Clinica Chimica Acta, 187 (1990) 265-272 Elsevier
265
CCA 04677
Sensitive enzyme immunoassay for human aldolase A Kazuki Okajima r, Naomi Kurobe
I, Kikuo Shimizu 2 and Kanefusa
Kato t
’ Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya, Kasugai, Aichi, and 2 Medical and Biological Laboratories (Received
31 July 1989; revision received
Key worak Aldolase;
31 October
Isozyme;
Co. Ltd., Ina, Nagano (Japan)
1989; accepted
3 November
1989)
Immunoassay
A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A,, was developed using purified antibodies specific to the A subunit of aldolase. The antibodies were raised in sheep being immunized with purified aldolase A, and then purified by immunoaffinity chromatography on a column of aldolase A,-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab’), fragments and the same antibody F(ab’), fragments labeled with P-D-galactosidase from Escherichia coli. The assay was sensitive enough to detect 10 pg/tube of aldolase A,. The assay was specific to the A subunit of aldolase (aldolase A). It cross-reacted about 40% to aldolase A&, 7% to A& and 0.3% to AC,, but not cross-reacted with C, nor B4. Coefficients of variation in intra- and inter-assay were less than 16%. Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml. The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined. High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.
Introduction
One of the glycolytic enzyme, aldolase (EC 4.1.2.13) cleaves fructose 1,6-diphosphate to dihydroxyacetone phosphate and glycelaldehyde 3-phosphate. Its activity was found in various tissues and varied across the tissues. It has a tetrameric form
Correspondence and requests for Developmental Research,
0009-8981/90/$03.50
for reprints to: Dr. Kanefusa Kato, Department of Biochemistry, Institute Aichi Prefectural Colony, Kamiya, Kasugai, Aichi 480-03, Japan.
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
266
with three immunologically distinct subunits; A, B and C [l]. In skeletal muscle, heart and spleen aldolase A is predominant and presents as the A, form. Aldolase B is predominant in the liver, kidney and intestine, while aldolase C is mainly distributed in the brain as the C, or A-C hybrids (A$, A& and AC,) [2]. Aldolase A, the fetal form of enzyme, has been studied as a bio-marker for several neoplasms [3,4,5]. The transition of isozyme pattern of aldolase has also been studied in various neoplasms [6,7,8]. In order to evaluate aldolase A as a serum bio-marker for specified diseases, we now developed a highly sensitive and specific immunoassay method for measurement of aldolase A. Recently, Royds et al. [5] and Asaka et al. [9] developed a radioimmunoassay method for aldolase A, of which sensitivity was 1 ng/tube and 2 ng/tube, respectively. Materials and methods Reagents
CNBr-activated Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden); P-D-galactosidase from Escherichia coli, from Boehringer Mannheim (Mannheim, FRG); pepsin from porcine stomach mucosa, from Sigma Chemical Co. (St. Louis, MO); bovine serum albumin, from Organon Teknika (The Netherlands); N-succinimidyl 4-( N-maleimidomethyl) cyclohexane-1-carboxylate, from Zieben Chemicals Co. Ltd. (Tokyo, Japan); polystyrene balls (3.2 mm in diameter), from Precision Plastic Ball Co. (Chicago, IL). All other reagents are of highest grade available. Buffer G used for the immunoassay was composed of 10 mmol/l sodium phosphate buffer (pH 7.0) containing 0.3 mol/l NaCl, 1 mmol/l MgCl z, 1 g/l NaN,, 1 g/l bovine serum albumin and 5 g/l protease-treated gelatin [lo]. Buffer A was composed of 10 mmol/l sodium phosphate buffer (pH 7.0) containing 0.1 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l NaN, and 1 g/l bovine serum albumin. Purification of aldolase A
Aldolase A, was purified from pectoral muscles obtained at surgical resection of breast cancer, as described by Penhoet et al. [ll] with modifications. The phosphocellulose fraction was passed through a column of CM-Sephadex, equilibrated with 20 mmol/l sodium phosphate buffer (pH 6.0) containing 2 mmol/l EDTA. After concentrated, this fraction was applied to a column of Sephacryl S-200. The final preparation had a specific activity of about 15 U on the basis of mg protein, and showed a single band (Fig. 1) on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Assay of aldolase activity and purification of aldolase B4, C, and A-C hybrids were already described elsewhere [12,13]. Antibodies
The antisera to aldolase A were raised in a sheep by subcutaneous injection the purified aldolase A., (1 mg/animal) emulsified with Freund’s complete juvant. The same immunizations were repeated 4 times every 2 wk and then sheep was bled 1 wk after immtmization. The specific antibody IgG was purified
of adthe by
267
Fig. 1. SDS-PAGE of the purified aldolase A D and a crude muscle extract, and immunoblots with purified anti-aldolase A antibodies. Lane 1, a human muscle extract containing about 10 pg proteins; lane 2, 0.3 ag of purified aldolase A4. a, polyacrylamide gel stained with Coomassie blue; b, nitrocellulose sheet stained with anti-aldolase A.
