Radioimmunoassay of rat liver metallothionein

4NAI.YTlCAL.

98,

RIOCHEMISI’RY

89-94

(1979)

Radioimmunoassay FRANK

A simple. has liver

been zinc

inexpensive developed. thionein

protein: the displacement

0.

and

of Rat Liver Metallothionein’~’ BRADY:’

AND

Received

February

convenient

The double-antibody with trace amounts

binding of this

of this antigen

L.

RICHARD

I?.

1979

radioimmunoassay assay of “‘*Cd(H)

KAFKA

involves the to a specific

for

rat

labeling activity

liver

metallothionein

of homogeneous. rat of l-2 x IO” cpmipg

antigen by rabbit anti-rat liver metallothionein by unlabeled zinc thionein or cadmium.

antiserum: zinc-thionein;

the the

precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of IO-500 ng protein. The assay is accurate as compared in extracts of rat liver. A radial

with a previous immunodiffusion

Metallothioneins are low molecular weight, inducible cytoplasmic proteins synthesized in certain eucaryotic tissues in response to transition metal ions. Metallothionein has been implicated as functioning in cadmium and mercury detoxification and in zinc homeostasis. Several reviews are available which deal with the properties and potential functions of metallothionein ( l-4). During our studies on metallothionein it became evident that a more sensitive quantitative assay for metallothionein needed to be developed. The usual assay was to determine the amount of metal (zinc or cadmium) bound to metallothionein after chromatography on Sephadex G-75 (5,6). This assay is sensitive to about 5- 10pg of protein, which is sufficient for the determination of metallothionein in liver and in kidney. It is I Supported by a research grant from NIEHS, USPHS (ES 01288) with funds provided by an interagency grant from the EPA (IAG-DSE772). ” A preliminary report of this work was presented at the First International Symposium on Metallothionein and Other Low Molecular Weight Metal Binding Proteins. Ziirich. Switzerland. July 17-22. 1978. .j Recipient of a Research Career Development Award (ES 00022~ from NIEHS. USPHS.

technique of quantitating assay for metallothionein

metallothionein is also described.

not sufficiently sensitive for assaying metallothionein in other tissues or when sample volumes are small, such as with bile or serum samples. We report herein the development of a simple. inexpensive, and convenient radioimmunoassay for rat liver metallothionein, which is useful in the range of lo-500 ng of protein. A radial immunodiffusion assay for metallothionein, useful in the range of 25-250 pg protein, is also described. Other immunoassays for metallothionein have been reported by Vander Mallie (7,8), by Madapallimatam and Riordan (9). and by Tohyama and Shaikh (IO). METHODS

Metallothionein was purified from rat liver after induction by zinc ( 10 mgikg body weight daily for 3 days) or after induction by cadmium (0.25 mg/kg body weight daily for 3 days). Male Sprague-Dawley rats (300-350 g), obtained from Sasco, Inc., Omaha, Nebraska, were used for the preparation of metallothionein. Zinc thionein and cadmium. zinc-thionein were purified according to established procedures (5,6,11,12). through the DEAE-cellulose chromatog-

