J BIOCHEM TOXICOLOGY Volume 7, Number 4, 1992

Heterogeneity of Antibodies to Metallothionein Isomers and Development of a Simple EnzymeLinked Immunosorbent Assay Hing Man Chan, Gordon A . Pringle, and M . George Cherian Department of Pathology, The University of Western Ontario, London, Ontario, Canada

ABSTRACT: A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1-100 ng for MT-2 and 10-1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1%Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our antiserum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%)for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl, (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.


Metallothionein, ELISA, Plasma, Urine,



Received March 20, 1992. Address correspondence to Dr. M. G. Cherian, Department of Pathology, The University of Western Ontario, London, Ontario N6A 5C1, Canada. Tel. (519) 661-2030; Fax (519) 661-3370.

Metallothionein (MT), an ubiquitous protein of low molecular weight (6000-8000), with two main isoforms, has been isolated from various tissues and biological fluids in vertebrates, invertebrates, and microorganisms (1). The synthesis of MT is inducible by various metal ions and hormones, and this protein is generally believed to play an important role in metal homeostasis or detoxification. However, Cd-MT can be nephrotoxic if injected into animals (2). Therefore, a specific and sensitive method for quantification of MT is necessary for the investigation of its physiological functions and its role in metal toxicity. Since the initial isolation and characterization of MT (3), several methods have been developed to quantify MT in tissues and biological fluids. The widely used metal binding assays determine MT concentration indirectly by measuring the amount of bound metal ions following saturation of the sample with a metal possessing high affinity for MT, such as Hg (4), Cd (5), or Ag (6). These assays are, however, limited by their lack of sensitivity, which is typically in the range of microgram/milliliter (pg/mL). Immunoassays have the potential of greater sensitivity (ng/mL), and, unlike the metal-binding assays, may provide greater specificity by directly measuring the protein moiety of MT. Several radioimmunoassays (HAS) have been developed for MT (7-9). The RIAs were found to be comparable to the metal-binding assays in terms of precision and recovery and provided better sensitivity (10). However, following induction of MT synthesis by Cd and Zn, tissue MT levels determined by RIA were 2-3 times lower than those derived by the

0 1992 VCH Publishers, Inc.


+ .25




metal-saturation methods (11).On the other hand, a recently developed enzyme-linked immunosorbent assay (ELISA) reported human liver MT levels that were much higher than those obtained with a Cd-hem assay (12). Thus far, no immunoassay provided results consistently comparable to the metal-saturation assays. The purpose of this study, therefore, was to develop a simple and sensitive competitive ELISA to measure accurately MT in biological fluids and tissues in the range of 0.1-100 ng.

MATERIALS AND METHODS Preparation of Antigens Metallothionein was isolated from livers of rats that had been injected ip with CdC1, (1 mg Cd/ kg) daily for 2 weeks. The purification of MT-1 and MT-2 was similar to that described previously (13). Briefly, liver samples were homogenized in a 50 mM sodium phosphate buffer (pH 7.4), and following centrifugation at 12,000 x g for 10 min at 4"C, the supernatants were heated at 80°C for 2 min. The denatured proteins were removed by centrifugation at 27,000 X g for 10 min (4"C), and the supernatant was applied to a 5 x 90 cm Sephadex G-75 column. Fractions containing Cd were pooled and lyophilized. The two MT isoforms were separated by polyacrylamide gel electrophoresis (PAGE) in a 7.5% gel under nondenaturing conditions, eluted from the gel, and lyophilized. The Apo-MT was prepared as described (14). Briefly, Zn was removed from Zn-MT-1 or -2 by gel filtration chromatography using a 5 x 45 cm Sephadex G-25 column equilibrated with 0.01 N HCI. Silver, Cd-, Cu-MT isoforms were prepared by dialyzing Apo-MT overnight at 4°C against phosphate buffered saline (0.05 M Na phosphate, 0.15 M NaC1, pH 7.4, phosphate-buffered saline (PBS)) containing 4 mol equiv of the corresponding metal. The MT-1 and MT-2 isolated from the blue crab Callinectes sapidus were kindly provided by Dr. M. Brouwer (15).

