Immunology Letters, 32 (1992) 21 -26

Elsevier IMLET 01747

Monoclonal antibodies against metallothioneins from the human liver Kristina Van H o u d t , Inge Nicasi, Els Van Mechelen, Bea Veulemans a n d M a r c D e Ley Katholieke Universiteit Leuven, Laboratorium voor Biochemie, Dekenstraat 6, Leuven, Belgium

(Received 10 October 1991; accepted 11 November1991)

1. Summary Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One antibody, K5A6, was specific for MT-1, the others recognized all isoMTs present in the human fetal liver. Each of these antibodies showed a unique cross-reactivity pattern when tested with MTs from the livers of different mammals. A double-antibody sandwich enzyme-linked-immunosorbent-assay (ELISA) was developed with the antibodies L2E3 and L5G2. This assay allows the detection of 60 pg human fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and Hg-MT. 2. Introduction Metallothioneins (MTs) are low molecular weight, cytosolic heavy metal binding proteins found in eukaryotic species and in some prokaryotes. They bind Zn 2÷, Cd 2÷, Cu ÷, Hg +, Ag ÷ and Au ÷ , and are induced by these heavy metals [1, 2]. In addition, MT is also induced by glucocorticoid hormones, by cytokines and by various physical Key words: Monoclonalantibody; Metallothionein;ELISA Correspondence to: Marc De Ley, Laboratorium voor Biochemie, Dekenstraat6, B-3000Leuven, Belgium.Tel.: +32-16285701; Fax- + 32-16-201215. Abbreviations: MT. metallothionein; ELISA, enzyme-linked-

immunosorbent-assay; RIA, radioimmunoassay; PBS, Dulbecco's phosphate-bufferedsaline; HRPO, horseradish peroxidase.

and chemical stress conditions [3, 4]. MTs are characterized by a high cysteine content and by the lack of aromatic amino acids. The protein sequence data show remarkable homology in MTs from different species [1]. They have been found in nearly all mammalian tissues with the highest concentrations present in liver, kidney and intestine. Compared to the adult mammalian tissues, fetal and neonatal liver contain very high amounts of MT. MT isolated from most tissues is present as a mixture of two isoforms, designated MT-1 and MT-2, and in the human fetal liver a third isoform was found, MT-F. All mammalian MTs are single chain polypeptides of 61 residues [1; 51. A radioimmunoassay (RIA) and an enzymelinked-immunosorbent assay (ELISA) were developed for the quantitation of MT. The conventional antisera used in these assays, show crossreactivity between isoforms and among MTs derived from various animals [6, 7]. Using monoclonal antibodies prepared against rabbit MT and against MT from Cd-resistant fibroblasts of the Chinese hamster lung, it was possible to distinguish between different isoforms and MT from different species. Some of these antibodies not only bind to MTs but also to other metal proteins [8, 9]. In this paper we describe monoclonal antibodies generated against purified human MTs isolated from the fetal liver, and their reactivity with MTs from several other mammalian tissues. With these monoclonal antibodies a highly sensitive and specific ELISA was developed. These monoclonal antibodies will also permit the cloning of the human fetal MT genes and the immunohistochemical localisation of different MTs.

0165- 2478 / 92 / $ 5.00 © 1992 ElsevierSciencePublishers B.V. All rights reserved

21

3. Materials and Methods

3.1. Experimental animals and organs Human fetal livers from autopsy and adult kidneys from surgery were kindly put at our disposal by Professor J. Lauweryns (Department of Biomedical Research, Katholieke Universiteit Leuyen, K.U. Leuven) and by Professor L. Baert (St. Pieters University Hospital, K.U. Leuven), baboon and dog livers by Dr. T. M611hoff (Center for Experimental Surgery and Anesthetics, K.U. Leuven). All organs were washed with phosphate buffered saline (PBS) and kept frozen at - 8 0 °C until

3.3. Generation o f hybridomas

use.

