New competitive enzyme-linked immunosorbent assay for determination of metallothionein in tissue and sera

Talanta 46 (1998) 325 – 333

New competitive enzyme-linked immunosorbent assay for determination of metallothionein in tissue and sera Margarita Apostolova a,*, Choudomir Nachev b, Milena Koleva c, Panayot R. Bontchev d, Ivan Kehaiov e a

Department of Chemistry and Biochemistry, Medical Academy, 2, Zdra6e Strasse, Sofia 1431, Bulgaria b Department of Internal Medicine, Clinic of Cardiology, Medical Academy, Sofia 1431, Bulgaria c Department of Biochemistry, Faculty of Biology, Sofia Uni6ersity, Sofia 1421, Bulgaria d Department of Analytical Chemistry, Faculty of Chemistry, Sofia Uni6ersity, Sofia 1126, Bulgaria e Laboratory of Molecular Immunology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 4 November 1996; received in revised form 10 June 1997; accepted 12 June 1997

Abstract Very little information is available concerning the relationship between metallothionein (MT) and diseases in humans. Several methods to measure MT levels exist but many of these assays are not sensitive to measure MT in human sera. A new sensitive competitive ELISA system has been developed using MT labeled with horseradish peroxidase as a conjugate and high-titre polyclonal antibodies obtained from rabbit immunoglobulin G for MT determination in human sera. The cELISA proposed here permits a reliable determination of MT in the range 10–2 000 000 pg ml − 1. The method was compared with Cd-hem assay and showed good agreement of results. The recovery of the assay was determined by spiking rat MT into rat and human sera, and comparing it with spiked diluent controls. The overall recoveries of the added MT were 101% for rat sera and 89% for human sera. The variation within-assay and between assay were 3 and 6%, respectively. A significant difference (PB 0.001) was found between the MT-level in human sera from patient with essential hypertension (646 9 223 ng ml − 1, n=90) and normotensive subjects (21 9 18 ng ml − 1, n=236). A correlation between arterial hypertension and MT-level seems possible. A very sensitive new cELISA method was presented for determination of MT in sera and tissues. It enables investigation of possible correlations between sera MT-concentration and certain diseases. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Metallothionein; Hypertension; Competitive ELISA; Western blotting

1. Introduction

* Corresponding author. Tel.: + 359 2 9515664; fax: + 359 2 517278; e-mail: [email protected]

Metallothionein (MT) is a low molecular weight protein with a very important role in heavy metal detoxification and metal homeostasis [1,2]. This protein contains 20 cysteine (CYS)

0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 3 4 1 - X

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residues and no aromatic aminoacids [3]. MT genes are highly conserved and have strong inducible promoters [4]. The synthesis of MT is induced by various metal ions (Cd2 + , Zn2 + , etc.) [3], glucocorticoids [4], catecholamines, IL-1 and IL-6 and interferons [5 – 7]. Structural analysis of MT shows two domains, the first one includes the carboxylterminal part and binding four bivalent metal ions and the second domain containing three bivalent metal ions. Immunological studies of MT have suggested that the N-terminal region is the most immunogenic domain [8]. An epitope consists of a region of five to seven aminoacids residues [9]. Different methods for quantitative determination of MT have been described [10 – 12]. MT can be measured indirectly, based on the known binding stechiometries between metals and MT, by quantifying the number of atoms the Cd- or Hg bound to the protein [13,14]. Radioimmunoassays (RIA) and an enzyme linked-immunoassays (ELISA) have been developed for determination of MT [15–18]. Immunological studies using different antisera against MT showed that the antibodies were crossreactive with various kinds of mammalian MT [19]. These methods have a typical detection limit of about 0.1 – 1 ng of MT and determination of MT in body fluids is difficult due to low MT concentrations. In previous studies [20], we have found that the basal MT levels in liver from spontaneously hypertensive rats (SHR) were higher than that in the Wistar–Kyoto rats (WKY). The close similarity of the SHR-model and human hypertension disease makes the investigation of the relation between MT homeostasis and arterial hypertension in humans interesting. In order to investigate physiological functions and roles of metallothionein in humans, the aim of this study was to develop a new competitive ELISA system for MT determination in human sera with low detection limit.

2. Experimental

2.1. Reagents Cadmium

chloride,

Tris-(hydroxymethyl)-

amino-metan, Na2HPO4.12H2O, NaH2PO4.6H2O were obtained from Sigma (St. Louis, MO). The acrylamide, bis-acrylamide and sodiumlaurilsulfate (SDS) were acquired from Merck (Darmstadt, Germany). All other reagents were purchased from Aldrich Chemical (Windsor, ON, Canada). The aqueous solutions were prepared with highly purified water (B 18 MV) using Milli-Q reagent water system (Millipore, Milford, MA, USA).

