comparison of highly sensitive sandwich enzyme immunoassay and radioimmunoassay for human ferritin

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Comparison Of HighlySensitive SandwichEnzyme Immunoassay AndRadioimmunoassay ForHuman FerritinMasayoshi Imagawa a , Shinji Yoshitake a , EijiIshikawa b a , Yoshiro Niitsu a , Ichiro Urushizakic a , Reizo Kanazawa a , Seiji Tachibana a ,Nobuhiko Nakazawa a & Hiroshi Ogawa d aa Department of Biochemistry , Medical Collegeof Miyazaki , Kiyotake, Miyazaki, 889-16b Department of Internal Medicinec Sapporo Medical College , S-l, W-16, Sapporo,060d Daiichi Radioisotope Labs , 3-10-5, Nihonbashi,Tokyo, 103Published online: 05 Dec 2006.

To cite this article: Masayoshi Imagawa , Shinji Yoshitake , Eiji Ishikawa , YoshiroNiitsu , Ichiro Urushizaki , Reizo Kanazawa , Seiji Tachibana , Nobuhiko Nakazawa& Hiroshi Ogawa (1981) Comparison Of Highly Sensitive Sandwich EnzymeImmunoassay And Radioimmunoassay For Human Ferritin, Analytical Letters,14:20, 1679-1692, DOI: 10.1080/00032718108059832

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ANALYTICAL LETTERS, 14(B20), 1679-1692 (1981)

COMPARISON OF HIGHLY SENSITIVE SANDWICH ENZYME IMMUNOASSAY

AND WIOIMMUNOASSAY FOR HUMAN FERRITIN

KFX WORDS: 8-D-Galactosidase, Sandwich enzyme immunoassay,

Sandwich radioinnnunoassay, F e r r i t i n

Masayoshi Imagaua, S h i n j i Yoshi take, E i j i Ishikawaa,

Yoshiro Ni i t su , I c h i r o Urushizaki , Reizo Kanazava, S e i j i

Tachibana, Nobuhiko Nakazawa and Hi rosh i Ogawa'

Department of Biochemistry, Medical College of Miyazakia,

Kiyotake, Miyazaki 889-16

Department of I n t e r n a l Medicine, Sapporo Medical Col lege , b

S-1, W-16, Sapporo 060 C Dai ich i Radioisotope Labs. , 3-10-5. Nihonbashi, Tokyo 103

ABSTRACT

A h ighly s e n s i t i v e sandwich enzyme immunoassay (EIA) f o r

human f e r r i t i n w a s developed us ing r a b b i t a n t i - f e r r i t i n IgG-

coated polys tyrene b a l l s and a f f i n i t y - p u r i f i e d r a b b i t a n t i -

f e r r i t i n Fab ' l a b e l l e d wi th 8-D-galactosidase from Escher ich ia

- c o l i and compared wi th t h e corresponding sandwich radioimmuno-

Copyright 0 1982 by Marcel Dekker, Inc.

1680 IMAGAWA ET AL.

a s s a y ( R I A ) . The s p e c i f i c and n o n s p e c i f i c b inding of l a b e l l e d

a n t i - f e r r i t i n t o t h e polys tyrene b a l l s w e r e examined i n r e l a t i o n

t o t h e amount of l a b e l l e d a n t i - f e r r i t i n used p e r tube , and t h e

h i g h e s t s e n s i t i v i t y of each immunoassay (0.2 amol/ tube i n E I A and

2.5 amol/ tube i n RIA) was obta ined by u s i n g t h e minimal amount of

t h e cor responding l a b e l l e d a n t i - f e r r i t i n (0.71 fmol i n EIA and

4.5 fmol = 4436 cpm i n RIA) which gave a r e l i a b l e c a l i b r a t i o n

curve. The sandwich R I A w a s less s e n s i t i v e , l a r g e l y because

t h e s p e c i f i c r a d i o a c t i v i t y of 1251-label led a r t t i - f e r r i t i n used

w a s n o t s u f f i c i e n t l y h igh .

