Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

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  • This article was downloaded by: [Ohio State University Libraries]On: 22 April 2013, At: 01:28Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered 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 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

    To link to this article: http://dx.doi.org/10.1080/00032718808055502

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  • whatsoever or howsoever caused arising directly or indirectly in connectionwith or arising out of the use of this material.

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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 30C 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 30C 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 30C 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

    30C 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 20C 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

    20C for 2 h, The two incubation mixtures were mixed and

    incubated with hTSH standards in 0.09 ml of buffer A at 20C

    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

    20C 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 20C 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 30C 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 30C 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 20C 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 20C 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 30C 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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