169
Clinica Chimica Acta, 191 (1990) 169-174 Elsevier
CCA04812
A simple enzyme-linked immunoassay of human carbonic anhydrase I for the study of developing erythroid cells Takahito Kondo ‘, Kazuhiro Murakami I, Hiroshi Isobe ‘, Naoyuki Taniguchi ’ and Yoshikazu Kawakami ’ ’ The First Department of Medicine, School of Medicine, Hokkaido University, Sapporo and ’ Department (Received
of Biochemisfry, Medical School, Osaka University, Osaka (Japan)
15 March
Key words: Carbonic
1990; revision anhydrase
received 22 June 1990; accepted I; Enzyme-linked
immunoassay;
25 June 1990) Erythroid
cell
A simple and rapid enzyme-linked immunoassay for human erythrocyte carbonic anhydrase isozyme I was developed. The assay was found to be sensitive enough for the detection of nanogram amounts of the enzyme in incubation mixtures. The first incubation with anti-human carbonic anhydrase I IgG was carried out for 6 hours at room temperature. The second incubation of the enzyme was carried out in the presence of goat plasma Clq coupled with peroxidase for 1 h at room temperature. The enzymatic reaction was performed for 30 min using 2,2’-azino-di(3-ethyl-benzthiazoline sulfonate) as a substrate, and absorbance at 414 nm was recorded. Carbonic anhydrase I was assayed on the range of 1 to 200 ng/ml using this method. The levels of carbonic anhydrase I in K562 cells induced by erythropoietin and in other blood cells were determined. This enzyme-linked immunoassay has application for the study of developing erythroid cells.
Introduction Carbonic anhydrase (EC 4.2.1.1) plays an important role in acid-base equilibrium within the tissues and in the transport of carbon dioxide. There are three
Abbreviations: Enzyme-linked immunoassay, EIA; carbonic anhydrase CA-II: carbonic anhydrase III, CA-III; 2,2’-azino-di(3-ethyl-benzthiazoline Correspondence to: T. Kondo, The First Department of Medicine, University, Sapporo, 060 Japan. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
I, CA-I; carbonic anhydrase II, sulfonate) ABTS. School of Medicine, Hokkaido
B.V. (Biomedical
Division)
170
major isozymes designated as carbonic anhydrase I (CA-I), II (CA-II), and III (CA-III). These isozymes are distinguishable immunologically and genetically [l]. A quantitative immunochemical estimation for them was established by Funakoshi and Deutsch [l]. The levels of CA-I change considerably under some pathological or physiological conditions [2-41. The detectable limit of the amount of CA-I by radial immunodiffusion is in the range of 10 to 1000 pg/ml, and a more sensitive method is required for the estimation of CA-I in various body fluids. Previously we found that iodination of CA-I both by the peroxidase method and by the chloramine T method resulted in loss of the antigenicity of CA-I, and we developed a sensitive and specific radioimmunoassay method for CA-I using a solid-phase technique with labeled antibody [5]. However, this method requires a 3-day procedure and a radioisotope. The present study describes a rapid and simple EIA method for the estimation of low concentrations of CA-I in human erythroid cells. K562 cells are known to possess characteristics of erythroid cells, while HL60 cells to possess those of human myeloid cells. These cells were used in the present study to determine the levels of the enzyme during the differentiation of erythroid cells. Determination of CA-I may be useful as a marker of erythroid cell differentiation. Since the levels of CA-I is quite low in new born erythrocytes, the method described here enable us to study the changes of the enzyme in hereditary and metabolic disorders of erythrocytes [3], using small amounts of samples. Materials and methods Materials
m-Cellulose and microcrystalline cellulose were purchased from Sigma, St. Louis, MO, USA. DE52 and Sepharose 4B were from Pharmacia Fine Chemicals, Uppsala, Sweden. Goat Clq coupled with peroxidase was from Seikagaku Kogyo, Japan. RPM1 1640 and fetal serum were from GIBCO, Grand Island, N.Y., and ABTS from Zymed Laboratories, INC., San Francisco, CA, USA. Authentic erythropoietin was a gift from Chugai Pharmaceutical Co., Japan. K562 human erythroleukemia cells apd HL60 human leukemia cells were obtained from JCRB Cell Bank, Tokyo. Pwzar@ion of samples