Preparation of Antiserum Rabbit antiserum to rat liver MT-2 was obtained, as previously described (16). Two milliliters of a 1 mg mL-' solution of glutaraldehydepolymerized MT-2 (MW 50,000) in PBS was emulsified with an equal volume of Freunds complete adjuvant and injected subcutaneously into rabbits. The animals were boosted with injections of an-

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tigen once every 2 weeks for a period of 6 months. Antibodies to MT-2 were detected initially by immunodiffusion and antiserum obtained by exsanguination. An IgG fraction of the antiserum was prepared by precipitation at 33% saturation of ammonium sulfate at 4°C overnight, followed by DEAE ion exchange chromatography (Whatman DE-23) using a linear NaCl gradient to 500 mM. The purity of the IgG fraction was determined by SDSPAGE (17). Preliminary results suggested that the IgG fraction might contain heterogeneous populations of antibodies to MT isoforms. Therefore, MT-2 specific antibody was subsequently purified by removing immunoglobulins cross-reactive with MT1 from the polyclonal preparation by immunoaffinity purification (18). Seven milligrams of MT-1 were coupled to 0.3 g activated CH-Sepharose 4B (Pharmacia Fine Chemicals, Baie dUrfe, Quebec.) in a 0.1 M sodium bicarbonate, 0.5 M NaC1, pH 8.0 buffer for 2 h at room temperature. After washing with a 0.1 M tris-HC1, pH 8.0 buffer to block remaining active sites, the coupled ligand was re-equilibrated in PBS, then incubated overnight at 4°C with 1 mL of the IgG fraction. The supernatant containing the unbound anti-MT-2 IgG fraction was removed, and the coupled ligand was cleared of the bound antibodies by repeated washing with a 0.2 M glycine, pH 2.5 buffer. To ensure complete removal of the bound antibodies, the supernatant was reincubated with the washed coupled ligands. Immunoglobulin fractions were stored in PBS (1 mg mL-') at -20°C.

Enzyme Linked Immunosorbent Assay The ELISA technique used was similar to the method of Rennard et al. (19), as modified by Pringle et al. (20), for connective tissue components. Flat bottom, polystyrene plates (Costar, Cambridge, MA) were coated (200 ng/well) with rat MT2 in a 100 mM sodium carbonate/bicarbonate, pH 9.6 buffer and stored, covered, overnight at 4°C. In a separate set of round bottom microtiter plates (Dynatech Lab, Chantilly, VA), 100 p1 of a 1:2000 dilution of purified IgG fraction was added to 100 pl of serially diluted (twofold) sample or MT-standard and the mixtures were incubated overnight at 4°C. Concentrations of MT-standards were measured by the silver-saturation (Ag-hem) method (6). Once the MT-coated plates were warmed to room temperature and given three 3 min washes with PBS containing 0.05% Tween 20 (PBS/T), the inhibitor/primary antibody mixtures in the round bottom plates were transferred well to well to the

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MT-coated plates and allowed to incubate for 30 min at room temperature. After washing with PBS/ T, the MT-coated plates were incubated in turn with (1) a 1:2000 dilution of biotinylated goat anti-rabbit IgG (Vector Lab, Burlingame, CA) in PBS/T for 60 min, (2) a 1:2000 dilution of horseradish peroxidase conjugated Avidin D (Vector Lab) in PBS/ T for 60 min, and (3) an aqueous solution of 0.01% 1,2-phenylenediamine dihydroxide (Aldrich Chemical Co., Windsor, ON), 0.003% hydrogen peroxide (BDH, Toronto, ON) for the time necessary to generate a suitable level of color, typically 30 min. Each step was preceded by three washes with PBS/T. The enzyme reaction was terminated by adding 50 pl of 4N sulfuric acid, and A492nmwas measured with a SLT-Labinstruments EAR400 plate reader (Fisher Scientific, Ottawa, ON). All ELISA conditions (concentrations of coating antigen, primary and secondary antibody, and substrate) were optimized by a series of checkerboard titrations using MT-standards (21). The concentration of MT in unknown samples was determined by linear regression of the inhibition curve after logit Y transformation: log,, (100 X [l - (A, - A,)/(A, - A,)]}, where A, is the absorbance caused by nonspecific binding (normal rabbit serum used instead of anti-MT serum), A, is the absorbance of the sample, and A, is the maximum absorbance (no competing sample or standard added).

Treatment of Tissue Samples Liver and kidney were dissected from male Sprague-Dawley rats, male C3H syngeneic mice, and male Toxic milk (TX) mice, a mutant mouse strain with high Cu concentrations in liver (22). About 0.5 g of tissue were homogenized in 3 mL of a 0.25 M sucrose solution. The homogenate was centrifuged at 10,000 X g for 10 min at 4°C. The supernatant was diluted a hundred fold with PBS containing 1% Tween 20 and heated at 80°C for 10 min prior to its use as an inhibitor in the ELISA. A separate aliquot of the supernatant was used for Ag-hem assay.

