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Novel and UltrasensitiveSandwich Enzyme Immunoassay(Sandwich Transfer EnzymeImmunoassay) for AntigensSeiichi Hashida a , Koichiro Tanaka a , TakeyukiKohno a & Eiji Ishikawa aa Department of Biochemistry, Medical College ofMiyazaki, Kiyotake, Miyazaki, 889-16, JapanVersion of record first published: 06 Dec 2006.
To cite this article: Seiichi Hashida , Koichiro Tanaka , Takeyuki Kohno & Eiji Ishikawa(1988): Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich TransferEnzyme Immunoassay) for Antigens, Analytical Letters, 21:7, 1141-1154
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ANALYTICAL LETTERS, 21(7), 1141-1154 (1988)
NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY
(SANDWICH TRANSFER ENZYME IMMUNOASSAY) FOR ANTIGENS
KEY WORDS: Enzyme immunoassay, Antigen, B-D-Galactosidase,
Thyroid-stimulating hormone, Growth hormone
Seiichi Hashida, Koichiro Tanaka, Takeyuki Kohno and Eiji Ishikawa
Department o f Biochemistry, Medical College of Miyazaki, Kiyotake , Miyazaki 889-16, Japan
ABSTRACT
A novel and ultrasensitive sandwich enzyme imrnunoassay
(sandwich transfer enzyme immunoassay) for antigens is described.
Antigens were reacted with dinitrophenyl monoclonal mouse antibody
IgGl and rabbit antibody Fab'-8-D-galactosidase conjugates. The
complex formed of antigens with dinitrophenyl monoclonal mouse
anti body I gG1 and rabbit anti body Fab'-B-D-gal actos i dase
1141
Copyright 0 1988 by Marcel Dekker, Inc.
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1142 HASHIDA ET AL.
conjugates was trapped onto aff i ni ty-puri f ied rabbit (apti-
dinitrophenyl bovine serum a1 bumin) IgG-coated polystyrene balls.
After eliminating excess of the conjugates, the complex was eluted
from the polystyrene balls with dinitrophenyl-L-lysine and trans-
fered to clean polystyrene balls coated with affinity-purified
rabbit (anti-mouse IgG) IgG. R-D-Galactosidase activity bound
to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed
by fluorimetry. Nonspecifically bound R-D-galactosidase
activity considerably decreased with less decrease in specifically
bound R-D-galactosidase activity. As a result, the detection
limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol)
and human growth hormone (10 fg, 0.5 amol) by the present enzyme
immunoassay were 30-fold lower than those by the conventional
enzyme immunoassay, in which antigens were incubated with monoclo-
nal mouse antibody IgG1-coated polystyrene balls and rabbit anti-
body Fab'-B-D-galactosidase conjugates,
INTRODUCTION
In the conventional sandwich enzyme immunoassay, antigens are
trapped onto antibody-coated solid surfaces and reacted with
antibody-enzyme conjugates. The immunoreaction on solid
surfaces efficiently takes place so that attomole amounts of
antigens can be measured using affinity-purified Fab'-enzyme
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY 1143
conjugates prepared by the hinge method, in which Fab' molecules
are conjugated to enzyme molecules by selective use of thiol
groups in the hinge. However, the nonspecific binding of
antibody-enzyme conjugates to solid surfaces is still one of the major factors limiting the sensitivity of the conventional sandwich
enzyme inmunoassay.
This paper describes a novel and ultrasensitive sandwich
enzyme immunoassay (sandwich transfer enzyme immunoassay) , in
which the sensitivity was improved by reduction of the nonspecific
binding of antibody-enzyme conjugates.
MATERIALS AND METHODS
Buffer
The regularly used buffer was 10 mmol/l sodium phosphate
buffer, pH 7.0, containing 1 g/1 bovine serum albumin (fraction V ,
Armour Pharmaceutical Co. , Kankakee, Illinois), 0.1 mol/l NaCl , 1 mmol/l MgC12 and 1 g/1 NaN3 (buffer A ) .
