to samples, though the authors attempted TFA derivatization intended to augment affinity. However, the increase in affinity remained rather low. The approach of Hammar et al. (1985) was also inappropriate since t-Met-histamine was recognized better than histamine itself. Table 1 summarizes the various approaches that have been used.

995

A. MDREL

996 Table AUTHORS

1, Synopsis

et

al.

of histamine

IMMUNOGEN

TRACERS

HAYDIK (1983)

3H-Hfstam,ne

BUCKLER et al. (1967)

lz51 HisSGTA

cww~cMYC~Cww-C~c0-x

GUESDON at al (1966)

I?galactosldase antibody

PEYRETet al. (1986)

C”WW+CO-NH-(CM,);

-lAMMAR et al C1985) represents

W-CO-X

lz51

HIS HD-GT

3H- Histamine

WITAet al. [1984)

CMrcnyNH-CO-CnrCHrssnrCO-NM-X

CONVERSION NO

Kd (nM) 30

3 H-Histamine

c+WWWC+Ctirco-x

MOREL and DELAAGE (1984)

X

immunoanalysis

HIS-I?gal

No

500

SGA

0.1

Benzoquinone ovalbumin

0.7

Hexamethylene (HD) diisocyanate + acetyl lysine Met-amide

3

Trifluoro-acetate thio-ethylester

65

NO

100

._

the earner I! any.

Since only the succinyl glycinamide approach has become popular and has been exploited commercially, it seemed to deserve a detailed physicochemical analysis. Clearly the superiority of the succinyl-glycinamide approach reflects the choice of a derivatization employing chemical groups already proven in immunoanalysis and acting synergistically. This paper aims to analyse the mechanism of this synergy which is closely linked to the ionization state of imidazole, and to clarify the thermodynamical aspects of affinity enhancement in hapten systems. MATERIALS AND METHODS

(a) Chemicals

Standard chemical reagents were of analytical grade and purchased from Merck. Histamine, commercially available metabolites, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and bovine serum albumin (BSA) were purchased from Sigma. Original derivatives based-upon succinylated histamine were synthesized as follows: ‘H histamine was used as tracer to monitor the individual steps of the synthesis. Histamine was succinylated at an alkaline pH (Morel and Delaage, 1988). After anionic exchange chromatography the hemisuccinate was either transformed into histamine-succinylamide by adding an excess of ammonium in the presence of ethyl chloride carbodiimide or the hemisuccinate was coupled via the mixed anhydride method (Puizillout et al., 1982) to glycyl-ethyl-ester, to glycyl-glycyl-methyl-ester and to glycinamide. These later derivatives were purified by cationic exchange chromatography and the ester function hydrolyzed at a slightly alkaline pH. All acidic functions were subsequently transformed to amide as described.

The synthesis of immunogenic histamine-succinylglycyl albumin, tracer histamine-succinyl-glycyl ‘251tyrosinamide (‘*‘His-SGTA) and acylating agent have been described elsewhere (Morel and Delaage, 1988).

(6) Monoclonal antibodies

Mice, rats and rabbits were immunized with histamine-succinyl-glycyl-albumin (Morel and Delaage, 1988). Spleen cells of immune mice were fused with X63 myeloma cells in the presence of PEG 4000 as described by Galfre et al. (1977). Culture supernatants were tested as previously described (Morel and Delaage, 1988). Twenty five out of 960 supernatants bound 1251-labelled histamine. Three monoclonal antibodies A, B, C, were selected for their ability to bind 1251-His-SGTA. All selected hybrids were grown in ascitic fluid by intraperitoneal injection of cell suspensions into pristane-primed BALB/c mice (Potter et al., 1972). The determination of Ig classes was performed by immunodiffusion assay on SEBIA (Issy-les-Moulineaux, France) immuno-film using DAK0 (Copenhagen, Denmark) specific antisera.

(c) Antibody afJinity analysis

The Scatchard analysis was performed on histamine-succinyl-glycyl-tyrosinamide (His-SGTA) giving the dissociation equilibrium constant Kd. Other equilibrium constants were deduced from this value using the following formula (Morel and Delaage, 1988): Kd x ro KA = (1 - ro)c,,z - ___

2 - ro

Immunorecognition

of histamine

997

Table 2. Effect of pH on recognition of several histamine analogues by three monoclonal antibodies Antibody (Ig subclass)

Analogue pH = 8.5

A (71)

B (71)

C (rl)