Application of ELISA to Measure MT in Urine and Plasma Male Sprague-Dawley rats (300-350 g) were injected iv with saline (control), or 0.3 mg Cd/kg body weight, as either CdC12 or Cd-MT and kept individually in metabolic cages. Urine samples were collected every 24 hours for 7 days. About 0.5 mL heparinized plasma was collected from each rat by venipuncture of the tail vein 2, 24, 48, 72, 96, and



3.0 2.5



--O 1 :3200

/' :6400





0.50.0 0.1

1 :12800

1 .o 10.0 100.0 1000.0 Coating Ag (MT 11) conc (ng/well)


FIGURE 1. Titration curve for optimization of concentration of coating antigen (MT-2) and primary antibody (purified antiwas measured rat liver MT-2 rabbit serum). The value of A49Znm after 1 h incubation of a series of twofold antibody dilutions with eight rat MT-2 coating concentrations (0.5 to 1000 ng/ well). Coating concentration of 200 ng/well and antibody dilution of 1:2000 were chosen for subsequent measurements. Both biotinylated secondary antibody and avidin peroxidase were diluted 1:2000 to give optimum response (titer curve not shown). The MT concentrations used were based on measurement by the Ag-hem method.

168 hours after injection. The MT concentrations in urine and plasma were measured by ELISA using the purified IgG fraction of the antiserum.

Statistical Analysis Means of two groups of samples were compared by a two-tailed Student's t-test, as described by Sokal and Rolf (23). Five percent was used as the level of significance unless otherwise specified.

RESULTS The results of the checkerboard titrations to optimize the concentration of the primary antibody and the rat MT-2 used to coat the ELISA plates are presented in Figure 1. In order to reach saturation at a suitable level of absorbance in a reasonable period of time with a dilution of primary antibody that would minimize unwanted crossreactivity, a 1:2000 dilution of primary and secondary antibodies with an MT-2 coating concentration of 200 ng/well was selected for subsequent experiments. Using Cd-MT-2 coated plates under these conditions, inhibition curves (percentage inhibition vs. log concentration) were obtained with Cd-MT-1 and Cd-MT-2 as inhibitors of the IgG fraction of the rabbit antiserum to rat MT-2 (Figure 2(a)). The concentrations of MT-2 and MT-1 re-

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80 --






70-- 0-OMT

TABLE l. Effect of Metals on the Cross-Reactivity of MT with the IgG Fraction of Rabbit Anti-MT-2 Antiserum M T Concentration Required fur 50% Inhibition (nq/rnL) (mean ? SEM, n = 6 ) MT-7 1970 2 2450 t 2700 ? 2670 ? 2870 2





0 '





108 0. 0.1

- /. /


20 --






100.0 1000.0

, 1.OE4

230" 349 128 342 312

98 -+ 134 C 173 2 123 t141 4

10" 13 43 17 32

See the Materials and Methods section for preparation of Apo-MT and other metal-bound MTs. "Denotes values significantly lower than those of Cd-MT (p < 0.05; df = 10).

MT conc (ng/ml)





MT conc (ng/ml)

FIGURE 2. (a) and (b)Inhibition curves (inhibition percentage vs. log MT concentrations) of competitive ELISA using rat liver Cd-MT-1 and Cd-MT-2 as antigens (0.1-10 000 pg in 100 pL). The ELISA plates were coated with Cd-MT-2. Percentage of inhibition was calculated by 100 (1 - AJA,), where A, and A , are absorbance of sample and maximum response, respectively, after correction of background reading (a) using a 1:4,000 dilution of the IgG fraction and (b) using a 1:800 dilution of the immunoaffinity purified, MT-2 specific antibodies.