Hormones
Human thyroid-stimulating hormone (hTSH) and human growth
hormone (hGH) used as standard were preparations included in
radioimmunoassay kits (hTSH kit "Daiichi", Daiichi Radioisotope
Labs., Ltd., Tokyo, Japan and HGH RIA KIT, Dainabot Co., Ltd.,
Tokyo, Japan). hCG coupled to CNBr-activated Sepharose 4B was
obtained from Calbiochem-Behring Corporation, La Jolla, Califor-
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1144 HASHIDA ET AL.
nia. hGH used for immunization was obtained from KabiVitrum
Ab, Stockholm, Sweden.
An ti bodies
Monoclonal mouse anti-hTSH R-subunit IgGl was obtained from
Mallinckrodt, Inc., St. Louis, Missouri. Monoclonal mouse
anti-hGH IgGl was prepared as described previously. Rabbit
anti-hCG IgG was obtained from Dakopatts a/s, Glostrup, Denmark.
Rabbit (anti-dinitrophenyl bovine serum albumin) serum was
obtained from Miles Laboratories, Inc., El khart, Indiana.
Rabbit (anti-mouse IgG) IgG was obtained from Medical Biological
Laboratories Co., Ltd., Nagoya, Japan. Anti-hGH serum was 3 prepared in rabbit as described previously.
IgG, F(ab')il and Fab'
IgG were prepared from serum by fractionation with Na2S04 4 followed by passage through a column of DEAE-cellulose.
F(ab')p was prepared by digestion of IgG with pepsin, and Fab' was
prepared by reduction of F(ab'),! The amount of IgG, F(ab')2 4 and Fab' was calculated from the absorbance at 280 nm.
Dinitrophenyl Monoclonal IgGl and Bovine Serum Albumin
1. Mercaptosuccinylated monoclonal IgGI. Monoclonal IgGl
(0.25 mg) in 0.45 ml of 0.1 mol/l sodium phosphate buffer, pH 7.5,
was incubated with 0.05 ml of 40 mmol/l S-acety lmercaptosucc in ic
anhydride (Nakarai Chemicals, Ltd., Kyoto, Japan) in N,N-dimethyl-
formamide at 30°C for 30 min. After incubation, the mixture
was incubated with 0.05 m l of 1 mol/l Tris-HC1 buffer, pH 7.0,
0.03 ml of 0.1 mol/l EDTA, pH 7.0, and 0.06 ml of 1 mol/l
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY 1145
hydroxylamine, pH 7.0, at 30°C for 5 min. The reaction mixture
was subjected to gel filtration on a column (1.0 x 30 cm) of
Sephadex 6-25 using 0.1 mol/l sodium phosphate buffer, pH 6.0,
containing 0.5 mmol/l EDTA. The average numbers of thiol
groups introduced per IgGl molecule were 10.5 (anti-hTSH) and 6.2 4 (anti-hGH).
2. Maleimide-dinitrophenyl-L-lysine. An aliquot (0.9 ml)
of 5.5 mmol/l dinitrophenyl-L-lysine-HC1 (Tokyo Kasei Kogyo, Co.,
Ltd., Tokyo, Japan) in 0.1 mol/l sodium phosphate buffer, pH 7.0,
containing 5 mmol/l EDTA was incubated with 0.1 ml of 5 mmol/l
N-succi nimi dyl -6-ma1 eimi dohexanoate (Doj i rid0 Laboratories , Kumamoto, Japan) in N,N-dimethylformamide at 30°C for 30 min.
