This article was downloaded by: [Carnegie Mellon University]On: 05 December 2014, At: 14:44Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UK

Analytical LettersPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lanl20

Novel and SensitiveNoncompetitive EnzymeImmunoassay (Hetero-Two-Site Enzyme Immunoassay)for Arginine VasopressinSeiichi Hashida a , Koichiro Tanaka a , NaokoYamamoto a , Takeshi Uno b , Ken'Ichi Yamaguchic & Eiji Ishikawa aa Department of Biochemistry , Medical Collegeof Miyazaki , Kiyotake, Miyazaki, 889-16, Japanb Department of Anesthesiology , MedicalCollege of Miyazaki , Kiyotake, Miyazaki, 889-16,Japanc Department of Physiology , Niigata UniversitySchool of Medicine , Asahimachi, Niigata, 951,JapanPublished online: 23 Oct 2006.

To cite this article: Seiichi Hashida , Koichiro Tanaka , Naoko Yamamoto ,Takeshi Uno , Ken'Ichi Yamaguchi & Eiji Ishikawa (1991) Novel and SensitiveNoncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay)for Arginine Vasopressin, Analytical Letters, 24:7, 1109-1123, DOI:10.1080/00032719108052957

To link to this article: http://dx.doi.org/10.1080/00032719108052957

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of allthe information (the “Content”) contained in the publications on our

platform. However, Taylor & Francis, our agents, and our licensorsmake no representations or warranties whatsoever as to the accuracy,completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views ofthe authors, and are not the views of or endorsed by Taylor & Francis.The accuracy of the Content should not be relied upon and should beindependently verified with primary sources of information. Taylor andFrancis shall not be liable for any losses, actions, claims, proceedings,demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, inrelation to or arising out of the use of the Content.

This article may be used for research, teaching, and private studypurposes. Any substantial or systematic reproduction, redistribution,reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of accessand use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

ANALYTICAL LETTERS, 24(7), 1109-1123 (1991)

NOVEL AND S E N S I T I V E NONCOMPETITIVE ENZYME IMMUNOASSAY

(HETERO-TWO-SITE ENZYME IMMUNOASSAY) FOR ARGININE VASOPRESSIN

I N PLPSMA

KEY WORDS: Enzyme immunoassay, A r g i n i n e vasopress in, B i o t i n ,

B i o t i n y l a t i o n , Plasma

2 S e i i c h i Hashidal , K o i c h i r o Tanaka', Naoko Yamamoto', Takeshi Uno , I K e n ' i c h i Yamaguchi' and E i j i Ish ikawa

'Department o f B iochemis t r y , Mec$ical Co l l ege o f M iyazak i , Kiyotake, M iyazak i 889-16, Japan, Department o f Anes thes io logy , y e d i c a l Co l l ege o f Miyazaki , K iyo take , M iyazak i 889-16, Japan and

Asahimachi , N i i g a t a 951, Japan Department o f Phys io logy, N i i g a t a U n i v e r s i t y School o f Medic ine,

ABSTRACT

A novel and s e n s i t i v e noncompe t i t i ve enzyme immunoassay

( h e t e r o - t w o - s i t e enzyme immunoassay) f o r a r g i n i n e vasopress in i n

plasma i s desc r ibed . Plasma (0.3 ml) was d i l u t e d 1 . 3 - f o l d w i th

an a p w o p r i a t e bu f fe r and f i l t e r e d by c e n t r i f u g a t i o n i n a m i c r o -

1109

Copyright 0 1991 by Marcel Dekker, Inc.

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1110 HASHIDA ET AL.

concen t ra to r w i t h po l ysacchar ide membrane t o e l i m i n a t e plasma

p r o t e i n s . A r g i n i n e vasopress in i n plasma f i l t r a t e s was b i o -

t i n y l a t e d and t rapped on to a n t i - a r g i n i n e vasopress in IgG-coated

po lys ty rene b a l l s . A f t e r washing t h e p o l y s t y r e n e b a l l s t o

e l i m i n a t e o t h e r b i o t i n y l a t e d substances, t h e b i o t i n y l a t e d a r g i n i n e

vasopress in was e l u t e d f rom t h e p o l y s t y r e n e b a l l s w i t h HC1 and was

r e a c t e d w i t h a n t i - a r g i n i n e vasopress in Fab ' -perox idase conjugate.

