Clinica Chimica Acta, 212 (1992) 89-102 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/$05.00
Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody Tetsuo Adachi”, Hideki Ohtaa, Harutaka Yamadab, Arao Futenma”, Katsumi Katob and Kazuyuki Hiranoa
(Received 1 June 1992; revision received 4 September 1992; accepted 5 September 1992)
Key words: Extracellular-su~roxide
dismutase; Monoclonal antibody; Enzyme-linked immunosor~nt assay
Summary The superoxide anion has been implicated in a wide range of diseases. The major protector against superoxide anion in the extracellular space is extracellularsuperoxide dismutase (EC-SOD). EC-SOD is the major SOD isozyme in plasma and forms an equilibrium between the plasma phase and heparan sulfate proteoglycan on the surface of the endothelium. An ELBA method for the measurement of human EC-SOD with monoclonal antibody was established. The proposed method had a high sensitivity (assay range, 0.05-50 r&ml), good recovery (recovery percentage, 96.9 f 5.6%) and reproducibility (within-day assay, C.V. = 8.6 - 10.2%; between-day assay, C.V. = 6.5 - 11.7%)).EC-SOD levels in sera from healthy persons are clearly divided into two groups: a lower group (Group I, below 120 @ml, n = 146) and higher group (Group II, above 400 ng/ml, n = 10). The EC-SOD in Group I were almost normally distributed and the mean level was 55.8 f 18.8 ng/ml. The serum EC-SOD level assayed by ELISA correlated well with serum SOD activity. The serum EC-SOD in Group I is heterogeneous with regard to affinity for heparin-Sepharose and could be separated into three approximately equal fractions, whereas the EC-SOD in Group II is mainly one fraction with a high affinity for the column. The apparent molecular weight and carbohydrate structure of serum ECCorrespondence to: Kazuyuki Hirano, Department of Pharmaceutics, Gifu Pharmaceutical University, 5-
6-1 Mitahora-higashi, Gifu 502, Japan.
SOD in Group II are identical to those in Group I. The high EC-SOD level in sera from some individuals may reflect the excessive stimulation of EC-SOD synthesis in vivo or the growth of selected cells in vivo, because EC-SOD is known to be expressed by a few cell types in vivo as a high-heparin-affinity subtype.
Introduction Reactive oxygen metabolites have been implicated in a number of disease processes [ 11.Oxidative stress in intracellular spaces is efficiently inhibited by enzymatic defense mechanisms such as superoxide dismutase (SOD), catalase and glutathione peroxidase, because most cells and tissues contain these enzymes . The NADPHdependent oxidase system on the surface of neutrophils and xanthine oxidase in or on the surface of endothelial cells are major sources of the superoxide anion [3-51. Extracellular fluids such as blood plasma, synovial fluid and cerebrospinal fluid contain little or no catalase and only low levels of SOD and glutathione peroxidase . Extracellular-SOD (EC-SOD) is the major SOD isozyme in extracellular fluids [7,8], whereas this isozyme occurs in tissues at much lower levels than copper, zincSOD (Cu, Zn-SOD) or manganese-SOD (Mn-SOD) [9,10]. EC-SOD is a secretory tetrameric copper- and zinc-containing glycoprotein with a subunit molecular weight of about 30 kDa [11,12]. EC-SOD in plasma is heterogeneous and can be divided into three fractions, A which lacks affinity, B with weak affmity and C with relatively strong affinity for heparin-Sepharose [13,14]. It appears that EC-SOD is primarily synthesized in the body as type C [ 12,151 and is distributed between the plasma phase and the heparan sulfate proteoglycan of glycocalyx of endothelial cell surfaces [16,17]. Endothelial cells have been proposed as the initial site of tissue injury, since they are ubiquitous and are located at the blood-tissue barrier where the extracellular ability to degrade reactive oxygen species is lower than that within cells. Moreover, endothelial cells are a rich source of xanthine oxidase . For these reasons, it is speculated that EC-SOD in plasma forms an equilibrium with the enzyme on endothelial cell surfaces and reflects the situation at the cell surface. The plasma and synovial fluid levels of EC-SOD have been reported [8,18,19]. However, no significant differences from controls were demonstrated in samples from patients with insulin dependent diabetes mellitus [ 181 and juvenile neuronal ceroid-lipofuscinosis . Chromatography with antibody-conjugated or concanavalin A (Con A)Sepharose has been used in previous work to separate EC-SOD from Cu, Zn-SOD and Mn-SOD. Direct determination of EC-SOD by enzyme-linked immunosorbent assay (ELISA) appears feasible because anti-EC-SOD antibody does not cross-react with the other SOD isozymes [20,21]. Some immunochemical assays of Cu,Zn-SOD [22-261 and Mn-SOD [27-301 have been developed for clinical applications. This report describes the development of an ELISA for human EC-SOD using a monoclonal antibody (MAb) and its application for determining human EC-SOD levels in serum.
