Enzyme immunoassay for ceruloplasmin: application to cancer patient serum

Clinica Chimica Acta, 158 (1986) 281-292 Elsevier

287

CCA 03542

Brief technical

note

Enzyme immunoassay for ceruloplasmin: application to cancer patient serum Robert Department

A. DiSilvestro

and Elizabeth

A. David

of Foorls and Nutrition, Purdue Universiiy> West Lafayette, IN 47907 (USA) (Received

March 3rd, 1986; revision received May lOth, 1986; accepted after revision May 12th, 1986)

Key words: Enzyme immunoassay;

Ceruloplasmin;

Cancer

Introduction

Ceruloplasmin (Cp), a glycoprotein of molecular weight 132 000 contains most of the copper found in human serum [I]. Cp has been proposed to act as a copper transport agent and to carry out enzymatic functions [2]. Serum or plasma copper concentrations rise during many disease states such as cancer [3], arthritis [4] and infection [3,5] as well as during nonpathological stresses such as pregnancy [5], lactation [7] and chronic exercise [8]. Generally, increases in serum or plasma copper have been found to correspond to elevated levels of Cp oxidase activity toward one of several substrates [3-81. In the case of cancer, Cp oxidase reaches highest levels during the most severe stage of the disease [3]. On the other hand, one study found that successful treatment of several types of cancer was accompanied by a return of Cp oxidase activity concentrations toward normal [9]. Despite these various studies, no functional or diagnostic significance has been firmly established for stress-induced elevations in serum or plasma copper and Cp oxidase activity. It is very possible that the mechanisms regulating activity levels as well as the functional consequences of changes may vary for different stress conditions. Further characterization of the different stress-induced increases in serum copper and Cp oxidase activity levels requires comparison of these parameters with immunoreactive Cp levels. Recently, work in this laboratory [lo] showed that lactation raised immunoreactive Cp concentrations to a greater extent than serum copper or Cp oxidase activity. Immunoreactive Cp levels were measured using commercially available radial immunodiffusion (RID) plates (Calbiochem-Behring Diagnostics). The present paper describes an enzyme-linked immunoadsorbant assay (ELISA) for ceruloplasmin. The ELISA is cheaper and faster than the RID approach and utilizes commercially available antibodies. No conjugation synthesis is required of the user. The ELISA was applied to serum from cancer patients and controls but should be sensitive enough for application to appropriate cell cultures. 0009-8981/86/$03.50

0 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

288

Materials and methods Materials

All commercially obtained reagents were from companies in the USA. Ceruloplasmin standards were obtained from Atlantic Antibodies (3-times concentrate) in Scarborough, MN. Goat anti-Cp IgG was purchased from U.S. Biochemical Corp. of Cleveland, OH. Rabbit anti-Cp was brought from Boehringer-Mannheim, Indianapolis, IN. Goat IgG F(ab), fragments from antisera to rabbit IgG, conjugated to alkaline phosphatase, phosphatase substrate and bovine serum albumin (RIA grade) were purchased from Sigma Chemical Co., St. Louis, MO. Polystyrene culture tubes were obtained from American Scientific Products, McGaw Park, IL. Standard human serum in lyophilized form along with Cp RID plates were purchased from Calbiochem-Behring Diagnostics, La Jolla, CA. Control serum was obtained from 9 employees at the Purdue University Health Center. Cancer serum was obtained from 13 patients at a local clinic following positive diagnosis for some form of cancer, primarily in breast, lung, or colon. All serum was stored frozen until analysis for Cp oxidase activity, serum copper and Cp protein. The Cp standard, which also contained some other serum proteins, was stored at 4°C and used within 3 wk after arrival. Enzyme immunoassays

All steps were carried out at room temperature unless noted otherwise. Goat anti-Cp IgG was freshly diluted l/4000 into 0.05 mol/l Na,CO,, 1 mmol/l MgCl,, pH 9.8. Then, l-ml aliquots were incubated overnight in polystyrene tubes. The IgG solution was removed and the tubes washed twice with 1 ml phosphate-buffered saline (PBS) plus 0.05% Tween 20. All subsequent wash steps also used this buffer. Each tube, except for blanks, was exposed to serum samples or Cp standards. Serum (10 ~1) was freshly diluted into 2 ml deionized water and further diluted l/100 into PBS-Tween 20 containing 10 mg/ml high-quality bovine serum albumin (BSA). Exactly 1 ml of each diluted serum sample was added to three tubes. Standard Cp was diluted into PBS-Tween 20-BSA to provide a final concentration of either 50, 40, 30, 20, or 10 ng Cp/ml. Blanks contained buffer only. After a 90-min incubation, the standards and samples were removed, the tubes were washed twice and then exposed to 1 ml rabbit anti-Cp IgG. The IgG was diluted l/500 in PBS-Tween 20-BSA. After 1 h, this solution was removed, the tubes again washed twice and then exposed to 1 ml of goat IgG, F(ab), fragment, from antisera to rabbit IgG, conjugated to alkaline phosphatase. This conjugate was diluted l/500 in PBS-Tween 20-BSA. After ‘1 h, the conjugate was removed and the tubes washed three times. To each tube was added 1 ml Sigma phosphatase substrate (pnitrophenyl phosphate - 0.1% w/v in 0.05 mol/l Na,CO,, 1 mmol/l MgCl,, pH 9.8). The tubes were incubated with substrate for 10 min at 37°C. The yellow color formed was measured as A,, nm. Other methods