INTRODUCTION

F ive y e a r s ago, t h e f u t u r e of enzyme immunoassay w a s d i s -

cussed i n comparison w i t h radioimmunoassay, and i t w a s sugges ted

t h a t radioimmunoassay might be t h e only method f o r t h e a n a l y s i s

of femtomole (fmol = 1 x 10-15m01) amounts of s t e r o i d s , hormones

I n c o n t r a s t , our exper ience on a sandwich enzyme and so on . immunoassay suggested t h a t enzyme immunoassay would be as s e n s i -

t i v e as o r more s e n s i t i v e than radioimmunoassay and provide a t t o -

mole (am01 = 1 x 1 0 - ~ ~ m o l ) s e n s i t i v i t i e s * . A number of enzyme

immunoassays have s i n c e been developed and r e p o r t e d t o be as

s e n s i t i v e as radioimmunoassays. However, no r e p o r t h a s de-

s c r i b e d how t h e s e n s i t i v i t i e s f o r t h e s e immunoassays are l i m i t e d .

T h i s paper d e s c r i b e s a sandwich enzyme immunoassay f o r human

f e r r i t i n , which is a t l eas t 100 f o l d more s e n s i t i v e t h a n t h o s e

p r e v i o u s l y r e p o r t e d 3-8, comparing w i t h t h e cor responding

SANDWICH ENZYME IMMUNOASSAY 1681

sandwich radioimmunoassay wi th s p e c i a l r e fe rence t o t h e i r sensi-

t i v i t i e s .

MATERIALS AND METHODS

Prepa ra t ion of Human F e r r i t i n and Rabbit An t i -Fe r r i t i n Serum

F e r r i t i n w a s prepared from human l i v e r as described previ-

F e r r i t i n prepara t ion obtained was e l ec t rophore t i -

c a l l y pure. The amount i n g of p u r i f i e d f e r r i t i n were esti-

mated by the method of Lowry e t a1 us ing bovine serum albumin

as standard, and its amount i n mol w a s ca l cu la t ed by taking its

10 molecular weight t o be 450,000 .

3 ous ly .

A n t i - f e r r i t i n sera were ra i sed i n r a b b i t s as described

An t i - f e r r i t i n sera obtained shared no cross- previously . r e a c t i v i t y wi th o the r serum pro te ins o r w i th c e l l u l a r components

on examination by a double-immunodiffusion technique and immuno-

e l ec t rophores i s .

P repa ra t ion of Af f in i ty -Pur i f i ed Ant i -Fer r i t in IgG

A n t i - f e r r i t i n IgG w a s p rec ip i t a t ed by t r e a t i n g a n t i - f e r r i t i n

The serum (8 ml) w i th Na2S04 as described previous ly . p r e c i p i t a t e w a s d i sso lved i n and d ia lysed aga ins t 0.01 M sodium

phosphate bu f fe r , pH 7.4 con ta in ing0 .15 M NaC1. The d i a l y s a t e

(117 mg) w a s appl ied t o a fe r r i t in -Sepharose 4B column (1.5 x 2.8

em), and the column w a s washed wi th t h e same buf fe r . A n t i -

f e r r i t i n IgG w a s e lu t ed wi th 0.3 M potassium thiocyanate and

d ia lysed aga ins t t h e same buffer . The amount of a f f i n i t y -

pu r i f i ed a n t i - f e r r i t i n IgG obtained w a s 5.1 mg.

1682 IMAGAWA ET AL.

F e r r i t i n (2 mg) was coupled t o CNBr-activated Sepharose 4B

(1 g) (Pharmacia F ine Chemicals AB, Uppsala) fo l lowing t h e

i n s t r u c t i o n of t h e manufacturer .

Assay of 8-D-Galactosidase A c t i v i t y

f3-D-Galactosidase a c t i v i t y w a s assayed by f luor imet ry us ing

4-methylumbelliferyl-~-D-galactoside as a s u b s t r a t e i n 0.01 M

sodium phosphate b u f f e r , pH 7.0, con ta in ing 0.1 M N a C l , 1 mM

MgC12, ( b u f f e r A) as

One u n i t of t h e enzyme a c t i v i t y was desc r ibed p rev ious ly . def ined as t h a t which hydrolysed 1 pmol of t he s u b s t r a t e pe r min.

