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Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay a
a
Yasuo Ohe , Tomoyoshi Nishino , Sadahito Shin a
a
b
, Hisanobu Hirano , Kazuya Higashino , Yoshiki b
b
b
Amuro , Shinsuke Tamura , Toshikazu Hada & Hiroshi Fujioka
b
a
Otsuka Assay Laboratories, Otsuka Pharmaceutical Co. Ltd., Kawauchi-cho 224–18, Tokushima, 770, Japan b
The Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya Hyogo, 663, Japan Version of record first published: 23 Oct 2006.
To cite this article: Yasuo Ohe, Tomoyoshi Nishino, Sadahito Shin, Hisanobu Hirano, Kazuya Higashino, Yoshiki Amuro, Shinsuke Tamura, Toshikazu Hada & Hiroshi Fujioka (1990): Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay, Journal of Immunoassay, 11:4, 459-475 To link to this article: http://dx.doi.org/10.1080/01971529008055045
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/termsand-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan,
sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
JOURNAL OF IMMUNOASSAY, 11(4), 4 5 9 - 4 7 5 (1990)
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MONOCLONAL ANTIBODY TO PSEUDOURIDINE USED TO DEVELOP A RADIOIMMUNOASSAY Yasuo Ohe' , Tomoyoshi Nishino' , Sadahito Shin' , Hisanobu H'irano' , Kazuya Higashino2 ,Yoshiki Amuro2 , Shinsuke Tamura2, Toshikazu Hada2, and Hiroshi Pujioka2
.
Otsuka Assay Laboratories, Otsuka Pharmaceutical Co. L t d ' , Kawauchi-cho 224-18, Tokushima 770, and The T h i r d Department of I n t e r n a l Medicine, Hyogo College of Medicine2, Mukogawa-cho 1- 1, Nishinomiya Hyogo 663, Japan
ABSTRACT Pseudouridine, a component of t R N A , was modified t o y i e l d t h e d e r i v a t i v e s : succinyl, palmitoyl pseudouridine, and protein conjugates. These d e r i v a t i v e s were used i n preparation of monoclonal a n t i b o d i e s s p e c i f i c a l l y d i r e c t e d t o pseudouridine. MAbs from t h r e e hybridomas (OAL 881,812 and 814) were e s t a b l i s h e d and shown t o b e d i r e c t e d t o pseudouridine, u r i d i n e and u r a c i l . OAL 881 was equally r e a c t i v e w i t h t h e t h r e e s u b s t r a t e s , OAL 812 showed the same r e a c t i v i t y f o r pseudouridine and u r a c i l , while OAL 814 showed a r e a c t i v i t y mainly f o r pseudouridine. MAb OAL 814 was used i n a radioimmunoassay(RIA1 system unique f o r A good dose-response curve was observed i n t h e pseudouridine. I n t r a - and i n t e r - a s s a y range between 31.3 and 2000 nmole/ml. CV values were below 4.1 X and 7.4 % respectively, and good r e s u l t s were obtained from recovery of added material and dilution tests. The r a t i o of pseudouridine/creatinin i n u r i n e was s i g i f i c a n t l y higher i n p a t i e n t s w i t h cancer than i n normal subjects.
KEY WORDS: Pseudouridine, RIA, Monoclonal Antibody 459 Copyright 0 1990 by Marcel Dekker, Inc.
460
OHE ET AL.
INTRODUCTION Modified ribonucleosides, derived primarily from t r a n s f e r ribonucleic a c i d ( t R N A ) ,
a r e excreted i n abnormal amounts i n t h e
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
urine of cancer p a t i e n t s ( 1 - 2 ) .
These excretion products include
pseudouridine and methylated nucleosides(3).
Interest i n these
substances a s p o t e n t i a l tumor markers (4) was stimulated by r e p o r t s t h a t t R N A produced by c e r t a i n cancer t i s s u e s had increased a c t i v i t y when compared t o t h a t from t h e corresponding normal t i s s u e s .
Borek e t a l ( 5 ) showed t h a t t R N A from cancer
tissue had a much higher turnover r a t e than t h a t from t h e
corresponding nornal t i s s u e , and t h a t pseudouridine i s not catabolized f u r t h e r b u t excreted i n u r i n e a s the i n t a c t molecule. Some of the present authors(8,7) have r e c e n t l y reported an a n a l y t i c a l method f o r urinary nucleosides u s i n g reversed-phase h i g h perfornance l i q u i d chromatography following concentration b y
means of boronated g e l ( 8 ) .