The complex formed was t rapped on to s t r e p t a v i d i n - c o a t e d p o l y s t y -

rene b a l l s . Perox idase a c t i v i t y bound t o t h e p o l y s t y r e n e b a l l s

was assayed by f l u o r o m e t r y . The d e t e c t i o n l i m i t o f a r g i n i n e

vasopress in was 11 f g (10 amol) / tube. T h i s was 4 5 - f o l d l ower

than t h a t by c o m p e t i t i v e enzyme immunoassay u s i n g t h e same

ant iserum as used i n t h i s s tudy and 9 t o 4 0 0 - f o l d l ower t h a n those

p r e v i o u s l y r e p o r t e d by c o m p e t i t i v e radioimmunoassays. The

assay range o f a r g i n i n e vasopress in i n plasma was 0.14-140 n g / l

u s i n g 100 u l o f plasma f i l t r a t e s co r respond ing t o 75 u1 o f plasma.

Plasma l e v e l s o f a r g i n i n e vasopress in i n 8 h e a l t h y s u b j e c t s aged

25-41 y r w i t h l i b i t u m wa te r i n t a k e and normal a c t i v i t y

approx imate ly 4 h a f t e r b r e a k f a s t were 0.72 f 0.22 ( S D ) n g / l

( range, 0.42-1.04 n g / l ) .

INTRODUCTION

A r g i n i n e vasopress in, t h e a n t i d i u r e t i c hormone sec re ted by

t h e p o s t e r i o r p i t u i t a r y , i s a n i n e amino a c i d s i n g l e c h a i n p e p t i d e

w i t h an i n t r a m o l e c u l a r d i s u l f i d e b r i d g e . The c o n c e n t r a t i o n o f

t h i s hormone i n t h e c i r c u l a t i o n has been determined by c o m p e t i t i v e

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1111

r a d i o i m m u n ~ a s s a y ~ - ~ and c o m p e t i t i v e enzyme immunoassay6 o n l y a f t e r

2 e x t r a c t i o n f r o m 1-5 m l o f p lasma u s i n g magnesium s i l i c a t e ,

o c t a d e c y l o r acetone’ y 3 y 6 f o l l o w e d by c o n c e n t r a t i o n .

T h i s i s p a r t l y due t o i n s u f f i c i e n t s e n s i t i v i t i e s o f t h e s e

c o m p e t i t i v e immunoassays and p a r t l y due t o t h e r e a c t i v i t y o f

I a n t i - a r g i n i n e v a s o p r e s s i n a n t i b o d i e s w i t h some o f plasma p r o t e i n s .

T h i s paper d e s c r i b e s a n o v e l and s e n s i t i v e n o n c o m p e t i t i v e

enzyme immunoassay ( h e t e r o - t w o - s i t e enzyme immunoassay) , w h i c h c a n

measure l ow l e v e l s o f a r g i n i n e v a s o p r e s s i n be low t h o s e i n plasma

o f h e a l t h y s u b j e c t s a f t e r removal o f p lasma p r o t e i n s by f i l t r a t i o n

o f 0.3 m l o f p lasma t h r o u g h a p o l y s a c c h a r i d e membrane w i t h o u t

c o n c e n t r a t i o n .

MATERIALS AND METHODS

B u f f e r

The r e g u l a r l y used b u f f e r was 10 mmol / l sodium phospha te

b u f f e r , pH 7.0, c o n t a i n i n g 0 . 1 m o l / l NaCl and 1 g / l b o v i n e serum

a l b u m i n ( c r y s t a l l i z e d , M i l e s I n c . , D i a g n o s t i c s D i v i s i o n ,

Kankakee,, I l l i n o i s , USA) ( b u f f e r A).

Pep t i des

8 [ A r g I - v a s o p r e s s i n ( a r g i n i n e Vasopress in ) , o x y t o c i n ,

[ A s u l Y 6 , A r g I - v a s o t o c i n and [ A r g I - v a s o t o c i n were o b t a i n e d f rom

P e p t i d e I n s t i t u t e , I n c . , Osaka, Japan. [Lys I - v a s o p r e s s i n was

o b t a i n e d f r o m P e n i n s u l a L a b o r a t o r i e s , I n c . , Belmont , C a l i f o r n i a ,

USA.

8 8

8

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1112 HASHIDA ET AL.