K562 cells were grown in RPM1 1640 medium supplemented with 10% fetal calf serum. In order to induce differentiation, K562 cells were treated with 2 U/ml of erythropoietin. The culture medium was drawn on the first to seventh days, and the cells were collected by centrifugation at 500 X g for 10 minutes, and washed three times with isotonic saline. Five hundred milliliters of ice-cold water was added to 1 x lo6 cells and the cells were lysed by freezing at - 20°C and thawing at 37°C. The stroma was removed by centrifugation at 10000 X g for 30 min. Fresh venous blood collected in 1 mg of EDTA/ml was freed of leukocytes and most platelets by passage through a small column of a-cellulose and microcrystalline cellulose (1: 1 w/w) [6]. The erythrocytes were washed three times with isotonic saline. Three volumes of ice-cold water were then added to the erythrocytes and the materials were frozen at -20°C and thawed at 37°C to effect hemolysis. The stroma was
171
removed by centrifugation at 10000 x g for 30 min. Young and old erythrocytes were separated on the basis of specific gravity according to the method described by Murphy [7]. Lymphocytes, and polynuclear leukocytes were prepared by the Ficoll-Paque gradient method. Purification of CA-I
Erythrocyte CA-I was purified from healthy subjects as previously described [8]. Briefly, 100 ml of packed red cells was used as a starting material. Three volumes of ice-cold water were added to the red cells and then they were treated with 3.6 g of NaCl at -4°C. Then 85 ml of 95% ethanol and 85 ml of chloroform previously cooled at - 20°C were added slowly according to the method of Tsuchihashi [9]. After centrifugation of the mixture, the supematant was dialyzed against 1.8 mmol/l Tris-HCl buffer, pH 8.0. The extracts were then loaded onto a DE52 column which had been previously equilibrated with the same buffer. The CA-I containing fractions were further applied to an affinity chromatography column of Sepharose 4B coupled with p-aminobenzene sulfonilamide. The purity of the protein was checked by sodium dodecyl sulfate gel electrophoresis as described by Weber and Osbom [lo]. Preparation of monospecific antiserum
Specific polyclonal antiserum to CA-I was obtained by immunization of a horse with an antigen being employed as in previous studies [ll] . The specificity of the antiserum was checked by double immunodiffusion and immunoelectrophoresis and the antiserum was found to be monospecific as judged by these techniques. Assay procedure
The assay was carried out in disposable polystyrene microtest tissue culture plates. All procedures were performed in triplicate at room temperature. Unless otherwise indicated, 10 pg of anti-human CA-I IgG in 100 ~1 of 50 mmol/l NaHCO,, pH 9.6, was added to each well. The plate was incubated for 6 h, the supematant decanted, and each well washed two times with 200 ~1 of 50 mmol/l potassium phosphate, pH 7.4, containing 0.139 mol/l NaCl, 0.05% (v/v) Tween 20, and 0.1% bovine serum albumin (Buffer A) with shaking. Then 200 ~1 of Buffer A was added to each well, which then stood for 1 h, after which it was washed once with Buffer A. Addition of 20 ng of goat Clq labeled with peroxidase in 50 ~1 of Buffer A was followed by the addition of 50 ~1 of samples or standards in Buffer A. The plate was incubated for 1 h. After washing four times with Buffer A to remove unbound materials, 100 ~1 of 0.04% 2,2’-azino-di(3-ethyl-benzthiazoline sulfonate) (ABTS) in 0.1 mol/l citrate buffer, pH 4.0, containing 0.15% hydrogen peroxide was added to each well. The reaction was performed for 30 min and the change of absorbance at 414 nm was recorded. The standard
Purified human CA-I was dissolved in the above incubation buffer and the concentration of CA-I was adjusted to l-200 ng/ml. The concentration of the enzyme was determined photometrically at 280 nm using El%/cm = 16.3 for CA-I.