P r e p a r a t i o n of 6-D-Galactosidase-Labelled A n t i - F e r r i t i n

0.1 X bovine serum albumin and 0.1 X N a N 3 11

Aff in i ty -pur i f i ed a n t i - f e r r i t i n IgG w a s converted t o Fab'

and l a b e l l e d wi th f3-D-galactosidase as descr ibed p rev ious ly . The average number of Fab' molecules conjugated per B-D-galacto-

s i d a s e molecule w a s 2.3-2.5, which w a s c a l c u l a t e d a s descr ibed

The amount o f t h e conjugate w a s expressed i n p rev ious ly . u n i t s of f3-D-galactosidase a c t i v i t y .

P r e p a r a t i o n of Anti-Ferr i t in-Coated Po lys ty rene Balls

A n t i - f e r r i t i n IgG w a s prapared by f r a c t i o n a t i o n of a n t i s e r a

11 wi th Na SO

and polys tyrene b a l l s (3.2 mm i n diameter , P r e c i s i o n P l a s t i c

Balls Co., Chicago) w e r e coated wi th a n t i - f e r r i t i n IgG (no t

The concent- af f i n i t y - p u r i f i e d ) by phys ica l adso rp t ion . r a t i o n of a n t i - f e r r i t i n IgG s o l u t i o n used w a s 0.1 mgfml.

Sandwich Enzyme Imunoassay and Sandwich Radioimmunoassay

and chromatography wi th d ie thylaminoethyl c e l l u l o s e 2 4

An a n t i - f e r r i t i n IgG-coated polys tyrene b a l l w a s incubated

wi th pu r i f i ed f e r r i t i n in 0.15 m l of bu f fe r A a t 37'C f o r 6 h

wi th shaking. Af te r t h e incubation, t h e polystyrene b a l l w a s

washed twice wi th 1 m l of bu f fe r A and incubated wi th 50 Uunits

of B-D-galactosidase-labelled a n t i - f e r r i t i n in 0.15 m l of bu f fe r

A a t room temperature (24-26'C) f o r 16 h without shaking and then

a t 37'C f o r 6 h wi th shaking. The polys tyrene b a l l w a s washed

twice wi th 1 m l of bu f fe r A and t r ans fe red t o another test tube

which w a s f r e e from B-D-galactosidase a c t i v i t y . 8-D-Galacto-

s i d a s e a c t i v i t y bound w a s assayed as described above.

Radioimmunoassay w a s performed in t h e same way as the

sandwich enzyme imhunoassay except t h a t a f f in i ty -pur i f i ed an t i -

f e r r i t i n IgG which w a s l abe l l ed wi th L251 by using chloramine-T

was used.

was 5.4 uCi/ug (1790 dpm - 976 cpm/fmol).

Calcu la t ion of t h e Amount of Labelled Ant i -Fer r i t in Bound

The s p e c i f i c r a d i o a c t i v i t y of t he l a b e l l e d IgG used

To compare the two immunoassays, t he amount of l abe l l ed

a n t i - f e r r i t i n bound t o t h e polystyrene b a l l s was ca l cu la t ed by

d iv id ing 8-D-galactosidase a c t i v i t y bound ( u u n i t s ) o r t he radio-

a c t i v i t y bound (cpm) by the s p e c i f i c a c t i v i t y of l abe l l ed an t i -

f e r r i t i n (70 puni t s / fmol o r 976 cpm/fmol). The amount of

l abe l l ed a n t i - f e r r i t i n s p e c i f i c a l l y bound t o f e r r i t i n which was

adsorbed on ant i - fe r r i t in -coa ted polys tyrene b a l l s w a s ca l cu la t ed

by sub t r ac t ing t h e amount of l abe l l ed a n t i - f e r r i t i n nonspecifi-

c a l l y bound in t h e absence of f e r r i t i n from the amount bound i n

t h e presence of f e r r i t i n . The average number of l abe l l ed

a n t i - f e r r i t i n molecules s p e c i f i c a l l y bound per f e r r i t i n molecule

1684 IMAGAWA ET AL.

added was c a l c u l a t e d by d i v i d i n g t h e amount of l a b e l l e d a n t i -

f e r r i t i n s p e c i f i c a l l y bound by t h e amount of f e r r i t i n added.