Materials and Methods Materials
Human recombinant EC-SOD C (r-EC-SOD C) prepared as previously described  was kindly provided by SYMBICOM AB (Umei, Sweden). BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). X63-Ag8-6,5,3 mouse myeloma cells were purchased from Dainippon Pharmaceutical Company, Ltd. (Tokyo, Japan). Horseradish peroxidase (HRP, Type IV) was purchased from Sigma Chemical Company (St. Louis, MO, USA). Immunoplates (96-well) and tissue culture flasks were from Nunc (Roskilde, Denmark). Heparin-Sepharose CLdB and Con A-Sepharose were products of Pharmacia LKB Biotechnology (Uppsala, Sweden). Lentil lectin (LCA) agarose and wheat germ agglutinin (WGA) agarose were purchased from Seikagaku Co. (Tokyo, Japan). TSKgel G3000 HPLC column was purchased from Tosoh (Tokyo, Japan) Subjects
The subjects studied were healthy laboratory personnel or healthy persons who had participated in an annual health checkup, 68 men (age: median, 41.5; range, 15-62) and 88 women (age: median, 46.5; range, 16-68). They were free of any abnormality on physical examination, blood chemical screening, electrocardiogram, chest X-ray or urinalysis. Blood samples were taken without anti-coagulant. Blood samples for the assay of antithrombin III were collected into siliconized glass tubes containing CPD solution (sodium citrate 26.3 g/l, citric acid 3.27 g/l, glucose 23.2 g/l and sodium dihydrogenphosphate dihydrate 2.51 g/l). After centrifugation (1,500 x g for 15 min) the serum and plasma were kept at -30°C until use. Preparation of monoclonal antibodies
BALB/c mice (female, 6 weeks old) were immunized 4 times at 2-week intervals with 50 pg of r-EC-SOD C. Spleen cells were fused with the mouse myeloma cell line X63-Ag8-6,5,3 at a ratio of lO:l, with 50% polyethylene glycol (PEG) 4000 as described previously [23,29]. Wells with hybridoma growth were screened for ECSOD-specific antibodies by ELISA. Following the identification of positive hybridomas, hybrid cells were subcloned by the limiting dilution technique. Selected subclones were then expanded and grown as ascitic tumors in pristane-primed BALBlc mice. Antibodies were purified from ascitic fluids by ammonium sulfate precipitation (40% saturated) followed by DEAE-Sepharose column chromatography. Preparation of the HRP-labeled monoclonal antibody
The HRP-labeled MAb was prepared using glutaraldehyde reagent by the method described previously [23,29].