172
Effect of antibody concentration The effects of different concentrations of antibody on the binding of the antigen were studied. Anti-CA-I IgG ranging from 1 pg to 10 pg was used for coating each well. The binding capacity depended on the quantity of the antibody used as shown in Fig. 1. The results suggested that the 10 pg of antibody in each well possessed sufficient capacity in the present study. Effect of time on the antiboe coating of wells The effect of time on the first incubation was studied. The incubation was run for 1, 2, 4, 6 and 12 h. Binding of antigen reached steady levels within 6 h at three different antigen ~n~ntrations as shown in Fig. 2. An overnight incubation at 4°C was convenient for the first incubation. Sensitivity of the assay The lower detectable level of the antigen was 1 ng/ml and the working range was l-200 ng/ml as shown in Fig, 1. The SD over the assay range was within 4%. The cellular levels of CA-I This isozyme has been reported to be undetectable in K562 cells [ll]. No CA-I protein was detected in K562 cells in the present study. Incubation of K562 cells with erythropoietin (2 U/ml) resulted in the induction of 19 + 2 ng CA-I/lo6 cells (mean f SD of four different expe~ments) at 12 hours and the levels of CA-I increased to 175 I)r 13 ng CA-I/lo6 cells at 24 hours, and 375 + 17 ng CA-I/lo6 cells at 48 hours as shown in Table I. No CA-I protein was detected in HL60 cells, polynuclear leukocytes, or lymphocytes. Mature erythrocytes from normal subjects were separated into three groups on the basis of specific gravity. The level of CA-I
0.4 -
0.3 z z 8 .E = 0.23
z 9
- ;;Is::: a.': 0 / 12
5
IO 20
50
100 200
Fig. 1. Effect of differ&t concentrations of anti-CA-1 horse IgG on the standard curve. 0, 10 anti-CA-I-&G in each well; A, 2 pg of anti-CA-I-&C, X : 1 pg of anti-CA-I-IgG.
E”gof
173
0
’ 0
I
I
2
4
6 Incubation
8
I
I
10
12
(h)
Fig. 2. Effect of the first incubation time on the uptake of CA-I by antibody-coated wells. The incubation system was comprised of 10 pg (0) and 2 gg (0) of anti-CA-I-IgG, and 50 pg of CA-I. The value represents a mean of two experiments with tripricate analysis.
in young erythrocytes (the top 20% of the fractionated cells) was 810 f 20 ng/106 cells, that in middle aged erythrocytes (the middle 404&of the fractionated cells) was 710 + 14 ng/106 cells, and in old erythrocytes (the bottom 20% of the fractionated cells) it was 590 * 22 ng/106 cells. The antibody coated wells are stable at 4°C for 7 days. The incubation with enzyme samples and goat Clq was carried out for 1 h. Further addition of the substrate, ABTS, showed a reaction in 30 min. This EIA method is very simple and rapid.
TABLE I Levels of CA-I in blood cells Cells
Incubation
CA-I (ng/106 cells)
K562
control erythropoietin for 12 h erythropoietin for 24 h erythropoietin for 48 h
n.d. a 19* 2 175 f 13 375 f 17
HL60
control erythropoietin 24 h
n.d. n.d.
Lymphocytes
n.d.
Leukocytes
n.d.
Erythrocytes young middle old
81Ok20 710*14 590+22
a n.d., not detected; values are mean f SD of the four different experiments.
174
Discussion In the present study, we developed a method for the estimation of CA-I. Clq specifically binds to antigen-antibody complexes [12]. Addition of Clq bound with peroxidase to anti-CA-I-IgG-coated wells resulted in the formation of immune complexes in the presence of CA-I. Previously we found that the specific activity of CA-I is appro~mately one fifth of that of CA-II in normal human erythrocytes. The concentration of CA-I is approximately 14 mg/g Hb and that of CA-II is approximately 3.2 mg/g Hb in these cells [2]. Since the activity of CA-I decreases in the presence of physiological concentrations of electrolytes [13], the physiological role of CA-I has not been fully elucidated. Weil et al. [14] reported that CA-I is expressed in erythroid cells during e~~opoiesis in vitro and, recently, Villeval et al. ]lS] reported that CA-I is an early specific marker of normal human erythroid differentiation. In the present study the amount of CA-I expressed in response to erythropoietin was determined in erytbroid cells. Further study on the regulatory mechanism of the expression of CA-I in these cells may provide an explanation of the physiological significance of CA-I. References 1 Funakoshi S, Deutsch HF. Human carbonic anhydrase. J Biol Chem 1970;245:2852-2856, 2 Kondo T, Taniguchi N, Murao M, Takakuwa E. Estimation of active and inactive carbonic anhydrase B in human red cells. Clin Chim Acta 1975$X347-353. 