quired to yield 50% inhibition were 150 ng mL-' and 2500 ng mL-' or 15 ng and 250 ng, respectively. Immunoaffinity purification of the IgG fraction effectively removed cross-reactivity with CdMT-1 (Figure 2(b)). The species of metal bound to rat MT had little influence on the antigenicity of either MT-1 or MT2 (Table 1). However, removal of the metal from the protein increased the affinity of the IgG fraction for both isoforms. Preliminary results using the IgG fraction revealed that the ELISA significantly underestimated the concentration of MT in tissue extracts known to contain high concentrations of MT, as determined by the Ag-hem method. Addition of 2-mercaptoethanol or dithiothreitol to the extracts (up to 100 pM) either had no effect or inhibited the antigen-antibody reaction altogether. How-

ever, increasing the concentration of Tween 20 in the tissue homogenate supernatant to 1%,prior to heating the sample, yielded values averaging 90% of those obtained by the Ag-hem method (Figure 3 ) . Heating the sample was found to greatly reduce background to less than 5% of maximum absorbance. Employing these conditions, sequential dilutions of tissue cytosol (rat liver and kidney and human liver) or physiological fluid (rat urine and serum and human serum) samples yielded inhibition curves that paralleled a typical standard curve produced with rat liver Cd-MT-2 (Figure 4). Addition of known concentrations of MT-2 to the samples resulted in good recoveries by ELISA (Table 2). The coefficients of variation for reproducibility within and between assays were generally less than 5% and lo%, respectively. The concentration of MT in tissue extracts from various species was measured by both the Ag-hem











0 0.0






% Tween 20 in PBS

FIGURE 3. Effect of Tween 20 concentration on ELISA. A rat liver cytosol sample (125 pg MT per mL 0.25 M sucrose as measured by the Ag-hem method) was diluted with seven concentrations of Tween 20 (0.125-5%) in PBS, and its MT concentrations (expressed as a percentage of 125 pg mL-') were measured by ELISA. Mean ? SEM, n = 3.

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MT conc (ng/ml) FIGURE 4. A typical standard (logit-log plot) curve using rat liver Cd-MT-2 as the standard (100 pL). The regression line covers a linear range between 0.1-100 ng; y = -2.25 + 2 . 5 8 ~ ; r = 0.99 ( p < 0.001, df = 19). Each point represents the geometric mean of three replicate measurements. The value of lm (MT required to give 50% inhibition) was calculated from Xprediction of the regression analysis.

method and ELISA (Table 3). Both antibody preparations (antiserum and affinity purified antibodies) cross-reacted with human and mouse MT but not with MT present in crab hepatopancreas. The ELISA and Ag-hem method provided very similar mean values of MT concentration (about 5-10 pg g-') in normal murine liver and kidney as well as in TX mouse kidney. However, in tissues containing higher levels of MT, produced by induction of MT synthesis by injection of Cd into rats and mice or by a genetic disorder in TX mouse liver or as is normally the case in human liver, the ELISA consistently provided lower mean MT concentrations than the metal-binding assay. Regression analysis of the total MT concentrations in Table 3 obtained by Ag-hem method and TABLE 2. Recovery by ELISA of Known Concentration of Rat Liver MT-2 Percentage Recove@

M T Added (ng/well)b


83 28 9.2 3.1 1.0 0.3

99.6 98.2 103 83.9 62.0 140

106 112 109 140




Liver 106




109 101 98.1 98.4 118 90.3

108 99.0 99.4 104 97.8 104



'Data shown are geometric means of triplicates. Basal MT levels are 98, 145, 30, and 8 ng for kidney, liver, plasma, and urine samples, respectively. m e amount of rat liver MT-2 added was based on measurement by the Ag-hem method.


ELISA using the IgG fraction showed a good linear relationship (Figure 5(a)), with a slope of regression line b = 0.883, which is significantly less than 1, p < 0.001, df = 71. Similar analysis of values obtained by the Ag-hem method and ELISA using the immunoaffinity purified MT-2 specific antibody preparation also showed a good linear relationship (Figure 5b), with the slope of the regression line b = 0.721, which is significantly higher than 0.5 ( p < 0.001, df = 71), suggesting that over 50% of the tissue cytosolic MT is MT-2. The sensitivity of the ELISA also permitted us to measure the quantity of MT in plasma and urine. The MT concentration in plasma of control rats was about 30 ng mL-' (Table 4). Two days after injection of CdCl,, plasma MT concentrations increased significantly and remained high for the following 5 days. In contrast, rats injected with CdMT exhibited significantly higher plasma MT concentrations within 2 hours of injection, which remained high for the next 2 days then decreased to the control level after 3 days. In metabolic studies with rats, volume of urine excreted by all rats was quite uniform and averaged about 20 mL per day. Mean MT concentrations in urine from control rats and rats injected with CdC12 were not significantly different and ranged from 30-150 ng/day (Table 5). Urine MT concentrations from Cd-MT injected rats decreased with time but remained sigruficantly higher than those of the control and CdC12 injected rats (Table 5).