2.2. Animals and human sera Male Wistar rats and guinea pigs were obtained from the animals laboratory (Medical Academy, Sofia, Bulgaria). Rats weighing 170–190 g (10 weeks old) were housed in groups of five per cage in an environmentally controlled room (light 7–19 h, 239 1.5°C). Four male New Zealand rabbits (3 kg) were received from the laboratory of molecular immunology (Institute of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria). Animals were supplied with water and a commercial laboratory chow, ad libitum. The human sera from patients with essential hypertension (n=90) and normotensive subjects (n= 236) were received from the Department of Internal Medicine II, Clinic of Cardiology (Medical Academy, Sofia, Bulgaria) and National Institute for Blood Collection (Sofia, Bulgaria).

2.3. Purification of MT-antigen Fresh or frozen liver from rats, in which the MT concentration has been induced by CdCl2 were cut into small pieces and homogenized in 25% (w/v) of 0.1 M Tris–HCl buffer pH 8.6, containing 0.25 M with Teflon-glass homogenizer (8–10 strokes min − 1). The homogenate was centrifuged at 10 000× g for 10 min. The resulting supernatant was heated for 1 min at 100°C. The precipitate was removed by centrifugation at 100 000×g for 30 min and 5 ml from the supernatant were applied into a Sephadex G-75 gelfiltration column (2.6 cm/1 m, Pharmacia,

M. Apostolo6a et al. / Talanta 46 (1998) 325–333

Uppsala, Sweden). Elution was carried out with 50 mM Tris – HCl buffer, pH 7.4 at a flow rate of 1 ml min − 1. The absorbency of elute at 254 and 280 nm was continuously recorded using a flow cell and UV detector (Pharmacia, Uppsala, Sweden). Fractions of 5 ml were collected. All fractions corresponding to MT were combined and concentrated under nitrogen on an Amicon (Danvers, MA) YM 2 membrane. Aliquots of the concentrated solution (300 ml) were used for HPLC separation of MT isoforms (MT1 and MT2). HPLC was performed on a semi-preparative mBondapak C18 column (300 mm/7.8 mm/5 mm) obtained from Waters (Milford, MA). The details concerning the MT separation by HPLC systems have been discussed in previous papers [20,21]. The MT concentration was determined by Bradford assays [22]. We used as a standard MT produced by Sigma (St. Louis, MO, Cat c M7641). The Zn and Cd contents were determined in all samples by electrothermal atomic absorption spectrometry (Perkin-Elmer Zeeman 5000) with an HGA-500 graphite furnace, AS-40 autosampler and ASDS-10 data station.

327

2 weeks interval. The amount of MT in the conjugates were 6, 4, 2 and 2 mg per each interval. The final injection, after 2 months, consisted of MT emulsified with mixture from complete and incomplete adjuvants. Blood was drawn from rabbits before immunization (in order to obtain preimmune serum) and 7 days after each injection to obtain antiserum. It was kept at 37°C for 1 h and at 4°C for 2 h. After the serum was separated by centrifugation, the immunoglobulin fractions were separated by ammonium sulfate precipitation. The serum was stored at − 80°C until ready to use.

2.5. Characterization of the rabbit anti-rat MT antibodies The antibodies formation was checked by double diffusion with 1% agar on a glass plate at room temperature. The antigenic reactivities of various MT and lysozymes and the potency of

2.4. Preparation of antibodies Fig. 1 shows the homogeneity of MT1 and MT2 used as immunogen. The conjugation of MT with lysozyme (Sigma, St. Louis, MO) was carried out essentially using the method of Reichlin et al. [23]. Rat MT (mixture from MT1 and MT2, 0.5 mg ml − 1 for each one) and lysozyme (2 mg ml − 1) were dissolved in 500 ml of 0.1 M potassium phosphate buffer, pH 7.0. A total of 250 ml of 1% glutaraldehyde was added dropwise to the protein solution while constantly stirring and the reaction mixture was incubated for 60 min at room temperature. Then 1 ml of physiological saline was added to conjugated MT solution and the mixture was emulsified with 2 ml of Freund’s complete or incomplete adjuvant (DIFCO Laboratories, Detroit). Male New Zealand rabbits were injected s.c. with 2 ml from the emulsion 2 – 6 mg of the conjugates. The first four injections of the antigen emulsified with Complete Freund’s adjuvant were given at

Fig. 1. SDS-PAGE of metalothionein 1 (line 2 and 4) and metallothionein 2 (line 3 and 5). Line 2 and 3 were developed without b-mercaptoethanol in the sample buffer, line 4 and 5 with b-mercaptoethanol.