3 Kondo I’, Tar&u&i N, Tan&u&i K, Matauda I, Murao M. Inactive form of erythrooyte carbonic anhydrase isozyme B in patients with primary renal tubular acidosis. J Chn Invest 197~42:610-617. 4 Kondo T, Murakami K, Ohtsuka Y, et al. Estimation and characterization of glycosyiated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin Chim Acta 1987;166:227-236. 5 Taniguchi N, Kondo T, Ishikawa N, Ohno H, Takakuwa E, Matsuda I. A solid-phase radioimmunoassay for the human carbonic anhydrase. Anal Biochem 1976;72:144-155. 6 Beutler E, West C, Blume G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med 1976;88:328-333. 7 Murphy JR. Influence of temperature and method of ~nt~fugation on the separation of erythrocytes. J Lab CIin Ned 1973;82:334-341. 8 Kondo T, Taniguchi N, Hirano T, Kawakami Y. A novel lowactivity form of carbonic anhydrase I in erythrocytes of patients with primary aldosteronism: evidence for the presence of a mixed disulfide with glutathione. J Biol Chem 1984,259:15517-15522. 9 Tsuchihasbi M. Zur Kenntnis der Blutkatalase. Biochem Z 1923;40:63-112. 10 Weber K, Osbom M. The re~abi~ty of molecular weight dete~nation by dodecyl sulfate-polyacrylamide gel electrophoresis. J Bid Chem 1%6;244:4406-4412. 11 Rutherford TR, Clego JB, Weatherall DJ. K562 human leukaemic cells synthesise embryonic haemoglobin in response to haemin. Nature 1979;280:164-165. 12 Kolb WP, Kolb LM, Podack ER. Clq: Isolation from human serum in high yield by affinity chromato@yaphy and development of a highly sensitive hemolytic assay. J Biol Chem 1979;122:21032111. 13 Wistrand PJ. The importanceof carbonic anhydrase B and C for the loading of CO, by the human erythrocytes. Acta Physiol Stand 1981;113:417-426. 14 Weil SC, Walloch J, Frankel SR, Hirata RK. Expression of carbonic anhydrase and globin during erythropoiesis in vitro. Ann NY Acad Sci 1984,429:335-337. 15 Villeval JL, Testa U, Vinvi G, et al. Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation. Blood 1985;66:1162-1170.
Clinica Chimica Acta, 191 (1990) 169-174 Elsevier
CCA04812
A simple enzyme-linked immunoassay of human carbonic anhydrase I for the study of developing erythroid cells Takahito Kondo ‘, Kazuhiro Murakami I, Hiroshi Isobe ‘, Naoyuki Taniguchi ’ and Yoshikazu Kawakami ’ ’ The First Department of Medicine, School of Medicine, Hokkaido University, Sapporo and ’ Department (Received
of Biochemisfry, Medical School, Osaka University, Osaka (Japan)
15 March
Key words: Carbonic
1990; revision anhydrase
received 22 June 1990; accepted I; Enzyme-linked
immunoassay;
25 June 1990) Erythroid
cell
A simple and rapid enzyme-linked immunoassay for human erythrocyte carbonic anhydrase isozyme I was developed. The assay was found to be sensitive enough for the detection of nanogram amounts of the enzyme in incubation mixtures. The first incubation with anti-human carbonic anhydrase I IgG was carried out for 6 hours at room temperature. The second incubation of the enzyme was carried out in the presence of goat plasma Clq coupled with peroxidase for 1 h at room temperature. The enzymatic reaction was performed for 30 min using 2,2’-azino-di(3-ethyl-benzthiazoline sulfonate) as a substrate, and absorbance at 414 nm was recorded. Carbonic anhydrase I was assayed on the range of 1 to 200 ng/ml using this method. The levels of carbonic anhydrase I in K562 cells induced by erythropoietin and in other blood cells were determined. This enzyme-linked immunoassay has application for the study of developing erythroid cells.