the immunoaffinity column chromatography using aldolase A,-coupled Sepharose 4B as described previously for the purification of antibodies to enolase isozymes [14]. Purified antibody IgG fractions obtained (about 15 mg/lOO ml serum) were neutralized and concentrated with Amicon Centriflo CF-25 (Amicon Corp. Danvers MA). Specificity of antibodies was tested on SDS-PAGE of a crude extract of muscle, followed by immunoblot with the antibodies. As shown in Fig. 1, antibodies reacted with a single band at a position corresponding to the A subunit of aldolase. The purified sheep antibody IgG was digested with pepsin and resultant F(ab’), fragments of antibodies were isolated by the chromatography on a Sephadex G-150 column. In order to decrease the background value of immunoassay, and to protect the assay from non-specific interference by substances in biological samples, we prepared the assay system with the antibody F(ab’), fragments [15]. Polystyrene balls with immobilized antibody F(ab’), fragments The antibody Ffab’), fragments were coupled non-covalently 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 [16]. Antibody F(ab’lt fragments labeled with /3-D-galactosidase Antibody F(ab’), fragments were labeled with &B-D-galactosidase using N-succinimidyl4-( ~-m~ei~domethyl)cyclo~ex~e-l-c~boxylate (S-MCC) [17]. In brief, 5 ml of 0.1 mol/l sodium phosphate buffer (pH 7.0) containing antibody F(ab’), (AXI + 1) were mixed with 25 ~1 of S-MCC dissolved in dioxane (10 mg/ml) at room temperature, and the mixture was incubated at 30°C for 1 h. Then the F(ab’), was separated from free S-MCC by passing the mixture through a column of Sephadex G-25 ~~~brated with 0.1 mot/l sodium phosphate buffer (pH 7.0). Antibody F(ab’), fragments thus prepared contained 1.5 to 2 maleimide
268
residues/F(ab’)~. The pooled maleimide F(ab’), fraction (A,, i 0.6, about 6 ml) were mixed with 1 mg of /3-D-galactosidase from E. cob, and the mixture was kept overnight at 4°C. The mixture was concentrated to about 2 ml with Amicon B15 at room temperature. After adding 20 ~1 of 0.1 mol/l 2-mercaptoethylamine to block the maleimide residues possibly remaining on the F(ab’),, the mixture was applied on a column of Sepharose 6B equilibrated with buffer A to separate the galactosidase-labeled antibodies from unreacted free antibody F(ab’), fragments. The fractions containing the peak of galactosidase activity were pooled (about 12 ml) and used as the galactosidase-labeled antibody preparation. The amounts of labeled antibody are expressed in units of the galactosidase activity, and 1 U of the activity is defined as that which produces 1 pmol 4_methylumbelliferone/min under the conditions described previously f16].
Serum and tissue samples
Adult control serum samples were obtained from a local blood center. All serum samples were kept frozen at - 20°C until analysis. Various adult tissues were obtained at autopsy within 24 h postmortem. All samples were stored at -8O*C until analysis. Each tissue (0.5-l g) was homogenized at 4°C with a glass homogenizer in 10 vol (v/w) of 50 mmol/l Tris-HCl buffer (pH 7.5) containing 5 mmol/l MgSO,. The homogenate was centrifuged at 4°C at 20000 x g for 30 mm, and the soluble fraction was used for the analysis. lo-p1 portions of extract diluted 100 to 1000 times with buffer G were subjected in duplicate to the assay.
Immunoassay procedures
A polystyrene ball with antibody was incubated at 30°C unless otherwise specified, with 10 ~1 of the sample or standard aldolase A, solution in a final volume of 0.5 ml with buffer G in a test tube (10 x 75 mm) for 5 h with shaking. Then the buffer was aspirated, and the ball was washed twice with 1 ml of ice-cold buffer A in each test tube. The ball was then transferred to a fresh test tube (7.5 x 75 mm) containing 0.2 ml of buffer A with 1 mU of the antibody F(ab’), fragments labeled with galactosidase, and the mixture was shaken at 4°C overnight. The galactosidase activity bound to the ball was assayed with 4-methylumbellife~lfi-D-galactoside as a substrate [16]. The results were expressed as aldolase A, equivalent ng/ml or mg protein.
Other methods
SDS-PAGE and i~unoblot were performed as described previously [13]. Protein concentration of the extract was determined with the Bio-Rad Protein Assay kit which utilizes the principle of protein-dye binding. Concentrations in mg of the standard aldolase A,, B4, C, and A-C hybrids were estimated spectrophotometritally using values AO,Jz= 0.91, 0.89 and 0.88 for A,, B4 and C,, respectively 1181.
269
Results Detection limit and specificity of the assay
A standard curve of the assay of aldolase A, and the cross-reactivity of the assay with other forms of aldolase are shown in Fig. 2. The detection limit, defined as the lowest concentration giving a galactosidase activity significantly different from that of the zero standard at 99% confidence, was 10 pg/tube. The assay did not cross-react with aldolase C, nor aldolase B4, while it cross-reacted with the A-C hybrids that contain the A subunit of aldolase. The cross-reactivity of A,C, A&, and AC, were about 40, 7 and 0.38, respectively, when assessed by comparing the concentrations of aldolase A, and each A-C hybrid required to display a fluorescence intensity of 100 or 30 U. These results indicate that the present assay system is specific to the A subunit of aldolase. Precision and accuracy of the assay with serum samples
The precision of the assay was tested by assaying three serum samples (containing 211, 551 and 809 ng aldolase A/ml) 20 times in one assay (intra-assay) or three serum samples (containing 309, 738 and 886 ng aldolase A/ml) in 10 consecutive assays (inter-assay). The coefficients of variation (CV) of both assay were between 9 and 16%. Diluted serum samples were also employed to confirm the linearity of the assay (p < 0.01). Accuracy of the assay was determined by recovery experiments with serum samples containing 180-250 ng aldolase A/ml. Ten microliters of
Aldolase isozymes
(ng)
Fig. 2. Standard curve of the assay for aldolase A and its cross-reactivity with other aldolase isozymes. Various amounts of aldolase A, (O), A,C (A), A& (v), AC, (H), B4 (0) and C, (0) were subjected to the immunoassay in duplicate. Fluorescence intensity, 1000 = 1 pmol/l4-methylumbelliferone.