T

pH = 7

pH = 5.5

4 x 10-11 2 x 10-10 6.6 x IO-* 2.7 x lo-6

1.5 x 10-9 6.6 x lo.9 1.1 x 10-6 5.5 x 10-4

His-SGTA His-SGA 4-Met-Hi&GA t-Met-His-SGA

10-11 8X lo-” 6 x 10-9 1.3 x 10-6

His-SGTA His-SGA 4-Met-His-SGA t-Met-His-SGA

4 x 10-10 10-8 2.7 x 1O-8 2.4 x 1W4

3 x W’O

9 x 10-9

3;yx1;;.: 3.8 x lO-(j

2.1 1.2 x 10-8 10”’ 1.2 x 10-4

His-SGTA His-SGA 4-Met-Hi&GA t-Met-His-SGA

4 x lo-10 7.6 x 10-l’ 4.5 x 10-8 2.7 x 1O-7

6 x lo-lo 7.5 x 10-10 7.8 x 1O‘9 2.3 x IO-7

:: ND ND

Competition of iodinated tracer with acylated histamine analogues for their binding to three monoclonal antibodies performed at three different pH, (PEG pr~ipitation).

where: K,, = dissociation constant of tracer K$ = dissociation constant of the analogue ro = B/T of tracer without analogue c,,* = concentration of analogue which displaces half of bound tracer. RESULTS (a)

of anti-histamine

Specijicity

antibody

Monoclonal antibodies selected out on the basis of high affinity for ‘251-His-SGTA have in common a higher affinity for the tracer at pH 8.5 than at pH 5.5, indicating that the imidazole ring is recognized in its Table

I

3. Increment

NAME

in affinity

by

neutral rather than in its cationic form. They differ by their cross reactivity with t-Met-histamine and by their optimal pH for binding (Table 2). (b) E&et of che~jcul ~od~catiun

of the side chain

Table 3 illustrates the increase in antibody recogni tion of histamine analogues as the lengthening of the side chain makes them increasingly resemble the immunogen. The increase in affinity clearly depends on two factors, i.e. the size and the net charge of the linker added. The recognition of analogues is impaired when a free carboxylic group is present and changes the molecule into a zwitterionic ligand. For neutral increasing resemblance

to immunogen

& fW 8.3 x 10-5

Histamine-hemisuccinate

8.7 Y10-6

Histamine

succinyl amide

1.7 x 10-b

Histamine (His-SC)

wccinyi

2.5 x IO-’

glyclne

Histamine succinyl glycinami~e

I.1 x lo-‘*

(His-SGA)

Histamine succinyl glycyl

6.8 x lV”

glycyl (His SCG)

Histamine succinyl glycyl

3.x x lo-‘0

giycinamide (His-SGGA) Histamine succinyl glycyl tyro\inamide (‘I Ii\-.xi’rA)

10-1’

-Histamine was chemically derivatized in order to increase its resemblance to the immunogen. All anaiogues were allowed to compete with the tracer in antibody coated tubes.

A. MOREL et al.

998

analogues binding properties appear to depend only on the size of the linker. Thus, it seems that the antibody-binding site encompasses histamine, the succinyl group, glycine and part of the lysine residue of the carrier. The higher affinity for His-SGA represents a small additional effect due to an aromatic contribution.

Ln Kd

(c) Effect of pH The curve presented in Fig. 3 shows the binding of the iodinated derivative to antibody A as a function of pH. The striking characteristic is the decrease at mild acidic pH which is unlikely to be due to a structural transition in the antibody structure. The curve is consistent with an exclusive recognition of the uncharged form of the tracer. The pK ascribed to the iodinated derivative is 6.7, close to that expected for imidazole ring (6.8) (detailed calculations not shown). (d) Effect of temperature on ligand-antibody equilibrium

The equilibrium between antibody A and the main histamine derivatives was studied at four temps in order to determine the enthalpy and entropy of binding in every case. The & values for His-SGTA were obtained by Scatchard analysis (Fig. 1). The dissociation constants for histamine and His-SGA were deduced using the formula given in “Methods”. Enthalpy was determined from an Arrhenius plot for the four compounds tested at various temps (Fig. 2). Results are summarized in Table 4. B/F 2

,

I,

I,

I

3.45

I

I

I

I

I

I

3.5

I

I

I,

I

I

3.55

(1/T)x103

I 3.6

K”

Fig. 2. Arrhenius plot for histamine and three analogues. Histamine and analogues were allowed to compete 22 hr with “‘1-his-SGTA in coated tubes at various temps. Kd, were either determined by Scatchard analysis or calculated as described in the Method.

From this table we may calculate the increments brought about by SGA derivatization in terms of thermodynamical parameter of binding as follows: 6.7 kcal/mole to free energy, 9.7 kcal/mole to enthalpy and 10.5 Cal/o for entropy. The significance of these values will be discussed. DISCUSSION

The recognition of imidazole derivatives by monoclonal antibodies throws light on several problems of general interest in ligand-receptor interaction.

. 6’C il B’C . 13-c

1

0 16-c

BIT

1

6

7

8

9

10

PH 0

50

rpfulr u

Fig. 1. Scatchard analysis of His-SGTA binding. His-SGTA was allowed to compete 22 hr with the iodinated tracer in coated tubes incubated at various temps.

Fig. 3. Effects of pH on the binding of “‘I-His-SGTA to antibody A. Tracer was incubated overnight at 4°C at various pH, in coated tubes. Bound radioactivity is expressed as function of pH.