An ti bod i es

A n t i - a r g i n i n e v a s o p r e s s i n serum was r a i s e d i n ma le New

Zealand w h i t e r a b b i t s b y e i g h t i n t r a c u t a n e o u s i n j e c t i o n s o f

a r g i n i n e vasopress in -bov ine t h y r o g l o b u l i n c o n j u g a t e a t 2-3 week

i n t e r v a l s . A r g i n i n e v a s o p r e s s i n ( 5 mg) was c o n j u g a t e d t o b o v i n e t h y r o -

g l o b u l i n ( 5 0 mg, Sigma Chemical Company, S t . Lou is , M i s s o u r i , USA)

b y t h e c a r b o d i i m i d e method' u s i n g l-ethyl-3-(3-dimethylamino-

p r o p y l ) c a r b o d i i m i d e (Calb iochem-Behr ing, La J o l l a , C a l i f o r n i a ,

USA). F o r i n j e c t i o n , t h e c o n j u g a t e i n s a l i n e (0.4 mg/ml) was

e m u l s i f i e d w i t h an equa l volume o f F r e u n d ' s comp le te a d j u v a n t

( M i l e s I n c . , P i a g n o s t i c s D i v i s i o n , Kankakee, I l l i n o i s , U S A ) .

The amount o f t h e c o n j u g a t e i n j e c t e d was 0.2 rng p e r r a b b i t .

B lood was c o l l e c t e d 13 days a f t e r t h e l a s t immun iza t i on , and t h e

a n t i s e r u m was s t o r e d a t -20°C.

IgG was p repared f r o m serum by f r a c t i o n a t i o n w i t h Na2S04

f o l l o w e d b y passage t h r o u g h a column o f d i e t h y l a m i n o e t h y l

F ( a b ' ) 2 was p repared by d i g e s t i o n o f IgG w i t h 9 c e l l u l ose.

was p repared by r e d u c t i o n o f F ( a b ' ) 2 . The

i t s f ragmen ts was c a l c u l a t e d f r o m t h e absorbance

peps in , and Fab'

amount o f IgG and

9 a t 280 nm.

A r g i n i n e Vasopress in-Sepharose 48

A r g i n i n e v a s o p r e s s i n (0.61 mg) was coup led t o a c t i v a t e d

CH-Sepharose 4B (0.17 g, Pharmacia F i n e Chemicals AB, Uppsala,

Sweden) a c c o r d i n g t o t h e i n s t r u c t i o v s o f Pharmacia.

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1113

A f f i n i t y - P u r i f i ed A n t i - A r g i n i ne Vasopress in Fab' -Perox idase

Conjuga tt?

A n t i - a r g i n i n e vasopress in Fab' was con juga ted t o h o r s e r a d i s h

perox idase (Grade I, RZ=3.0, Boehr inge r Mannheim GmbH, Mannheim,

FRG) u s i n g N-succinimidyl-6-maleimidohexanoate ( D o j i n d o Labo-

r a t o r i e s , Kumamoto, Japan). lo The con juga te was a f f i n i t y - p u r i -

f i e d by e l u t i o n f rom a column o f a r g i n i n e vasopressin-Sepharose 48

a t pH 2.5. 1 1 y 1 2 The amount o f t h e con juga te was c a l c u l a t e d f rom

9 pe rox idase a c t i v i t y .

Prote in-Coated Po lys ty rene B a l l s

Po lys ty rene b a l l s (3 .2 mm i n d iameter , Immunochemical Co.,

Okayama, Japan) were coated w i t h r a b b i t a n t i - a r g i n i n e vasopress in

I g G (0.1 g / l ) and b i o t i n y l n o n s p e c i f i c r a b b i t IgG (0.1 g / l ) by

p h y s i c a l adso rp t i on . l3 B i o t i n y l n o n s p e c i f i c r a b b i t IgG was

prepared by t h e r e a c t i o n o f ma le im ide -nonspec i f i c r a b b i t IgG w i t h

N-biotinyl-2-mercaptoethylamine. l4 S t r e p t a v i d i n - c o a t e d p o l y s t y -

rene b a l l s were prepared by i n c u b a t i o n o f b i o t i n y l n o n s p e c i f i c

r a b b i t IgG-coated p o l y s t y r e n e b a l l s w i t h s t r e p t a v i d i n (0.1 g / 1 )

(Bethesda Research L a b o r a t o r i e s , Inc. , Maryland, USA) i n 0.1 m o l / l

sodium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 1 g/1 NaN3 a t 30°C f o r

4 h. The p r o t e i n - c o a t e d p o l y s t y r e n e b a l l s were s t o r e d i n

b u f f e r A c o n t a i n i n g 1 g/1 NaN3 a t 4°C.