210
TABLE I Aldolase A concentrations
in sera of patients
with muscular
dystrophy
Type of muscular dystrophy
No. of samples
Aldolase
A (q/ml)
Mean f 1 SD
Range
Duchenne Limb-girdle Facioscapulohumeral Myotonic Congenital
5 4 2 2 1
164Of 610 883 + 1320 204 190 11200
829-2440 117-2850 129,279 171,208
samples with or without additional 2 or 8 ng standard aldolase A were subjected to immunoassay in duplicate. The recoveries of aldolase A added were 108 to 133%. Serum aldolase A concentrations in normal samples The concentrations of aldolase A in serum samples of healthy adults (16-64 yr old) were 198 * 51 ng/ml (mean f SD) ranged from 85 to 409 ng/ml in male, 143 + 43 ng/ml ranged from 51 to 253 ng/ml in female. There is no age-related difference. However, the average value of male was significantly ( p < 0.001) higher than that of female. High concentrations of aldolase A were found in sera of patients with muscular dystrophy (Table I). Distribution of immunoreactive aldolase A in various human tissues The concentrations of immunoreactive aldolase A in human tissues were determined in samples obtained at autopsy from 2 to 6 subjects aged 45-68 yr. Aldolase A was detected in almost all tissues examined (average values of 155 to 26 500 ng aldolase A/mg protein). It was present at high concentrations in skeletal muscle (26 500 ng/mg protein), heart muscle (7040 ng/mg protein), cerebrum (10100 ng/mg protein) and lymphatic tissue (1600 ng/mg protein). Liver and kidney, in which aldolase B was predominant [12], also contained a significant amount of aldolase A (370 and 530 ng/mg protein, respectively). Discussion
In order to evaluate the applicability of isozymes as bio-markers for specified diseases, we have been developing sensitive immunoassay methods for measurements of various isozymes, including enolase (ar, B and -r) [14,19], creatine kinase (M and B) [20,21] and aldolase (B and C) [12,13] isozymes, by the use of purified rabbit antibodies. In those methods rabbits antibody Fab’ fragments were selectively coupled with /3-D-galactosidase by the use of o-phenylenedimaleimide (oPDM) as a coupling reagent [22,23]. The assays were highly sensitive and the minimum detection limit was 1 to 3 pg/assay tube. In the present paper we described the assay system for human aldolase A with use of sheep antibodies, because antigenicity of human aldolase A to rabbits seemed to be low, and we could
271
not raise sufficient antibodies in rabbits. Royds et al. [5] and Asaka et al. [9,24] prepared antisera to human aldolase A in the sheep and chicken, respectively. In the present assay system, we labeled sheep antibody F(ab’), fragments with galactosidase by the use of N-succinimidyl 4-( N-maleimidomethyl) cyclohexane-lcarboxylate (S-MCC) [17], because several trials for labeling the sheep Fab’ with galactosidase by the use of o-PDM with the same manner as for rabbit Fab’ showed poor results. Although the reason remains to be clarified, it was always found difficult to introduce maleimide residues in the sheep Fab’ with o-PDM. On the other hand, maleimide residues could be introduced in the sheep F(ab’), (1.5 to 2 residues/F(ab’),) with S-MCC under the conditions described. Because the maleimide residues are introduced randomly via amino residues of antibody fragments, some of the antibodies might become inactive after conjugation with the galactosidase. A relatively low sensitivity of the present assay (minimum detection limit 10 pg/tube) as compared with that of aldolase B [12] or C [13] may depend in part on the reason described above. However, the sensitivity of the present method was much higher than those of Royds et al. [5] or Asaka et al. [9], because detection limit of their assays were l-2 rig/assay tube. Aldolase A concentrations of normal adult serum measured by the present method were similar to those determined by Royds et al. [5] or Asaka et al. [9], and the concentrations in males were significantly higher than those of females (p < 0.001). Serum aldolase A was enhanced in patients with muscular dystrophy. The quantitative distribution of aldolase A in various tissues was also determined. The result confirmed the previous report [5], indicating aldolase A is distributed at high levels in the skeletal and heart muscles, brain and lymphatic tissue. The predominant form of aldolase in the undifferentiated tumor tissue is aldolase A [6]. Transition of the expression of aldolase isozyme to the A form is reported in the renal cell carcinoma [6] and hepatoma [4,5]. In addition, the anaerobic glycolysis is generally enhanced in the neoplastic tissues, which is accompanied by the increased production of aldolase A protein. These facts suggest a possible applicability of serum aldolase A as a tumor marker as reported by Asaka et al. [25,26]. The present assay system, together with the systems established previously for aldolase B [12] and aldolase C [13], may be useful to clarify the clinical applicability of aldolase isozymes as serum bio-markers for the specified tumors. Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas, Ministry of Education, Science and Culture, Japan and by a research grant from the JAMW Ogyaa Donation foundation. References 1 Penhoet EE, Rutter WJ. Catalytic and immunological properties of homometric and heterometric combinations of aldolase subunits. J Biol Chem 1971;246:318-323.
