Clinica Chimica Acta, 192 (1990) 171-180 Elsevier
Sensitive enzyme immunoassay for human Mn superoxide dismutase Naomi Kurobe
2 and Kanefusa
’ Depariment of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya, Kasugai, Aichi and 2 Department
of Internal Medicine, Geriatric Hospital, Nagoya-shi Kosein, Nagoya (Japan)
1990; revision received 27 June 1990; accepted
Key wor&: Superoxide
28 June 1990)
A sensitive sandwich-type enzyme immunoassay for measurement of human Mn superoxide dismutase (Mn SOD) was developed using purified antibodies specific to Mn SOD. The antisera were raised in rabbits by injecting Mn SOD purified from human liver. The antibody IgG, purified by the use of Mn SOD-coupled Sepharose, showed a single band on the immunoblotting test with a crude liver extract. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab’), fragments and the same antibody Fab’ fragments labeled with P-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg human Mn SOD/assay tube. Serum Mn SOD concentrations of healthy adults (77.5 & 18.0 “g/ml (1 SD), n = 120, 16-64 yr old) were not related to age or sex. Immunoreactive Mn SOD was detectable in most tissues examined except for erythrocytes. The concentrations of immunoreactive Mn SOD and Cu/Zn SOD in the cerebral cortex were not different among the patients with Alzheimer’s disease, and the age matched and young patients without neurological disorders.
Superoxide dismutase (SOD) catalyzes dismutation of superoxide radicals to 0, and H,O, [l], and animal species have two forms of SOD . One form is a dimer of about 32-kDa molecular weight containing Cu and Zn (Cu/Zn SOD), and is
Correspondence and requests for Developmental Research,
for reprints to: Dr. Kanefusa Kato, Department of Biochemistry, Aichi Prefectural Colony, Kamiya, Kasugai, Aichi 480-03, Japan.
0 1990 Elsevier Science Publishers
located in the cytosol. The other form is a tetramer of 80-kDa molecular weight containing Mn (Mn SOD), and is found predo~nantly in the mitochondria. Because a number of pathological processes have been suggested to involve free oxygen superoxide radicals , the significance of SODS in various diseases has been a topic in medical science. The enzymatic SOD activity is reported to decrease in various tissues during aging [4,5]. In order to determine the SOD concentrations in serum and tissue samples, we have recently established a highly sensitive enzyme immunoassay method for the measurement of human Cu/ Zn SOD . In this report, we describe a similarly sensitive immunoassay method for the assay of human Mn SOD, together with the determination of Mn SOD and Cu/Zn SOD in the cerebral cortices of autopsy specimens obtained from patients with Alzheimer’s disease and those without neurological diseases. Materials and methods Reagents
CNBr-activated Sepharose 4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); ~-D-g~actosidase from ~sc~e~c~ja co&, from Boehringer Mannheim (Mannheim, FRG); pepsin from porcine stomach mucosa, from Sigma Chemical Co. (St. Louis, MO, USA); bovine serum albumin (fraction V), from Organon Teknika (Holland); polystyrene balls (3.2 mm in diameter), from Immuno Chemical Inc., Okayama, Japan. Buffer G used for the immunoassay and immunoblot was composed of sodium phosphate buffer 10 mmol/l (pH 7-O), containing 0.3 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l NaN,, 1 g/l bovine serum albumin and 5 g/l protease-treated gelatin . Buffer A was composed of sodium phosphate buffer 10 mmol/l (pH 7.0), containing 0.1 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l bovine sernm albumin and 1 g/l NaN,. Superoxide
Human Cu/Zn SOD was purified from erytlsrocytes as described previously . Human Mn SOD was purified from livers obtained at autopsy. In brief, the frozen liver was thawed and homogenized in a 5-volume (vol/wt) of 0.15 mol/l KC1 in a Waring blender. The supematant obtained by centrifugation of the homogenate was heated at 70” C for 15 min, and then the soluble fraction was fractionated with (NH4)$Oe. The precipitate collected by cent~fugation (between 50-90% saturation) was dissolved in and dialyzed against 40 mmol/l potassium acetate buffer (pH 5.9, and applied to a CM-Sephadex C-50 column, equilibrated with the potassium acetate buffer. The column was washed with the same buffer containing 0.05 mol/l NaCl, and then Mn SOD was eluted with a linear concentration gradient of 0.04-0.3 mol/l potassium acetate buffer (pH 5.5) containing 0.05 mol/l N&l. The CM-Sephadex fraction with ~y~de-insensitive SOD activity was further chromatographed on a column of hydroxylapatite, equilibrated with 20 mmol/l potassium phosphate buffer (pH 7.0). The SOD activity was eluted with a linear concentration gradient of 0.02-0.4 mol/l potassium phosphate buffer (pH 7.0). The hydroxylapatite fraction was chromatographed on a column of Toyopearl HW-55 (Tosoh,
Fig. 1. SDS-PAGE of the purified human Mn SOD and a crude liver extract (A), and irmnunoblots with purified anti-Mn SOD antibodies (B). Lane 1, a human liver extract containing about 1.1 pg of immunoreactive Mn SOD with 74 ng proteins; lane 2, 1.2 pg of purified human Mn SOD; lane 3, standard proteins with molecular mass in kDa. A, Poiyacrylamide gel stained with Coomassie blue; B, nitrocellufose sheet stained with anti-human Mn SOD.