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various rabbit antisera were determined by the direct ELISA systems [17].

2.6. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting SDS-PAGE was performed by the method of Laemmli [24]. The separating gel containing 15% (w/v) acrylamide was 6×9 cm in size and 1 mm thick. Samples were dissolved in a sample buffer (125 mM Tris – HCl, pH 6.8 containing 15 (w/v) SDS, 10% (w/v) glycerol and 0.01% bromphenol blue with or without b-mercaptoethanol) and incubated in a water bath at 80°C for 5 min. After the electrophoresis, the gel was soaked in a transfer buffer (25 mM Tris/192 mM Glycin/20% (v/v) methanol) and the proteins in the gel were transferred electrophoretically to a nitrocellulose membrane (Bio-Rad, Richmond, CA) using the modified method of Towbin et al. [25].

2.7. Detection of MT on the nitrocellulose membrane The membrane was soaked in 10 mM PBS (KCl, KH2PO4, NaCl, Na2HPO4.12H2O) pH 7.4 containing 10% (v/v) Tween-20 for 1 h. The MT on the membrane were detected immunochemically using rabbit anti-rat MT serum (1:10 000) diluted in TBS for 1 h, at room temperature. After the membrane was washed with TBS (PBS +0.05% (v/v) Tween-20) buffer, it was incubated in a peroxidase conjugated goat anti-rabbit IgG and the membrane method was developed according to the manufacturer’s instructions.

2.8. Conjugation of enzyme (horseradish peroxidase, HRPO) to antigen (MT*) HPLC-purified rat MT1 and MT2 (4 mg each) were dialyzed against 0.1 M carbonate buffer pH 7.0, overnight at 4°C. A total of 250 ml from 4 mg ml − 1 HRPO (dissolved in 0.1 M carbonate buffer, pH 8.7) was mixed with 200 ml of freshly prepared 0.1 M NaIO4 and incubated at room temperature for 20 min in the dark. After that, the mixture was dialyzed against 1 mM acetate buffer, pH 4.4. Immediately after dialysis the pH

was adjusted to 9.0–9.5 with 0.2 M carbonate buffer and MT (mixture from MT1 and MT2, 8 mg) was added. This solution was stirred gently for 2 h at room temperature. Freshly prepared 0.1 ml NaBH4 (4 mg ml − 1) solution was added and incubated for 2 h at 4°C. The excess of unbounded HRPO was separated by gel chromatography (Superose 12™ column, FPLC, Pharmacia, Sweden) gel-chromatography. The HRPO labeled MT (MT*) was mixed with glycerol as a cryoprotector mixed with glycerol and stored at − 20°C until ready to use.

2.9. Competiti6e ELISA system (cELISA) The 50 ml from MT-antibodies diluted in 10 mM carbonate buffer, pH 9.7 was adsorbed on the flat type microwell plates overnight at 4°C. After 1 h incubation with PBS plus 1% (v/v) Tween-20 (200 ml), the MT* (25 ml, 1:400 dil.) and the samples (25 ml) to be studied were added and incubated for 2 h at 37°C. The reaction was visualized by means of ortophenylenediamine in phosphate citrate buffer, pH 5.5, 0.006% H2O2 and stopped with 10% H2SO4. The absorption at 492 nm was measured by ELISA reader (Dynatech, Germany). After each step the plates were washed three times with TBS for 5 min.

2.10. Statistical analysis The statistical evaluation of the obtained results was performed by regression and ANOVA analysis, PB 0.001 (Microcal Origin, version 3.5). The data were expressed as mean9 SD.

3. Results

3.1. De6elopment and optimization of a sensiti6e competiti6e ELISA for human sera In order to develop a competitive ELISA, rat metallothioneins were coupled to HRPO and different combinations between MT* and Ab were tested in order to obtain an optimal cELISA condition all combination. A highly sensitive ELISA was obtained by the absorption of MT

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Table 1 Quantification of MT in different tissue cytosols by cELISA and Cd-hem assay Type MT

Fig. 2. Competitive ELISA, using absorption of antigen (MT) on the microwell plate and MT* (MT conjugated with horseradish peroxidase) as a conjugate. Rat liver MT was assayed in the range 10– 2 000 000 pg ml − 1.

antibodies on the microwell plate in combination with the analyzed samples and MT* (see Section 2) as a conjugate. This cELISA permitted determination of MT as low as to 0.5 pg well − 1 MT (Fig. 2). MT concentrations of 10 – 2 000 000 pg ml − 1 can produce changes in the percentage binding that are linearly related to the logarithm of the MT concentrations. Metallothionein concen-