3. Dinitrophenyl monoclonal IgG1. An aliquot (0.5 ml) of
the maleimide-dinitrophenyl-L-lysine solution was incubated with
the mercaptosuccinylated monoclonal IgGl (0.1 mg) in 4.5 ml of 0.1
mol/l sodium phosphate buffer, pH 6.0, containing 5 mmol/l EDTA at
30°C for 30 min. The reaction mixture was subjected t o gel
filtration on a column (1.0 x 30 cm) of Sephadex 6-25 using 0.1
mol/l sodium phosphate buffer, pH 7.0. The average numbers o f
dinitrophenyl groups introduced per IgGr molecule were 7.2
(anti-hTSH) and 5.7 (anti-hGH) , which were calculated from the
absorbance at 360 nm by taking the molar extinction coefficient to
be 17,400 mol-l. 1 i ter .cm-’.
Dinitrophenyl bovine serum albumin was prepared in the same
way, and the number of dinitrophenyl groups introduced per bovine
serum albumin molecule was 7.0.
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1146 HASHIDA ET AL.
Protein-Sepharose 48
hCG (2.5 mg), dinitrophenyl bovine serum albumin (10 mg),
nonspecific mouse IgG (20 mg) and nonspecific mouse serum protein
(10 mg) were coupled to CNBr-activated Sepharose 48 (1 g,
Pharmacia Fine Chemicals AB, Uppsala, Sweden) according to the
instructions of Pharmacia. Nonspecific mouse serum protein for
coupling was obtained as follows. Nonspecific mouse serum
(0.5 ml) was subjected to gel filtration on a column (1.0 x 30 cm)
of Sephadex 6-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden)
using 0.1 mol/l sodium borate buffer, pH 8.0, containing 0.5 mol/l
NaCl . The amount of serum protein was calculated from the
absorbance at 280 nm using bovine serum albumin as standard.
Aff i ni ty-Puri f icat ion of Anti bodies
Rabbi t anti - hCG F ( a b ' ) , rabbi t (anti -din i trophenyl bovine
serum albumin) IgG and rabbit (anti-mouse IgG) IgG were
affinity-purified by elution at pH 2.5 from columns of hCG-,
I gG- dinitrophenyl bovine serum albumin and nonspecific mouse 6 coupled Sepharose 4B.
Fab' -R-D-Gal actosi dase Conjugate
Affinity-purified rabbit anti-hCG Fab' and rabbit ant
Fab' were conjugated to B-D-galactosidase from Escherichia
using N,N'-o-phenylenedimaleimide! The average numbers o f
mo7ecules conjugated per B-D-galactosidase molecule were
-hGH
Fab'
2.1
(anti-hCG Fab') and 4.1 (anti-hGH Fab'). The rabbit Fab'-R-D-
galactosidase conjugates (0.8 mg) in 0.5 ml of buffer A were
passed through a column (0.55 x 1.0 cm) of nonspecific mouse serum
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY 1147
protein-Sepharose 48 using buffer A . The amount of the 4 conjugate was calculated from B-D-galactosidase activity.
IgG-Coated Polystyrene Bal Is
Polystyrene balls ( 3 . 2 mm in diameter, Precision Plastic Ball
Co., Chicago, Illinois) were coated by physical adsorption with
affini ty-purified rabbit (anti-dini trophenyl bovine serum a1 bumin)
IgG, affinity-purified rabbit (anti-mouse IgG) IgG, monoclonal r anti-hTSH R-subunit IgGl and monoclonal anti-hGH IgGl (0.1 g/l).
Polystyrene balls coated with (anti-dinitrophenyl bovine serum
albumin) IgG had been marked with a black dot for discrimination
from (anti-mouse IgG) IgG-coated polystyrene balls.