as a cross-linking
ELISA for human EC-SOD
An 80-~1 portion of 50 mg/l MAb (1ElO) dissolved in sodium carbonate buffer, 50 mmol/l, pH 9.5, containing sodium azide, 200 mg/l, was added to each well of the immunoplates and left to stand overnight at 4°C. Each well was washed with sodium phosphate buffer, 10 mmol/l, pH 7.4 containing NaCl 150 mmol/l, Tween 20 500 mg/l and merthiolate 200 mg/l (washing buffer). The remaining protein-binding sites were blocked with 300 ~1 of sodium phosphate buffer, 10 mmoliI, pH 7.4, containing NaCll50 mmol/l, BSA 10 g/l, Tween 20 500 mg/l and merthiolate 200 m@l (blocking buffer). The plate was then left to stand at 4°C until use. Seventy microliters of sample or standard diluted with the blocking buffer were added to the wells. The plate was incubated for 2 h at room temperature and washed three times with the washing buffer. Then 80 ~1 of HRP-labeled MAb (1ElO) diluted with the blocking buffer was added to each well and the plate was incubated for 2 h at room temperature, followed by washing three times with the washing buffer. Substrate solution (150 ~1 of McIlvain buffer (disodium hydrogenphosphate-citric acid buffer) 100 rnrnol!l, pH 6.5, containing o-phenylenediamine 3 g/l and H202 200 rngfl) was then added to each well and the plate was incubated for 30 min at room temperature. The enzyme reaction was stopped by the addition of 50 ~1 of H2S04 4.5 mol/l and the absorbance at 492 nm was measured. Heparin-Sepharose column chromatography
Serum and urine were dialyzed against sodium phosphate buffer, 25 mmol/l, pH 6.5, before application. Dialyzed samples (4 ml of serum or 2 ml of urine) were applied to a heparin-Sepharose column (vol. = 6 ml) equilibrated with the above buffer and washed with the same buffer. The bound fractions were then eluted with a linear gradient of NaCl (0 - 1 M) in the buffer. Serial lectin affinity technique
Structure studies of asparagine-linked carbohydrate chains were performed using the method of Cummings and Kornfeld . Serum (300 ~1) was applied to a Con saline A-Sepharose column (vol. = 2 ml) equilibrated with phosphate-buffered (PBS) containing BSA, 10 pg/ml and washed with the same buffer. The bound enzyme was then eluted by stepwise addition of two different concentrations (10 mrnol/l and 500 mmol/l) of a-methyl-D-mannoside (a-MM). Three fractions, an unbound fraction (Fr. I), a weakly bound fraction (Fr. II) and a strongly bound fraction (Fr. III), were dialyzed against PBS, followed by concentration with YM-10 membrane filter. Fr. II and Fr. III were further applied to lentil lectin (LCA, vol. = 1 ml) and wheat germ agglutinin (WGA, vol. = 1 ml) columns, respectively, equilibrated with the above buffer. The unbound and the bound fractions on the respective columns were separated by using (r-MM, 200 mmol/l and N-acetyl-Dglucosamine (GlcNAc), 100 mmol/l, respectively, as elution buffers. Five fractions were obtained by this method: I, the fraction not bound to the Con A-column; IIA, the fraction bound weakly to the Con A-column which then passed through the
LCA-column; IIB, the fraction bound weakly to the Con A-column which was then bound clearly to the LCA-column; IIIA, the fraction bound strongly to the Con Acolumn which then passed through the WGA-column; IIIB, the fraction bound strongly to the Con A-column which was then bound to the WGA-column. TSKgel G3000 HPLC
TSKgel G3000 was equilibrated with PBS and the column was operated at a flow rate of 0.4 ml/min.
Production of monoclonal antibodies and their properties
After the fusion step, 41 out of 144 wells showed hybridoma growth and 21 wells were screened positively by ELISA. Some cultures which exhibited high activities were cloned by limiting dilution of thymus cell-feeder layers. Two clones were selected for further culture and ascites production. Both MAbs were IgG2&y2t,Y2t,KK). Development of ELISA for human EC-SOD (1) Construction of ELBA
A two-step ELISA for EC-SOD was set up with two MAbs, named lEl0 and 2C7. Dose-response curves were prepared by using the combination of HRP-labeled lEl0 or 2C7 and solid-phase adsorbed lEl0 or 2C7. With the use of a single MAb, lEl0
SOD (pg/ml) Fig. 1. Calibration curve for EC-SOD determined by the proposed method and cross reactivities with other SOD isozymes. 0, EC-SOD, 0, Cu,Zn-SOD, A, M-SOD.
