THEJOURNAL OF BIOLOGICAL CHEMISTRY
Communication
Isolation of Human Erythropoietin
with Monoclonal Antibodies*
(Received for publication, October 3,1983)
Shin-ichi Yanagawa, Kumiko Hirade, Hideki
Ohnota, Ryuzo SasakiS, HidmChiba, Masatsugu
Uedag, and Masaaki Gotog
From the Department of Food Scienceand Technology,
Faculty of Agriculture, Kyoto University, Kyoto 606, Japan
and the §Research Institute of Life Science, SnowBrand
Milk Products Co., Ltd. 519,Shimoishibashi, Zshibashi-
machi, Shirnotsuga-gun, Tochigi 329-05,Japan
Human erythropoietin was isolated from urine of
aplastic anemic patients in a high yield with a simple
purification procedure using animmunoadsorbent col-
umn of monoclonal antibodies and a Sephadex G - 1 0 0
column. About 6 mg of erythropoietin was isolated
from 700 liters of urine and the specific activity was
estimated to be 81,600 unitslmg of protein with an in
vivo 68Fe incorporation assay method, using starved
rats. Activity measurement of the extracts from sliced
gels after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and the Western blotting technique re-
vealed heterogeneityofthe isolated erythropoietin,
which is probably caused by variable amounts of car-
bohydrates attached to the polypeptide chain. Thirty
amino acids inthe NH2-terminal portion of the isolated
hormone were sequenced.
Erythropoietin is a sialoglycoprotein which is believed to
play an important role in regulating, by stimulating, eryth-
ropoiesis. Purification of erythropoietin from the urine of
patients with aplastic anemia,using conventional purification
procedures, has been reported (1, 2). However, laborious pu-
rification procedures with low yields and limited supplies of
starting material have prevented us from obtaining amounts
of pureerythropoietinsuitable for studying its structure,
mechanism of action, and metabolism. We have prepared a
stable hybridoma clone that secretes monoclonal antibody
against human urinary erythropoietin.’ We describe here a
very simple isolation procedure with a high yield of erythro-
poietin from humanurine by the use of this monoclonal
antibody.
EXPERIMENTALPROCEDURES
Materials-Erythropoietin (38,000units/mg of protein with in vivo
assay method) was purified from human urine by a combination of
conventional methods, immunoadsorbent columns against contami-
nants and preparative SDS’ polyacrylamide gel electrophoresis as a
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
$ T o whom reprint requests and correspondence should be ad-
dressed at, Department of Food Science and Technology, Faculty of
Agriculture, Kyoto University, Kyoto 606, Japan.
‘S. Yanagawa, S . Yokoyama, K. Hirade, R. Sasaki, H. Chiba, M.
Ueda, and M. Goto, submitted for publication.
’ The abbreviations used are: SDS, sodium dodecyl sulfate; PBS,
10 mM phosphate-buffered saline.
2707
Vol. 259, No. 5. Issue of March 10, pp. 2707-2710,1984
0 1984 by The American Society of Biological Chemists, Inc.
Printed in U.S.A.
final step. The hybridoma secreting monoclonal antibody against
erythropoietin was prepared by fusion of mouse myeloma cells with
spleen cells from a mouse receiving the first immunization with the
erythropoietin antigen in SDS-polyacrylamidegel suspension; the gel
after SDS-polyacrylamide gel electrophoresis for the final step of
erythropoietin purification was sliced and the sliced gel containing
erythropoietin protein was ground. The animal was boosted with the
purified erythropoietin. A large amount of the monoclonal antibody
(IgG,) against erythropoietin was purified from mouse ascitic fluids.