90

BRADY

AND

raphy step. Zinc thionein II and cadmium. zinc-thionein II were used for immunization of rabbits, as these proteins were the easiest to obtain in electrophoretically homogeneous quantities. New Zealand white rabbits were immunized by multiple intradermal injections on their shaved backs with a homogenized mixture of 1.0 ml Freund’s complete adjuvant (Cappel Laboratories, Downingtown, Pa.) and 1.O ml of metallothionein (0.5 mg in 0.02 M Tris.HCl, pH 8.6), using 0.4 ml per site. Subsequent injections were spaced out at 7- to IO-day intervals, using the same method, but substituting Freund’s incomplete adjuvant (Cappel Laboratories) for the complete adjuvant. Seven to 10 days following the fourth series of injections rabbits were bled (40-45 ml) by cardiac puncture, and an equal volume of saline was injected intraperitoneally. Immunized rabbits were given booster injections 4-6 weeks after bleeding and re-bled 7- 10 days after being injected. Blood was collected in glass centrifuge tubes and was allowed to clot for 60 min at 37°C and then overnight at 4°C. Antiserum was then harvested by centrifugation and stored frozen at -20°C. The presence of immunoglobulins specific for metallothionein was assessed by Ouchterlongy double diffusion on 1.5%~ agar plates. Goat antirabbit IgG immunoglobulins were a gift of Dr. Kowk-Ming Chan of this Division. They were prepared by immunization of goats with ammonium sulfate-purified rabbit immunoglobulins. The goat antibodies were purified by repeated precipitation with 50% saturated ammonium sulfate. Bolton-Hunter reagent (iz51 1500 Ci/mmoI), ‘@+CdCl, (3.7 mCi/mg), “i”ZnCl, (3.9 mCi/mg), and [“5S]cystine (51.9 Ci/mmol) were all obtained from New England Nuclear Corporation, Boston, Massachusetts. In vitro labeling of metallothionein (5 pg) with the BoltonHunter reagent was attempted according to recommended procedures (13). III \gitro labeling with ““Zn and with ‘OQCdis described in Table 1. In t’ibw labeling with IYd was

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by injecting a rat (400 g) with zinc (5 mgikg body wt) at time 0 and with zinc (10 mg/kg body wt) plus 10 PCi “‘“Cd at 18 h. The rat was killed at 23 h, its liver excised and frozen, and metallothionein purified as described above (5,6,11~ 12). E.v \,ir~~ labeling with [““Slcystine was performed in an isolated liver perfusion system (6), using 100 &i of cystine, 75 pg Cd(II), and a perfusion time of 2 h. After perfusion the liver was frozen, and metallothionein was subsequently purified as above. Details of the radioimmunoassay of metallothionein, using ‘““Cd-labeled rat liver metallothionein, are described in the legend to Fig. 2. Oxoid cellulose actate filters, used in this assay, were manufactured by Oxoid Ltd., Basingstoke, England, and were obtained from Med-Ox Chemicals Ltd., Ottawa, Canada. Liquid scintillation counting was done in a Packard Tri-Carb, using 3a70B cocktail (Research Products International, Elk Grove Village, Ill.). Gamma counting was done in a Packard AutoGamma spectrometer. Radial immunodiffusion was performed according to published procedures (14,15). All chemicals were reagent-grade quality or better and were commercially obtained.

done

RESULTS

Our initial attempts at developing a quantitative immunoassay for metallothionein were focused on a radial immunodiffusion assay (14,15). With the antimetallothionein prepared from immunized rabbits, we indeed could assay metallothionein quantitatively (Fig. l), but the sensitivity range of this assay (25-250 pg) was two or three orders of magnitude greater than was useful for measuring metallothionein in biological samples, such as blood, bile, and tissues other than liver. Because greater sensitivity was required, attempts were then made to develop a radioimmunoassay for metallothionein of sufficient sensitivity to measure nanogram

RADIOIMMUNOASSAY RAJIAL

OF RAT

IMMUNODIFFUSION

I”“‘“”

FIG. 1. Radial immunodiffusion assay of rat liver zinc thionein. The technique of radial immunodiffusion (14.15) was used to quantitatively assay zinc thionein (ZnMT). Agar plates (1.5%, 45 x 90 mm, purchased from Miles Labomratories. Elkhart, Ind.), containing 15 mg of ammoniulm sulfate-purified rabbit anti-rat zinc thionein II immunoglobulins, were used in this assay. Three-millimeter-diameter wells were cut in the agar, and known amounts of rat liver zinc thionein II were pipetted into the wells. Diffusion and development of radial immunoprecipitates were allowed to continue for 36-48 h in a humidified desiccator. The diameters of the immunoprecipitates were measured, using a calibrating viewer (Transidyne General Corp., Ann Arbor. Mich.), which accurately measures *O. 1 mm. The data are presented as the averages of duplicate determinations.