Present Sandwich Transfer Enzyme Imunoassay for hTSH and hGH
For hTSH assay, affinity-purified rabbit anti-hCG Fab'-R-D-
galactosidase conjugate (200 fmol) in 0.01 ml of buffer A was
incubated with nonspecific mouse IgG (0.1 ug) in 0.01 ml of buffer
A at 20°C for 2 h. Dinitrophenyl monoclonal anti-hTSH
6-subunit IgGl (150 fmol) in 0.02 ml of buffer A was incubated
with nonspecific rabbit F(ab')* (0.1 mg) in 0.02 ml of buffer A at
20°C for 2 h, The two incubation mixtures were mixed and
incubated with hTSH standards in 0.09 ml of buffer A at 20°C
overnight. Subsequently, two aff ini ty-purif i ed rabbit (anti - dinitrophenyl bovine serum a1 bumin) IgG-polystyrene balls marked
with a black dot were added, and the incubation was continued at
20°C for 4 h. After removal of the incubation mixture, the
marked polystyrene balls were washed twice by addition and
aspiration of 2 ml of buffer A and incubated with 0.15 ml of
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1148 HASHIDA ET AL.
b u f f e r A c o n t a i n i n g 1 mmol/ l d i n i t r o p h e n y l - L - l y s i n e , 0.66 g/1
n o n s p e c i f i c r a b b i t F ( a b ' ) * and two a f f i n i t y - p u r i f i e d r a b b i t
(anti-mouse IqG) IgG-po lys ty rene b a l l s w i t h no mark a t 20°C f o r 4
h. The p o l y s t y r e n e b a l l s w i t h no mark were washed t w i c e as
desc r ibed above, and bound O-D-galactosidase was assayed by
f l u o r i m e t r y a t 30°C f o r 16 h u s i n g 4 - m e t h y l u m b e l l i f e r y l B-D-
g a l a c t o s i d e as s u b s t r a t e . F luorescence i n t e n s i t y was measured
r e l a t i v e t o mol/l 4-methy lumbe l l i f e rone i n 0.1 m o l / l g l y c i n e -
NaOH b u f f e r , pH 10.3 u s i n g a Shimadzu f l uo rospec t ropho tomete r
(RF-510, Shimadzu Seisakusho, L td. , Kyoto, Japan). ( I n some
exper iments, 0-D-galactos idase a c t i v i t y bound t o t h e marked
p o l y s t y r e n e b a l l s b e f o r e i n c u b a t i o n w i t h d i n i t r o p h e n y l - L - l y s i n e
was assayed a t 30°C f o r 1 h.)
hGH was assayed as desc r ibed above, excep t t h a t d i n i t r o p h e n y l
monoclonal a n t i -hGH IgGl and a n t i -hGH Fab'-8-D-gal ac tos idase
con juga te were s u b s t i t u t e d f o r d i n i t r o p h e n y l monoclonal anti-hTSH
& s u b u n i t IgGl and a f f i n i t y - p u r i f i e d anti-hCG Fab'-O-D-galacto-
s idase con juga te .
Convent ional Sandwich Enzyme Immunoassay f o r hTSH and hGH
For hTSH assay, one monoclonal anti-hTSH O-subuni t IgG1-
coa ted p o l y s t y r e n e b a l l was i ncuba ted w i t h hTSH standards i n 0.15
m l o f b u f f e r A a t 20°C o v e r n i g h t . A f t e r removal o f t h e
i n c u b a 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 was washed t w i c e as
desc r ibed above and incuba ted w i t h a f f i n i t y - p u r i f i e d r a b b i t
anti-hCG Fab'-D-D-galactosidase cop juga te (200 fmol ) and nonspeci -
f i c r a b b i t F ( a b ' ) * (0.1 mg) i n 0.15 m l o f b u f f e r A a t 20°C f o r 4 h
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY 1149
a n t
Fab
a n t
w i t h con t inuous shaking. A f t e r removal o f t h e i n c u b a 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 was washed t w i c e as d e s c r i b e d above
and t r a n s f e r e d t o ano the r c l e a n t e s t tube, and bound O-D-galacto-
s idase a c t i v i t y was assayed hy f l u o r i m e t r y a t 30°C f o r 1 h as
d e s c r i b e d above.
hGH was assayed as d e s c r i b e d above, excep t t h a t monoclonal
-hGH IgG1-coated p o l y s t y r e n e b a l l s and r a b b i t anti-hGH
-O-D-galactosidase con juga te were s u b s t i t u t e d f o r monoclonal
-hTSH O-subuni t IgG1-coated p o l y s t y r e n e 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 anti-hCG Fab ' -0-D-gal a c t o s i d a s e con j u g a t e .