Further details of all mentioned above have been described elsewhere.’
The antibody wasfixed on Affi-Gel 10 (3). Urine, collected from
anemic patients, was filtered under suction, and concentrated by
ultrafiltration on a hollow-fiber device (Amicon) with a nominal M,
cutoff of 10,000, The urine concentrate was lyophilized. Standard
erythropoietin (sheep plasma erythropoietin step 111, 4 units/mg of
protein) was obtained from Connaught Medical Research Laborato-
ries, Willowdale, Canada.
Assay for Erythropoietin-For selection of urine from anemic pa-
tients with high erythropoietin titers, the activity was assayed by
counting erythroid colonies (4). Erythropoietin was assayed in uitro,
utilizing the stimulatory effect of erythropoietin on incorporation of
59Feinto heme (5) or [3H]thymidine into DNA (6) in cultured mouse
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fetal liver cells. Each value shown in this paper is the mean of
triplicate cultures. The erythropoietin activity was determined also
in uiuo using the starved rat (4 rats/sample) (7).
SDS-PolyacrylamideGel Electrophoresisand Erythropoietin Actiu-
ity in Gel Extracts-SDS-polyacrylamide gel electrophoresis was per-
formed by the method of Laemmli (8) and stained with silver accord-
ing to the manufacturer’s directions (Bio-Rad). In some cases, eryth-
ropoietin activity of extracts from sliced gels was measured. The gel
without staining was sliced into 1-mm lengths. The gel pieces were
putinto test tubes containing PBS,pH 7.4, 0.1% bovine serum
albumin (0.5 mllsliced gel) and ground with a glass rod. After incu-
bating the suspensions overnight to extract protein, they were cen-
trifuged to obtain the clear supernatants. The activity in the super-
natants was measured with an in uitro [3H]thymidinemethod ( 6 ) .
Amino Acid Sequence-Amino acid sequence of pure erythropoietin
was determined with an Applied Biosystems 470A automatic sequen-
ator, a gas phase sequenator (9).
RESULTS
Purification of Erythropoietin from Urine Concentrate-A
concentrate from about 700 liters of urine was applied on a
column in which erythropoietin-directed monoclonal antibody
fixed on Affi-Gel 10 was packed. The column was extensively
washed and thendeveloped with the pH2.5 buffer. As shown
in Fig. 1,purification with the immunoadsorbent column was
tremendously effective; most of the protein in the urine con-
centrate emerged in the flow-through fractions without being
adsorbed, and erythropoietin was eluted sharply by the pH
2.5 buffer. About 2600-fold purification was achieved by this
single step and 75% of the activity was recovered (Table I).
The presence of SDS in heat treatment of the urine con-
centrate is needed for erythropoietin to be retained on the
column. Erythropoietin mostly appeared in the flow-through
fractions on the heat treatment in the absence of SDS. Activ-
ity measurement after dilution of the SDS-treated urine con-
centrate with PBS or the erythropoietin assay medium
showed little loss of activity during the SDS treatment;since
the activity was measured with the SDS-treatedurine concen-
trate diluted more than lOOO-fold, effects of SDS on the target
cells (fetal mouse liver cells) were avoided. Neither erythro-
poietin in the concentrate diluted back to the original urine
volume nor that in the urine before concentration was ad-
sorbed on the column unless the sample was subjected to heat
treatment in the presence of SDS. Erythropoietin eluted from
2708 Isolation of Human Erythropoietin
1 2 3 4 5 6 .