quantities of the protein. The results of several different approaches to radiolabeling of rat liver metallothionein are shown in Table 1. Most successful and reproducible was the in rzitro labeling of zinc thionein 11 by “‘“Cd in tsitro in the presence of 0.5 mM dithiothreitol. Yields of 45-76s of label incorporated were attained with specific activities of 0.64-1.85 x 10” cpmigg protein. Use of less protein (~5.5 pg) reduced the yield. Use of more dithiothreitol (~0.5 mM) reduced the specific activity of the product, altlhough the complete absence of dithiothreitol greatly reduced both the yield and the specific activity. Routinely, we now use 5.5 pg zinc thionein 11, 0.5 mM

LIVER

91

METALLOTHIONEIN

dithiothreitol, 10 FCi “‘“CdCl,, 0.02 M Tris. HCl, pH 8.6, in an incubation mixture for 18-24 h at 4°C to obtain freshly radiolabeled metallothionein. Atomic absorption analysis of the product indicated that less than 0.01% of the metal bound to the protein was cadmium. the rest being zinc. In tjir~ labeling of zinc thionein with “‘“Cd during zinc induction yielded a product of low yield and low specific activity. E-x ~li\w labeling of cadmium, zinc -thionein with [:‘“S]cystine during cadmium induction also resulted in a product of low yield and low specific activity. Labeling with ‘““I-BoltonHunter reagent, which is specific for the ERIA

FOR

METALLOTHIONEIN

i ‘F

-

IO

1000

I00 ZnMT .

or

Cd.i!nMT. I

nq

FIG. 2. Radioimmunoassay for metallothionein. 0. rat hepatic zinc thionein I or II. X. rat hepatic cadmium, zinc-thionein I or II. The data are presented as the means of three replicate determinations and are plotted as bound/free (B/F) vs log wg protein in an assay. 0 indicates the data obtained for control assays in which unlabeled metallothionein was not included. Each assay contained in a total volume of 200 PI 20.00025,000 cpm “‘“Cd-labeled zinc thionein II (IO- 15 ng). 1: 10.000 dilution of rabbit antisera. known amounts of unlabeled metallothionein, and 0.03 M potassium phosphate, pH 7.4. The assay mixtures were incubated at 4°C for 14-16 h. Goat anti-rabbit IgG immunoglobulins (45 pg) were then added, and incubation was continued for S-10 h at 4°C. The assay mixture was then filtered on cellulose acetate filters, the tubes were rinsed with 2 ml of 0.03 M potassium phosphate. pH 7.4. and the washes were passed through the same filters. The filters were air-dried, placed in test tubes, and counted in a gamma counter.

92

BRADY

AND

separation in the two curves may only reflect differences in the quantitation of the different proteins. The primary antibody was tested inthisassayat l:lO-, l:lOO-. l:lOOO-, l:lO,OOO-, and 1: lOO,OOO-fold dilutions. while all other components were held constant. The best and most reproducible results were obtained with the 1: 10.000 dilution of the rabbit antisera. A maximum of 55-60(1r of the counts could be precipitated in this assay. A routine assay now consists of incubation of the primary antibody (1: 10,000 dilution) with 20,000-25,000 cpm l”yCd-labeled zinc thionein II (lo-15 ng) in 0.03 M potassium phosphate, pH 7.4, with unknowns for 1416 h at 4°C. Goat anti-rabbit IgG immunoglobulins (45 pgl are then added, and the incubation is continued for 8- 10 h. The mixture is then filtered through cellulose acetate disks, which are washed with 0.03 M potassium phosphate, pH 7.4. dried and counted in a gamma counter. The usefulness of this radioimmunoassay is predicated on its ability to quantitatively

amino group of lysines. resulted in a product with low yield, but with a relatively high specific activity. Unfortunately, in our hands this *zSI-labeled zinc thionein was not precipitable by our antisera preparations, so this technique was abandoned. Vander Mallie has reported the successful use of this technique in developing a radioimmunoassay for cadmium thionein (7,s). Attempts were also made to label metallothionein with ““Zn, using the same in r-itro procedures as we used successfully with ‘@Cd. All of these attempts to label rat liver zinc thionein II with ““Zn failed. Using ‘O”Cd-labeled zinc thionein II, a double-antibody radioimmunoassay was developed for metallothionein. Figure 2 illustrates the type of results obtained with this assay. The closed circles are unlabeled zinc thionein I or II and the x’s are unlabeled cadmium, zinc-thionein I or II. All four of these proteins successfully competed with roYCd-labeled zinc thionein II for rabbit anti-zinc thionein II antibodies. The slight TABLE RADIOLABELED