The level of modified nucleosides i n
urine is an important c l i n i c a l marker f o r cancer diagnosis (9,101
In the present s t u d y , we prepared d e r i v a t i v e s of pseudouridine, e s t a b l i s h e d s p e c i f i c MAbs against these d e r i v a t i v e s , and developed an RIA system f o r pseudouridine based on one(0AL 814) of these MAbs.
MONOCLONAL ANTIBODY TO PSEUDOURIDINE
461
MATERIALS AND METHODS Materials used were: pseudouridine, and bovine l i p o p r o t e i n (Sigma Chemical Co., St.Louis, Mo.USA), uridine, u r a c i l , other
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
nucleosides, anhydride s u c c i n a t e and ovalbumin (WAKO PURE Chemical Co., Osaka, Japan), peroxidase-goat a n t i mouse IgG + IgA t
IgM (H+L), Iodogen ( P i e r c e Chemical Co., Rockford, IL, USA) and
water s o l u b l e carbodiimide (The Peptide I n s t i t u t e , Inc., Osaka, Japan).
NMR was by Brucker WH-400 NMR Spectrometer
(Rheinstettn W-Germary), and i n f r a r e d spectroscopy by Nippon Bunko I n d u s t r i e s (Tokyo, Japan).
Urine samples were c o l l e c t e d
f o r 24 hours and s t o r e d a t -20 "C u n t i l a n a l y s i s .
Modification of Pseudouridine Pseudouridine (1 mM) was dissolved i n 5 m l pyridine, 1.5 mM anhydride s u c c i n a t e was added t o t h i s s o l u t i o n , and t h e r e a c t i o n mixture kept overnight a t room temperature.
The r e a c t a n t was
concentrated u n d e r reduced pressure and purified on HPLC w i t h DEAE-5PW (TOSOH Co., Osaka, Japan) on a l i n e a r g r a d i e n t from 10
mM (pH 4.5) t o 0.5 M ammonium a c e t a t e (pH 6.8).
Pseudouridine
(1 mM) was dissolved i n 5 n l pyridine containing 2 mM palmitoyl chloride.
After standing overnight, t h e r e a c t a n t was
successively p r e c i p i t a t e d w i t h excess d i e t h y l e t h e r , f i l t e r e d , washed w i t h d i e t h y l e t h e r , dissolved i n hot ethanol,
OHE ET AL.
462
r e c r y s t a l l i z e d a t 4 “C, f i l t e r e d , again washed w i t h d i e t h y l e t h e r , and d r i e d under reduced pressure. BSA (1 pM) and pseudouridine s u c c i n a t e (20 pMM) were
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
dissolved i n 5 m l of 50 mM PBS and supplenented w i t h 100 p H water s o l u b l e carbodiimide.
After standing f o r s e v e r a l hours,
the r e a c t i o n mixture was dialyzed extensively a g a i n s t d i s t i l l e d water and lyophilized.
Immunization of Balb/c mice and preparation of hybridomas
Three groups of 5 week old male Balb/c mice were immunized subcutaneously w i t h 10 p g palmitoyl pseudouridine emulsified i n complete Preund’s adjuvant w i t h 5 % m e t h y l BSA(11).
Second
and t h i r d immunizations were given a t 2 weeks i n t e r v a l s .
Spleen
c e l l s were prepared 3 days a f t e r t h e t h i r d immunization, fused
w i t h P3Ul mouse myeloma c e l l s ( l 0 spleen c e l l s per myeloma c e l l ) i n t h e presence of 35% polyethylene glycol-1500, and plated i n 24 -well t i s s u e c u l t u r e p l a t e s ( 1 - 2 x lo6 c e l l s / w e l l ) .
Cell growth
was followed b y microscopic examination and c u l t u r e supernatants were c o l l e c t e d d a i l y f o r screening of antibody production.
Screening o f monoclonal antibody Pseudouridinyl BSA was dissolved i n 0.1M sodium bicarbonate a t 5pg/ml, and 100 p 1 a l i q u o t s of s o l u t i o n were dispensed i n t o
463
MONOCLONAL ANTIBODY TO PSEUDOURIDINE
96 wells and s t o o d o v e r n i g h t .