Rabbits (New-Zealand), mice (Balb/C) and rats (Wistar) were obtained from the animal breeding center of the K.U. Leuven. Mice and rats were injected intraperitoneally with 15 mg Zn/kg and 10 mg Zn/kg body weight, respectively, every other day for 14 days. MT induction in rabbits was accomplished by intradermal injection of 15 mg Zn/kg every other day for 2 weeks. For these administrations, ZnCI 2 was dissolved in 0.15 M NaC1. Forty-eight hours after the last injection the animals were sacrificed, livers and kidneys were immediately washed with PBS and frozen at - 80 °C. 3.2. Isolation and purification o f M T from mam-

malian tissues All tissues were homogenized in 10 mM TrisHCI, 0.25 M sucrose, pH 7.4, at 4 °C (20% w/v) in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 100000 g for 2 h at 4 °C (LKB, Ultrospin centrifuge, Bromma, Sweden). Proteases were inactivated by heating the supernatants for 2 min in a water bath at 80°C [5]. The precipitate was removed by centrifugation at 10000 x g for 20 rain at 4°C, and the absence of proteases was confirmed by the Hide Powder Azure Assay [10]. The homogenates were applied to a Sephadex G-75 column (63 x 6 cm) and the proteins were eluted at a flow rate of 25 ml/h with 10 mM TrisHC1, pH 7.4, at 4 °C. The absorbance of each fraction was measured both at 218 nm and 280 nm. The zinc and cadmium content of the fractions was determined by atomic absorption spectrome22

try. Zinc or cadmium containing fractions with a high 218 nm/low 280 nm absorption ratio were pooled for MT. The pooled samples were applied to a DEAESephacel anion exchange column ( 1 5 x l . 7 cm) equilibrated with 10 mM Tris-HCl, pH 7.4. The column was eluted with 50 ml starting buffer followed by a linear 10-300 mM Tris-HCl, pH 7.4, gradient. Fractions (1.5 ml) were collected at a flow rate of 21 ml/h and MT was detected by atomic absorption spectrometry (Perkin-Elmer 372 Atomic Absorption Spectrophotometer, Ueberlingen, Germany).

Balb/C mice were immunized with purified human MT-1 and MT-2 isolated from human fetal liver. Four injections of 50 #g MT each were given intraperitoneally. The first injection was administered with complete Freund's adjuvant 39 days before the fusion, the second one two weeks later with Freund's incomplete adjuvant. The last two injections were given four days before the fusion, when the animals were injected intraperitoneally and intravenously, and 24 h before the fusion. The mice were killed by cervical dislocation and the spleens were removed aseptically. Spleen cells were fused with P3X63-Ag8-653 myeloma ceils as described by K6hler and Milstein [11] using PEG 4000 as the fusing agent. After HAT-selection positive clones were identified by ELISA on human MT-1 and MT2 coated microwell plates and cloned by limiting dilution. Hybridoma and mYeloma cells were cultured in RPMI-1640 medium supplemented with 10O7o v/v fetal bovine serum, 0.25 g/l glucose, 2 mM glutamine and 50 mg/1 gentamycin (Gibco, Paisley, Scotland). Immunoglobulins were produced as ascites fluid in Balb/C mice and the antibodies were isolated by (NH4)2SO 4 precipitation. The immunoglobulin subclass was determined by a "Line-Immuno-Assay" (Innogenetics, Antwerp, Belgium). 3.4. Coupling o f horseradish peroxidase (HRPO)

to antibodies Fluorodinitrobenzene (0.1 ml of 1% (w/v) in ethanol) was added to a solution of HRPO (5 mg in

1 ml of 0.3 M NaHCO 3, pH 8.1) and the mixture was stirred at room temperature for 1 h. Then 1 ml of 0.06 M NalO 4 was added, the mixture was stirred again for 30 min, followed by the addition of 1 ml of 0.16 M ethyleneglycol and 1 h stirring. Finally the solution was dialyzed against 0.01 M sodium carbonate buffer, pH 9.5 [12]. Equimolar amounts of HRPO and IgG (also dialysed against the same carbonate buffer) were mixed and stirred for 3 h. Finally 0.5 mg NaBH4/ mg IgG was added, the mixture was stored overnight at 4 °C. After dialysis against PBS the conjugate was purified by gel chromatography on an UItrogel AcA 34 column (90×2.5 cm) eluted with 0.1 M NH4HCO 3.

the reaction was visualized with orthophenylenediamine (0.04% (w/v) in phosphate citrate buffer, pH 6, 0.006% (w/v) H202).