A r g i n i n e Vasopress in Standards, Plasma and Plasma F i l t r a t e s

A r g i n i n e vasopress in (0.61 mg) was d i s s o l v e d i n 0.5 m l o f 0.2

m o l / l a c e t i c a c i d and d i l u t e d w i t h 0.1 m o l / l sodium phosphate

b u f f e r , pH 7.0, c o n t a i n i n g 0.1 m o l / l NaC1, 1 g/1 bov ine serum

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1114 HASHIDA ET AL.

albumin ( c r y s t a l l i z e d , Miles I n c . ) and 1 mmol/l EDTA. The

amount of a r g i n i n e vasopress in was c a l c u l a t e d by t a k i n g the

molecular weight a s 1,084. 15

Blood was withdrawn i n t o c h i l l e d g l a s s tubes c o n t a i n i n 9 EDTA

(Venoject VT-07ONA EDTA-2Na, Terumo Corp., Tokyo, Japan) and

c e n t r i f u g e d a t 1,500 x g f o r 20 min t o s e p a r a t e plasma. The

f i n a l concent ra t ion of EDTA i n blood was 1 . 5 g/1.

Plasma (0.3 ml o r 0.2 ml) was mixed wi th 0 . 1 ml of 0.4 mol/l

sodium phosphate b u f f e r , pH 7 . 0 , o r 0 .2 ml of 0 .2 mol/l sodium

phosphate b u f f e r , pH 7 .0 , and f i l t e r e d by c e n t r i f u g a t i o n i n a

microconcent ra tor with polysacchar ide membrane (CENTRICON-30,

Amicon Divis ion , W . R . Grace & Co., Beverly, Massachuset ts , USA) a t

4°C a t 5,000 x g f o r 20 min. The f i l t r a t e s (0 .15 ml) were

mixed w i t h 1/99 volume of 10 g/1 bovine serum albumin ( c r y s t a l -

l i z e d , Miles I n c . ) .

The c o n c e n t r a t i o n s of amino groups i n plasma and plasma 16 f i l t r a t e s were determined by f luorometry using f luorescamine.

L-Leucine was used a s s t a n d a r d .

Bi o t i nyl a t i on

B i o t i n y l a t i o n was performed i n two d i f f e r e n t ways.

D i r e c t b i o t i n y l a t i o n . - A 100 ul a l i q u o t of the d i l u t e d

a r g i n i n e vasopress in was incubated with 10 ul of 66 mmol/l

N-hydroxysuccinimidobiotin (Zymed L a b o r a t o r i e s , I n c . , San

Franc isco , C a l i f o r n i a , USA) i n d imethylsu l foxide a t 20°C f o r 1 h .

A f t e r i n c u b a t i o n , the r e a c t i o n mixture was incubated with 10 ul o f

1 mol/l glycine-NaOH, pH 7.0, a t 20°C f o r 30 m i n , fol lowed by

a d d i t i o n of 30 u l of b u f f e r A conta in ing 5 g/1 NaN3.

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1115

I n d i r e c t B i o t i n y l a t i o n . - A 100 p1 a l i q u o t G f t h e d i l u t e d

a r g i n i n e vasopress in o r plasma f i l t r a t e s was incuba ted w i t h 5 v l

of 63 mmol/l N-succinimidyl-6-maleimidohexanoate ( D o j i n d o Lab.) i n

d i m e t h y l s u l f o x i d e a t 20°C f o r 1 h. A f t e r i n c u b a t i o n , t h e

r e a c t i o n m i x t u r e was incuba ted w i t h 5 p1 o f 99 mmol/l g l u t a t h i o n e

(reduced form, K o h j i n Co.

phosphate b u f f e r , pH 7.0,

and subsequent ly w i t h 5

b i o t i n (Zymed Lab., I n c .

L td. , Tokyo, Japan) i n 0 .1 m o l / l sodium

c o n t a i n i n g 1 mmol/l EDTA a t 20°C f o r 1 h

p1 of 138 mmol/l N-hydroxysucc in imido-

i n d i m e t h y l s u l f o x i d e a t 20°C f o r 1 h.

F i n a l l y , 5 p1 o f 2 m o l / l glycine-NaOH, pH 7.0, was added, and t h e

i n c u b a t i o n was con t inued a t 20°C f o r 30 min, f o l l o w e d by a d d i t i o n

o f 30 u l o f b u f f e r A c o n t a i n i n g 5 g/1 NaN3.