DISCUSSION The purpose of this report is to describe the development and usefulness of a simple ELISA capable of reliably measuring MT concentration in tissues and biological fluids in the range of 0.1100 ng. After optimizing the concentrations of the reagents, the incubation times, and temperatures, the IgG fraction of our antiserum provided a level of sensitivity comparable to the lowest range reported (100 pg) by RIA (9,24) and fluorometric ELISA (26) and significantly better than an ELISA reported for human MT (15 ng) (12). Moreover, it offers relative simplicity over the reported assays; over 250 samples (six plates with two series of known concentrations of standards in each plate) can be processed within 6 hours without automation. The precautions and disposal problems associated with handling of radioisotope can be avoided.


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TABLE 3. Comparison of ELISA and Ag-Hem in Quantification of MT in Various Tissue Cytosol Samples ELlSA Tissues and Treat men t Rat liver Control Cd injected" Rat kidney Control Cd injected Mouse liver Control Cd injected Mouse kidney Control Cd injected Human liver TX mouse liverb TX mouse kidney Crab hepa topancreas

Affinity Purified Antibody

4s Ax-Hem 4.6 586


7.5 195



* 23 f

1.9 21

9.6 2 7.5 110 & 23 f 1.6 85 f 34 150 2 30 1646 f 381 5.3 f 1.3 67 f 21


Fraction 6.3 383

? f

2.1 (140) 52 (65)

4.9 f 1.1 (106) 303 e 28 (52)

10 f 3.4 (139) 163 f 10 (84)

8.5 132


2.1 (113)

8.9 98

7.2 80


2.2 (75) 30 (73)



1.6 (93) 14 (89)

4.4 f 0.4 (142) 69 -C 13 (81) 133 f 84 (89) 1728 f 485 (105) 5.5 f 0.4 (104) 0.08 k 0.02

* 34 (68) f

2.3 t 1.1 (74) 59 f 12 (69) 109 f 37 (73) 965 f 234 (59) 4.9 f 2.0 (92) 0.01 f 0.01

"Denotes rats injected with 1.0 mg Cd/kg (sc) 24 h before sacrifice. 'TX mice are transgenic mice with high Cu concentrations in liver. 'Values in parenthesis are average percentages of the Ag-hem value. Mean ? SD ( n = 12, triplicate measurement of tissues from four animals).

Because our rabbit antiserum was raised against rat liver MT-2, the IgG fraction of the antiserum contained antibodies of considerably greater affinity for rat liver MT-2 than MT-1. Most rabbit or sheep anti-MT antisera raised against either MT-1 or MT-2 have been reported to show total crossreactivity to both isoforms (7,8,10,12). Leibbrandt et al. (25) reported complete cross-reactivity between commercial rabbit MT-1 and MT-2 using our antiserum. This may be due to the low affinity of the antiserum (from rabbit) to rabbit MT and/or impurities of the commercial MT isoforms used in the assay as standards. Only one study (9) has previously reported the production of an MT-1 specific antiserum in sheep. These results suggest that there may be two specific populations of antibody for MT-1 and MT-2. This hypothesis is proven by the isolation of MT-2 specific antibodies by immunoaffinity purification in our study. The difference in sensitivity for the two isoforms may therefore be due to a higher proportion of antibodies to MT-2 in IgG fraction of our antiserum or to the affinity of antibodies to MT-2 isomer being higher than that of the antibodies to MT-1 isomer. The antibodies bound to the MT-1 coated affinity column, however, were found to cross-react with both MT-1 and MT-2. It has been suggested that

MT may have two antigenic epitopes (27). Our results indicate that there are at least two epitopes on MT-2; one of them is common between the two isoforms and the other is specific and recognized by our MT-2 specific antibodies. Moreover, the presence of MT-1 specific antibodies prepared by Mehra and Bremner (9) suggests that there is also an isoform-specific epitope on MT-1. However, it will be necessary to identify the residue sequences which constitute these different epitopes before they can be confirmed. Based on the primary and tertiary structure of the isoform, it seems reasonable to speculate that the major epitopes recognized by the IgG fraction are on exposed portions of the protein where the amino acid sequence is not strictly conserved (i.e., not in the vicinity of metal binding sites) and differs between the isoforms. These differences are apparently shared by mouse and human and possibly other mammalian isoforms, as the IgG fraction cross-reacted very well with MT from these species. The lack of crossreactivity with crab MT confirms other data (8) that the immunological properties of invertebrate MTs are very different from those of mammals. The ELISA also demonstrated that the affinity of the IgG fraction toward purified rat MT-1 and MT-2 was independent of the species of bound