FRACTION NUMBER
FIG.1. Purification of erythropoietin from human urine
concentrate with an immunoadsorbent column containing
monoclonal antibody against erythropoietin. 0, absorbance at
280 nm; 0, erythropoietin activity assayed with in uitro ['Hlthymidine

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incorporation method. The lyophilized urine concentrate (350g) was
dissolved in 700 ml of PBS, pH7.4,and dialyzed extensively. Insoluble
materials were removed by centrifugation and the supernatant (690 FIG.2. Erythropoietin purified with the immunoadsorbent
ml) was heated at 100 "C for 3 min after the addition of solid SDS column was analyzed with SDS-polyacrylamide gel electro-
(final concentration 2%). The SDS-treated solution was cooled and phoresis. Lanes I and 6,M, standards (94,000,phosphorylase b;
left overnight at 0 "C. The precipitated SDS was removedby centrif- 67,000,bovine serum albumin; 43,000,ovalbumin; 30,000,carbonic
ugation and the supernatant (680 ml) was loaded on an immunoad- anhydrase; 20,000,soybean trypsin inhibitor; 14,400,a-lactoalbumin).
sorbent column (3.2 X 2.5 cm) equilibrated with the PBS containing Lane 2, urine sample applied on the immunoadsorbent column; lane
716 mg of the erythropoietin-directed monoclonal antibody fixed on 3, flow-through fractions; lane 4, eluted erythropoietin; lane 5,eryth-
Affi-Gel 10. The column was extensively washed with 1 liter of the ropoietin purified by a combination of conventional methods and
PBS, 500 ml of 10 mM Napi, pH7.4,0.5 M NaCI, and 500 ml of 0.15 immunoadsorbents against contaminants.
M NaCI, in this order, and theneluted by reverse flow of0.2 M acetate,
pH 2.5,0.15 M NaCI. The eluted fractions (112ml) were immediately
neutralized by adding 5.3 ml of 3.4 M Tris. The flow rate was 24 ml/ vealed two peaks of protein (Fig. 3A). It was found by meas-
h duringapplication of the sample and elution with the pH 2.5 buffer, uring the activity in the pooled preparation of each peakthat
while it was 200 ml/h during washing of the column. The volume of most of the erythropoietin activity was recovered in the sec-
one fraction was 8 ml. ond peak (Table I) and that about 5% of the total recovered
activity appeared in the void fractions (the first peak). The
TABLE I faint brown appeared in the void fractions, while the pooled
Purification of erythropoietinfrom urine concentrate erythropoietin preparation was quite transparent. Fig. 3B
Urine concentrate from about 700 liters of human urine was used shows the analytical results of the Sephadex G-100fractions
as the startingmaterial. Protein was measured with Coomassie bril- on SDS-polyacrylamidegel electrophoresis. Contaminants in-
liant blue binding assay (lo),using ovalbumin as a standard. Eryth- cluding the main contaminant, the MI20,000 protein, which
ropoietin activity was measured with an in uitro ['Hlthymidine in- were found in the erythropoietin preparation purified with
corporation using fetal liver cells as the target (4,6) or with an in
uiuo 59Fe incorporation method, using starved rats (7). the immunoadsorbent column, were not detected inany frac-
tions of the second peak (Fig. 3B, lanes 43-53). As is most
Specific Purifi- Specific
Activity
in vitro ield activity actiyity clearly seen in fraction 48 (Fig. 3B, lane 481, however, they
in vitro m vwo contained faint but detectable protein components on the
mg units X 10" % units/mg units/mg leading sideof the erythropoietin main band with MI35,000.
Urine concentrate 34 X lo3 792 100 23 1 14 Activity measurements in the extracts from sliced gels indi-
Immunoadsorbent cated that there is erythropoietin activity in the leading side
column 59,400
59,000
2,580
7559410 as well as in the main band (Fig. 3C), suggesting heterogeneity
Sephadex G-100 of the erythropoietin protein. It was proved withthe Western
5.7 500
63 8
8
O,O
O 3.830 81,600
blotting technique that the components in the leading side
are indeed erythropoietin protein; the monoclonal antibody
the immunoadsorbent column was no longer adsorbed on a against erythropoietin reacted with these components (Fig. 4,
newly madecolumn but when subjectedto theSDS treatment lane 3 ) . The erythropoietin protein with M, 35,000 binds
it was adsorbed again. properly with the antibody. Protein species which migrated
Erythropoietin purified withthe immunoadsorbentcolumn somewhat faster thanthe erythropoietin main band and
wasrecognized as a main band with MI 35,000 on SDS- bound with the antibody were also present in the erythropo-
polyacrylamide gel electrophoresis, but some other protein ietin preparation before SephadexG-100column chromatog-
bands, including a MI 20,000 protein whichwas a main raphy (Fig. 4, lane 2). Thus, the final erythropoietin prepa-
contaminant, were seen (Fig. 2, lane 4 ) . Furthermore, this ration contains erythropoietin species with mobilities larger
preparation was faintly tinged with brown. than theM,35,000 erythropoietin, although at only 510% of
Further purification with a Sephadex G-100 column re- the amount of MI 35,000 erythropoietin protein, based on
 Isolation
Erythropoietin
of Human 2709