Exp ress ion o f t h e D e t e c t i o n L i m i t o f hTSH and hGH
The d e t e c t i o n l i m i t o f hTSH and hGH by t h e p r e s e n t and
conven t iona l enzyme immunoassays was taken as t h e min imal amount
of hTSH and hGH which gave a bound O-0-galactos 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 of hTSH and hGH (background) . The e x i s t e n c e o f a
s i g n i f i c a n t d i f f e r e n c e f rom t h e background was con f i rmed by t h e
- t - t e s t ( p < 0.01, n=5).
C a l c u l a t i o n o f S p e c i f i c a l l y Bound O-D-Galactosidase A c t i v i t y
S p e c i f i c a l l y bound O-D-galactosidase a c t i v i t y was c a l c u l a t e d
by s u b t r a c t i n g D-D-galactos idase a c t i v i t y 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 n t i g e n s f rom D-D-galactos idase a c t i v i t y bound i n
t h e presence o f an t i gens .
RESULTS AND DISCUSSION
I n t h e p r e s e n t sandwich t r a n s f e r enzyme immunoassay, a n t i g e n s
were r e a c t e d w i t h d i n i t r o p h e n y l monoclonal mouse a n t i b o d y IgGl and
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11 50 HASHIDA ET AL.
rabbit antibody Fabl-R-D-galactosidase conjugates, and the complex
formed was trapped onto affinity-purified rabbit (anti-dinitro-
phenyl bovine serum albumin) IgG-coated polystyrene balls.
After eliminating excess of the conjugates by washing, the complex
was eluted from the (anti-dinitrophenyl bovine serum albumin) IgG-
coated polystyrene balls with dinitrophenyl-L-lysine and trans-
fered to clean polystyrene balls coated with affinity-purified
rabbit (anti-mouse IgG) IgG. Finally, bound 0-D-galactosidase
activity was assayed by fluorimetry. In the conventional
sandwich enzyme immunoassay, antigens were reacted with monoclonal
mouse antibody IgG1-coated polystyrene balls and subsequently with
rabbit antibody Fabl-R-D-galactosidase conjugate. hTSH and hGH
were subjected to both the present enzyme imunoassay and the
conventional enzyme immunoassay (Figs. 1 and 2).
In the conventional enzyme immunoassay, B-D-galactosidase
activity nonspecifically bound to monoclonal antibody IgG1-coated
polystyrene balls in the absence of antigens (background) was
0.0011 % (hTSH) and 0.0014 % (hGH) of O-D-galactosidase activity
added. In the present enzyme immunoassay, R-D-galactosidase
activity nonspecifically bound to (anti-dinitrophenyl bovine serum
albumin) IgG-coated polystyrene balls was 0.0026 % (hTSH) and
0.012 % (hGH) , and R-D-galactosidase activity nonspecifical ly
bound to (anti-mouse IgG) IqG-coated polystyrene balls (back-
ground) was 0.000017 % (hTSH) and 0.00012 % (hGH). And bound
B-D-galactosidase activity was assayed for 1 h in the conventional
enzyme immunoassay and for 16 h in the present enzyme immunnacca**
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY
7000
1000
100
10 u al m n
- -
*
:
-
-
:
L 0 c
1151
I 1 I 1 I I I
0.01 0.1 1 10 100 lL, ' "
hTSH (nU/tube 1
Fig. 1 Dose-response curves o f hTSH by the present enzyme immuno- assay ( c i r c l e s ) and the conventional enzyme immunoassay ( t r i a n g l e s ) . Squares i n d i c a t e 8-D-galactosidase a c t i v i t y spec i f i c a l l y bound t o ( a n t i - d i n i t rophenyl bovine serum albumin) IgG-coated po lys ty rene b a l l s .