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1500 1

served increase in immunological affinity for both apo-MT isoforms suggests that bound heavy metal ions maintain protein conformation, as indicated by physicochemical data (28). However, this result contrasts with earlier reports that removal of metals from MT results in a decrease (9) and no difference (7) in its antigenicity. The increase in affinity for apo-MT suggests that the change in conformation caused by removal of metal ions increases the number and/or accessibility of epitopes. When the ELISA was initially employed to measure MT concentrations in tissue homogenates without any pretreatment, the results were comparable to those obtained by the Ag-hem method when the tissues contained low (resting) levels of MT. The ELISA, however, consistently underestimated MT concentration in tissue samples collected from animals that had been injected with CdC1, to induce MT synthesis. Others have experienced similar problems with immunoassays (11) and have speculated that the discrepancy may be due to (1)overestimation of MT by the metal-binding assays because of a parallel induction of nonMT metal-binding peptides (lo), (2) the presence of cross-reacting substances in MT-induced tissues (29), and (3) some structural change in MT causing decreased antigenicity (30). A potential structural change in MT during sample preparation was oxidation of sulfhydryl groups to disulfide bonds with the formation of polymers. Addition of reducing agents to the sample did not increase cross-reactivity. However, increasing the concentrations of Tween 20 to 1.0% in the sample diluent followed by heating the sample gave MT estimates by ELISA (using the total IgG fraction) consistently at 80100% of those of the metal-binding assay. Because Tween 20 is a nonionic detergent used to dissociate hydrophobic interactions, these results suggest that the induced MT, which is present in high concentrations in liver and kidney samples, is likely


A 1500



Y = 21.5 0.721 X b = 0.721 k 0.019






Ag saturation method MT cone ( P d d

FIGURE 5. (a) and (b) Cytosolic MT concentrations from rat liver and kidney, mouse liver and kidney, and human liver obtained by ELISA (ordinate) plotted against those obtained with the Ag-hem method (abscissa) from the same sample (a) using the IgG fraction and (b) using the immunoaffinity purified MT-2 specific antibodies.

metal ion. This indicates that the epitopes consist largely, if not exclusively, of protein. The data also suggest that the conformation of both isoforms, at least in the vicinity of the epitopes, is also independent of the species of bound metal. The ob-

TABLE 4. Effects of Injection of CdClz and Cd-MT on Plasma MT Concentration MT Concentrations in Plasma (nx/rnLi ~~

Day after lnjection

Control 34 ? 22 ? 33 ? 38 ? 25 ? 31 ?


11 14 15 23 13 18


48 59 ? 73 ? 96 ? 95 ? f_



18 23 23 29" 35" 34"

Cd-MT 114 t 23" 125 2 46" 137 ir 32' 51 2 29 18 2 9 24 2 21

Rats were injected iv with 0.3 mg Cd/kg body weight as CdClz or Cd-MT. Control rats were injected with an equivalent volume of saline. Mean k SD ( n = 3 ) . 'Denotes values significantly higher than the control value ( p < 0.05; df = 4).


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TABLE 5. Effects of Injection of CdC12, and Cd-MT on Excretion of MT in Urine M T Excreted in Urine (nglday)

Day after Injection 1 2 3 4 7



140 t 64 98 t 45 70 t 45 110 t 56 74 i 38

53 ? 34 34 & 25 31 t 19 61 t 45 50 ? 29

Cd-MT 3611 t 1106 -+ 1224 t 962 ? 423 t

367' 289" 39W 452" 210"

Rats were injected iv with 0.3 mg Cd/kg body weight as CdCl, or Cd-MT. Control rats were injected with an equivalent volume of saline. Mean * SD (n = 3). 'Denotes values significantly higher than the control value ( p < 0.05; df = 4).