272 2 Lebherz HG, Rutter WJ. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 1969;8:109-121. ED, Stoye JP. A classification of tumor development based on an analysis of enzymes in 3 Wachsmuth tissue sections of hypemephroid carcinoma in man. Beitr Path Bd 1976;159:229-248. 4 Asaka M, Nagase K, Alpert E. Biochemical and clinical studies of aldolase isozymes in human cancer. Isozymes: current topics in biological and medical research. Vol. 11: Medical and other applications. New York: Alan R. Liss Inc., 1983;183-195. 5 Royds JA, Kennedy HJ, Little PV, Taylor CB, Triger DR. Serum aldolase isoenzymes in benign and malignant liver disease. Clin Chim Acta 1987;167:237-246. 6 Kellen JA, Chan A, Caplan B, Malkin A. Aldolase isoenzyme patterns during human ontogeny and in lung, kidney and breast cancer. Enzyme 1980;25:228-235. 7 Schapira F, Hatzfeld A, Reuber MD. Fetal pattern of aldolase in transplantable hepatomas. Cancer Res 1971;31:1224-1230. 8 Sato S, Sugimura T, Chien TC, Takakura K. Aldolase isozyme patterns of human brain tumors. Cancer 1971;27:223-227. 9 Asaka M, Nagase K, Miyazaki T, Shiraishi T, Alpert E. Radioimmunoassay for human aldolase A. Clin Chim Acta 1981;117:289-296. 10 Kato K, Umeda Y, Suzuki F, Kosaka A. Improved reaction buffers for solid-phase enzyme immunoassay without interference by serum factors. Clin Chim Acta 1980;102:261-265. 11 Penhoet EE, Kochman M, Rutter WJ. Isolation of fructose diphosphate aldolases A, B and C. Biochemistry 1969;8:4391-4395. 12 Haimoto H, Kurobe N, Hosoda S, Kato K. Sensitive enzyme immunoassay for human aldolase B. Clin Chim Acta 1989;181:27-36. 13 Haimoto H, Kato K. Highly sensitive enzyme immunoassay for human brain aldolase C. Clin Chim Acta 1986;154:203-212. 14 Kato K, Asai R, Shimizu A, Suzuki F, Ariyoshi Y. Immunoassay of three enolase isozymes in human serum and in blood cells. Clin Chim Acta 1983;127:353-363. in a solid-phase enzyme immunoassay system 15 Kato K, Umeda Y, Suzuki F, Kosaka A. Interference by serum factors. J Appl Biochem 1979;1:479-488. Y, Okaya S, Ishikawa E, Kobayashi K, Katunuma N. Use of rabbit IgG-loaded 16 Kato K, Hamaguchi silicone piece for the sandwich enzyme immunoassay for macromolecular antigens. J Biochem (Tokyo) 1977;81:1557-1566. S, Imanaga M, Ishiiawa E, et al. Mild and efficient conjugation of rabbit Fab’ and 17 Yoshitake horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J Biochem (Tokyo) 1982;92:1413-1424. and isolation of mammalian fructosediphosphate aldolases. 18 Penhoet EE, Rutter WJ. Detection Method Enzymol 1975;42:240-249. of human muscle 19 Kato K, Okagawa Y, Suzuki F, Shimizu A, Mokuno K, Takahasbi Y. Immunoassay enolase subunit in serum: a novel marker antigen for muscle diseases. Clin Chim Acta 1983;131:75-85. T. Highly sensitive enzyme 20 Kato K, Shimizu A, Ishiguro Y, Mokuno K, Ariyoshi Y, Nakajima immunoassay for human creatine kinase BB isozyme. Clin Chim Acta 1985;150:31-40. for human creatine kinase MM and MB 21 Kato K, Shimizu A. Highly sensitive enzyme immunoassay isozymes. Clin Chim Acta 1986;158:99-108. Y, Ishikawa E. Enzyme-linked immunoassay: conjugation of the Fab 22 Kato K, Fukui H, Hamaguchi fragment of rabbit IgG with /?-D-galactosidase from E. coli and its use for immunoassay. J Immunol 1976;116:1554-1560. in a separation method for enzyme immunoassay. Methods 23 Kato K. Use of activated thiol-Sepharose Enzymol 1983;92:345-359. solid-phase radioimmunoassay for 24 Asaka M, Nagase K, Miyazaki T, Alpert E. A non-competitive human aldolase A. Clin Chim Acta 1982;125:31-40. of muscular 25 Asaka M, Nagase K, Shiraishi T, Miyazaki T. Diagnosis of cancer by radioimmunoassay type of aldolase. Gann 1980;71:433-440. of aldolase A, Determination of 26 Asaka M, Nagase K, Miyazaki T, Alpert E. Radioimmunoassay normal serum levels and increased serum concentration in cancer patients. Cancer 1983;51:1873-1878.