Tokyo, Japan) equilibrated with 50 mmol/l sodium phosphate buffer (pH 7.0). As shown in Fig. lA, the final SOD preparation showed a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Rat Mn SOD was purified from frozen rat livers with the procedures similar to those described above, except that the CM-Sephadex c~omato~aphy was performed with imidazole-HCl (0.02-0.2 mol/l) buffer, pH 6.5. The final rat preparation also showed a single band on SDS-PAGE (not shown). Cyanide-insensitive SOD activity was assayed by the method described by Beauchamp and Fridovich , with 5 mmol/l cyanide. The purified Mn SODS had a specific activity of > 2 500 U on the basis of mg protein. Antibodies The antisera to human Mn SOD were raised in Japanese white rabbits by intracutaneus injection of the purified SOD (0.5 mg/rabbit) in Freund’s complete adjuvant. The same i~u~tion was repeated 4 times every 2 weeks, and then rabbits were bled 1 wk after duration. The antibody IgG was purified by immunoaffinity column chromatography with the use of Mn SOD-coupled Sepharose 4B as described previously . The purified antibody fractions were pooled, neutralized and concentrated. About 30 mg of the monospecific antibody IgG were obtained per 100 ml of the antisera. Specificity of antibodies thus prepared was tested on SDS-PAGE of a crude extract of liver followed by i~unoblot with the purified antibodies. As shown in Fig. lB, the crude extract transferred to the nitrocellulose showed an immunoreactive single band at position corresponding to Mn SOD. These results indicate that the purified antibodies were specific to Mn SOD. The purified antibody IgG was digested with pepsin from porcine stomach mucosa to obtain the F(ab’), fragment of the antibody [lO,ll]. We have prepared the ~uno~say system using antibody fragments, because the pepsin treatment aided the removal of a trace amount of contaminated antigen which co-eluted from
the antigen-coupled column, and the assay system prepared with the antibody fragments was more sensitive and less affected by the sample-interference . Polystyrene
balls with immobilized
The antibody F(ab’), fragments were coupled noncovalently on polystyrene balls as described previously , and the balls were stored at 4” C in buffer A for at least 2 days to stabilize the assay. Antibody
labeled with /3-D-galactosidase
Antibody Fab’ fragments were labeled with /3-D-galactosidase from Escherichia coli by the use of N,N’-o-phenylenedimaleimide [lO,ll]. The amounts of labeled antibody are expressed in units of galactosidase activity, and one unit of activity is defined as that which produces 1 pmol 4-methylumbelliferone/min under the conditions described previously [ 131. Serum and tissue samples
Adult control serum samples were obtained from a local blood bank and kept frozen at - 80° C until analysis. Two-microliter portions of serum samples were subjected to the immunoassay. Various adult (45-97 yr old) tissues including brain samples of Alzheimer’s disease were obtained at autopsy within 12 h postmortem and stored at - 80 o C. The diagnosis of Alzheimer’s disease was confirmed by histological examination of brain sections after autopsy. Each tissue (0.5-1.0 g) was homogenized at 0°C with a glass homogenizer in 10 vol (v/w) of 50 mmol/l Tris-HCl buffer (pH 7.5) containing 5 mmol/l EDTA. The homogenate was centrifuged at 4 o C at 20 000 X g for 20 min, and the soluble fraction was used for the analysis. Ten-microliter portions of extract diluted lOO- to lOOOO-fold with buffer A were subjected to the immunoassay in duplicate. Immunoassay
A polystyrene ball with antibody was incubated at 30” C in duplicate, unless otherwise specified, with 2-10 ~1 of the sample or standard SOD solution in a final volume of 0.5 ml with buffer G. After 5 h of incubation with shaking, the ball was washed twice with 1 ml of chilled buffer A in each test tube. The ball was then incubated with shaking at 4°C overnight in a fresh test tube containing 0.2 ml buffer A with 1 mU of the antibody Fab’ fragments labeled with galactosidase. The galactosidase activity bound to the ball was assayed with 4-methylumbelliferyl+ D-galactoside as substrate as described previously . Other methods
Concentrations in pg of the purified SOD were determined by Lowry’s Folin method  using bovine serum albumin as a standard. Protein concentrations of the tissue extracts were determined with the Bio-Rad Protein Assay kit, which utilizes a principle of protein-dye binding . The SDS-PAGE was performed by the method of Laemmli . Immunoblots were carried out as described by Towbin
175 et al.  with modifications . Immunoreactive Cu/Zn SOD concentrations in the extract of cerebral cortex were determined by the method described recently .