16.7 12.5 10
Fig. 2. Effect of dilution of serum (a) and urine (b) on calculated EC-SOD values. The sera from five individuals and urine from four individuals were diluted IO-50-fold or 2-16-fold, respectively and then aliquots of the dilutions were assayed.
for solid phase-coating and HRP-labeling was found to give a good dose-response curve. A typical standard curve of human EC-SOD is shown in Fig. 1. The lower limit of detection was 50 pg/ml and the working range was 50 pg/ml - 50 rig/ml. The proposed ELBA showed no cross-reactivities with other SOD isozymes, Cu,ZnSOD purified from human erythrocyte  or Mn-SOD purified from human placenta  (Fig. 1). (2) Precision and accuracy of the assay with samples The accuracy of the assay was determined by recovery experiments with ten serum samples containing 33.8 - 96.0 rig/ml of EC-SOD. When two different amounts of
EC-SOD (ng/ml) Fig. 3. Histogram of the distribution of serum EC-SOD in normal healthy persons.
r-EC-SOD C (1.0 and 5.0 ng/well) were added, the average recovery of the r-ECSOD added was 96.9 f 5.6%. The effect of dilution of the sample was also investigated with five serum samples and four urine samples. As shown in Fig. 2, the calculated EC-SOD values were linear with dilution. The precision of the assay was tested by assaying five serum samples (38.1 - 572 @ml of EC-SOD). The coefficients of within-day assay and between-day assay were 8.6 - 10.2% (n = IO) and 6.5 - 11.7% (n = lo), respectively. EC-SOD levels in normal healthy subjects The levels of EC-SOD in sera from 68 male and 88 female healthy individuals were examined. The distribution of serum EC-SOD is discontinuous (Fig. 3). All but 10 of the 156 subjects had levels below 120 ng/ml (Group I). The 10 subjects with higher levels (Group II) showed ranges of 400 @ml to 1000 @ml. EC-SOD levels in Group I were almost normally distributed with a mean of 55.8 @ml and S.D. of 18.8 ng/ml. There was no significant difference between males (52.7 f 18.4 ng/ml) and females (58.1 i 18.9 ngml) in this group. Within one individual the high ECSOD was maintained when plasma samples were obtained at 6 months interval. There was no correlation between serum EC-SOD levels and any other blood chemical analyses. Comparison with serum SOD activity The serum EC-SOD concentration measured by ELISA and total serum SOD activity measured by the cytochrome c method  correlated closely (Fig. 4, r = 0.908).
EC-SOD (ng/ml) Fig. 4. Correlation
concentration in serum measured SOD activity.
by the proposed
method and total
Fraction number (1 mi~ube)
Fraction number (2 ml/tube) LCA
Fig. 5. Serial leetin affinity column chromatographies of sera from an individual in Group I(a) and an individual in Group II(b). Chromatographies ceeded by the methods described in Materials and Methods.