265
CCA 04677
Sensitive enzyme immunoassay for human aldolase A Kazuki Okajima r, Naomi Kurobe
I, Kikuo Shimizu 2 and Kanefusa
Kato t
’ Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya, Kasugai, Aichi, and 2 Medical and Biological Laboratories (Received
31 July 1989; revision received
Key worak Aldolase;
31 October
Isozyme;
Co. Ltd., Ina, Nagano (Japan)
1989; accepted
3 November
1989)
Immunoassay
A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A,, was developed using purified antibodies specific to the A subunit of aldolase. The antibodies were raised in sheep being immunized with purified aldolase A, and then purified by immunoaffinity chromatography on a column of aldolase A,-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab’), fragments and the same antibody F(ab’), fragments labeled with P-D-galactosidase from Escherichia coli. The assay was sensitive enough to detect 10 pg/tube of aldolase A,. The assay was specific to the A subunit of aldolase (aldolase A). It cross-reacted about 40% to aldolase A&, 7% to A& and 0.3% to AC,, but not cross-reacted with C, nor B4. Coefficients of variation in intra- and inter-assay were less than 16%. Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml. The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined. High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.
Introduction
One of the glycolytic enzyme, aldolase (EC 4.1.2.13) cleaves fructose 1,6-diphosphate to dihydroxyacetone phosphate and glycelaldehyde 3-phosphate. Its activity was found in various tissues and varied across the tissues. It has a tetrameric form
Correspondence and requests for Developmental Research,
0009-8981/90/$03.50
for reprints to: Dr. Kanefusa Kato, Department of Biochemistry, Institute Aichi Prefectural Colony, Kamiya, Kasugai, Aichi 480-03, Japan.
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
266
with three immunologically distinct subunits; A, B and C [l]. In skeletal muscle, heart and spleen aldolase A is predominant and presents as the A, form. Aldolase B is predominant in the liver, kidney and intestine, while aldolase C is mainly distributed in the brain as the C, or A-C hybrids (A$, A& and AC,) [2]. Aldolase A, the fetal form of enzyme, has been studied as a bio-marker for several neoplasms [3,4,5]. The transition of isozyme pattern of aldolase has also been studied in various neoplasms [6,7,8]. In order to evaluate aldolase A as a serum bio-marker for specified diseases, we now developed a highly sensitive and specific immunoassay method for measurement of aldolase A. Recently, Royds et al. [5] and Asaka et al. [9] developed a radioimmunoassay method for aldolase A, of which sensitivity was 1 ng/tube and 2 ng/tube, respectively. Materials and methods Reagents
CNBr-activated Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden); P-D-galactosidase from Escherichia coli, from Boehringer Mannheim (Mannheim, FRG); pepsin from porcine stomach mucosa, from Sigma Chemical Co. (St. Louis, MO); bovine serum albumin, from Organon Teknika (The Netherlands); N-succinimidyl 4-( N-maleimidomethyl) cyclohexane-1-carboxylate, from Zieben Chemicals Co. Ltd. (Tokyo, Japan); polystyrene balls (3.2 mm in diameter), from Precision Plastic Ball Co. (Chicago, IL). All other reagents are of highest grade available. Buffer G used for the immunoassay was composed of 10 mmol/l sodium phosphate buffer (pH 7.0) containing 0.3 mol/l NaCl, 1 mmol/l MgCl z, 1 g/l NaN,, 1 g/l bovine serum albumin and 5 g/l protease-treated gelatin [lo]. Buffer A was composed of 10 mmol/l sodium phosphate buffer (pH 7.0) containing 0.1 mol/l NaCl, 1 mmol/l MgCl,, 1 g/l NaN, and 1 g/l bovine serum albumin. Purification of aldolase A
Aldolase A, was purified from pectoral muscles obtained at surgical resection of breast cancer, as described by Penhoet et al. [ll] with modifications. The phosphocellulose fraction was passed through a column of CM-Sephadex, equilibrated with 20 mmol/l sodium phosphate buffer (pH 6.0) containing 2 mmol/l EDTA. After concentrated, this fraction was applied to a column of Sephacryl S-200. The final preparation had a specific activity of about 15 U on the basis of mg protein, and showed a single band (Fig. 1) on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Assay of aldolase activity and purification of aldolase B4, C, and A-C hybrids were already described elsewhere [12,13]. Antibodies
The antisera to aldolase A were raised in a sheep by subcutaneous injection the purified aldolase A., (1 mg/animal) emulsified with Freund’s complete juvant. The same immunizations were repeated 4 times every 2 wk and then sheep was bled 1 wk after immtmization. The specific antibody IgG was purified
of adthe by
267
Fig. 1. SDS-PAGE of the purified aldolase A D and a crude muscle extract, and immunoblots with purified anti-aldolase A antibodies. Lane 1, a human muscle extract containing about 10 pg proteins; lane 2, 0.3 ag of purified aldolase A4. a, polyacrylamide gel stained with Coomassie blue; b, nitrocellulose sheet stained with anti-aldolase A.