Introduction Carbonic anhydrase (EC 4.2.1.1) plays an important role in acid-base equilibrium within the tissues and in the transport of carbon dioxide. There are three
Abbreviations: Enzyme-linked immunoassay, EIA; carbonic anhydrase CA-II: carbonic anhydrase III, CA-III; 2,2’-azino-di(3-ethyl-benzthiazoline Correspondence to: T. Kondo, The First Department of Medicine, University, Sapporo, 060 Japan. 0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
I, CA-I; carbonic anhydrase II, sulfonate) ABTS. School of Medicine, Hokkaido
B.V. (Biomedical
Division)
170
major isozymes designated as carbonic anhydrase I (CA-I), II (CA-II), and III (CA-III). These isozymes are distinguishable immunologically and genetically [l]. A quantitative immunochemical estimation for them was established by Funakoshi and Deutsch [l]. The levels of CA-I change considerably under some pathological or physiological conditions [2-41. The detectable limit of the amount of CA-I by radial immunodiffusion is in the range of 10 to 1000 pg/ml, and a more sensitive method is required for the estimation of CA-I in various body fluids. Previously we found that iodination of CA-I both by the peroxidase method and by the chloramine T method resulted in loss of the antigenicity of CA-I, and we developed a sensitive and specific radioimmunoassay method for CA-I using a solid-phase technique with labeled antibody [5]. However, this method requires a 3-day procedure and a radioisotope. The present study describes a rapid and simple EIA method for the estimation of low concentrations of CA-I in human erythroid cells. K562 cells are known to possess characteristics of erythroid cells, while HL60 cells to possess those of human myeloid cells. These cells were used in the present study to determine the levels of the enzyme during the differentiation of erythroid cells. Determination of CA-I may be useful as a marker of erythroid cell differentiation. Since the levels of CA-I is quite low in new born erythrocytes, the method described here enable us to study the changes of the enzyme in hereditary and metabolic disorders of erythrocytes [3], using small amounts of samples. Materials and methods Materials
m-Cellulose and microcrystalline cellulose were purchased from Sigma, St. Louis, MO, USA. DE52 and Sepharose 4B were from Pharmacia Fine Chemicals, Uppsala, Sweden. Goat Clq coupled with peroxidase was from Seikagaku Kogyo, Japan. RPM1 1640 and fetal serum were from GIBCO, Grand Island, N.Y., and ABTS from Zymed Laboratories, INC., San Francisco, CA, USA. Authentic erythropoietin was a gift from Chugai Pharmaceutical Co., Japan. K562 human erythroleukemia cells apd HL60 human leukemia cells were obtained from JCRB Cell Bank, Tokyo. Pwzar@ion of samples
K562 cells were grown in RPM1 1640 medium supplemented with 10% fetal calf serum. In order to induce differentiation, K562 cells were treated with 2 U/ml of erythropoietin. The culture medium was drawn on the first to seventh days, and the cells were collected by centrifugation at 500 X g for 10 minutes, and washed three times with isotonic saline. Five hundred milliliters of ice-cold water was added to 1 x lo6 cells and the cells were lysed by freezing at - 20°C and thawing at 37°C. The stroma was removed by centrifugation at 10000 X g for 30 min. Fresh venous blood collected in 1 mg of EDTA/ml was freed of leukocytes and most platelets by passage through a small column of a-cellulose and microcrystalline cellulose (1: 1 w/w) [6]. The erythrocytes were washed three times with isotonic saline. Three volumes of ice-cold water were then added to the erythrocytes and the materials were frozen at -20°C and thawed at 37°C to effect hemolysis. The stroma was
171
removed by centrifugation at 10000 x g for 30 min. Young and old erythrocytes were separated on the basis of specific gravity according to the method described by Murphy [7]. Lymphocytes, and polynuclear leukocytes were prepared by the Ficoll-Paque gradient method. Purification of CA-I
Erythrocyte CA-I was purified from healthy subjects as previously described [8]. Briefly, 100 ml of packed red cells was used as a starting material. Three volumes of ice-cold water were added to the red cells and then they were treated with 3.6 g of NaCl at -4°C. Then 85 ml of 95% ethanol and 85 ml of chloroform previously cooled at - 20°C were added slowly according to the method of Tsuchihashi [9]. After centrifugation of the mixture, the supematant was dialyzed against 1.8 mmol/l Tris-HCl buffer, pH 8.0. The extracts were then loaded onto a DE52 column which had been previously equilibrated with the same buffer. The CA-I containing fractions were further applied to an affinity chromatography column of Sepharose 4B coupled with p-aminobenzene sulfonilamide. The purity of the protein was checked by sodium dodecyl sulfate gel electrophoresis as described by Weber and Osbom [lo]. Preparation of monospecific antiserum