Immunorecognition Table 4. Effect of temperature ‘emperature

“C

of histamine

on binding

999

of histamine

analogues

4 CM) His SGTA

His SGA

His-S

His

2.1 x 10-11

1 x 10-10

0.9 x 10-5

2.2 x 10.5

3x lo-11

1.6 x 10-l’

1.3 x 10-5

2.6 x 1V5

6.2 x 1O-11

2.1 x lo-10

2.3 x KY5

3.5 x 10.5

10.3 x lo.11

3.9 x 10-10

2.7 x 1O-5

3.6 x lo-’

Go (Kcal/mole)

13.3

12.5

6.3

5.8

Ho (Kcal/mole)

20.9

16.8

11.5

7.1

26.5

14.9

18.7

4.4

A So d/o

After incubation of histamine analogues with the tracer in antibody-coated tubes at various temps, the dissociation constant of the tracer was determined by Scatchard analysis. Dissociation constants for other compounds were calculated as shown in the Method. AGO and ASo and AHo were determined from an Arrhenius plot (Fig. 2). They were computed in the direction of dissociation.

The first one is the effect of charge on the binding. We have seen that only uncharged imidazole is recognized by selected antibody A. (It is noteworthy that all antibodies, raised in either mice or rabbits, show the same behavior). Furthermore considering a series of histamine derivatives modified on the side chain, it may be seen that every time there is a free carboxyl group there is a loss in activity. The carboxylate is likely to induce a charge effect on imidazole, stabilizing the cationic form. The hemisuccinate derivative (His-S) is 5.1 times less recognized (Kd ratio) than the corresponding amide (His-SA). The effect is even more pronounced for the succinyl glycine derivative; in this case the Kd ratio of the carboxylate form of histamine to the amide SGA, increases up to 2280! (Table 3). Such a difference reflects an optimal cyclization of the zwitterionic carboxylate form. The second question concerns the theoretical limit of affinity enhancement by chemical derivatization applied to haptens. The problem arose in the context of cyclic AMP. Some researchers believed that the lOO-fold increase in a affinity brought about by succinylation might lead to cross reactivity by unrelated compounds. Delaage er al. (1979) showed that this was not the case. But what about the histamine system in which affinity enhancement reaches 105-103 (Morel and Delaage, 1988; Guesdon et al., 1986). The question can be addressed by both experimental and theoretical analysis. Experiments with biological fluids and numerous unrelated amines never showed such interference. This can best be understood through a thermody-

namical analysis of the phenomenon. Basically, for two molecules (e.g. ligand and receptor) to bind to one another and independently of the nature of the molecules, an “entropy fee” has to be paid; it represents the loss of degree of freedom of the system. We can obtain an idea of the amount of entropy by considering the entropy variation associated with the dissociation of a pair of atoms in a gas, i.e. the loss of one degree of freedom. Deduced from the classical Sackur-Tetrode formula we find: AS - = 0.5 log T + 0.5 log M + log V R

- 2 log d - 40.216

Where T is the temp in K, M the molecular ratio of the atom, V the volume in cm3, d the interatomic distance in cm. Taking a numerical example T = 277”K, M = 100, V = 1000 cm3, d=2x lo-’ cm, it becomes: AS = 14.6 Cal/o. In terms of free energy this value represents a negative contribution of -4.1 kcal. By contrast the affinity enhancement yields a positive contribution of 6.7 kcal. The resulting value is AGO = 6.74.1 = 2.6 kcal corresponding to a K,, value of 8.9 x 1O-3 M. This is the order of magnitude expected for irrelevant amines after SGA derivatization. Thus the cross reactivity ratio with histamine-SGA is 1.1 x lo-‘O/8.9 x 10m3= 1.2 x lo-’ which is barely observable in experimental situations. In natural specimens, the amine concentration is too low to interfere with histamine. The thermodynamical analysis of SGA affinity enhancement provides additional information. By

A. Moat3L ef al.

1000

comparing enthalpic and entropic terms for histamine and histamine-SGA we can conclude that SGA contributes 9.7 kcal enthalpy and lOScal/o entropy, resulting in an overall contribution of 6.7 kcal/mole to the free energy. Thus the SGA derivatization has essentially an enthalpic character, partially cancelled by an entropic effect. The imidazole ring also provides a major enthalpic contribution so that the SGA derivative binding represents a loss of entropy approximately equal to that of one degree of freedom (14.9 Cal/o versus 14.6 Cal/o). Affinity enhancement by non-specific chemical derivatization is a principle of general applicability, not limited to hapten systems. Although initially developed for immunoanalysis it is now applied in nucleic acid probes in which the use of intercalating molecules makes it possible to achieve high affinity while keeping the advantage of short sequences (Helene et al., 1985). The theoretical limit of affinity enhancement in hapten immunoanalysis is probably not reached in the histamine system. In favourable situations in which no large excess of reacting material occurs and a charge effect is present, an affinity enhancement of 10’ is possible. Acknowledgements-We thank H. V. Rickenberg assistance with the English version of this paper.

for his

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