the immunoaffinity column chromatography using aldolase A,-coupled Sepharose 4B as described previously for the purification of antibodies to enolase isozymes [14]. Purified antibody IgG fractions obtained (about 15 mg/lOO ml serum) were neutralized and concentrated with Amicon Centriflo CF-25 (Amicon Corp. Danvers MA). Specificity of antibodies was tested on SDS-PAGE of a crude extract of muscle, followed by immunoblot with the antibodies. As shown in Fig. 1, antibodies reacted with a single band at a position corresponding to the A subunit of aldolase. The purified sheep antibody IgG was digested with pepsin and resultant F(ab’), fragments of antibodies were isolated by the chromatography on a Sephadex G-150 column. In order to decrease the background value of immunoassay, and to protect the assay from non-specific interference by substances in biological samples, we prepared the assay system with the antibody F(ab’), fragments [15]. Polystyrene balls with immobilized antibody F(ab’), fragments The antibody Ffab’), fragments were coupled non-covalently 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 [16]. Antibody F(ab’lt fragments labeled with /3-D-galactosidase Antibody F(ab’), fragments were labeled with &B-D-galactosidase using N-succinimidyl4-( ~-m~ei~domethyl)cyclo~ex~e-l-c~boxylate (S-MCC) [17]. In brief, 5 ml of 0.1 mol/l sodium phosphate buffer (pH 7.0) containing antibody F(ab’), (AXI + 1) were mixed with 25 ~1 of S-MCC dissolved in dioxane (10 mg/ml) at room temperature, and the mixture was incubated at 30°C for 1 h. Then the F(ab’), was separated from free S-MCC by passing the mixture through a column of Sephadex G-25 ~~~brated with 0.1 mot/l sodium phosphate buffer (pH 7.0). Antibody F(ab’), fragments thus prepared contained 1.5 to 2 maleimide
268
residues/F(ab’)~. The pooled maleimide F(ab’), fraction (A,, i 0.6, about 6 ml) were mixed with 1 mg of /3-D-galactosidase from E. cob, and the mixture was kept overnight at 4°C. The mixture was concentrated to about 2 ml with Amicon B15 at room temperature. After adding 20 ~1 of 0.1 mol/l 2-mercaptoethylamine to block the maleimide residues possibly remaining on the F(ab’),, the mixture was applied on a column of Sepharose 6B equilibrated with buffer A to separate the galactosidase-labeled antibodies from unreacted free antibody F(ab’), fragments. The fractions containing the peak of galactosidase activity were pooled (about 12 ml) and used as the galactosidase-labeled antibody preparation. The amounts of labeled antibody are expressed in units of the galactosidase activity, and 1 U of the activity is defined as that which produces 1 pmol 4_methylumbelliferone/min under the conditions described previously f16].
Serum and tissue samples
Adult control serum samples were obtained from a local blood center. All serum samples were kept frozen at - 20°C until analysis. Various adult tissues were obtained at autopsy within 24 h postmortem. All samples were stored at -8O*C until analysis. Each tissue (0.5-l g) was homogenized at 4°C with a glass homogenizer in 10 vol (v/w) of 50 mmol/l Tris-HCl buffer (pH 7.5) containing 5 mmol/l MgSO,. The homogenate was centrifuged at 4°C at 20000 x g for 30 mm, and the soluble fraction was used for the analysis. lo-p1 portions of extract diluted 100 to 1000 times with buffer G were subjected in duplicate to the assay.
Immunoassay procedures
A polystyrene ball with antibody was incubated at 30°C unless otherwise specified, with 10 ~1 of the sample or standard aldolase A, solution in a final volume of 0.5 ml with buffer G in a test tube (10 x 75 mm) for 5 h with shaking. Then the buffer was aspirated, and the ball was washed twice with 1 ml of ice-cold buffer A in each test tube. The ball was then transferred to a fresh test tube (7.5 x 75 mm) containing 0.2 ml of buffer A with 1 mU of the antibody F(ab’), fragments labeled with galactosidase, and the mixture was shaken at 4°C overnight. The galactosidase activity bound to the ball was assayed with 4-methylumbellife~lfi-D-galactoside as a substrate [16]. The results were expressed as aldolase A, equivalent ng/ml or mg protein.
Other methods
SDS-PAGE and i~unoblot were performed as described previously [13]. Protein concentration of the extract was determined with the Bio-Rad Protein Assay kit which utilizes the principle of protein-dye binding. Concentrations in mg of the standard aldolase A,, B4, C, and A-C hybrids were estimated spectrophotometritally using values AO,Jz= 0.91, 0.89 and 0.88 for A,, B4 and C,, respectively 1181.
269
Results Detection limit and specificity of the assay
A standard curve of the assay of aldolase A, and the cross-reactivity of the assay with other forms of aldolase are shown in Fig. 2. The detection limit, defined as the lowest concentration giving a galactosidase activity significantly different from that of the zero standard at 99% confidence, was 10 pg/tube. The assay did not cross-react with aldolase C, nor aldolase B4, while it cross-reacted with the A-C hybrids that contain the A subunit of aldolase. The cross-reactivity of A,C, A&, and AC, were about 40, 7 and 0.38, respectively, when assessed by comparing the concentrations of aldolase A, and each A-C hybrid required to display a fluorescence intensity of 100 or 30 U. These results indicate that the present assay system is specific to the A subunit of aldolase. Precision and accuracy of the assay with serum samples
The precision of the assay was tested by assaying three serum samples (containing 211, 551 and 809 ng aldolase A/ml) 20 times in one assay (intra-assay) or three serum samples (containing 309, 738 and 886 ng aldolase A/ml) in 10 consecutive assays (inter-assay). The coefficients of variation (CV) of both assay were between 9 and 16%. Diluted serum samples were also employed to confirm the linearity of the assay (p < 0.01). Accuracy of the assay was determined by recovery experiments with serum samples containing 180-250 ng aldolase A/ml. Ten microliters of
Aldolase isozymes
(ng)
Fig. 2. Standard curve of the assay for aldolase A and its cross-reactivity with other aldolase isozymes. Various amounts of aldolase A, (O), A,C (A), A& (v), AC, (H), B4 (0) and C, (0) were subjected to the immunoassay in duplicate. Fluorescence intensity, 1000 = 1 pmol/l4-methylumbelliferone.