limit of the immunoassay
for Mn SOD
A standard curve for the immunoassay of human Mn SOD and cross reactivity with other forms of SODS are shown in Fig. 2. The detection limit, define as the lowest concentration giving a galactosidase activity significantly different from that of the zero standard at 0.99 confidence, was < 1 pg/assay tube. When Cu/Zn SOD purified from human erythrocytes was subjected to the above immunoassay system, there was no increase (up to 10 ng/tube) in the galactosidase activity bound on the polystyrene ball, indicating there is no cross-reactivity of the present assay with human Cu/Zn SOD. The present assay, however, reacted also with rat Mn SOD (Fig. 2). The cross reactivity of rat Mn SOD was about 60%, when assessed by comparing the concentrations of human and rat Mn SODS required to display a fluorescence intensity of 100 U.
Fig. 2. Standard curve of the assay for human Mn SOD and its cross-reactivity with other forms of SOD. Indicated amounts of human Mn SOD (o), rat Mn SOD (A) or human Cu/Zn SOD (0) were subjected in duplicate to the immunoassay. /3-D-Galactosidase activity bound on the polystyrene ball is expressed as the fluorescence intensity of 4-methylumbelliferone produced in a 20-min reaction with 0.1 mmol/l 4-methylumbelliferyl-/3-D-galactoside. In the fluorescence intensity scale, 1000 equals 1 pmol/l Cmethylumbelliferone.
176 TABLE I Serum Mn SOD concentrations of healthy subjects Age (yr)
No. of samples
16-19 20-29 30-39 40-49 50-59 60-64
10 10 10 10 10 10
Mn SOD (mean + 1 SD) (ng/ml) Male 79.5 90.1 86.1 84.5 87.5 67.3
f f f f f f
Female 12.2 25.3 15.8 19.2 19.8 18.0
62.9+ 9.0 61.5 k 12.7 71.9k11.2 76.5k13.7 76.8+ 7.4 77.3 + 25.1
Precision and accuracy of the assay with serum samples
Accuracy of the assay was determined by recovery experiments with five normal serum samples which contained 42.3-57.5 ng Mn SOD/ml. Two ~1 of serum samples were subjected in duplicate with or without 1 ng standard Mn SOD. The average recovery of the Mn SOD added was 95%. The precision of the assay was tested by assaying four serum samples (containing 37.9, 61.4, 69.7, and 130 ng Mn SOD/ml, respectively) 20 times in one assay (within-run) or the same serum samples in duplicate in ten consecutive assays (between-run). The coefficients of variation in each assay were < 12% (3-12s). Serum h4n SOD concentrations in normal adults The concentrations of Mn SOD in healthy adults (16-64 yr old) are shown in
Table I. Serum Mn SOD levels were not related to age and sex. The serum concentration in male and female ranged from 46.3 to 149 and 42.3 to 137 ng/ml, respectively. The average values f SD of males and females (n = 60) are 83.0 + 19.4 and 72.1 + 15.0 ng/ml, respectively, and the average value f SD of total healthy adult samples (n = 120) is 77.5 & 18.0 “g/ml. Distribution of immunoreactive Mn SOD in human tissues
Concentrations of immunoreactive Mn SOD in human tissues were determined in samples obtained at autopsy (Table II). The Mn SOD was present at high concentrations in heart, liver, kidney and adrenal gland. Relatively high concentrations of Mn SOD were observed in lung, spleen, digestive tract, bladder, and skeletal muscle. Other tissues, including prostate, testis, thyroid, mammary gland, adipose, and aorta, contained significant levels of Mn SOD, but there was no detectable amount in erythrocytes (data not shown). Concentrations of Mn SOD and Cu/ Zu SOD in the cerebral cortices of patients with Alzheimer disease, control age-matched and young patients The concentrations of Mn SOD and Cu/Zn SOD were determined in the