Label ‘O”CdCI,”

NBCdc,,” [%]Cystine” ‘““I-BoltonHunter ““ZnCl2“

reagent”

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1

METALLOTHIONEIN

Method

Yield f%ji)

DTT (mM) 0 0.5 2.5 5.0 In ~iLW Ex viva

7 4.5-76 65 54 4.1 0.3

In vitro DTT (mM) 0 0.5 5.0

1.2 0 0 0

Specific (cpm/pg

0.64-1.85

activity protein1

5.9 x 10’ x 10” 5.5 x lo” 4.8 x 10” 190-460 990-1210 2.2 x lo” 0 0 0

n Rat liver zinc thionein II (5.5 pg) was incubated with varying concentrations of dithiothreitol (DTT). 10 &i LUSCdCI,, 0.02 M Tris.HCl, pH 8.6. in a total volume of 100 ~1 for 18-24 h at 4°C. The labeled protein was separated from unbound ‘“Vd by chromatography on a column (0.9 x 12 cm) of Sephadex G-25 fine. equilibrated, and developed with 0.02 M Tris’HCl, pH 8.6. h.c.‘f See Methods for details. ” Incubation with 10 /*Ci “%ZnCl, was performed in a manner identical to that done with ‘““CdCI,.

RADIOIMMUNOASSAY

OF

RAT

LIVER

TABLE

2

93

METALLOTHIONEIN

COMPARISON OF ASSAYS FORMETALLOTHIONEIN

Radioimmunoassay (pg protein/liver)

Sample Zn induced” l8R 18G 18B 18W Mean i

Sephadex G-751 atomic absorption (5,6) (pg protein/liver)

SE

2416 2542 2080 2325 2341 t 981

2510 2112 2680 1987 2322 -c 163”

Cd induN:edh 18G 18W 18B Mean & SE

1404 1542 1890 1612 2 144”

1287 1715 1640 1547 k 132”

’ Rats received IO mg zinc/kg body weight at time 0; they were killed at 18 h. and each liver was processed individually. ” Rats received 0.25 mg cadmium/kg body wt at time 0: they were killed at 18 h, and each liver was processed individually. ‘.” Not statistically significantly different, using Fisher’s t test.

measure zinc thionein and cadmium, zincbe the technique of choice to use in the thionein in tisssue extracts and in blood and future. Vander Mallie (7,s) has described an bile samples. The accuracy of the assay is assay in which rat cadmium, zinc-thioneins compared in Table 2 with a prior technique I and II have been labeled with lZsI, using the (5.6) used in this laboratory to assess quantiBolton-Hunter reagent. A high specific tatively the content of metallothionein in activity (> IO6 cpm/pg protein) radiolabeled rat liver. As can be seen, agreement between antigen was obtained, and the sensitivity of the radioimmunoassay and column chromathis radioimmunoassay was quite good tography/atomic absorption method is rea- (5- 100 ng protein). This assay, however, is sonably good, leading us to conclude that expensive due to the high cost of the this radioimmunoassay is a useful technique Bolton-Hunter reagent, as compared to the and can be utilized on a routine basis when cost of l”SCdCl,. Madapallimatam and the levels of metallothionein are too low to Riordan (9) have prepared antisera from be analyzed by the column chromatography/ rabbits, immunized against rat liver low atomic absorption method. molecular weight copper binding proteins. This antisera also precipitated rat liver cadmium-zinc thionein. No attempts were DISCUSSION reported by these authors concerning the We have described herein a sensitive, use of this antisera to develop a quantitative convenient, and inexpensive radioimmunoimmunoassay. Tohyama and Shaikh (IO) assay for rat zinc thionein and rat cadmium, have reported the preparation from rabbits zinc-thionein using “%Zd-labeled zinc thi- of anti-rat liver cadmium. zinc-thionein II. onein II as 1:he radiolabeled antigen in the This antisera cross-reacted with rat liver double-antibody assay. Comparing our cadmium. zinc-thionein I, as well as with rat assay with other published immunoassays and human renal cadmium, zinc-thioneins for metallothionein indicates that ours may and rabbit hepatic cadmium, zinc-thioneins.