210
TABLE I Aldolase A concentrations
in sera of patients
with muscular
dystrophy
Type of muscular dystrophy
No. of samples
Aldolase
A (q/ml)
Mean f 1 SD
Range
Duchenne Limb-girdle Facioscapulohumeral Myotonic Congenital
5 4 2 2 1
164Of 610 883 + 1320 204 190 11200
829-2440 117-2850 129,279 171,208
samples with or without additional 2 or 8 ng standard aldolase A were subjected to immunoassay in duplicate. The recoveries of aldolase A added were 108 to 133%. Serum aldolase A concentrations in normal samples The concentrations of aldolase A in serum samples of healthy adults (16-64 yr old) were 198 * 51 ng/ml (mean f SD) ranged from 85 to 409 ng/ml in male, 143 + 43 ng/ml ranged from 51 to 253 ng/ml in female. There is no age-related difference. However, the average value of male was significantly ( p < 0.001) higher than that of female. High concentrations of aldolase A were found in sera of patients with muscular dystrophy (Table I). Distribution of immunoreactive aldolase A in various human tissues The concentrations of immunoreactive aldolase A in human tissues were determined in samples obtained at autopsy from 2 to 6 subjects aged 45-68 yr. Aldolase A was detected in almost all tissues examined (average values of 155 to 26 500 ng aldolase A/mg protein). It was present at high concentrations in skeletal muscle (26 500 ng/mg protein), heart muscle (7040 ng/mg protein), cerebrum (10100 ng/mg protein) and lymphatic tissue (1600 ng/mg protein). Liver and kidney, in which aldolase B was predominant [12], also contained a significant amount of aldolase A (370 and 530 ng/mg protein, respectively). Discussion
In order to evaluate the applicability of isozymes as bio-markers for specified diseases, we have been developing sensitive immunoassay methods for measurements of various isozymes, including enolase (ar, B and -r) [14,19], creatine kinase (M and B) [20,21] and aldolase (B and C) [12,13] isozymes, by the use of purified rabbit antibodies. In those methods rabbits antibody Fab’ fragments were selectively coupled with /3-D-galactosidase by the use of o-phenylenedimaleimide (oPDM) as a coupling reagent [22,23]. The assays were highly sensitive and the minimum detection limit was 1 to 3 pg/assay tube. In the present paper we described the assay system for human aldolase A with use of sheep antibodies, because antigenicity of human aldolase A to rabbits seemed to be low, and we could
271
not raise sufficient antibodies in rabbits. Royds et al. [5] and Asaka et al. [9,24] prepared antisera to human aldolase A in the sheep and chicken, respectively. In the present assay system, we labeled sheep antibody F(ab’), fragments with galactosidase by the use of N-succinimidyl 4-( N-maleimidomethyl) cyclohexane-lcarboxylate (S-MCC) [17], because several trials for labeling the sheep Fab’ with galactosidase by the use of o-PDM with the same manner as for rabbit Fab’ showed poor results. Although the reason remains to be clarified, it was always found difficult to introduce maleimide residues in the sheep Fab’ with o-PDM. On the other hand, maleimide residues could be introduced in the sheep F(ab’), (1.5 to 2 residues/F(ab’),) with S-MCC under the conditions described. Because the maleimide residues are introduced randomly via amino residues of antibody fragments, some of the antibodies might become inactive after conjugation with the galactosidase. A relatively low sensitivity of the present assay (minimum detection limit 10 pg/tube) as compared with that of aldolase B [12] or C [13] may depend in part on the reason described above. However, the sensitivity of the present method was much higher than those of Royds et al. [5] or Asaka et al. [9], because detection limit of their assays were l-2 rig/assay tube. Aldolase A concentrations of normal adult serum measured by the present method were similar to those determined by Royds et al. [5] or Asaka et al. [9], and the concentrations in males were significantly higher than those of females (p < 0.001). Serum aldolase A was enhanced in patients with muscular dystrophy. The quantitative distribution of aldolase A in various tissues was also determined. The result confirmed the previous report [5], indicating aldolase A is distributed at high levels in the skeletal and heart muscles, brain and lymphatic tissue. The predominant form of aldolase in the undifferentiated tumor tissue is aldolase A [6]. Transition of the expression of aldolase isozyme to the A form is reported in the renal cell carcinoma [6] and hepatoma [4,5]. In addition, the anaerobic glycolysis is generally enhanced in the neoplastic tissues, which is accompanied by the increased production of aldolase A protein. These facts suggest a possible applicability of serum aldolase A as a tumor marker as reported by Asaka et al. [25,26]. The present assay system, together with the systems established previously for aldolase B [12] and aldolase C [13], may be useful to clarify the clinical applicability of aldolase isozymes as serum bio-markers for the specified tumors. Acknowledgements
This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas, Ministry of Education, Science and Culture, Japan and by a research grant from the JAMW Ogyaa Donation foundation. References 1 Penhoet EE, Rutter WJ. Catalytic and immunological properties of homometric and heterometric combinations of aldolase subunits. J Biol Chem 1971;246:318-323.