Hetero-Two-Site Enzyme Immunoassay

The b i o t i n y l a t e d m i x t u r e (150 p l ) was i ncuba ted w i t h t w o

a n t i - a r g i n i n e vasopress in IgG-coated p o l y s t y r e n e b a l l s a t 4°C

o v e r n i g h t w i t h o u t shaking. A f t e r i n c u b a t i o n , t h e p o l y s t y r e n e

b a l l s were washed t w i c e by a d d i t i o n and a s p i r a t i o n o f 2 m l o f

10 mmol/l sodium phosphate b u f f e r , pH 7.0, c o n t a i n i n g 0 .1 m o l / l

NaCl and were i ncuba ted w i t h t h e m i x t u r e o f 100 u1 o f b u f f e r A

c o n t a i n i n g 1 rnmol/l EDTA and 20 ~l o f 1 m o l / l HC1 a t 4°C f o r 1 h

w i t h o u t shak ing. A f t e r removal o f t h e p o l y s t y r e n e b a l l s , t h e

rema in ing s o l u t i o n was n e u t r a l i z e d by a d d i t i o n o f t h e m i x t u r e o f

10 p1 o f 1 m o l / l sodium phosphate b u f f e r , pH 7.0, and 20 p1 of 1

m o l / l NaOH. The n e u t r a l i z e d m i x t u r e was incuba ted w i t h

a f f i n i t y - p u r i f i e d a n t i - a r g i n i ne vasopress in Fab' -pe rox idase

con juga te (200 f m o l ) and n o n s p e c i f i c r a b b i t F ( a b ' ) ? (0.1 mg) i n 20

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1116 HASHIDA ET AL.

u l o f b u f f e r A a t 4°C o v e r n i g h t w i t h o u t shak ing. Subsequently,

two s t r e p t a v i d i n - c o a t e d p o l y s t y r e n e b a l l s were added, and t h e

i n c u b a t i o n was con t inued a t 4°C f o r 5 h w i t h o u t shak ing. A f t e r

removal o f t h e r e a c t i o n m i x t u r e , t h e p o l y s t y r e n e b a l l s were washed

t w i c e as desc r ibed above, and perox idase a c t i v i t y bound t o t h e

po lys ty rene b a l l s was assayed a t 30°C f o r 60 min u s i n g 3-(4-hydro-

F1 uorescence xyphenyl ) p r o p i o n i c a c i d as hydrogen donor.

i n t e n s i t y was measured r e l a t i v e t o 0.2 mg/l q u i n i n e i n 50 mmol/l

H2S04 u s i n g 320 nm f o r e x c i t a t i o n and 405 nm f o r emiss ion w i t h a

Shimadzu spect rof luorophotometer (RF-510, Shimadzu Seisakusho,

L t d . , Kyoto, Japan).

Express ion o f t h e D e t e c t i o n L i m i t o f A r g i n i n e Vasopressin

17

The d e t e c t i o n l i m i t o f a r q i n i n e vasopress in was taken as t h e

minimal amount o f a r g i n i n e vasopress in which gave a bound

perox idase a c t i v i t y s i g n i f i c a n t l y i n excess o f t h a t n o n s p e c i f i c a l -

l y bound i n t h e absence o f a r g i n i n e vasopress in (background).

The ex i s tence o f a s i g n i f i c a n t d i f f e r e n c e from t h e background was

conf i rmed by t h e J - t e s t (p<O.OOl, n.5).

RESULTS AND DISCUSSION

D e t e c t i o n L i m i t o f A r g i n i n e Vasopress in

A r g i n i n e vasopress in, a n i n e amino a c i d p e p t i - i e w i t h an

i n t r a m o l e c u l a r d i s u l f i d e b r i d g e ( F I G . l ) , was b i o t i n y l a t e d

d i r e c t l y w i t h N-hydroxysuccinimidobiotin and, a f t e r e l i m i n a t i n g

o t h e r b i o t i n y l a t e d substances, was measured u s i n g a n t i - a r g i n i n e

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1117

.CONH2

[ Arg8]-Vasopressin [ ArgB l-~asotocin

Oxytocin [ ~ysBl-~asopressin

F I G . 1 Pr imary s t r u c t u r e s o f a r g i n i n e vasopress in and r e l a t e d pep t ides .

vasopress in Fab ' -perox idase con juga te and s t r e p t a v i d i n - c o a t e d

p o l y s t y r e n e b a l l s . The d e t e c t i o n l i m i t o f a r g i n i n e vasopress in

was 54 f g ( 5 0 amol) / tube. From t h e p r i m a r y s t r u c t u r e o f t h e

p e p t i d e , however, t h e f o l l o w i n g p o s s i b i l i t y was suggested.