Mr 1 2 3 4 5
0.4 1

43K-

30K

2 OK-

FRACTION NUMBER FIG. 4. Identification of erythropoietin with the Western

blotting technique, using erythropoietin-directed monoclonal
antibody. Western blotting was carried out toidentify erythropoietin
( B) on SDS-polyacrylamide gel electrophoresis according to the method
of Burnette (16). Samples (about 2 pg of protein of each) were
subjected to the electrophoresis and the fractionated proteins were
transferred to nitrocellulose paper. The blot was reacted with the

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monoclonal antibody against erythropoietin (20 pglml), followed by
the peroxidase-conjugated anti-mouse IgG. The erythropoietin pro-
tein was visualized by incubating the blot with the substrates (Hz02
and 4-chloro-1-naphthol) of peroxidase. Lane 1 , erythropoietin puri-
fied by a combination of conventional methods and immunoadsorbent
against contaminants;lane 2, fraction eluted from the immunoadsor-

.
I
20 10 0
bents column with the pH 2.5 buffer (see Fig. 1); lane 3, the second
peak from the Sephadex G-100 column chromatography (see Fig. 3A 1;
lane 4, the first peak; lane 5, the sialidase-treated second peak. The
second peak (0.4 ml) from the Sephadex G-100 column was brought
ACTIVlTY(ulml) to about pH 5.5 by adding 0.4 M acetate, pH 5.2, and incubated with
FIG.3. Sephadex 6-100chromatography of erythropoietin 0.1 unit of sialidase for 5 h at 37 "C.The sialidase was inactivated by
fraction purified withthe immunoadsorbent column. The heating at 100 "C for 5 min.
erythropoietin fraction eluted from the immunoadsorbent column
(Fig. 1) was extensively dialyzed against distilled water and lyophi-
lized. The dried material was dissolved in 2 ml of PBS, pH 6.9, and that themain component in the void fractions was MI35,000
the clear solution was loaded on aSephadex G-100 column (1.2 X 130 protein. Furthermore, it was shown that thiscomponent was
cm) equilibrated with the PBS and the column was developedat 4 "C indeed erythropoietin protein; erythropoietin activity was
with a speed of 6 ml/h. The volume of one fractionwas 2 ml. A shows
the elution pattern of protein based on absorbance a t 280 nm. The found inthe extracts from sliced gels containingthe MI35,000
horizontal lines with arrowheads indicate fractions pooled after elu- component after SDS-polyacrylamide gel electrophoresis of
tion. The pooled fractions were designated the first peak (or the void fraction 36 (not illustrated) and Western blotting revealed
fraction) and the second fraction (or the erythropoietin fraction). B the binding of this component with the monoclonal antibody
shows SDS-polyacrylamide gel electrophoresis of fractions (20 pl of against erythropoietin (Fig. 4, lane 4 ) . Interestingly, the MI
each). M,,M,standards (same as Fig. 2); lane numbers correspond to 20,000protein did not bind withthe antibody. Activity meas-
the fractions of Sephadex G-100 chromatography. C shows erythro-
poietin activity of the extracts from sliced gels. The othergel to which urement of fraction 36, in which there should be no contam-
fraction 48 was applied, was sliced into 1-mm lengths without stain- ination of erythropoietin from the second peak, showed about
ing. The gel pieces were put into test tubes containing 0.5 ml (per '/I6 of the specific activity of the secondpeak,basedon
sliced gel) of PBS, 0.1% bovine serum albumin and ground with a absorbance at 280 nm.More than 50% of the protein of
glass rod. After incubating the suspensions overnight to extract fraction 36,however, appears to be the M,35,000protein (Fig.
protein, they were centrifuged to obtain the clear supernatants. The 3B,lane 36) from the intensity of protein staining. Therefore,
activity in the supernatants was measured with the in uitm [3H]
thymidine incorporation method. it seems that erythropoietin molecules in the void fractions
have much lower activity than those in the second peak (see
"Discussion"). In spite of the Occurrenceof the undefined