Each well was washed twice w i t h
d i s t i l l e d water, blocked with 50 mM PBS with 0.2 (4 g e l a t i n , and stored a t
4 "C.
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The c u l t u r e f l u i d s were each t e s t e d a g a i n s t a well c o a t e d w i t h p s e u d o u r i d i n y l BSA and a well c o n t a i n i n g 10 ug p s e u d o u r i d i n e a s an i n h i b i t o r .
Each well was i n c u b a t e d f o r 2 hours a t room
t e m p e r a t u r e , washed twice w i t h d i s t i l l e d water, supplemented w i t h 1 0 0 ~ of 1 p e r o x i d a s e - g o a t antimouse I g G + 1gA + IgM (H+L), s t o o d f o r 1 hour, and then each washed twice w i t h d i s t i l l e d w a t e r . P e r o x i d a s e c o n j u g a t e r e a c t i n g with the well was c o l o r e d by a s o l u t i o n of 0.25 % o -phenylenediamine / 0.05% hydrogen percxide. The r e a c t i o n was stopped by 1
M s u l f o n i c a c i d , and t h e
absorbance of t h e r e a c t i o n mixture was measured a t 490 nm ( T i t e r t e k Multiskan
MCC, Abbott, Chjcago Co. USA).
MAbs from
three hybridomas were e s t a b l i s h e d (OAL 881, 812, 8141, and t h e i r s u b c l a s s determined by Ouchterlony's method.
R a d i o l a b e l l i n n of monoclonal antibody (OAL 814) F i f t y f i g MAb d i s s o l v e d i n 200 p 1 of 0.1 M sodium b o r a t e , pH
8.2, c o n t a i n i n g c o a t e d w i t h 40
1 m C i of Na'251 was t r a n s f e r r e d t o a g l a s s t u b e ,ug
of Iodogen and s t o o d f o r 5 m i n a t 0 "C.
The
i o d i n a t e d p r o d u c t was p u r i f i e d by g e l chromatography on a column
(1.0 x 30 cm) of Sephacryl S-300 developed w i t h 50 mM PES
464
OHE ET AL.
containing 0.1 X g e l a t i n and 0.02 X NaN3.
The s p e c i f i c
r a d i o a c t i v i t y of iodinated HAb was about 5 p C i / p g protein.
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Assay Procedure f o r urinary Pseudouridine One thousand polystyrene beads (8.3 am diameter) were soaked
i n 200 m of 0.11 sodiua bicarbonate b u f f e r (containing l O p g / a l of pseudouridinyl BSA) overnight a t 4°C.
The buffer was drained
off and the beads were soaked i n 50 mH PBS w i t h 0.2 X g e l a t i n t o
block the s u r f a c e not covered, and s t o r e d a t 4 "C.
25 p l of
sample and a bead were added t o the t e s t tube, supplemented with
200 "C.
p l of
1 2 5 1 OAL 814 (ca 50000cpd, and stood overnight a t 4
The bead was uashed 3 times w i t h d i s t i l l e d water and i t s
r a d i o a c t i v i t y was measured i n an auto 7 -counter.
Procedure f o r urinary Pseudouridine by HPLC Determination of urinary pseudouridine was performed according t o the method of Aauro e t a l ( 7 ) .
The a n a l y t i c a l systes was a
combined HPLC system w i t h an a f f i n i t y and reversed-phase column and both columns were e q u i l i b r a t e d w i t h 0.1 role/l sodiumpotassium phosphate buffer(pH 8.7).
The chromatography was
i n i t i a t e d by i n j e c t i n g the sample i n t o t h e a f f i n i t y column of Boronate 5PW (7.5 mm x 7.5cr,TOSOH), and the elute containing pseudouridine f r o n the a f f i n i t y column was separated on the
465
MONOCLONAL ANTIBODY TO PSEUDOURIDINE
reversed-phase column of Octadecyl 4PW (4.6 mm x 15 cn,TOSOH) and was detected b y UV spectrophotonetry.
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RESULTS AND DISCUSSION The mixture of pseudouridine s u c c i n a t e were separated and p u r i f i e d with DEAF, 5PU column (TOSOH Co., Tokyo, Japan) on a l i n e a r g r a d i e n t from l0.n (pH 6.8).