3.5. Direct ELISA

4. Results

The antigen (5 #g/ml in 10 mM carbonate buffer, pH 9.7) was adsorbed to microwell plates (Immunosorb, Nunc, Roskilde, Denmark) overnight at 4 °C. After each step the plates were washed three times with 0.05°7o (v/v) Tween 20 in PBS. After 1 h incubation with 1°70 (w/v) casein in PBS the samples of monoclonal antibodies were added and incubated for 2 h. Diluted rabbit-anti-mouse serum conjugated to HRPO (1/1000 (v/v) in PBS, Dako, Copenhagen, Denmark) was incubated for 1 h and

4.1. Identification and cloning of anti-MT immunoglobulin secreting hybridomas

TABLE

The buffers were as described above. Microwell plates were coated overnight with a purified monoclonal antibody (10/~g/ml in carbonate buffer). The plates were then incubated with casein solution for 1 h. In the next step, antigen was added in a sequential dilution series and incubated for 2 h. A monoclonal antibody conjugated to HRPO was then incubated for 1 h and the reaction visualized with orthophenylenediamine.

Using a direct ELISA the presence of immunoglobulins reacting with fetal liver MT was demonstrated in the sera of Balb/C mice, immunized with MT-1 and MT-2. The supernatants of the fusion products were also screened by ELISA and among all specific hybrids, six were selected for cloning and further characterization. The immunoglobulin

1

lmmunoglobulin

subclass and interspecies cross-reactivity pattern of monoclonal

Subclass Human Human

3.6. Double-antibody sandwich ELISA

fetal L MT-1 a fetal L MT-2

antibodies generated against human

fetal liver MT.

L5G2

L2E3

K5A6

L3D4

L6F6

L 2 B 10

IgG l

IgG 1

IgG 1

IgGEa

IgG 1

IgG2b

+ + + +

+ + + +

+ + -

+ + + +

+ + + +

+ + + +

Human

R MT Ia b

N.D. c

N.D.

-

N.D.

N.D.

N.D.

Human

R MT Ib

N.D.

N.D.

-

N.D.

N.D.

N.D.

Human

R MT II

N.D.

N.D.

+ +

N.D.

N.D.

N.D.

+ + + + + ++

+ + + + + ++

+ + + + -

+ + +

+ + + + ++

+ + + + +

Dog L MT Rabbit L MT

+ + / -

+/-

-

+ + / -

+/-

+ -

Rat L MT Mouse L MT

+/+ / -

-

+/-

+/+

+ / -

+ -

Baboon L MT Baboon L MT-1 BaboonLMT-2,

"

a L MT, liver MT; b R MT, renal MT; c N.D.,

not determined.

23

subclass was determined and the specificities of the 6 monoclonal antibodies were examined by an ELISA on microwell plates coated with the purified isoforms (Table 1). From these experiments we concluded that only K5A6 was specific for one isoform, namely MT-1. 4.2. Optimization of a sensitive ELISA for human liver M T In order to develop a double-antibody sandwich ELISA, all monoclonal antibodies were coupled to H R P O and all combinations were tested. A highly sensitive ELISA was obtained by the adsorption of L2E3 on the microwell plate in combination with L5G2-HRPO as the conjugate. This ELISA permitted the quantitative determination of 60 pg MT (Fig. 1). In order to assess the metal-dependency of the ELISA, signal different derivatives, prepared from human fetal liver MT by metal exchange, were compared. As shown in Fig. 2, on an equal molar base the Zn-loaded form yielded the highest signal, followed by the Hg- and finally the Cd-loaded form. A

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0 0.01

I I **llltl

| ii i ~ 0.1

1 ng MT

10

l I 1 Jllll 100

Fig. 2. Metal-dependence of the double-antibody sandwich ELISA using adsorption of L2E3 on the microwell plate in combination with L 5 G 2 - H R P O as the conjugate. MTs derived from the h u m a n fetal liver were assayed after substitution of Zn with different metals: + , CdMT; v , HgMT; n , ZnMT.