Concentration of MT (mg g−1 tissue) Cd-hema

cELISAa

Rat liver Control Cd-injected

6.6 91.4 511 9 17

5.29 1.7 504 923

Rat kidney Control Cd-injected

6.1 91.3 174 9 11

9.7 92.3 182 9 25

Guinea pig Control Cd-injected

6.99 2.1 497 9 32

8.9 9 1.5 475 9 14

a

Mean 9 SD of 66 determinations.

trations were determined by linear regression (Fig. 3). Regression analysis of the total MT concentrations in Table 1 obtained by Cd-hemoglobin assay and cELISA using the MT* show good linear relationship, with the slope of the regression line=0.843. The sensitivity of the competitive ELISA assay, permitted us to measure MT concentration in human and animal sera with low detection limits. In order to assess the metal-dependency of the ELISA, single different derivatives, prepared from rat hepatic MT by metal exchange Cd5.33Zn1.33MT, Cd6.54MT, (Zn6.22Cu0.52MT, AMT), were compared. As shown in Fig. 4, the Zn-loaded and apometallothionein (AMT) forms yield the highest signal on equal molar ratio.

3.2. Specificity, reco6ery, reproducibility and sensiti6ity of the assay

Fig. 3. MT standard curve (linear regression) from competitive ELISA, using absorption of antigen (MT) and MT* conjugated with horseradish peroxidase, as a conjugate.

The specificity of the antisera was examined by the Ouchterlony’s method. A single precipitation line was observed between the rat hepatic MT and the antiserum (after the antiserum was immunoaffinity purified, data not shown). As shown in Fig. 5 similar competitive inhibition patterns were obtained when the rabbit anti-rat MT serum reacted with MTs from mammalian organs: rat hepatic MT, guinea pig hepatic MT, human

330

M. Apostolo6a et al. / Talanta 46 (1998) 325–333 Table 2 Recovery of serum MT by cELISA MT pg well−1

1.5 2.5 5.5 25.5 50.5 75.5

Ratsa

Humanb

Recovery (%)

Recovery (%)

96 95 97 97 103 94 99 9 6 105 95 111 912

89 9 7 97 9 5 83 97 91 93 88 92 90 99

The recovery in the cELISA has been determined by spiking rat MT in rat and human sera and comparing with spiked standard diluent controls. a b , Mean9 SD of 15 experiments. Fig. 4. Metal-dependence of competitive ELISA using MT*, as a conjugate. MTs derivatives (CdMT: Cd6.24MT; ZnMT: Zn6.22Cu0.52MT; CdZnMT: Cd5.33Zn1.33MT, AMT) were obtained from rat liver.

serum MT. Lysozyme solution used for conjugation of MT did not show any cross reactivity (Fig. 5). The recovery of the assay has been determined by spiking normal rat and human sera with known amounts of rat MT and comparing them with the controls (Table 2). The overall average

recoveries of the added MT* were 101% for rat sera and 89% for human sera. Table 3 summarizes the within-assay and between-assay reproducibility obtained by cELISA system. The assay gave reliable results for analyses of MT in both sera and tissue cytosols. The within-assay coefficient of variation was less than 3% and the between-assay coefficient of variation was less than 6%. From the standard curves and all the data described above, it appears that reliable results can be obtained for MT in sera and tissue in the range 10–2 000 000 pg ml − 1, using the cELISA method described here.

3.3. Detection of MT from rat and guinea pig li6er by Western blotting technique MT was isolated from the liver of several mammals. The MT fraction (10 kDa) from all cytosols Table 3 Reproducibility of MT in human sera by the cELISA MT pg well−1

Within-assay

Between-assay

Coefficient of varia- Coefficient of variation (%) tion (%) 10 20 50 Fig. 5. Binding (%) of different MTs and lysozyme with the rabbit anti-rat MT antiserum by competitive ELISA.

n = 30.

1.3 2.4 3.3

5.9 5.7 6.1

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Fig. 6. Detection of different MT forms by Western blotting. Line 1 — molecular weight standard markers: 71 kDa, BSA; 42 kDa, carbonic anhydrase; 31 kDa, soybean trypsin inhibitor; 18 kDa, lysozyme; 7 kDa, aprotinin. Lines 2–6, cytosols from: rat liver in which MT synthesis was induced by (2) CdCl2, (3) ZnCl2, (4) control. Lines 5–6, cytosol from guinea pig liver, (5) CdCl2, (6) control

were separated by gel chromatography and investigated by an immunoblotting technique. The results from Fig. 6 indicate that different amounts of MT can be detected by Western blotting. The limit of detection for each MT isoform from the animals studied was 4 ng. The control MT content or MT induced in the rat liver after Cd or Zn administration were detected by the method described above.