(PH 4.5) t o 0.5 H amnoniun a c e t a t e
Four major f r a c t i o n s uere obtained and i d e n t i f i e d by
chemical analysis: (1) non-react-ed; ( 2 ) 5’-mono-succinyl d e r i v a tives; (3) 2’ -and 3’ -mono-succinyl d e r i v a t i v e s ; (4) 2’
S’-di-succinyl d e r i v a t i v e s . 19.0 %, 21.2 X
, 29.9 X , and
-, 3’ -, and
The y i e l d s of the 4 f r a c t i o n s were 29.9% r e s p e c t i v e l y .
Infrared
s p e c t r a of a l l succinate d e r i v a t i v e s showed an absorbance peak a t 1750 cm-’ due t o the carboxyl ester group.
NHR a n a l y s i s
showed chemical s h i f t s a t t r i b u t a b l e t o aethylene protons on s u c c i n y l groups: 5’-CH2OSuc a t 4.21 and 4.31 (d,d 2H H-5’1, 2’-
CHOSuc a t 5.23 ( t 1 H H-2’1, and 3’-CHOSuc a t 5.08 ( t 1H H-2’)ppm. Methylene on succinate caused s h i f t a t 2.38 and 2.56 (t 4H) ppm.
These NHR and IR finding uere a t t r i b u t e d t o succinylation of
pseudouridine.
P a l a i t o y l pseudouridine(yie1d 86 X I was
i d e n t i f i e d chemically a s follows.
In i n f r a r e d a n a l y s i s , t h e
absorbance peaks appearing a t 1750 and 2900 cm-I were due t o carboxyl ester and a l k y l groups; i n NHR a n a l y s i s , s h i f t s d u e t o
OHE ET A L .
466
TABLE 1 C r o s s r e a c t i v i t y of t h r e e MAbs w i t h v a r i o u s i n h i b i t o r s Inhibition
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MAbs
Pseudouridine
OAL 881 OAL 812 OAL 814
100 100 100
(%I
Uracil
Uridine
120 50 20-30
55
other inhibitor
25
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay a
a
Yasuo Ohe , Tomoyoshi Nishino , Sadahito Shin a
a
b
, Hisanobu Hirano , Kazuya Higashino , Yoshiki b
b
b
Amuro , Shinsuke Tamura , Toshikazu Hada & Hiroshi Fujioka
b
a
Otsuka Assay Laboratories, Otsuka Pharmaceutical Co. Ltd., Kawauchi-cho 224–18, Tokushima, 770, Japan b
The Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya Hyogo, 663, Japan Version of record first published: 23 Oct 2006.
To cite this article: Yasuo Ohe, Tomoyoshi Nishino, Sadahito Shin, Hisanobu Hirano, Kazuya Higashino, Yoshiki Amuro, Shinsuke Tamura, Toshikazu Hada & Hiroshi Fujioka (1990): Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay, Journal of Immunoassay, 11:4, 459-475 To link to this article: http://dx.doi.org/10.1080/01971529008055045
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/termsand-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan,
sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
JOURNAL OF IMMUNOASSAY, 11(4), 4 5 9 - 4 7 5 (1990)
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
MONOCLONAL ANTIBODY TO PSEUDOURIDINE USED TO DEVELOP A RADIOIMMUNOASSAY Yasuo Ohe' , Tomoyoshi Nishino' , Sadahito Shin' , Hisanobu H'irano' , Kazuya Higashino2 ,Yoshiki Amuro2 , Shinsuke Tamura2, Toshikazu Hada2, and Hiroshi Pujioka2
.