associated with itself or some other molecule(s) and that this association decreases the availability of epitopes. Whether this association is of biological significance during induction or merely an artifact of sample preparation is currently being investigated. The reason that the IgG fraction of our antiserum does not achieve 100% of the value of the metal-binding assay for total MT concentration is likely due to the underestimation of the MT-1 component in the sample because of the intrinsic characteristics of our antiserum which cause it to have higher affinity/concentration for MT-2 than MT-1. The purified MT-2 specific antibodies gave MT concentration values that were generally 6873% of the metal-binding assay values. These results suggest that the remaining 30% of induced MT is MT-I. Little is known about the proportion of the h4T isoforms in tissues (31). The normal basal tissue MT concentrations in rat and mouse, as determined by ELISA using the MT-2 specific antibodies, matched the values of the Ag-hem method and the ELISA using the IgG fraction, indicating that the predominant form of basal MT in control tissues is MT-2, as has been suggested by Lehman-McKeeman and Klaassen (32) from the result of their high-pressure liquid chromatography study. Induction of MT by zinc is associated with an increase in both isoforms but at different rates (33). The MT isoform genes have been shown to be differentially induced by both metals and glucocorticoids (34), and cell type-specific expression of mRNA for MT isoforms has also been documented (35). The MT-2 specific antibodies, therefore, can be used to quantify and/or localize MT-2 in tissues of animals under various physiological and pathological conditions. The results of MT estimation in rat plasma and urine suggest that the intracellular hepatic Cd-MT or its degradation can be leached out extracellularly and transported to the kidney in the plasma.

The presence of this extracellular Cd-h4T after CdC1, injection, although at relative low concentration as compared to that after Cd-MT injection, may cause increase in Cd accumulation in kidney. The urinary excretion of Cd-MT in rats injected with Cd-MT and not with CdC1, suggests that urinary excretion of Cd-MT may be related to the renal toxicity. These findings are in agreement with results of other immunological studies (8,36). In summary, the ELISA reported in this study provides a simple, specific, and sensitive method of MT estimation in various tissues and biological fluids from different mammalian species which have a wide range of MT concentrations. In addition, we report the isolation of MT-2 specific antibodies for the first time and this may provide an useful tool to study the potential biological functions of MT isoforms.

REFERENCES 1. J. H. R. Kagi and M. Nordberg (1979). Metallothionein, Birkhauser Verlag, Basel. 2 . M. G. Cherian, R. A. Goyer, and L. DelaquerriereRichardson (1976). Cadmium metallothionein induced nephropathy. Toxicol. A p p l . Pharmacol., 38, 399-408. 3. J. H. R. Kagi and B. L. Vallee (1960). Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem., 235, 3460-3465. 4. J. K. Piotrowski, W. Bolanowsaka, and A. Sapota (1973). Evaluation of metallothionein content in animal tissues. Actu. Biochim. Pol., 20, 207-215. 5. S. Onasaka, K. Tanaka, M. Doi, and K. Okahara (1978). A simplified procedure for determination of metallothionein in animal tissues. Eisei. Kugaku., 24, 128-133. 6. A. M. Scheuhammer and M. G. Cherian (1986). Quantification of metallothionein by a silver-saturation method. Toxicol. Appl. Phmzacol., 82, 417-425. 7. R. J. Vander Mallie and J. S. Garvey (1979). Radioimmunoassay of metallothioneins. J. Biol. Chem., 254, 8416-8421.