( F e r r i t i n w a s a lmost completely adsorbed t o a n t i - f e r r i t i n - c o a t e d

p o l y s t y r e n e b a l l s under t h e c o n d i t i o n d e s c r i b e d above, which was

confirmed as d e s c r i b e d p r e v i o u s l y ".)

RESULTS

The s p e c i f i c and n o n s p e c i f i c b indings of l a b e l l e d a n t i b o d i e s

t o antibody-coated s o l i d phase are c l o s e l y r e l a t e d t o t h e s e n s i -

t i v i t y and accuracy of sandwich immunoassays. So, t h e s e were

examined i n r e l a t i o n t o t h e amount of l a b e l l e d a n t i - f e r r i t i n

When t h e amount of B-D-galactosidase-labelled a n t i - f e r r i t i n

used per tube w a s i n c r e a s e d 10 f o l d , i ts amount n o n s p e c i f i c a l l y

bound t o a n t i - f e r r i t i n - c o a t e d p o l y s t y r e n e b a l l s i n t h e absence of

f e r r i t i n (background) w a s a l s o i n c r e a s e d 10 f o l d b u t its amount

s p e c i f i c a l l y bound was increased only 1.3-2.1 f o l d (TABLES 1 and

2 ) . S i m i l a r r e s u l t s were obta ined i n t h e sandwich rad io-

immunoassay (TABLES 1 and 2 ) . A s a r e s u l t , t h e p r o p o r t i o n of

t h e amount of l a b e l l e d a n t i - f e r r i t i n s p e c i f i c a l l y bound t o t h a t

n o n s p e c i f i c a l l y bound w a s decreased w i t h i n c r e a s i n g amounts of

l a b e l l e d a n t i - f e r r i t i n used per tube. These r e s u l t s i n d i c a t e

t h a t bo th sandwich immunoassays can be improved i n s e n s i t i v i t y

and accuracy by us ing t h e minimal amount of l a b e l l e d a n t i -

f e r r i t i n t h a t g i v e s a r e l i a b l e c a l i b r a t i o n curve.

SANDWICH ENZYME IMMUNOASSAY

TABLE 1

Amount of Labelled Anti-Ferritin Nonspecifically Bound in Relation to the Amount Used

Labelled anti-ferritin Labelled anti-ferritin

used nonspecifically bound

(fmol /tube) (amol/tube) (I) ~

p-D-galactosidase-labelled

(I) 0.71 (50) 0.29 (0.020) 0.041

(11) 2.8 (200) 1.15 (0.082) 0.041

(111) 7.1 (500) 3.1 (0.22) 0.044

251-label led

(I) 4.5 (4436) 13.4( 13) 0.30

(11) 9.0 (8805) 28.5 (28) 0.32

(111) 18.0(17557) 58.6 (57) 0.33

Values in parentheses indicate 6-D-galactosidase activity in punits of B-D-galactosidase-labelled anti-ferritin and

radioactivity in cpm of 1251-labelled anti-ferritin.

The minimal amount of B-D-galactosidase-labelled anti-

ferritin that could be determined by 30 min-assay was 0.016 punit

which gave a reading of 2 when the fluorescence intensity of

M 4-methylumbelliferone as standard was adjusted to a scale

of 100. The amount of B-D-galactosidase-labelled anti-ferritin

nonspecifically bound was 0.02 punits (0.29 amol) and its amount

specifically bound with 0.2 amol of ferritin was .0.032 punit

(0.45 amol), when 50 Uunits (0.71 fmol) of 6-D-galactosidase-

1686 IMAGAWA ET AL.

TABLE 2

Amount of Labelled Anti-Ferritin Specifically Bound in Relation to the Amount Used

Ferr i t in Labelled anti-ferritin specifically bound

added (amoljtube)

(amol/tube) (1) (11) (111)