4.3. Detection o f liver M T from other mammals

0.35

0.3

0.25

0.2

0.15

0.1

0.05

I

1

i

i

1 IIII

|

10

i

I

I

i1111

1

100 pg MT

Fig. 1. Double-antibody sandwich ELISA using adsorption of L2E3 on the microwell plate in combination with L 5 G 2 - H R P O as the conjugate. H u m a n fetal liver MT was assayed in a range of 4 - 200 pg.

24

492

492

0.4

0

A 1.8

MT was isolated from the liver of several mammals. All cytosolic fractions, separated by gel chromatography yielded a major Zn-containing fraction characterized by a high absorption at 218 nm with low absorbance at 280 nm and an apparent molecular weight of 13 000. The livers of Zn induced animals showed significantly higher levels of Zn-MT. In the double-antibody sandwich ELISA, as described above, only MTs from the investigated primates, i.e. human fetal liver MT and baboon liver MT, were readily detected. The reactivities of all monoclonal antibodies with the various liver MTs were also examined in a direct ELISA and the results are summarized in Table 1. The elution pattern of the different isoforms present in baboon liver MT, when separated on DEAE-Sephacel exhibited two peaks, designated MT-1 and MT-2 in the order of elution. The monoclonal antibody K5A6 specifically recognized MT-1 (Table 1), while none of the other antibodies allowed the identification of different isoforms.

mM Tris

IJg Z n / m l

350

0.2

/= 300 t

0.15 /

Ib la / ~

ol

250

/ /

I

2OO

t/-'~ 150 100

0.05

'50

0

10

20

30

40

50

60

70

0 80

fraction number

Fig. 3. DEAE-Sephacelfractionation of human renal MT obtained from SephadexG-75. The gel (7 x 1.7 cm) was eluted with a gradient of 10 - 300 mM Tris-HCl (---), pH 7.4 at a flow rate of 21 ml/h. MT was detected by atomic absorption spectrometry (--). 4.4. Detection o f renal M T The elution pattern of the cytosolic fraction of h u m a n kidney on Sephadex G-75 was very similar to that of the h u m a n fetal liver. A characteristic high 218 n m / l o w 280 nm absorbance peak eluted in the molecular weight range of about 13 000. Metal analysis of this fraction by atomic absorption spectroscopy revealed the presence of mainly Zn and Cd. In a double-antibody sandwich ELISA human renal MT exhibited a lower reactivity than human fetal liver MT. The renal M T pool obtained from Sephadex G75 was further separated on a DEAE-Sephacel anion exchange column. The elution pattern showed two major Zn-containing fractions, designated I (a and b) and II in the order of elution. These forms eluted at the same ionic strengths as MT-1 and MT2 from the human fetal liver, but the size and form of the elution peaks were different (Fig. 3). In a direct ELISA, it was found that K5A6 showed a high reactivity towards peak II, but did not recognize peak I (Table 1). This indicated that both isoforms are immunologically different compared to the ones from the human fetal liver. 5. Discussion Several hybridomas secreting antibodies directed against h u m a n fetal liver MT were obtained, six of