Otsuka Assay Laboratories, Otsuka Pharmaceutical Co. L t d ' , Kawauchi-cho 224-18, Tokushima 770, and The T h i r d Department of I n t e r n a l Medicine, Hyogo College of Medicine2, Mukogawa-cho 1- 1, Nishinomiya Hyogo 663, Japan
ABSTRACT Pseudouridine, a component of t R N A , was modified t o y i e l d t h e d e r i v a t i v e s : succinyl, palmitoyl pseudouridine, and protein conjugates. These d e r i v a t i v e s were used i n preparation of monoclonal a n t i b o d i e s s p e c i f i c a l l y d i r e c t e d t o pseudouridine. MAbs from t h r e e hybridomas (OAL 881,812 and 814) were e s t a b l i s h e d and shown t o b e d i r e c t e d t o pseudouridine, u r i d i n e and u r a c i l . OAL 881 was equally r e a c t i v e w i t h t h e t h r e e s u b s t r a t e s , OAL 812 showed the same r e a c t i v i t y f o r pseudouridine and u r a c i l , while OAL 814 showed a r e a c t i v i t y mainly f o r pseudouridine. MAb OAL 814 was used i n a radioimmunoassay(RIA1 system unique f o r A good dose-response curve was observed i n t h e pseudouridine. I n t r a - and i n t e r - a s s a y range between 31.3 and 2000 nmole/ml. CV values were below 4.1 X and 7.4 % respectively, and good r e s u l t s were obtained from recovery of added material and dilution tests. The r a t i o of pseudouridine/creatinin i n u r i n e was s i g i f i c a n t l y higher i n p a t i e n t s w i t h cancer than i n normal subjects.
KEY WORDS: Pseudouridine, RIA, Monoclonal Antibody 459 Copyright 0 1990 by Marcel Dekker, Inc.
460
OHE ET AL.
INTRODUCTION Modified ribonucleosides, derived primarily from t r a n s f e r ribonucleic a c i d ( t R N A ) ,
a r e excreted i n abnormal amounts i n t h e
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
urine of cancer p a t i e n t s ( 1 - 2 ) .
These excretion products include
pseudouridine and methylated nucleosides(3).
Interest i n these
substances a s p o t e n t i a l tumor markers (4) was stimulated by r e p o r t s t h a t t R N A produced by c e r t a i n cancer t i s s u e s had increased a c t i v i t y when compared t o t h a t from t h e corresponding normal t i s s u e s .
Borek e t a l ( 5 ) showed t h a t t R N A from cancer
tissue had a much higher turnover r a t e than t h a t from t h e
corresponding nornal t i s s u e , and t h a t pseudouridine i s not catabolized f u r t h e r b u t excreted i n u r i n e a s the i n t a c t molecule. Some of the present authors(8,7) have r e c e n t l y reported an a n a l y t i c a l method f o r urinary nucleosides u s i n g reversed-phase h i g h perfornance l i q u i d chromatography following concentration b y
means of boronated g e l ( 8 ) .
The level of modified nucleosides i n
urine is an important c l i n i c a l marker f o r cancer diagnosis (9,101
In the present s t u d y , we prepared d e r i v a t i v e s of pseudouridine, e s t a b l i s h e d s p e c i f i c MAbs against these d e r i v a t i v e s , and developed an RIA system f o r pseudouridine based on one(0AL 814) of these MAbs.
MONOCLONAL ANTIBODY TO PSEUDOURIDINE
461
MATERIALS AND METHODS Materials used were: pseudouridine, and bovine l i p o p r o t e i n (Sigma Chemical Co., St.Louis, Mo.USA), uridine, u r a c i l , other
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
nucleosides, anhydride s u c c i n a t e and ovalbumin (WAKO PURE Chemical Co., Osaka, Japan), peroxidase-goat a n t i mouse IgG + IgA t
IgM (H+L), Iodogen ( P i e r c e Chemical Co., Rockford, IL, USA) and
water s o l u b l e carbodiimide (The Peptide I n s t i t u t e , Inc., Osaka, Japan).
NMR was by Brucker WH-400 NMR Spectrometer
(Rheinstettn W-Germary), and i n f r a r e d spectroscopy by Nippon Bunko I n d u s t r i e s (Tokyo, Japan).
Urine samples were c o l l e c t e d
f o r 24 hours and s t o r e d a t -20 "C u n t i l a n a l y s i s .
Modification of Pseudouridine Pseudouridine (1 mM) was dissolved i n 5 m l pyridine, 1.5 mM anhydride s u c c i n a t e was added t o t h i s s o l u t i o n , and t h e r e a c t i o n mixture kept overnight a t room temperature.
The r e a c t a n t was
concentrated u n d e r reduced pressure and purified on HPLC w i t h DEAE-5PW (TOSOH Co., Osaka, Japan) on a l i n e a r g r a d i e n t from 10
mM (pH 4.5) t o 0.5 M ammonium a c e t a t e (pH 6.8).