silver-staining intensity. The erythropoietin preparation, erythropoietin species on Sephadex G-100 chromatography,
which we purified and used as an immunogen for raising purification of human urinary erythropoietin with an immu-
erythropoietin-directed hybridomas, also contained such noadsorbent and a Sephadex G-100 column provides pure
erythropoietin species (Fig. 4, lane 1 ). Treatment with siali- erythropoietin in a high yield (Table I).
dase converted the main MI35,000 band to one with larger Characterization of the Purified Erythropoietin-Protein
mobility (Fig. 4, lane 5 ) , which suggests that electrophoretic concentrations were determined with a Coomassie brilliant
heterogeneity of erythropoietin is, at least in part, due to its blue binding assay according to themethod of Bradford (10).
variable amount of sialic acid attached to erythropoietin pro- A value of E$&mm for pure erythropoietin was calculated to be
tein. 13.1 when a standard curve was made with ovalbumin and
The main M,20,000contaminant and othersin the eryth- 19.7 withbovine serum albumin. The formervalue was
ropoietin preparation eluted from the immunoadsorbent col- adopted here becauseof the unusual behavior of bovine serum
umn appeared in the void fractions on Sephadex G-100chro- albumin in this assay method. A value of EGnm = 12.6 was
matographv (Fig. 3B, lanes 36-38). It was rather surprising also obtained by the method of Lowry et al. (11)using bovine
2710 Isolation of Human Erythropoietin
serum albumin as standard. The specific activity of purified inaccessible to the antibody, but exposed in the presence of
erythropoietin was determined to be 88,000 units/mg of pro- SDS. It is also possible that the antibody recognizes a struc-
tein with an in vitro [3H]thymidineincorporation method and ture formed by the binding of SDS to erythropoietin. The
81,600 with an in vivo starved rat method (see Table I). Assay binding of erythropoietin to the immunoadsorbent column
with the in vitro 59Fe incorporation method gave a similar can occur only in the presence of SDS but there appears to
value. A specific activity of 70,400 units/mg of protein has be no problems concerning the eluted erythropoietin. The
been reported for erythropoietin isolated from human urine purified erythropoietin no longer binds with the antibody,
with conventional purification procedures by Miyake et al. (2) and has a specific activity comparable to the value for pure
using E;$”,,, = 8.5. erythropoietin (2). The immunoadsorbent column is so exten-
Sialidase treatment (see legend of Fig. 4) of our erythropo- sively washed before the elution of the erythropoietin that, if
ietin completely abolished in vivo activity, while in vitro SDS remains in theeluted erythropoietin,the amount would
activity increased 1.3-fold. Similar results have been reported be very small.
with erythropoietinpartially purified from the plasma of The presence of the erythropoietin molecule with a low
anemic sheep (12, 13). Loss of in vivo activity would becaused specific activity in the void fractions of Sephadex G-100
by the hepatic removal of asialoglycoproteins from the circu- chromatography is puzzling. The void fractions may contain
lation (14). associated forms of erythropoietin and those of the contami-
Thirty amino acids in the NH2-terminalportion of eryth- nant, M, 20,000 protein which has a high affinity for mouse
ropoietin were sequenced with a gas phase sequenator; the IgG (for instance, a component of compliments). Association
sequence was H2N-Ala-Pro-?-Arg-Leu-Ile-Leu-Asp-Ser-Arg- of erythropoietin may result inreducing the activity. It seems,
Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-?- however, that the binding of the M , 20,000 protein to the
Ile-Thr-Asp-Gly-Gly-Ala. Possible candidates for the uniden- antibody column is mediated by erythropoietin, since the M ,
tified positions are cysteins involved in disulfide bonds or 20,000 protein fractionated on the gel electrophoresis was
amino acids (probably Asn, from the carbohydrates unable to bind with the monoclonal antibody (see Fig. 4, lane