6-D-Galactosidase-labelled

0.45 (2 .3 ) 0.55 ( 2 . 8 )

0.77 ( 1 . 9 ) 1.3 (3 .3 )

2.2 (2 .2 ) 2.9 ( 2 . 9 )

3.9 ( 1 . 9 ) 4.7 ( 2 . 3 )

9.2 (2 .3 ) 11.0 ( 2 . 8 )

22.0 (2.2) 24.7 (2 .5 )

89.0 (2 .2 ) 117.4 (2 .9 )

1251-labelled

14.0 ( 5 . 6 ) 14.4 ( 5 . 7 )

25.7 ( 5 . 1 ) 27.3 (5 .5 )

38.5 (3 .9 ) 46.0 ( 4 . 6 )

82.2 (4 .1) 95.4 (4 .8 )

365.5 ( 3 . 7 ) 434.1 ( 4 . 3 )

0.87 (4 .4 )

1.6 (4 .0 )

3.1 (3.1)

5.9 (3 .0)

13.4 (3 .3 )

28.1 (2.8)

133.2 ( 3 . 3 )

19.5 (T.8)

30.7 ( 6 . 1 )

51.6 (5 .2 )

107.0 (5.3)

524.4 (5 .2 )

(I), (11) and (111) indicate the amounts of labelled anti- ferritin used as indicated in TABLE 1. Values in parentheses indicate the average numbers of labelled anti-ferritin molecules specifically bound per ferritin molecule added.

SANDWICH ENZYME 1MM"OASSAY 1687

l abe l l ed a n t i - f e r r i t i n per tube was used (TABLES 1 and 2) .

When the amounts of f e r r i t i n added were 0 , 0.2, 1, 10, 100 amol

per tube, t he v a r i a t i o n (s tandard dev ia t ion ) of B -D-galactosidase

a c t i v i t y bound by using 50 p u n i t s w a s 17.8, 12.5, 11.0, 5.0,

6.5 % (n=lO), r e spec t ive ly , being s a t i s f a c t o r i l y s m a l l (FIG. 1).

Judging from these va lues , 50 p u n i t s seemed t o be t h e minimal

t h a t could be used per tube, and the h ighes t s e n s i t i v i t y of t h e

sandwich enzyme hmunoassay was taken t o be about 0.2 amol of

f e r r i t i n p e r tube (FIG. 1 ) .

The amount of 1251-labelled a n t i - f e r r i t i n nonspec i f i ca l ly

bound w a s 13 cpm (13 amol) and its amount s p e c i f i c a l l y bound wi th

2.5 and 5.0 a m 0 1 of f e r r i t i n w a s 14 cpm (14 amol) and 25 cpm

(26 amol), r e spec t ive ly , when 4436 cpm (4.5 fmol) o f 1251-

l a b e l l e d a n t i - f e r r i t i n per tube was used (TABLES 1 and 2).

Therefore , the h ighes t s e n s i t i v i t y of the sandwich radioimmuno-

assay w a s taken t o be 2.5-5.0 am01 of f e r r i t i n per tube (FIG. 1).

DISCUSSION

The minimal amount of l a b e l l e d a n t i - f e r r i t i n t h a t could be

used per tube was 6.3 fo ld l a r g e r i n t h e radioimmunoassay (4.5

fmol) than i n the enzyme immunoassay (0.71 fmol) . Consequent-

ly . t he background was much h igher , l i m i t i n g t h e s e n s i t i v t y , i n

t h e radioimmunoassay than i n t h e enzyme immunoassay (FIG. 1).

This is obviously because t h e s p e c i f i c r a d i o a c t i v i t y of lZ5I-

l a b e l l e d a n t i - f e r r i t i n used was no t s u f f i c i e n t l y high.

The s p e c i f i c r a d i o a c t i v i t y of c a r r i e r - f r e e 1251 (R) is

ca lcu la t ed from its h a l f l i f e (60.2 days) as fol lows.