Specific polyclonal antiserum to CA-I was obtained by immunization of a horse with an antigen being employed as in previous studies [ll] . The specificity of the antiserum was checked by double immunodiffusion and immunoelectrophoresis and the antiserum was found to be monospecific as judged by these techniques. Assay procedure
The assay was carried out in disposable polystyrene microtest tissue culture plates. All procedures were performed in triplicate at room temperature. Unless otherwise indicated, 10 pg of anti-human CA-I IgG in 100 ~1 of 50 mmol/l NaHCO,, pH 9.6, was added to each well. The plate was incubated for 6 h, the supematant decanted, and each well washed two times with 200 ~1 of 50 mmol/l potassium phosphate, pH 7.4, containing 0.139 mol/l NaCl, 0.05% (v/v) Tween 20, and 0.1% bovine serum albumin (Buffer A) with shaking. Then 200 ~1 of Buffer A was added to each well, which then stood for 1 h, after which it was washed once with Buffer A. Addition of 20 ng of goat Clq labeled with peroxidase in 50 ~1 of Buffer A was followed by the addition of 50 ~1 of samples or standards in Buffer A. The plate was incubated for 1 h. After washing four times with Buffer A to remove unbound materials, 100 ~1 of 0.04% 2,2’-azino-di(3-ethyl-benzthiazoline sulfonate) (ABTS) in 0.1 mol/l citrate buffer, pH 4.0, containing 0.15% hydrogen peroxide was added to each well. The reaction was performed for 30 min and the change of absorbance at 414 nm was recorded. The standard
Purified human CA-I was dissolved in the above incubation buffer and the concentration of CA-I was adjusted to l-200 ng/ml. The concentration of the enzyme was determined photometrically at 280 nm using El%/cm = 16.3 for CA-I.
172
Effect of antibody concentration The effects of different concentrations of antibody on the binding of the antigen were studied. Anti-CA-I IgG ranging from 1 pg to 10 pg was used for coating each well. The binding capacity depended on the quantity of the antibody used as shown in Fig. 1. The results suggested that the 10 pg of antibody in each well possessed sufficient capacity in the present study. Effect of time on the antiboe coating of wells The effect of time on the first incubation was studied. The incubation was run for 1, 2, 4, 6 and 12 h. Binding of antigen reached steady levels within 6 h at three different antigen ~n~ntrations as shown in Fig. 2. An overnight incubation at 4°C was convenient for the first incubation. Sensitivity of the assay The lower detectable level of the antigen was 1 ng/ml and the working range was l-200 ng/ml as shown in Fig, 1. The SD over the assay range was within 4%. The cellular levels of CA-I This isozyme has been reported to be undetectable in K562 cells [ll]. No CA-I protein was detected in K562 cells in the present study. Incubation of K562 cells with erythropoietin (2 U/ml) resulted in the induction of 19 + 2 ng CA-I/lo6 cells (mean f SD of four different expe~ments) at 12 hours and the levels of CA-I increased to 175 I)r 13 ng CA-I/lo6 cells at 24 hours, and 375 + 17 ng CA-I/lo6 cells at 48 hours as shown in Table I. No CA-I protein was detected in HL60 cells, polynuclear leukocytes, or lymphocytes. Mature erythrocytes from normal subjects were separated into three groups on the basis of specific gravity. The level of CA-I
0.4 -
0.3 z z 8 .E = 0.23
z 9
- ;;Is::: a.': 0 / 12
5
IO 20
50
100 200
Fig. 1. Effect of differ&t concentrations of anti-CA-1 horse IgG on the standard curve. 0, 10 anti-CA-I-&G in each well; A, 2 pg of anti-CA-I-&C, X : 1 pg of anti-CA-I-IgG.
E”gof
173
0
’ 0
I
I
2
4
6 Incubation
8
I
I
10
12
(h)
Fig. 2. Effect of the first incubation time on the uptake of CA-I by antibody-coated wells. The incubation system was comprised of 10 pg (0) and 2 gg (0) of anti-CA-I-IgG, and 50 pg of CA-I. The value represents a mean of two experiments with tripricate analysis.
in young erythrocytes (the top 20% of the fractionated cells) was 810 f 20 ng/106 cells, that in middle aged erythrocytes (the middle 404&of the fractionated cells) was 710 + 14 ng/106 cells, and in old erythrocytes (the bottom 20% of the fractionated cells) it was 590 * 22 ng/106 cells. The antibody coated wells are stable at 4°C for 7 days. The incubation with enzyme samples and goat Clq was carried out for 1 h. Further addition of the substrate, ABTS, showed a reaction in 30 min. This EIA method is very simple and rapid.