Radial immunodiffusion (RID) was carried out on 5 ~1 of serum or diluted standards for 48 h as described in the manufacturer’s instructions. Serum copper

289

was assessed by atomic abso~tion sp~trophotomet~ on serum diluted l/l with deionized water. Cp oxidase activity was assessed toward p-phenylenediamine (PPD) by the method of Hot&in [ll] as modified by Rice [12]. Units were arbitrarily defined as A,,, nmx 103. Results

The results obtained with the ELISA showed strong consistency. Using standard human serum from Calbiochem-Behring Diagnostics, a coefficient of variance (CV) of less than 5% was obtained for 9 replicates in the same assay. A CV of less than 5% was also calculated for triplicates from three different assays. The amount of color produced in the ELISA was dependent on the amount of goat antibody used to coat the tubes. For a constant Cp concentration (50 ng,/ml), the Am3 nm increased with increasing concentration of coating antibody until a l/750 dilution was reached. Increasing the concentration further (l/500 dilution) actually produced less color than the l/750 dilution. A final antibody dilution of l/4000 was chosen for routine use. At this concentration, background color was low and a relatively high change in A, nm occurred between the lowest Cp standard and the highest. The coating buffer contained MgCl, although it was not included to enhance binding. Rather, this inclusion allowed a singIe buffer to be used for antibody coating and for alkaline phosphatase enzyme assay. Standard curves were derived from Cp concentrations between 0.01 and 0.05 ng, and typically yielded a line of y = 0.017x. Thus, changes in absorbance could exceed 0.15 for changes in Cp of 10 ng/ml. This fairly large difference in absorbance for a relatively small change in Cp concentration enhanced the accuracy of serum Cp determinations. Cp concentrations under the lowest standard of 10 ng/ml could have been accurately measured, particularly if the substrate incubation step was lengthened past 10 min. However, serum Cp. concentrations could be accurately assessed without standards lower than 10 ng/ml. A high degree of correlation was found between Cp concentrations calculated by ELISA versus those deduced from RID (Fig. 1). Both normal and cancer serums were used. A correIation coefficient (r value) of 0.97 was found with a p value of less than 0.001. On the. average, Cp values determined by ELISA were slightly higher than those determined by RID. Table I illustrates an application of the ELISA method to serum from 13 cancer patients, 9 controls and standard serum from a pool of healthy donors (Calbiochem-Behring Diagnostics). When compared to mean control values, serum from 7 of the 13 cancer patients showed a lower percent increase in Cp protein levels than in PPD oxidase activity. In 8 of 13 of the serums from cancer patients the percent increase in Cp protein levels was equal or greater than the percent increase for serum copper. The mean value for Cp protein for the 9 control serum donors was similar to the value found for the standard serum pool. A similar comparison using standard serum could not be made for PPD oxidase. The standard serum was stored as a lyophilized powder rather than as a frozen liquid. The method of serum storage can greatly affect the PPD oxidase activity [13]. In this laboratory,

290

Fig. 1. Correlation

TABLE

between

Cp determined

by ELISA versus Cp determined

by RID.

I

Comparison

of immunoreactive

Cancer patient

Cp levels with serum copper

and PPD oxidase

(pg/dl)

PPD oxidase (units “/dl)

1 2 3 4 5 6 7 8 9 10 11 12 13

174 145 191 192 191 175 191 145 118 145 196 174 118

371 302 410 311 481 367 434 268 207 243 349 422 265

Means Controls Standard

164k28 90529 87

Serum copper

serum

b

All means are f standard deviation. a Units are arbitrarily defined as As40nm X 103. b p -z 0.001 for differences from controls by Student’s ’ Accurate determination could not be made.