B i o t i n res idues bound t o t h e p e p t i d e molecules th rough N- te rm ina l

amino groups m i g h t have been f a i r l y c l o s e t o t h e e p i t o p i c s i t e s

recogn ized by a n t i - a r g i n i n e vasopress in Fab ' -pe rox idase con juga te .

There fo re , t h e b i o t i n y l a t e d p e p t i de molecules , wh ich had been

bound t o t h e con juga te molecules, m i g h t n o t have e f f i c i e n t l y

r e a c t e d w i t h s t r e p t a v i d i n - c o a t e d p o l y s t y r e n e b a l l s due t o s t e r i c

h indrance. T h i s p o s s i b i l i t y was t e s t e d by i n d i r e c t b i o t i n y -

l a t i o n as i l l u s t r a t e d i n F ig . 2. Male imide groups were

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1118 HASHIDA ET AL.

@O-z-(CH2)5 -$ 0

I N-succinimidyl-6-maleimidohexanoate

9 C-NH-CH2-COOH COOH I I

H S-C H 2-CH-NH-$-C H 2-C H p-C H-N H 0

Glutathione

H

Q-O-!-( CH2I4 NH

N-h ydroxysuccinimidobiotin

H

C-NH-CH2-COOH COOH I 0 S-CH,-CH-NH-$-CH2-CH2-CH-NH-C-(CH 2 4 )

? NH

I

I 0

FIG. 2 I n d i r e c t b i o t i n y l a t i o n o f a r g i n i n e vasopress in. A V P : a r g i n i n e vasopress in.

i n t r o d u c e d i n t o t h e p e p t i d e molecules u s i n g N-succ in i rn idy l -6-

maleimidohexanoate and subsequent ly r e a c t e d w i t h t h i o l groups o f

g l u t a t h i o n e molecules. F i n a l l y , amino groups o f g l u t a t h i o n e

res idues bound t o a r g i n i n e vasopress in molecules were reac ted w i t h

N-hydroxysuccinimidobiotin. A s a r e s u l t , t h e d e t e c t i o n l i m i t

o f a r g i n i n e vasopress in was lowered 5 - f o l d t o 11 f g (10 amo l ) / t ube

( F I G . 3 ) , which was 4 5 - f o l d l ower than t h a t by c o m p e t i t i v e enzyme

immunoassay u s i n g t h e same an t i se rum as used i n t h i s s t u d y and 9 6

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1119

i % c,

w c, U

r

.-

Q 1000 aJ v) m E

2 0 L

U c 3 0 m L 0 4-

% c, .r VI E aJ c, c - aJ V c aJ U VI aJ L 0 3

L c

lArg81-Vasopressin i n Plasma (ng/l)

0.1 1 10 100

10 50 100 1000 10000

aJ v) m E

2

2 100

0 L

U c

m L 0 4-

% c,

VI E aJ c, c

aJ V c aJ U VI aJ L 0 3

L

.r

- 10

c lArg81-Vasopressin i n Plasma (ng/l)

1 10 100

50 100 1000 10000 1

10

CArg8I-Vasopressin (amol/tube)

F I G . 3 Standard curves o f a r g i n i n e vasopress in by t h e p resen t enzyme irnmunoassay a f t e r t h e d i r e c t b i o t i n y l a t i o n (open c i r c l e s ) and t h e i n d i r e c t b i o t i n y l a t i o n ( c l o s e d c i r c l e s ) . Each p o i n t i s t h e mean o f 5 de te rm ina t ions .

t o 4 0 0 - f o l d l ower than those p r e v i o u s l y r e p o r t e d by c o m p e t i t i v e

radioimmunoassays. 1-5 T h i s suppor ted t h e above p o s s i b i l i t y and

suggested t h a t haptens w i t h amino groups c o u l d a l s o be measured

w i t h h i g h s e n s i t i v i t y i n a s i m i l a r manner.

I n t h e subsequent exper iments, t h e r e f o r e , enzyme immunoassay

was performed a f t e r t h e i n d i r e c t b i o t i n y l a t i o n .

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1120 HASHIDA ET AL.