Fraction num~r (1 ml/tube)
Fraction number (2 ml/tube)
This result supports the finding that the appearance of high EC-SOD in some normal sera is unlikely to be a nonspecific reaction in ELISA. The intercept, which is significantly different from zero (P < 0.05), may be attributable to the presence of other SOD isozymes, Cu,Zn-SOD (normal range = 26.9 f 7.8 rig/ml , 14 ng/ml , 36.3 f 15.6 @ml , 46.1 f 21.6 ng/ml) and Mn-SOD (normal range = 84.5 f 30.0 rig/ml , 82 f 21 @ml , 88.8 f 20.8 ng/ml (female), 99.8 f 24.8 @ml (male) ), because it is impossible to separate these isozymes using a method which determines its enzymic activity. On the other hand, the proposed ELISA showed no cross-reactivities with other SOD isozymes, as shown in Fig. 1. Correlation between EC-SOD and antithrombin III level The major part of EC-SOD in the body seems to exist in association with extracellular sulfated glycosaminoglycans (SGAGs), in equilibrium between plasma and endothelial cell surfaces [ 13- 171. Antithrombin III is also known to have a high affinity for heparin-like substances. We assayed plasma antithrombin III level in 13 normal persons, 4 of whom have high EC-SOD, by the method of Yoshida . Antithrombin III levels were similar in all subjects (27.0 f 2.42 mg/dl) and did not correspond with the EC-SOD level. Properties of EC-SOD in serum The carbohydrate chains of serum EC-SOD from individuals in Group I and Group II were compared by using serial lectin affinity techniques. There was no
E 1.0 c $j 0.8
8' 08 s +i 0.4
1.0 0.8 0.6 0.4 0.2
i?J0.2 0.0 0
Fraction number (0.2 ml/tube)
Fraction number (0.2 ml/tube)
Fig. 6. TSKgel G3000 HPLC of sera from an individual in Group I(a) and an individual in Group II(b). Samples are the same as those used in Fig. 5.0, absorbance at 280 nm; 0, EC-SOD concentration assayed by ELBA.
difference in their chromatograms, as shown in Fig. 5. Con A-Sepharose binds certain biantennary glycoproteins (Fr. II), which can be fractionated further by chromatography on LCA-column on the basis of internal fucose residues, Fr. IIA without internal fucose residues and Fr. IIB with internal fucose residues. Con ASepharose also strongly binds high mannose-type glycoproteins and hybrid-type glycoproteins (Fr. III). The WGA-column has a high aflinity for the hybrid-type glycoprotein and thus can be used to separate the high mannose-type (Fr. IIIA) and hybrid-type (Fr. IIIB). Almost all of the EC-SOD (85%) was found in a weakly bound fraction (Fr. II) on the Con A-Sepharose column. Little EC-SOD was found in the unbound fraction (Fr. I, 5%) or in the strongly bound fraction (Fr. III, 10%). In order to evaluate the lectin-binding affinity, Fr. II and III obtained from the Con A Sepharose column were further applied to LCA- and WGA-columns, respectively. Almost all Fr. II bound to the LCA-column and Fr. III did not bind to the WGAcolumn. The possible core structure of the carbohydrate chain of EC-SOD revealed by the serial lectin affinity technique is a biantennary complex type without bisecting N-acetyl-D-glucosamine (GlcNAc). The high-affinity binding to LCA-agarose shows the presence of internal fucose residues attached to an asparagine-linked GlcNAc. The apparent molecular weight of serum EC-SOD in Group I was identical to that of EC-SOD in Group II, judged by TSKgel G3000 HPLC gel filtration as shown in Fig. 6. The affinities for heparin-Sepharose of EC-SOD from both groups were also compared. Figure 7a shows that EC-SOD in Group I was separated into three approximately equal fractions: A without affinity, B with weak affinity and C with relatively strong heparin-affinity as shown previously [13,14]. On the other hand, serum ECSOD in Group II consisted mainly of C-type (Fig. 7b). Since the absolute level of
Fraction number (4 ml/tube)
Fraction number (4 ml/tube)
Fig. 7. Heparin-Sepharose column chromatographies of sera from an individual in Group I(a) and an individual in Group II(b). Samples are the same as those used in experiments of Fig. 5. Chromatographies were proceeded by the method described in Materials and Methods. 0, absorbance at 280 nm; 0, ECSOD concentration assayed by ELISA; - - -, NaCl concentration in buffer.
" E g E
EC-SOD in serum (ng/ml) Fig. 8. Comparison of EC-SOD levels in serum and in urine.