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1152 HASHIDA ET A L .
Fig. 2 Dose-response curves o f hGH by the present enzyme immuno- assay (circles) and the conventional enzyme immunoassay (triangles). Squares indicate R-D-galactosidase activity specifically bound to (anti-dinitrophenyl bovine serum a1 bumin) IgG-coated polystyrene bal Is.
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NOVEL AND ULTRASENSITIVE SANDWICH ENZYME IMMUNOASSAY 1153
Therefore, fluorescence intensity of the backgroupd in the present
sandwich transfer enzyme immunoassay was 4.2-fold lower (hTSH) avd
1.1-fold higher (hGH) than that in the conventional enzyme immuno-
assay.
R-D-Galactosidase activity specifically bound to (anti-
dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls
in the present enzyme immunoassay was 2.7-fold (hTSH) and 7.1-fold
(hGH) higher than that specifically bound to monoclonal antibody
IgG1-coated polystyrene balls in the conventional enzyme immuno-
assay. This indicated that the complex of antigens with mono-
clonal antibody IgGl and rabbit antibody Fab'-8-D-galactosidase
conjugate was more efficiently formed in the present enzyme
immunoassay than in the conventional enzyme immunoassay.
However, the complex was lost 55 % (hTSH) and 41 % (hGH) during
transfer from (anti-dinitrophenyl bovine serum albumin) IgG-coated
polystyrene balls to (anti-mouse IgG) IgG-coated polystyrene
balls. And bound D-D-galactosidase activity was assayed for
1 h in the conventional enzyme immunoassay and for 16 h in the
present enzyme immunoassay. Therefore, fluorescence intensity
for specifically bound D-D-galactosidase activity in the present
enzyme immunoassay was 20-fold !hTSH) and 67-fold (hGH) higher
than that in the conventional enzyme immunoassay.
As a result, the detection limit o f hTSH and hGH was lowered
30-fold as compared with that by the conventional enzyme immuno-
assay. Using affinity-purified anti-hCG Fab'-D-D-galactosidase
conjugate, 0.01 nU (0.02 amol) o f hTSH could be measured, and,
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1154 HASHIDA ET A L .
using anti-hGH Fab'-O-0-galactosidase conjugate without affinity-
purification, 10 fg (0.5 amol) of hGH could be measured (Figs. 1
and 2).
The present sandwich transfer enzyme irnmunoassay technique
may be used t o measure most kinds of antigens at attomole levels
using antibody Fab'-enzyme conjugates without affinity-purifi-
cation and at lower levels using affinity-purified Fab'-enzyme
conjugates.
1.
2.
3 .
4.
5.
6.
7.
8.
REFERENCES
E. Ishikawa, M. Imagawa and S. Hashida, Develop. Immunol. 2, 219 (1983) S. Hashida, E. Ishikawa, Z. Mohri, T. Nakanishi, H. Noguchi and Y. Murakami, Endocrinol. Japon. 35, 171 (1988) S. Yoshitake, Y. Endo, K. Nakagawa, M. Imagawa, S. Ohtaki and E. Ishikawa, Clin. Chim. Acta. 125, 1 (1982) E. Ishikawa, M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamaguchi and T. Ueno, J. Immunoassay 4, 209 (1983) H.N. Eisen, M.E. Carsten and S. Belman, J. Immunol. 73, 296 (1954) K-h. Ruan, M. Imagawa, S. Hashida and E. Ishikawa, Anal. Lett. 17(B7), 539 (1984) E. Ishikawa and K. Kato, Sand. J. Immunol. 8(Suppl. 7), 43 (1978) M. Imagawa, S. Hashida, Y. Ohta and E. Ishikawa, Ann. Clin. Biochem. 21, 310 (1984)
Received March 21, 1988 Accepted April 6, 1988 D
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