S p e c i f i c i t y

Bound perox idase a c t i v i t y i n t h e presence o f o x y t o c i n (10

ng/ tube) , EAsu' y6,Arg I - v a s o t o c i n (1 ng / tube ) , CArg I - v a s o t o c i n ( 1

pg/ tube) and [Lys I - v a s o p r e s s i n ( 2 1 f g / t u h e ) was n o t s i g n i f i c a n t l y

h i g h e r than t h a t i n t h e absence o f a r g i n i n e vasopress in (nonspeci -

f i c a l l y bound perox idase a c t i v i t y ) and s i g n i f i c a v t l y l ower than

t h a t i n t h e presence o f 11 f g (10 amol, t h e d e t e c t i o n l i m i t ) o f

a r g i n i ne vasopress in. The c r o s s - r e a c t i o n s w i t h o x y t o c i n ,

AS^"^ ,Arg I - v a s o t o c i n , [Arg I - v a s o t o c i n ( a n t i d i u r e t i c hormone i n

v e r t e b r a t e s o t h e r than mammals) and CLys I - v a s o p r e s s i n ( a n t i -

d i u r e t i c hormone i n p i g and hippopotamus) on a mo la r b a s i s were

l e s s than 0.0001 %, l e s s t h a n 0.001 %, 0.3 I and 39 %, r e s p e c t i v e -

1Y. These r e s u l t s i n d i c a t e d t h a t a n t i - a r g i n i n e vasopress in

Fab ' -perox idase con juga te recognized t h e r i n g s t r u c t u r e c o n t a i n i n g

8 8

8

8 8

8

Phe' o f a r g i n i n e vasopress in (FIG. 1).

Recovery o f A r g i n i n e Vasopressin Added t o Plasma

When i n c r e a s i n g volumes o f plasma were sub jec ted t o b i o t i n y -

l a t i o n , t h e recovery o f a r g i n i n e vasopress in added t o plasma was

decreased. T h i s was due t o t h e presence o f amino groups a t

h i g h concen t ra t i ons i n plasma (approx ima te l y 60 mmo l / l ) , w h i l e t h e

concen t ra t i ons o f N-succinimidyl-6-maleimidohexanoate, g l u t a t h i o n e

and N-hydroxysuccinimidobiotin added t o t h e r e a c t i o n m i x t u r e f o r

b i o t i n y l a t i o n were 3-6 mmol/l. The maximal volume o f plasma

t h a t cou ld be used w i t h a s a t i s f a c t o r y recove ry was o n l y 5-10 u1.

I n o rde r t o overcome t h i s d i f f i c u l t y , plasma was s l i g h t l y

d i l u t e d and f i l t e r e d by c e n t r i f u g a t i o n i n a m i c r o c o n c e n t r a t o r w i t h

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1121

po lysacchar ide membrane t o separate pep t ides f rom plasma p r o t e i n s

as desc r ibed above. When plasma was d i l u t e d 1 .3 - fo ld , t h e

concen t ra t i ons o f amino groups i n plasma f i l t r a t e s o f h e a l t h y

s u b j e c t s aged 28-37 y r a t 9:00 a.m. b e f o r e b r e a k f a s t (n=4) and

approx ima te l y 4 h a f t e r b r e a k f a s t (n=8) were b o t h 1.7-2.1 mmol/ l .

When plasma was d i l u t e d 2 - f o l d , t h e c o n c e n t r a t i o n s were b o t h

1.0-1.3 mmol/ l . When 100 p l o f plasma f i l t r a t e s co r respond ing

t o 50 p1 and 75 ~1 o f plasma was sub jec ted t o b i o t i n y l a t i o n , t h e

r e c o v e r i e s o f a r g i n i n e vasopress in (8 n g / l ) added t o 4 plasma

samples c o n t a i n i n g 0.37-0.96 n g / l o f a r g i n i n e vasopress in were

78.3-90.5 % and 76.6-87.6 %, r e s p e c t i v e l y .

Assay V a r i a t i o n

The assay v a r i a t i o n was examined a t 4 d i f f e r e n t l e v e l s o v e r

t h e range o f 0.57-37.7 n g / l f o r w i t h i n - a s s a y and a t 3 d i f f e r e n t

l e v e l s over t h e range o f 0.63-41.9 n g / l f o r between-assay. The

volume o f plasma f i l t r a t e s used was 100 p l co r respond ing t o 75 p l

o f plasma, and t h e numbers o f d e t e r m i n a t i o n s a t each l e v e l were 10

f o r w i t h i n - a s s a y and 10 f o r between-assay. The v a r i a t i o n

c o e f f i c i e n t s o f w i th in -assay and between-assay were 5.6-9.4 % and

6.3-8.9 %, r e s p e c t i v e l y .