EC-SOD A in Group II was equal to that in Group I, the difference in EC-SOD level resulted from the content of EC-SOD C. In all ten serum samples belonging to Group II, EC-SOD consisted mainly of C-type. Comparison of EC-SOD
levels in serum and in urine
The EC-SOD levels in urine of healthy persons (n = 30) were also assayed and compared with the serum EC-SOD levels. As shown in Fig. 8, two out of the three Group II subjects had urine levels no greater than those in the Group I patients.
Fraction number (1 mlltube)
Fraction number (1 ml/tube)
Fig. 9. Hepa~n-~pharo~ column chromato~aphies of urine from an individual in Group I (a) and an individual in Group II (b). Chromatographies were proceeded by the method described in Materials and Methods. 0, EC-SOD concentration assayed by ELBA; . - ., NaCI concentration in buffer.
Heparin-affinity of EC-SOD
The affinity for heparin-Sepharose of EC-SOD in urine from normal persons in Group I and II was examined. EC-SOD consisted mainly of C-type in urine from both groups (Fig. 9). Discussion In this report, we developed an ELISA with a MAb, lE-10. The epitope in the EC-SOD molecule recognized by lE-10 could be four copies per molecule, because this enzyme is composed of four identical subunits each with the molecular weight of Ca 30,000. For Mn-SOD, also a tetrameric enzyme, ELISAs for this enzyme have been developed with a single MAb [29,30]. The proposed ELISA is sensitive enough to assay EC-SOD in serum and urine. Moreover, the recovery of r-EC-SOD C added to human serum and the reproducibility of the assay are good. These results indicate that the proposed method is suitable for clinical diagnosis. We examined the EC-SOD levels in sera of healthy normal persons by the proposed ELISA. Unexpectedly, some persons (about 6.5%) show a high serum EC-SOD level without any apparent physical or clinical abnormality. Whereas Cu,Zn-SOD and Mn-SOD, intracellular isozymes, are synthesized by virtually all mammalian cell types, the expression of EC-SOD occurs in only a few cell types in vitro, in tibroblast cell lines and glia-cell lines, but not in endothelial or epithelial cell lines. All of the above cell lines secreted EC-SOD of type C . Atype and B-type EC-SODS observed in serum are probably the results of moditication after secretion, for example, proteolytic cleavage in the extracellular space  or non-enzymic glycation . The present findings show that EC-SOD in the urine is scarcely degraded and consists mainly of C-type. EC-SOD can be demonstrated in all tissues and accounts for about 1 - 2% of the total tissue SOD activity . EC-SOD synthesis by cell lines in vitro varies lOO-fold even among Iibroblast cells . Moreover, the amount of EC-SOD expressed by fibroblast cell lines in vitro is too small to account for the tissue EC-SOD content, because Iibroblasts account for only a minor portion of tissue cells [ 151. Marklund has shown that cytokines influence EC-SOD expression by human dermal libroblasts in vitro . The expression was markedly stimulated by interferon-y, was stimulated or depressed by interleukin-lar and was depressed by tumor necrosis factor-a! and transforming growth factor-p. The present observation of high serum EC-SOD level in some persons (Group II) may reflect excessive stimulation of ECSOD synthesis. Another possibility is related to the ability of endothelial cells to regulate the metabolism of EC-SOD. We have found that an endothelial cell line internalized r-EC-SOD C which had bound to glycocalyx on the cell surface. A part of the internalized EC-SOD was released back into the medium and the remainder was degraded (H. Ohta et al., unpublished work). It is possible that endothelial cells regulate the turnover of EC-SOD and/or transport EC-SOD from its site of synthesis to its site of action like other proteins having heparin-binding ability [36,37]. We are at present trying to examine the quantitative and qualitative changes of EC-SOD in sera and urine of patients with various diseases.
Acknowledgments r-EC-SOD was provided by SYMBICOM AB, Ume& Sweden. This work was supported in part by Grants-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Science and Culture of Japan (03771748) to T.A. References 1 2
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13 14 15 16 17 18 19 20 21
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