TABLE I Levels of CA-I in blood cells Cells
Incubation
CA-I (ng/106 cells)
K562
control erythropoietin for 12 h erythropoietin for 24 h erythropoietin for 48 h
n.d. a 19* 2 175 f 13 375 f 17
HL60
control erythropoietin 24 h
n.d. n.d.
Lymphocytes
n.d.
Leukocytes
n.d.
Erythrocytes young middle old
81Ok20 710*14 590+22
a n.d., not detected; values are mean f SD of the four different experiments.
174
Discussion In the present study, we developed a method for the estimation of CA-I. Clq specifically binds to antigen-antibody complexes [12]. Addition of Clq bound with peroxidase to anti-CA-I-IgG-coated wells resulted in the formation of immune complexes in the presence of CA-I. Previously we found that the specific activity of CA-I is appro~mately one fifth of that of CA-II in normal human erythrocytes. The concentration of CA-I is approximately 14 mg/g Hb and that of CA-II is approximately 3.2 mg/g Hb in these cells [2]. Since the activity of CA-I decreases in the presence of physiological concentrations of electrolytes [13], the physiological role of CA-I has not been fully elucidated. Weil et al. [14] reported that CA-I is expressed in erythroid cells during e~~opoiesis in vitro and, recently, Villeval et al. ]lS] reported that CA-I is an early specific marker of normal human erythroid differentiation. In the present study the amount of CA-I expressed in response to erythropoietin was determined in erytbroid cells. Further study on the regulatory mechanism of the expression of CA-I in these cells may provide an explanation of the physiological significance of CA-I. References 1 Funakoshi S, Deutsch HF. Human carbonic anhydrase. J Biol Chem 1970;245:2852-2856, 2 Kondo T, Taniguchi N, Murao M, Takakuwa E. Estimation of active and inactive carbonic anhydrase B in human red cells. Clin Chim Acta 1975$X347-353. 3 Kondo I’, Tar&u&i N, Tan&u&i K, Matauda I, Murao M. Inactive form of erythrooyte carbonic anhydrase isozyme B in patients with primary renal tubular acidosis. J Chn Invest 197~42:610-617. 4 Kondo T, Murakami K, Ohtsuka Y, et al. Estimation and characterization of glycosyiated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin Chim Acta 1987;166:227-236. 5 Taniguchi N, Kondo T, Ishikawa N, Ohno H, Takakuwa E, Matsuda I. A solid-phase radioimmunoassay for the human carbonic anhydrase. Anal Biochem 1976;72:144-155. 6 Beutler E, West C, Blume G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med 1976;88:328-333. 7 Murphy JR. Influence of temperature and method of ~nt~fugation on the separation of erythrocytes. J Lab CIin Ned 1973;82:334-341. 8 Kondo T, Taniguchi N, Hirano T, Kawakami Y. A novel lowactivity form of carbonic anhydrase I in erythrocytes of patients with primary aldosteronism: evidence for the presence of a mixed disulfide with glutathione. J Biol Chem 1984,259:15517-15522. 9 Tsuchihasbi M. Zur Kenntnis der Blutkatalase. Biochem Z 1923;40:63-112. 10 Weber K, Osbom M. The re~abi~ty of molecular weight dete~nation by dodecyl sulfate-polyacrylamide gel electrophoresis. J Bid Chem 1%6;244:4406-4412. 11 Rutherford TR, Clego JB, Weatherall DJ. K562 human leukaemic cells synthesise embryonic haemoglobin in response to haemin. Nature 1979;280:164-165. 12 Kolb WP, Kolb LM, Podack ER. Clq: Isolation from human serum in high yield by affinity chromato@yaphy and development of a highly sensitive hemolytic assay. J Biol Chem 1979;122:21032111. 13 Wistrand PJ. The importanceof carbonic anhydrase B and C for the loading of CO, by the human erythrocytes. Acta Physiol Stand 1981;113:417-426. 14 Weil SC, Walloch J, Frankel SR, Hirata RK. Expression of carbonic anhydrase and globin during erythropoiesis in vitro. Ann NY Acad Sci 1984,429:335-337. 15 Villeval JL, Testa U, Vinvi G, et al. Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation. Blood 1985;66:1162-1170.