which were cloned and further characterized. These antibodies all recognized the different isoforms present in the h u m a n fetal liver, except for K5A6, which is specific for MT-1. A highly sensitive double-antibody sandwich E L I S A was developed with the antibodies L2E3 and L5G2. This assay allowed the detection of 60 pg of h u m a n fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and H g - M T derived from h u m a n fetal liver MT. Several methods for the determination of MT in biological materials have already been described. Except for the immunological assays, these procedures rely on rather unspecific parameters such as the content of metals (Hg- and Cd-saturation method [13, 14], metal determination by atomic absorption spectrometry) or thiolate groups [15]. Much more specific are the R I A [6] and fluorometric E L I S A [7] procedures, developed with a polyclonal rabbit-anti-MT serum. They allow the detection of 100 pg MT and are sufficiently sensitive to assess basal levels in biological materials. The described E L I S A is therefore an alternative method to the present RIA and fluorometric E L I S A with a detection limit of 60 pg MT. Liver MTs from other m a m m a l s were examined by this ELISA, but only MTs from primates were recognized by all antibodies. Each of the examined monoclonal antibodies had a different specificity pattern in reacting with the liver MTs from other mammals. Three of the mouse hybridomas secreted antibodies which bound to mouse liver MTs. Similar murine autoantibodies were already obtained by the preparation of monoclonal antibodies against rat and Chinese hamster M T [8, 16, 17]. However, the significance of this phenomenon remains to be determined. H u m a n renal M T was isolated and the isoforms separated. The two isoforms eluted at the same ionic strengths as MT-1 and MT-2 from the h u m a n fetal liver: it was therefore concluded that the charge densities of the respective renal isoforms and the liver isoMTs are identical. The antibody K5A6 however did not recognize peak I but was specific for peak II from renal MT. This ambiguity in serological properties and charge density led us to the assumption that renal MTs and fetal liver MTs are different forms. Renal and liver MTs probably have different primary structures which 25

does not result in a different charge density, but in an important alteration of the epitope for K5A6.

Acknowledgements The authors are grateful to the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw for research fellowships (K.V.H.) and thank Professor J. Lauweryns, Professor L. Baert, Dr. Ph. Moerman and Dr. T. MOllhoff for kindly providing biological material.

References [1] K~igi, J. H. R. and Kojima, Y. (1987) in: Metallothionein II (J. H. R. K~igi and Y. Kojima, Eds.), pp. 2 5 - 6 1 , Birkh~iuser Verlag, Basel. [2] Palmiter, R. D. (1987) in: Metallothionein II (J. H. R. K~igi and Y. Kojima, Eds.), pp. 6 3 - 8 0 , Birkh~iuser Verlag, Basel. [3] Oh, S. H., Deagan, J. T., Whanger, P. D. and Weswig, P. H. (1978) Am. J. Physiol. 235, E282. [4] Friedman, R. L. and Stark, G. R. (1985) Nature 314, 637.

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[5] Clough, S. R., Mitra, R. S. and Kulkarni, A. P, (1986) Biol. Neonate 49, 241. [6] Vander Mallie, R. J. and Garvey, J. S. (1979) J. Biol. Chem. 254, 8416. [7] Garvey, J. S., Thomas, D. G. and Linton, H. J. (1987) in: Metallothionein II (J. H. R. K~igi and Y. Kojima, Eds.), pp. 335 - 342, Birkh~iuser Verlag, Basel. [8] Masui, T., Utakoji, T. and Kimura, M. (1983) Experientia 39, 182. [9] Talbot, B. G., Bilodeau, G. and Thirion, J. P. (1986) Mol. Immunol. 23, 1133. [10] Rinderknecht, H., Geokas, M. C., Silverman, P. and Haverback, B. J. (1968) Clin. Chim. Acta 21, 197. [11] K6hler, G. and Milstein, C. (1975) Nature 256, 495. [12] Nakane, P. K. and Kawaoi, A. (1974) J. Histochem. Cytochem. 22, 1084. [13] Kotsonis, F. N. and Klaasen, C. D. (1977) Toxicol. Appl. Pharmacol. 42, 583. [14] Eaton, D. L. and Toal, B. (1982) Toxicol. Appl. Pharmacol. 66, 134. [15] Sokolowski, G. and Weser, U. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1715. [16] Kikuchi, Y., Wada, N., Irie, M., ikebuchi, H., Sawada, J.-I., Terao, T., Nakayama, S., Iguchi, S. and Okada, Y. (1988) Mol. Immunol. 25, 1033. [17] Nagel, W., Hartmann, H.-J. and Weser, U. (1990) lmmunol. Lett. 26, 291.

Monoclonal antibodies against metallothioneins from the human liver.

Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One an...
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