Pseudouridine
(1 mM) was dissolved i n 5 n l pyridine containing 2 mM palmitoyl chloride.
After standing overnight, t h e r e a c t a n t was
successively p r e c i p i t a t e d w i t h excess d i e t h y l e t h e r , f i l t e r e d , washed w i t h d i e t h y l e t h e r , dissolved i n hot ethanol,
OHE ET AL.
462
r e c r y s t a l l i z e d a t 4 “C, f i l t e r e d , again washed w i t h d i e t h y l e t h e r , and d r i e d under reduced pressure. BSA (1 pM) and pseudouridine s u c c i n a t e (20 pMM) were
Downloaded by [Duke University Libraries] at 03:14 20 September 2012
dissolved i n 5 m l of 50 mM PBS and supplenented w i t h 100 p H water s o l u b l e carbodiimide.
After standing f o r s e v e r a l hours,
the r e a c t i o n mixture was dialyzed extensively a g a i n s t d i s t i l l e d water and lyophilized.
Immunization of Balb/c mice and preparation of hybridomas
Three groups of 5 week old male Balb/c mice were immunized subcutaneously w i t h 10 p g palmitoyl pseudouridine emulsified i n complete Preund’s adjuvant w i t h 5 % m e t h y l BSA(11).
Second
and t h i r d immunizations were given a t 2 weeks i n t e r v a l s .
Spleen
c e l l s were prepared 3 days a f t e r t h e t h i r d immunization, fused
w i t h P3Ul mouse myeloma c e l l s ( l 0 spleen c e l l s per myeloma c e l l ) i n t h e presence of 35% polyethylene glycol-1500, and plated i n 24 -well t i s s u e c u l t u r e p l a t e s ( 1 - 2 x lo6 c e l l s / w e l l ) .
Cell growth
was followed b y microscopic examination and c u l t u r e supernatants were c o l l e c t e d d a i l y f o r screening of antibody production.
Screening o f monoclonal antibody Pseudouridinyl BSA was dissolved i n 0.1M sodium bicarbonate a t 5pg/ml, and 100 p 1 a l i q u o t s of s o l u t i o n were dispensed i n t o
463
MONOCLONAL ANTIBODY TO PSEUDOURIDINE
96 wells and s t o o d o v e r n i g h t .
Each well was washed twice w i t h
d i s t i l l e d water, blocked with 50 mM PBS with 0.2 (4 g e l a t i n , and stored a t
4 "C.
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The c u l t u r e f l u i d s were each t e s t e d a g a i n s t a well c o a t e d w i t h p s e u d o u r i d i n y l BSA and a well c o n t a i n i n g 10 ug p s e u d o u r i d i n e a s an i n h i b i t o r .
Each well was i n c u b a t e d f o r 2 hours a t room
t e m p e r a t u r e , washed twice w i t h d i s t i l l e d water, supplemented w i t h 1 0 0 ~ of 1 p e r o x i d a s e - g o a t antimouse I g G + 1gA + IgM (H+L), s t o o d f o r 1 hour, and then each washed twice w i t h d i s t i l l e d w a t e r . P e r o x i d a s e c o n j u g a t e r e a c t i n g with the well was c o l o r e d by a s o l u t i o n of 0.25 % o -phenylenediamine / 0.05% hydrogen percxide. The r e a c t i o n was stopped by 1
M s u l f o n i c a c i d , and t h e
absorbance of t h e r e a c t i o n mixture was measured a t 490 nm ( T i t e r t e k Multiskan
MCC, Abbott, Chjcago Co. USA).
MAbs from
three hybridomas were e s t a b l i s h e d (OAL 881, 812, 8141, and t h e i r s u b c l a s s determined by Ouchterlony's method.
R a d i o l a b e l l i n n of monoclonal antibody (OAL 814) F i f t y f i g MAb d i s s o l v e d i n 200 p 1 of 0.1 M sodium b o r a t e , pH
8.2, c o n t a i n i n g c o a t e d w i t h 40
1 m C i of Na'251 was t r a n s f e r r e d t o a g l a s s t u b e ,ug
of Iodogen and s t o o d f o r 5 m i n a t 0 "C.