1688 IMAGAWA ET AL.

0 . 2 00.1 1 10 100 1000

Human Ferritin (amol/tube)

FIG. 1 Sandwich Enzyme Immunoassay and Radioimmunoassay of Human Ferritin. The amounts of B-D-galactosidase-labelled

and 1251-labelled anti-ferritin used were 50 punits (0.71 fmol) and 4436 cpm (4.5 fmol), respectively. Vertical bars indicate standard deviations (n=10).

SANDWICH ENZYME 1Ml"OASSAY 1689

A N 3.7 x 10" x 60 x 6.02 x 10

1.44 x 3.7 x 10'' x T

R = - (Ci/gram atom)

23 - (dpmlgram atom)

= 4.82 x 10l8 dpm/gram atom = 4.82 dpmlamol

23 A: Avogadro's number = 6.02 x 10

N: number of r ad io i so top ic atoms per C i

T: ha l f l i f e in sec (60.2 x 24 x 60 x 60)

1 C i = 3.7 x 10'' x 60 dpm 1311 I n the same way, t h e s p e c i f i c r a d i o a c t i v i t y of ca r r i e r - f r ee

wi th a ha l f l i f e of 8.06 days is 36.1 dpmfamol. And t h e

s p e c i f i c r a d i o a c t i v i t y of 1251-labelled IgG used i n the present

experiment w a s 5.4 pCi/pg. t h a t is, 1.8 dpm/amol. Therefore,

i t may be poss ib l e t o prepare 1251- or 1311-labelled an t ibod ie s

wi th higher s p e c i f i c r a d i o a c t i v i t i e s and raise the s e n s t i v i t y .

although 1311-labelled an t ibod ie s r ap id ly l o s e t h e i r s p e c i f i c

a c t i v i t i e s because of i ts s h o r t ha l f l i f e .

The propor t ion of t he amount of l abe l l ed a n t i - f e r r i t i n non-

s p e c i f i c a l l y bound t o its amount used per tube was 0.32 % i n t h e

radioimmunoassay, while i t w a s on ly 0.042 4: i n t h e enzyme immuno-

assay. This i s another f a c t o r t o l i m i t t h e s e n s i t i v i t y of the

radioimmunoassay and may be due t o t h e presence of Fc por t ion i n

1251-labelled IgG. This p o s s i b i l i t y was supported by the f a c t

t h a t a much lower background w a s obtained wi th 1251-labelled

anti-TSH F(ab') i n t he sandwich radioimmunoassay for human TSH

than wi th 1251-labelled anti-TSH IgG. However, t h e s p e c i f i c

binding of 1251-labelled anti-TSH F(ab')2 w a s a l s o lowered, and

1690 IMAGAWA ET AL,.

t h e s e n s i t i v i t y w a s no t enhanced. It remains t o be examined

whether t h i s is gene ra l ly t h e case i n t h e sandwich radioimmuno-

assay f o r o the r an t igens .

The s e n s i t i v i t y of the present sandwich enzyme immunoassay

is at least 100 f o l d h igher than those prev ious ly r epor t ed . There may be a t l e a s t t h r e e reasons f o r t h i s . 1) A n t i - f e r r i t i n

used w a s a f f i n i t y - p u r i f i e d , whi le i t w a s not i n t h e prev ious

r epor t s . 2 ) Small po lys tyrene b a l l s ( 3 . 2 mm i n diameter) were

used, g iv ing a low background. In some of t he prev ious r e p o r t s

( 4 - 6 ) . a l a r g e su r face a r e a of po lys tyrene tube w a s used as an t i -

f e r r i t i n -coa ted s o l i d phase. 3) 0-D-galactosidase was used as

l a b e l , which was very s e n s i t i v e l y de t ec t ed by f luor imet ry . I n

o the r previous r e p o r t s , a l k a l i n e phosphatase 4Y7 and pe r -

used a s l a b e l were assayed by spectrophotometry oxidase

The present enzyme immunoassay can measure 0.9-4500 ng of

f e r r i t i n p e r ml of serum us ing 0.1 p 1 of serum and even 9 pg per

ml of serum us ing 10 p1 of serum wi thout serum i n t e r f e r e n c e , and

i ts c o e f f i c i e n t s of within- and between-assay v a r i a t i o n s w e r e

5.9-8.6 %. When f e r r i t i n l e v e l s i n 4 3 human sera were esti-

mated by both sandwich immunoassays us ing 0.1 p1 of serum, t h e

r eg res s ion equation and c o r r e l a t i o n c o e f f i c i e n t were Y (RIA)=

0.90X(EIA)-4.7 and 0.99, r e spec t ive ly .