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contained3) to which carbohydrate residues were attached. No 4 ) . An intriguing hypothesis is that the occurrence of high
significant amount of amino acids other than alanine was molecular weight species of erythropoietin with reduced activ-
detected as the NH,-terminal residue, and therefore, hetero- ity is caused by association of erythropoietin with the M ,
geneity of the purified erythropoietin found on SDS-polyac- 20,000 protein which is a physiological regulator of erythro-
rylamide gel electrophoresis (see Fig. 4) is presumably due to poiesis. Existence of the inactive erythropoietin species with
difference in carbohydrate residues attached to the erythro- a high molecular weight, which dissociates to produce the
poietin polypeptide chain. The putative 26 NH2-terminal active hormone, has been suggested (18). Analysis of the
amino acids of erythropoietin were recently described by Sue erythropoietin in the void fractions is in progress. With con-
and Sytokowski (15), i.e. H2N-Ala-Pro-Pro-Arg-Leu-Ile-Asn- ventional purification procedures, such species of erythropo-
Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys- ietin must in all likelihood have escaped detection due to its
Glu-Ala-Glu-Lys-Ile-Thr. Three amino acids, positions 3, 7, low activity.
and 24, differ from our sequence for unknown reasons.
REFERENCES
DISCUSSION 1.Espada, J., Langton, A. A., and Dorado,M. (1972) Biochirn.
A hybridoma secreting the monoclonal antibody against Biophys. Acta 285,427-435
2. Miyake, T.,Kung, C. K.-H., and Goldwasser, E. (1977) J. Biol.
human urinary erythropoietin has been raised (17) but the Chern. 252,5558-5564
paper presented here is the first report of the use of mono- 3. Staehelin, T.,Hobbs, D. S., Kung, H., Lai, C.-Y., and Pestka, S.
clonal antibody for the large-scale isolation of erythropoietin. (1981) J. Biol. Chem. 256,9750-9754
Rapid isolation of erythropoietin from human urinary con- 4. Yanagawa, S., Narita, H., Sasaki, R., Chiba, H., Itada, N., and
centrate with a high yield can be achieved by using an im- Okada, H. (1983) Agric. Biol. Chem. 4 7 , 1311-1316
munoadsorbent column packed with the monoclonal antibody 5. Stephenson, J. R., and Axelrad, A. A. (1971) Endocrinology 88,
1519-1520
and a Sephadex G-100 column. Adequate amounts of pure 6. Brandon, N. C., Cotes, P. M., and Espada, J. (1981) Brit. J.
erythropoietin will make possible further studies on the bio- Haematol. 47,461-468
chemistry and physiology of erythropoietin and its clinical 7. Goldwasser, E., and Gross, M. (1975) Methods Enzyml.
testing. XXXVII, 109-121
The immunoadsorbent column does not bind erythropoietin 8. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685
in the untreated urine concentrate but binds erythropoietin 9. Hunkapiller, M. W., and Hood, L. E. (1983) Science ( Wash. D.
C.) 219,650-659
in the SDS-treatedone. Treatment with SDS was needed for 10. Bradford, M. (1976) Anal. Biochem. 72,248-254
the purified erythropoietin to be retained again on the im- 11. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
munoadsorbent column. It appears, therefore, that the lack (1951) J. Biol. Chern. 193, 265-275
of binding of erythropoietin in the untreated urine concen- 12. Lukowsky, W. A., and Painter, R. H. (1972) Can. J. Bwchern.
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trate but to aproperty of the antibody. The hybridoma 14. Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H.,and
secreting this antibody was constructed by using spleen cells Ashwell, G. (1968) J. Bid. Chern. 243, 155-159
from a mouse receiving its primary immunization with an 15. Sue, J. M., and Sytokowski, A. J. (1983) Proc. Natl. Acad. Sci. U.
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fore, this antibody may be directed against an epitope which 16. Burnette, W. N. (1981) Anal. Biochern. 112,195-203
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R. Sasaki, S. Yanagawa, H. Ohnota, and H. Chiba, unpublished 18. Sytokowski, A. J. (1980) Biochern. Biophys. Res. Cornrnun. 93,
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 Isolation of human erythropoietin with monoclonal antibodies.
S Yanagawa, K Hirade, H Ohnota, R Sasaki, H Chiba, M Ueda and M Goto
J. Biol. Chem. 1984, 259:2707-2710.

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