272 2 Lebherz HG, Rutter WJ. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 1969;8:109-121. ED, Stoye JP. A classification of tumor development based on an analysis of enzymes in 3 Wachsmuth tissue sections of hypemephroid carcinoma in man. Beitr Path Bd 1976;159:229-248. 4 Asaka M, Nagase K, Alpert E. Biochemical and clinical studies of aldolase isozymes in human cancer. Isozymes: current topics in biological and medical research. Vol. 11: Medical and other applications. New York: Alan R. Liss Inc., 1983;183-195. 5 Royds JA, Kennedy HJ, Little PV, Taylor CB, Triger DR. Serum aldolase isoenzymes in benign and malignant liver disease. Clin Chim Acta 1987;167:237-246. 6 Kellen JA, Chan A, Caplan B, Malkin A. Aldolase isoenzyme patterns during human ontogeny and in lung, kidney and breast cancer. Enzyme 1980;25:228-235. 7 Schapira F, Hatzfeld A, Reuber MD. Fetal pattern of aldolase in transplantable hepatomas. Cancer Res 1971;31:1224-1230. 8 Sato S, Sugimura T, Chien TC, Takakura K. Aldolase isozyme patterns of human brain tumors. Cancer 1971;27:223-227. 9 Asaka M, Nagase K, Miyazaki T, Shiraishi T, Alpert E. Radioimmunoassay for human aldolase A. Clin Chim Acta 1981;117:289-296. 10 Kato K, Umeda Y, Suzuki F, Kosaka A. Improved reaction buffers for solid-phase enzyme immunoassay without interference by serum factors. Clin Chim Acta 1980;102:261-265. 11 Penhoet EE, Kochman M, Rutter WJ. Isolation of fructose diphosphate aldolases A, B and C. Biochemistry 1969;8:4391-4395. 12 Haimoto H, Kurobe N, Hosoda S, Kato K. Sensitive enzyme immunoassay for human aldolase B. Clin Chim Acta 1989;181:27-36. 13 Haimoto H, Kato K. Highly sensitive enzyme immunoassay for human brain aldolase C. Clin Chim Acta 1986;154:203-212. 14 Kato K, Asai R, Shimizu A, Suzuki F, Ariyoshi Y. Immunoassay of three enolase isozymes in human serum and in blood cells. Clin Chim Acta 1983;127:353-363. in a solid-phase enzyme immunoassay system 15 Kato K, Umeda Y, Suzuki F, Kosaka A. Interference by serum factors. J Appl Biochem 1979;1:479-488. Y, Okaya S, Ishikawa E, Kobayashi K, Katunuma N. Use of rabbit IgG-loaded 16 Kato K, Hamaguchi silicone piece for the sandwich enzyme immunoassay for macromolecular antigens. J Biochem (Tokyo) 1977;81:1557-1566. S, Imanaga M, Ishiiawa E, et al. Mild and efficient conjugation of rabbit Fab’ and 17 Yoshitake horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay. J Biochem (Tokyo) 1982;92:1413-1424. and isolation of mammalian fructosediphosphate aldolases. 18 Penhoet EE, Rutter WJ. Detection Method Enzymol 1975;42:240-249. of human muscle 19 Kato K, Okagawa Y, Suzuki F, Shimizu A, Mokuno K, Takahasbi Y. Immunoassay enolase subunit in serum: a novel marker antigen for muscle diseases. Clin Chim Acta 1983;131:75-85. T. Highly sensitive enzyme 20 Kato K, Shimizu A, Ishiguro Y, Mokuno K, Ariyoshi Y, Nakajima immunoassay for human creatine kinase BB isozyme. Clin Chim Acta 1985;150:31-40. for human creatine kinase MM and MB 21 Kato K, Shimizu A. Highly sensitive enzyme immunoassay isozymes. Clin Chim Acta 1986;158:99-108. Y, Ishikawa E. Enzyme-linked immunoassay: conjugation of the Fab 22 Kato K, Fukui H, Hamaguchi fragment of rabbit IgG with /?-D-galactosidase from E. coli and its use for immunoassay. J Immunol 1976;116:1554-1560. in a separation method for enzyme immunoassay. Methods 23 Kato K. Use of activated thiol-Sepharose Enzymol 1983;92:345-359. solid-phase radioimmunoassay for 24 Asaka M, Nagase K, Miyazaki T, Alpert E. A non-competitive human aldolase A. Clin Chim Acta 1982;125:31-40. of muscular 25 Asaka M, Nagase K, Shiraishi T, Miyazaki T. Diagnosis of cancer by radioimmunoassay type of aldolase. Gann 1980;71:433-440. of aldolase A, Determination of 26 Asaka M, Nagase K, Miyazaki T, Alpert E. Radioimmunoassay normal serum levels and increased serum concentration in cancer patients. Cancer 1983;51:1873-1878.