A p p l i c a t i o n

The assay range o f a r g i n i n e vasopress in i n plasma u s i n g 100

~1 o f plasma f i l t r a t e s corresponding t o 75 p1 o f plasma was 0.14-

140 n q / l ( F I G . 3). The c o n c e n t r a t i o n s o f a r g i n i n e vasopress in

i n plasma o f 8 h e a l t h y s u b j e c t s aged 25-41 y r w i t h l i b i t u m

water i n t a k e and normal a c t i v i t y anp rox ima te l y 4 h a f t e r b r e a k f a s t

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

1122 HASHIDA ET AL.

were 0.72 2 0.22 ( S D ) n g / l (range, 0.42-1.04 n g / l ) . These

va lues were l ower t h a n p r e v i o u s l y r e p o r t e d ones (1.2-5.2 n g / l )

which were determined by c o m p e t i t i v e immunoassays o n l y a f t e r

e x t r a c t i o n and c o n c e n t r a t i o n u s i n g 1-5 m l o f plasma. 1-6

ACKNOWLEDGEMENTS

T h i s work was suppor ted i n p a r t by Gran ts - i n -A id f o r

S c i e n t i f i c Research (Nos. 01580168 and 02808029) f rom t h e M i n i s t r y

o f Education, Science, and C u l t u r e o f Japan and a research g r a n t

f o r n a t u r a l sc iences f rom t h e M i t s u b i s h i Foundat ion.

REFERENCES

1.

2 .

3. 4.

5.

6.

7.

8 .

9.

10.

11.

12.

G.L. Robertson, E.A. Mahr, S. A tha r and T. Sinha, 3. C l i n . I n v e s t . 52, 2340 (1973) J . J . Morton, P.L. P a d f i e l d and M.L. F o r s l i n g , J . Endocr. 65, 411 (1975) M. Hammer, Scand. J . C l i n . Lab. I n v e s t . 38, 707 (1978) F.T. LaRochelle, J r . , W.G. N o r t h and P . E e r n , P f l u g e r s Arch. - 387, 79 (1980) K. Glanzer, M. Appenheimer, F. Kruck, W . V e t t e r and H. V e t t e r , Acta Endoc r ino log i ca 106, 317 (1984) T. Uno, K. Uehara, K. irlotomatsu, E. Ish ikawa and K . Kato, E x p e r i e n t i a 38, 786 (1982) G.L. Roberts=, L.A. K l e i n , J . Roth and P. Gorden, Proc. Nat. Acad. Sc i . U.S.A. 66, 1298 (1970) W.R. Skowsky and T . A . F i she r , J. Lab. Cl in, Med. 80, 134 (1972) E. Ish ikawa, M. Imagawa, S. Hashida, S. Yoshi take, Y . Hamaguchi and T. Uencl, J . Iamunoassav 4, 209 (1983) S. Hashida, M. Imagawa, S. Inoue, K-h, Ruan and E. Ish ikawa, J . Appl. Biochem. 6, 56 (1984) K-h. Ruan, S. H;shida, S. Yoshi take, E. Ish ikawa, 0. Wakisaka, Y . Yamamoto, T. I c h i o k a and K. Nakajima, C l i n . Chim. Acta 147, 167 (1985) S. H a s h i d a , T Ish ikawa, M. Mukoyama, K. Nakao and H. Imura, J . C l i n . Lab. Anal . 2, 161 (1988)

-

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014

HETERO-TWO-SITE ENZYME IMMUNOASSAY 1123

13. E. Ish ikawa and K. Kato, Scand. J . Immunol. 8(supp1.7) . 43 (1978)

14. T. Kohno, E. Ish ikawa, S. Sugiyama and S. Nakamura, J. C l i n . Lab. Anal. 2, 19 (1988)

15. M.O. DayhofT, L.T. Hunt, P.J. McLaughl in and W.C. Barker , I n : A t l a s o f P r o t e i n Sequence and S t r u c t u r e (M.O. Dayhof f , ed . ) , pp D192 (1972), N a t i o n a l B iomedica l Research Foundation, Washington, D.C.

16. S. Udenfr iend, S . S t e i n , P. Bohlen. W. Dairman, W. Le imgruber and M. Weigele, Science, 178, 871 (1972)

17. M. Imagawa, S. Hashida, E. Ish ikawa, H. Mor i , C . Nakai, Y . I c h i o k a and K. Nakajima, Anal . L e t t . 16(B19), 1509 (1983)

Received January 9, 1991 AccePted March 26, 1991

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

14:

44 0

5 D

ecem

ber

2014