341k83 200+42 _=

r test.

b b

Cp protein (mg/dl) 48.8 45.9 52.6 62.0 71.3 75.6 65.1 51.8 36.7 52.7 83.5 60.0 38.2 57.2f 14.1 b 30.8 + 8.0 34.7

291

the reconstituted standard serum yielded serums from the control donors.

a lower PPD oxidase value than any of the

Discussion The ELBA described here proved rapid, sensitive, consistent, and economical. Furthermore, it could be carried out entirely with commercially available antibodies and required no conjugation synthesis by the user. The low Cp concentrations used for the standard curve allowed a large dilution of the serum samples. This large dilution helped minimize the influence of nonspecific protein binding to the tubes. As noted in Results, Cp concentrations lower than 10 ng/ml could be accurately measured, especially if the substrate incubation step was lengthened past 10 min. Measurements of low Cp concentrations might prove useful for studying Cp production by cell cultures such as hepatomas. Increased serum or plasma copper and Cp oxidase activity levels have been clearly established for a number of pathological and nonpathological stress states [3-81. However, these changes have not been extensively compared to changes in immunoreactive Cp levels. The ELBA described here provides a relatively inexpensive and practical means for making these comparisons. Such comparisons should provide insight into the regulatory mechanisms and functional and diagnostic significance associated with elevations in serum or plasma copper and Cp oxidase concentrations. A previous study on 12 lactating females, 1 month postpartum, found that in each subject percent increases in Cp protein levels, measured by RID, exceeded those for PPD oxidase [lo]. Less than half of the cancer patients examined here (Table I) showed percent increases in Cp protein levels which exceeded those for serum copper or PPD oxidase. In the present study and in one by Andrews [15], the average serum copper value rose in cancer patients to about the same extent as the average Cp protein value (Table I). Acknowledgements The authors wish to thank Deanna Case for fine technical assistance, and Dr. J. Morre and Dr. R. Esposito for providing the cancer patient serum, The work was supported by a grant from private sources administered through the Purdue Cancer Center. References 1 Wirth PL, Linder MC. Distribution of copper among components of human serum. J Nat1 Cancer Inst 1985; 75: 277-284. 2 Frieden E. Ceruloplasmin: a multifunctional cupro-protein of vertebrate plasma. In: Sorenson JRJ, ed. Inflammatory Diseases and Copper. Clifton: Humana Press, 1982: 159-169. 3 Linder ML, Moor JR, Wright K. Ceruloplasmin analysis in diagnosis and treatment of human lung, breast, and gastrointestinal cancers. J Nat1 Cancer Inst 1981; 67: 263-275. 4 Scudder PR, Al-Timini D, McMurray W, White AG, Zoob BC, Dormandy TL. Serum copper and related variables in rheumatoid arthritis. Ann Rheum Dis 1978; 37: 67-70.

292 5 Markowitz H, Gubler CJ, Mahoney JP, Cartwright GE, Wintrobe MM. Studies on copper metabolism. XIV. Copper, ceruloplasmin, and oxidase activity in sera of normal human subjects, pregnant women and patients with infection, hepato-lenticular degeneration and nephrotic syndrome. J Clin Invest 19.55; 34: 1498-1508. in pregnancy. Am J Obstet Gynecol 1971; 6 Burrows S, Pekala B. Serum copper and ceruloplasmin 109: 907-909. 7 Krebs NF, Hambidge KM, Jacobs MA. Plasma copper and ceruloplasmin concentrations during lactation. Fed Proc (Abstr) 1984; 43: 1855. 8 Lukaski HC, Bolonchuk WW, Klevay LM, Mime DB, Sandstead HH. Maximal oxygen consumption as related to magnesium, copper and zinc nutriture. Am J Clin Nu!r 1983; 37: 407-415. PK, Ghosh A, Chowdhury JR. Ceruloplasmin in human malignancies. Acta Med 9 Chakraborty Okayama 1984; 38: 315-320. and other acute phase proteins in lactating women. 10 DiSilvestro RA. Plasma levels of ceruloplasmin Fed Proc (Abstr) 1986; 45: 355. BO. A rapid calorimetric method for the quantitative determination of copper oxidase 11 Houchin activity (ceruloplasmin). Clin Chem 1958; 4: 519-523. of ceruloplasmin activity in terms of international enzyme units. Anal 12 Rice EW. Standardization Biochem 1962; 3: 452-456. In: Lontie R, ed. Copper Proteins and Copper Enzymes, Vol. 3. Boca 13 Ryden L. Ceruloplasmin. Raton: CRC Press, 1984: 37-100. 14 Smith JC, Holbrook JT, Danford DE. Analysis and evaluation of zinc and copper in human plasma and serum. J Am Co11 Nutr 1985; 4: 627-638. and growth hormone. J Clin Path01 15 Andrews GS. Studies of plasma zinc, copper, caeruloplasmin 1979: 32: 325-333.