The sandwich radioimmunoassay f o r f e r r i t i n descr ibed above

is 10 f o l d more s e n s i t i v e than immunoradiometrlc assay previous ly

repor ted (14) and 30-40 f o l d more s e n s i t i v e than sandwich radio-

immunoassays prev ious ly reported (15-17). These previous

sandwich radioimmunoassays are more s e n s i t i v e than a number of

competit ive radioimmunoassays prev ious ly reported (e.g. 18-20).

ACKNOWLEDGEMENTS

This work w a s supported i n p a r t by research g r a n t s from the

Ministry of Education, Science and Culture of Japan (Nos. 237014,

387072 and 456580). from Environment Agency of Japan and from the

Ministry of Pub l i c Welfare of Japan. We are g r a t e f u l t o Miss

S. Tahara, N. Ishigami, Y. Koba and M. I sh ida f o r t h e i r secre-

tarial and t echn ica l a s s i s t ance .

10. 11.

REFERENCES

D. Watson, K. Kato, Y. Hamaguchi, S. Okawa, E. Ishikawa, K. Kobayashi. and N. Katunuma, N. Watanabe, Y. N i i t su , S. Ohtsuka, J. Koseki, Y. Kohgo, I. Urushizaki, K . Kato, and E. Ishikawa, Clin. Chem. 25, 80

L. ThBriault , and M. Page, F. M. J. Zuyderhoudt, W. Boers, C. L in tho r s t , G. G. A. J&ning, and P . Hengeveld, S. Anaokar, P. J. Garry. and J. C. Standefer, Clin. Chem. -' 25 1426 (1979). R. L. F o r t i e r , W. P. McGrath, and S. L. "bomey, Clin. Chem. -' 25 1466 (1979). J. D. Conradie. and B. E. L. Mbhele, S. Afr. Med. J. 2, 0. H. Lowry, N. J. Rosebrough, A. L. Far r , and R. J. Randall, J. Biol. Chem. ,193, 265 (1951). R. R. Crichton, New Engl. J. Med. 284, 1413 (1971). E. Ishikawa, and K. Kato, Scand. J. Immunol. 8, Suppl. 7, 43 (1978). S. Yoshitake, Y. Hamaguchi, and E. Ishikawa, Scand. J. Immunol. E, 81 (1979). F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. -' 89 114 (1963).

Lancet, G. 569 (1976).

Lancet, i, 40 (1977).

(1979). Clin. Chem. 23. 2142 (1977).

Clin. Chim. Acta 88, 37 (1978).

282 (1980).

1692 IMAGAUA ET AL.

14. G. M. Addison, M. R. Beamish, C. N. Hales, M. Hodgkins, A. J a c o b s , and P . L l e w e l l i n , J . Cl in . P a t h . 25, 326 (1972)

15. L. E. M. Miles, D. A. L i p s c h i t z , C. P . Bieber, and J. D. Cook, Anal. Biochem. 61, 209 (1974).

16. J . W. H a l l i d a y , K . L. Gera, and L. W. P o w e l l , C l i n . Ch im. A c t a 58, 207 (1975)

17. L. M. Gonyea, C l i n . Chem. 2, 234 (1977). 18. A. W. Luxton, W. H. C. Walker, J . G a u l d u e , M. A. M. A l i , and

C. P e l l e t i e r , C l in . Chem. 23, 683 (1977). 19. W. M. Deppe, S. M. J o u b e r t , and P . Naidoo, J. C l i n . Pa thol . - 31, 872 (1978).

20. B. M s r i a d e c , J. J o l u , and R. Henry, C l i n . Chem. 2, 1596 (1979).

Received September 8, 1981 Accepted September 15, 1981

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