94

BRADY

AND

Radiolabeled metallothionein was prepared by the in rlii~> induction of metallothionein in rats with cadmium in the presence of L’YZdCl,. or by ijr t‘ifro incubations of purified metallothionein with “‘“CdCl,. They used these radiolabeled metallothioneins in a single-antibody radioimmunoassay in which the antigen-antibody complex was precipitated with 50% saturated ammonium sulfate. The precipitate was washed and counted directly. No report of the specific activity of their labeled antigens or of the sensitivity of their radioimmunoassay was given. The techniques used for labeling, the requirement for only a lo-fold diluted antiserum, and the large amounts of radiolabeled metallothionein (0.95-5.47 pg/ assay) used, indicate that this assay is probably not of sufficient sensitivity to be useful for routine radioimmunoassay of metallothionein. The radioimmunoassays of choice for metallothionein are therefore the one reported above by us and the one reported by Vander Mallie (7,8). Our ‘@‘Cd assay may be more useful because of its lower cost and the relative ease of preparation of freshly radiolabeled antigen, as compared with the rz51 assay. Vander Mallie’s assay is more sensitive than our assay, but our assay is sufficiently sensitive to be of use in determining low levels of metallothionein in tissue extracts and in biological fluids, such as blood, bile, and urine. Note trcl&d irr pror$ Two additional papers have been published since the submission of this manuscript.

KAFKA concerning (16.17).

immunological

a\\aya

for

metallothioncin

REFERENCES I. Kojima. Y.. and Kagi, J. H. R. (1978) I’?cz/tc/r Bi,Khrr,l. .S(,i. 3, 90-93. 2. Webb. M. ( 1979)i~ Metallothionein(Khgi. J. H. R.. and Nordberg. M.. eds.). Birkhauser Verlag. BaseliBostonStuttgart. in press. 3. Vallee. B. L.. and Ulmer, D. D. ( 1972) ,4,1,1/r. K<,l,. Biochen1. 41, 91- 128. 4. Friberg. L.. Piscator, M.. Nordberg, G. F.. and Kjellstrom, T. (1974) Cadmium in the Environment, 2nd ed.. CRC Press, Cleveland. 5. Day, F. A., Coles. 9. J., and Brady, F. 0. (1978) Bioimq. Chrru. 8, 93- 105. Panemangalore. M.. and Brady. F. 0. (1978) J. Bid. Chew. 253, 7898-7904. Vander Mallie. R. J. (1977) Fed. Proc. 36, Abs. 4860. Vander Mallie. R. J. (1978) F<‘Lj. Pwc,. 37, Abs. 2989. 9. Madapallimatam. G.. and Riordan. J. R. (1977)Bb chrtn. Bioph,vs. RPS. Cornw~ur~. 77, 1286- 1293. IO. Tohyama, C.. and Shaikh. Z. A. (1978) Biochrm. Biophys. Res. Comrrrun. 84, 907-913. II. Winge, D. R..andRajagopalan. K. V. (197?)Ar(~/1. Bic~c~lrc~trr. Biophy.~. 153, 755-762. 12. Winge. D. R., Premakumar. R., and Rajagopalan. K. V. (1975) Arch. Biochem. Bioph.vs. 170, 242-252. 13. Bolton, A. E.. and Hunter, W. M. (1973)Bioc~ltrm. .I. 133, 5299539. 14. Mancini. G., Carbonara, 0. A.. and Heremans. J. F. ( 1965) frr~nrunoc./zrn~i.str~ 2, 235-254. 15. Fahey. J. L.. and McKelvey, E. M. (1965) .I. Inr/U/1170/. 94, 84-90. 16. Vander Mallie. R. J.. and Garvey. J. S. (1978) l,,~t~~/~t~o~.l~~,t~~i.\tl:\. 15, 857-868. 17. Cherian, M. G.. Singhal, S. K.. and Goyer. R. A. (1979) Abstracts of Xlth Inter. Cong. of Biochemistry. Toronto, Abs. 13.c-RI 15.