3.4. Detection of MT in the sera from patients with essential hypertension and normotensi6e subjects The MT concentrations in sera from patients with essential hypertension (n =90) and normotensive subjects (n =236) obtained with this competitive ELISA method showed that, the MT level in human serum from patients with essential hypertension is significantly higher than that in the sera from normotensive subjects (Fig. 7).

4. Discussion The purpose of this report is to describe the development and usefulness of a simple competitive ELISA, capable of reliable determination of MT concentration in sera and tissues in the range

331

as low as 0.5 pg well − 1 of MT. After optimizing the reagents concentration, the incubation times and temperature, the IgG fraction of our antiserum provided a level of sensitivity better than the lowest range reported up to now—by ELISA [16,26]. The IgG fraction of the antiserum containing antibodies showed greater affinity for rat MT than for human MT because our rabbit antiserum was obtained against rat liver MT1 and MT2. Most of the rabbit or sheep anti-MT antisera obtained against either MT1 or MT2 have been reported to show greater cross reactivity to both isoforms [18,27]. In the present study it is shown that MT from rat liver, guinea pig and human serum exhibit very similar inhibition patterns (Fig. 5). The complete cross reactivity with various mammalian MT is due to similarities in their primary structure and epitope sequence [9]. On the basis of the primary and the tertiary structure of the MT, it seems reasonable to speculate that the epitopes detected by our IgG fraction are not exposed to very essential stereochemical changes. The data also suggests that the conformation changes in the MT after binding with HRPO, at least in the vicinity of the epitopes, are also smaller. This cELISA method demonstrated that the affinity of the IgG fraction against the purified rat Zn6.22Cu0.52MT and Cd5.33Zn1.33MT was similar. The observed increase in the immunological affinity of the antibodies for AMT indicate that the metals which determine the MT conformation are not very important for reaction of the antibody obtained by us with MT. However, this result contrasts with earlier reports, showing that the removal of the metals from MT results in a decrease [18,28] or in a significant difference [26] in its antigenicity. A highly sensitive cELISA was developed with labeling of the antigen (MT) with HRPO. This assay allowed the detection of 0.5 pg well − 1 of human serum MT and exhibited a relatively smaller metal-dependent response for Apo-, Znand Cd-MT derived from rat liver. A potential change in MT during the sample preparation was checked by the Western blotting method. It was shown that the MT genes were differently induced by Cd and Zn [4]. The immunoblot results showed good correlation with these data and proved that

332

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Fig. 7. Metallothionein concentration determined by competitive ELISA in sera from patients with essential hypertension (n = 90) and normotensive subjects (n= 236).

the anti-MT antiserum is highly sensitive in detecting the basal level of MTs in rat liver. Several methods for MT determination in biological materials have already been described [10– 12]. These procedures rely on rather unspecified parameters such as the total metal content of determination of metals by atomic absorption spectrometry or HPLC analyses. The immunological studies of course, represent an exception in this respect. More specific are RIA [18] and ELISA with monoclonal antibodies [16,29]. They allow the detection of 60 pg well − 1 MT and are sufficiently sensitive to assess the basal MT level in biological materials. The cELISA method, here proposed, with it’s detection limit of 0.5 pg well − 1, is therefore an alternative to the existing RIA [15,18] and monoclonal ELISA [16,28]. The successful determination of MT in sera from animals and humans in normal and pathological conditions described in the present report show that this cELISA can be applied not only for determination of the basal MT level but also for investigation of minor deviations in MT concentrations. The present studies not only confirm the earlier observations on the appearance of MT

in the body fluids but also provides evidence for an increased of MT level in serum during the development of essential hypertension in humans. The MT concentration in patients with essential hypertension is significantly higher in comparison to the MT levels of the healthy controls. The results for the MT evaluation in human sera suggest that the intracellular MT can be leached out extracellularly and transported by the serum. Several key enzymes and biologically active substances which are connected with regulation of blood pressure are metal-containing and related to the zinc and copper homeostasis (dopamine-bhydroxylase, tyrosinase, angiotensine converting enzyme). The Zn level also, paralleled the metabolite-to-precursor fatty acid ratios, being constantly higher in hypertensive patients [30,31]. Zn ions have been identified as constitutive cofactors of desaturase activity in human, animal, and in vitro studies [32–35]. The MT level strongly affects Zn and Cu concentration and its very wide distribution, made MT a crucial factor in the metabolism of these metals. The possible causatative interrelation between MT and arterial hypertension can not be excluded.

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