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8. C. Tohyama and Z. A. Shaikh (1981). Metallothionein in plasma and urine of cadmium-exposed rats determined by a single-antibody radioimmunoassay. Fundam. A p p l . Toxicol., 1, 1-7. 9. R. K. Mehra and I. Bremner (1983). Development of a radioimmunoassay for rat liver metallothionein-1 and its application to the analysis of rat plasma and kidneys. Biochem. J., 213, 459-465. 10. H. H. Dieter, L. Muller, J. Abel, and K. H. Summer (1986). Determination of Cd-thionein in biological material: comparative standard recovery by five current methods using protein nitrogen for standard calibration. Toxicol. Appl. Pharmacol., 85, 380388. 11. M. P. Waalkes, J. S. Garvey, and C. D. Klaassen (1985). Comparison of methods of metallothionein quantification: cadmium radioassay, mercury radioassay and radioimmunoassay . Toxicol. Appl. Pharmacol., 79, 524-527. 12. A. Grider, L. B. Bailey, and R. J. Cousins (1990). Erythrocyte metallothionein as an index of zinc status in humans. Proc. Natl. Acad. Sci. U S A , 87, 12591262. 13. D. M. Templeton and M. G. Cherian (1984). Chemical modification of metallothionein. Biochem. J., 221, 569-575. 14. M. J. Stillman, W. Cai, and A. J. Zelazowski (1987). Cadmium binding to metallothioneins. 1. Bid. Chem., 262, 4538-4548. 15. D. W. Engel and M. Brouwer (1984). Trace metal binding proteins in marine molluscs and crustaceans. Mar. Environ. Res., 13, 177-194. 16. D. Banerjee, S. Onosaka, and M. G. Cherian (1982). Immunhistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidney. Toxicology, 24, 95-105. 17. E. Harlow and D. Lane (1988). Antibodies. A Laboratory Manual, 726 pp., Cold Spring Harbour Laboratory, New York. 18. Affinity Chromatography. Principles and Methods. Pharmacia Fine Chemicals, Baie d’Urfe, Quebec, Canada. 19. S. I. Rennard, R. Berg, G. R. Martin, J. M. Fordart, and P. Gehron Robey (1980). Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal. Biochem., 104, 205-214. 20. G. A. Pringle, C. M. Dodd, J. W. Osborn, C. H. Pearson, and T. R. Mosmann (1985). Production and characterization of monoclonal antibodies to bovine skin proteodermatan sultate. Collagen Re. Res., 5, 2339. 21. A. Voller, D. E. Bidwell, and Bartlett. (1976). Microplate ELISA and its application. In Immunoenzymatic Assay Techniques, R. Malvano, ed., pp. 104115, Martinus Nijhoff, The Hague. 22. H. Rauch (1983). Toxic milk, a new mutation affecting copper metabolism in the mouse. J. Heredity, 74, 141-144.



23. R. R. Sokal and F. J. Rolf (1969). Biometry, W. H. Freeman & Co., San Francisco. 24. J. S. Garvey, R. J. Vander Mallie, and C. C. Chang (1982). Radioimmunoassay of metallothioneins. Methods Enzynzol., 84, 121-138. 25. M. E. I. Leibbrandt, J. Koropatnick, J. F. Harris, and M. G. Cherian (1991). Radioimmunoassay of metallothionein in rabbit, rat, mouse, Chinese hamster and human cells. Biol. Trace Element Res., 30, 245256. 26. D. G. Thomas, H. J. Linton, and J. S. Garvey (1986). Fluorimetric ELISA for the detection and quantitation of metallothionein. 1. Irnmunol. Methods, 89, 239247. 27. K. Nakajima, K. Suzuki, N. Otaki, and M. Kimura (1991). Epitope mapping of metallothionein antibodies. Methods Enzymol., 205, 174-190. 28. M.Vasak, A. Galdes, H. A. 0. Hill, J. H. R. Kagi, I. Bremner, and B. W. Young (1980). Investigation of the structure of metallothioneins by proton nuclear magnetic resonance spectroscopy. Biochemisfy, 19, 416-425. 29. M. G. Cherian (1988). An evaluation of methods of estimation of metallothionein. In Cadmium, S. Safe and M. Piscator, eds., pp. 227-235, Springer-Verlag, Berlin. 30. C. V. Nolan and Z . A. Shaikh (1986). Determination of metallothionein in tissue by radioimmunoassay and by cadmium saturation method. Anal. Biochem., 154, 213-223. 31. I. Bremner (1987). Nutritional and physiological significance of metallothionein. In Metallofhionein 11, pp. 81-107, Birkhauser Verlag, Basel. 32. L. D. Lehman-McKeeman and C. D. Klaassen (1987). Induction of metallothionein-I and metallothionein I1 in rats by cadmium and zinc. Toxicol. Appl. Pharmacol., 88, 195-202. 33. L. D. Lehman-McKeeman, G. K. Andrews, and C. D. Klaassen (1988). Mechanisms of regulation of rat hepatic metallothionein-I and metallothionein I1 levels following administration of zinc. Toxicol. Appl. Pharmocol., 92, 1-9. 34. R. I. Richards, A. Heguy, and M. Karin (1984). Structural and functional analysis of the human metallothionein-I gene: differential induction by metal ions and glucocorticoids. Cell, 54, 93-103. 35. U. Varshney, N. Jahroudi, N. R. Foster, and L. Gedamu (1986). Structure, organization, and regulation of human metallothionein I gene: differential and cell-type-specific-expression in response to heavy metals and glucocorticoids. Mol. Cell Biol., 6, 26-37. 36. Z. A. Shaikh, K. J. Ellis, K. S. Subramanian, and A. Greenberg (1990). Biological monitoring for OCcupational cadmium exposure: the urinary metallothionein. Toxicology, 63, 53-62.

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The...
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