The
i o d i n a t e d p r o d u c t was p u r i f i e d by g e l chromatography on a column
(1.0 x 30 cm) of Sephacryl S-300 developed w i t h 50 mM PES
464
OHE ET AL.
containing 0.1 X g e l a t i n and 0.02 X NaN3.
The s p e c i f i c
r a d i o a c t i v i t y of iodinated HAb was about 5 p C i / p g protein.
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Assay Procedure f o r urinary Pseudouridine One thousand polystyrene beads (8.3 am diameter) were soaked
i n 200 m of 0.11 sodiua bicarbonate b u f f e r (containing l O p g / a l of pseudouridinyl BSA) overnight a t 4°C.
The buffer was drained
off and the beads were soaked i n 50 mH PBS w i t h 0.2 X g e l a t i n t o
block the s u r f a c e not covered, and s t o r e d a t 4 "C.
25 p l of
sample and a bead were added t o the t e s t tube, supplemented with
200 "C.
p l of
1 2 5 1 OAL 814 (ca 50000cpd, and stood overnight a t 4
The bead was uashed 3 times w i t h d i s t i l l e d water and i t s
r a d i o a c t i v i t y was measured i n an auto 7 -counter.
Procedure f o r urinary Pseudouridine by HPLC Determination of urinary pseudouridine was performed according t o the method of Aauro e t a l ( 7 ) .
The a n a l y t i c a l systes was a
combined HPLC system w i t h an a f f i n i t y and reversed-phase column and both columns were e q u i l i b r a t e d w i t h 0.1 role/l sodiumpotassium phosphate buffer(pH 8.7).
The chromatography was
i n i t i a t e d by i n j e c t i n g the sample i n t o t h e a f f i n i t y column of Boronate 5PW (7.5 mm x 7.5cr,TOSOH), and the elute containing pseudouridine f r o n the a f f i n i t y column was separated on the
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MONOCLONAL ANTIBODY TO PSEUDOURIDINE
reversed-phase column of Octadecyl 4PW (4.6 mm x 15 cn,TOSOH) and was detected b y UV spectrophotonetry.
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RESULTS AND DISCUSSION The mixture of pseudouridine s u c c i n a t e were separated and p u r i f i e d with DEAF, 5PU column (TOSOH Co., Tokyo, Japan) on a l i n e a r g r a d i e n t from l0.n (pH 6.8).
(PH 4.5) t o 0.5 H amnoniun a c e t a t e
Four major f r a c t i o n s uere obtained and i d e n t i f i e d by
chemical analysis: (1) non-react-ed; ( 2 ) 5’-mono-succinyl d e r i v a tives; (3) 2’ -and 3’ -mono-succinyl d e r i v a t i v e s ; (4) 2’
S’-di-succinyl d e r i v a t i v e s . 19.0 %, 21.2 X
, 29.9 X , and
-, 3’ -, and
The y i e l d s of the 4 f r a c t i o n s were 29.9% r e s p e c t i v e l y .
Infrared
s p e c t r a of a l l succinate d e r i v a t i v e s showed an absorbance peak a t 1750 cm-’ due t o the carboxyl ester group.
NHR a n a l y s i s
showed chemical s h i f t s a t t r i b u t a b l e t o aethylene protons on s u c c i n y l groups: 5’-CH2OSuc a t 4.21 and 4.31 (d,d 2H H-5’1, 2’-
CHOSuc a t 5.23 ( t 1 H H-2’1, and 3’-CHOSuc a t 5.08 ( t 1H H-2’)ppm. Methylene on succinate caused s h i f t a t 2.38 and 2.56 (t 4H) ppm.
These NHR and IR finding uere a t t r i b u t e d t o succinylation of
pseudouridine.
P a l a i t o y l pseudouridine(yie1d 86 X I was
i d e n t i f i e d chemically a s follows.
In i n f r a r e d a n a l y s i s , t h e
absorbance peaks appearing a t 1750 and 2900 cm-I were due t o carboxyl ester and a l k y l groups; i n NHR a n a l y s i s , s h i f t s d u e t o
OHE ET A L .
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TABLE 1 C r o s s r e a c t i v i t y of t h r e e MAbs w i t h v a r i o u s i n h i b i t o r s Inhibition
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MAbs
Pseudouridine
OAL 881 OAL 812 OAL 814
100 100 100
(%I
Uracil
Uridine
120 50 20-30
55
other inhibitor
25