cerebral co&es obtained at autopsy from patients with Alzheimer’s disease, and the age-matched and young patients without neurological diseases. As shown in
177 TABLE II Concentrations
Tissues Cerebral cortex Heart muscle Lung Spleen Liver Esophagus Stomach SmaB intestine Kidney Bladder Adrenal Pancreas Adipose Skeletal muscle
Mn superoxide dismutase in various human tissues
No. of samples
Im.munoreactive Mn SOD (pg/mg
11 5 2 5 4 5 5 5 4 4 3 4 4 4
0.61 f 0.24 4.844 1.48 1.43 1.04*0.58 10.9 rt4.7 1.45 f 0.32 1.06f0.11 1.07 rfI0.26 6.11 j, 2.68 1.41 rt:0.49 7.09 f 2.62 2.63 f0.55 0.99 * 0.34 2.67 f 1.49
TABLE III Concentrations of Mn SOD and Cu/Zn age-matched and young controls No. of
samples Alzheimer’s disease
(range) (0.23- 1.15) (3.41- 6.43) (1.18- 1.68) (0.43- 1.66) (14.9 - 5.61) (1.20- 1.85) (0.91- 1.22) (0.71- 1.39) (2.77- 9.31) (0.87- 1.96) (5.36-10.1) (2.06- 3.28) (0.66- 1.35) (1.43- 4.76)
SOD in human cerebral cortices of Alzheimer’s disease, and
(pg/mg protein) Mn SOD
88*7 a (81-98) 82*7 (74-92) 31*10 (25-43)
0.781 it.255 = (0.491-1.26) 0.610 & 0.243 (0.233-1.15) 0.606 & 0.388 (0.329-1.05)
4.28 ztO.68 a (3.38-5.24) 4.24 + 0.75 (3.27-5.49) 4.02k1.18 (2.99-5.31)
a AI1 values are mean Ifr1 SD with (range).
Table III, the ~ncentrations of Mn SOD were much lower (about l/6) than those of Cu/Zn SOD in the cerebral cortex, and there was a relatively large variation in the Mn SOD values. However, the concentrations of both SODS were not significantly different among the three groups; Alzheimer’s disease, the age-matched control, and the young control. Discussion
Because of difficulties in the enzymatic determination of each form of SOD in the biological samples, we recently developed a sensitive immunoassay method for human Cu/Zn SOD , and here we have described a similarly sensitive immunoassay method for human Mn SOD.
For the me~urement of i~unorea~tive human Mn SOD, Baret et al. ]lS] reported a competitive-type radioimmunoassay, and Nishimura et al.  described a sandwich-type enzyme immunoassay. However, the minimum detection limit of these assays was about 1 rig/assay tube. The present assay system is about lOOO-fold more sensitive than the reported methods, because the minimum detection limit is < 1 pg/assay tube. The assay was specific to Mn SOD with no cross-reactivity to Cu/Zn SOD, but it cross-reacted about 60% with rat Mn SOD. These results suggest that the present assay method may also be applicable to the assay of rat Mn SOD. Normal serum levels of Mn SOD determined with the present method were about 80 ng/ml, being similar to the values reported by Nishimura et al. . The serum concentrations of Mn SOD were not related to age and sex as those of Cu/Zn SOD 161. Mn SOD is distributed widely in various tissues with relatively high levels as is Cu/Zn SOD. It is reported that SOD activity is decreased in the liver  and brain [4,5] of aged rats. In contrast, Kellogg and Fridovich  reported that the total SOD activities of brain and liver did not diminish from 2.4 to 26 months of age in the rat. In order to clarify the effect of aging on brain levels of Mn SOD and Cu/Zn SOD, we determined the concentrations of immunoreactive two SODS in the cerebral cortices of aged and young subjects as well as of patients with Alzheimer disease. The results indicate that concentrations of both SODS were not significantly different among the three groups. The sensitive immunoassay system for Mn SOD described here and that for Cu/Zn SOD reported recently  might be useful tools to clarify the pa~ophysiolo~cal significance of the two forms of SOD in clinical research. Acknowledgements
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