a micro-scale preparation of affinity-purified fab'-enzyme conjugates with high purity for...

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A Micro-Scale Preparation of Affinity-Purified Fab'-Enzyme Conjugates with High Purity for EnzymeImmunoassayKe-He Ruan a , Seiichi Hashida a & Eiji Ishikawa aa Department of Biochemistry , Medical College of Miyazaki Kiyotake , Miyazaki, 889-16,JapanPublished online: 19 Dec 2006.

To cite this article: Ke-He Ruan , Seiichi Hashida & Eiji Ishikawa (1984) A Micro-Scale Preparation of Affinity-PurifiedFab'-Enzyme Conjugates with High Purity for Enzyme Immunoassay, Analytical Letters, 17:18, 2075-2090, DOI:10.1080/00032718408077199

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ANALYTICAL LETTERS, 17(B18), 2075-2090 (1984)

A MICRO-SCALE PREPARATION OF AFFINITY-PURIFIED FAB'-ENZYME

CONJUGATES WITH H I G H PURITY FOR ENZYME IMMUNOASSAY

KEY WORDS: A f f i n i t y - p u r i f i c a t i o n , Peroxidase, O-D-Galactosidase,

Fab ' , Enzyme-label ing, 2 ,4-Din i t rophenyl group, Enzyme

immunoassav

Ke-he Ruan, S e i i c h i Hashida and E i j i Ish ikawa

Department o f B iochemis t r y , Medica l Co l l ege o f M iyazak i , K iyotake, Miyazaki 889-16 Japan

ABSTRACT

A mic ro -sca le method was developed f o r c o n j u g a t i n g a smal l

amount o f a f f i n i t y - p u r i f i e d Fab' t o enzymes through t h i o l groups

i n t h e h inge o f Fab ' . 2 ,4 -D in i t ropheny l groups were i n t r o d u c e d

i n t o r a b b i t a n t i -hCG F( ab ' ) 2 b e f o r e a f f i n i t y - p u r i f i c a t i o n , and

t h e 2 ,4 -d in i t ropheny l F ( a b ' ) 2 (0.2-2.0 mg) 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 hCG-Sepharose 46 w i t h 0.1 M g l y c i n e -

HC1 b u f f e r , pH 2 . 5 , c o n t a i n i n g normal r a b b i t F ( a b ' ) 2 (1.0 mg) as

a c a r r i e r . The a f f i n i t y - p u r i f i e d 2,4-dini t ropheny l F ( ab ' ) 2

Copyright 0 1984 by Marcel Dekker, Inc.

2075

0003-2719/84/1718-2075$3.50/0

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2076 R U M , HASHIDA, AND ISHIKAWA

mixed w i t h normal F ( a b ' ) 2 was s p l i t i n t o Fab' by r e d u c t i o n ,

reac ted w i t h male imide groups i n t r o d u c e d i n t o enzymes and

sub jec ted t o ge l f i l t r a t i o n t o separa te t h e con juga tes f rom

unconjugated components. F i n a l l y , t h e a f f i n i t y - p u r i f i ed

2 ,4 -d in i t ropheny l anti-hCG Fab'-enzyme con juga tes were separated

f rom normal Fab'-enzyme conjugates and unconjugated enzyme, i f

any, by a f f i n i t y chromatography on a column o f ( a n t i - Z Y 4 - d i n i t r o -

phenyl ) IgG-Sepharose 46. The con juga te p r e p a r a t i o n s o b t a i n e d

by t h e m i c r o - s c a l e method were s a t i s f a c t o r y i n p u r i t y ,

a n t i g e n - b i n d i n g a c t i v i t y and use fu lness f o r sandwich enzyme

immunoassay.

Th is method i s a p p l i c a b l e t o t h e c o n j u g a t i o n n o t o n l y w i t h

h o r s e r a d i s h pe rox idase b u t w i t h o t h e r enzymes, w h i l e a p r e v i o u s l y

r e p o r t e d method i s a p p l i c a b l e o n l y t o t h e c o n j u g a t i o n w i t h horse-

r a d i s h perox idase.

INTRODUCTION

We developed methods f o r t h e c o n j u g a t i o n of Fab' t o enzymes

through t h i o l groups i n t h e h inge o f Fab' by t h e r e a c t i o n between

t h i o l and male imide groups o r by t h e t h i o l - d i s u l f i d e exchange

r e a c t i o n ( t h e h inge methods) . The Fab ' -ho rse rad ish p e r o x i -

dase con juga te prepared by t h e h inge method was demonstrated t o be

s u p e r i o r i n b o t h sandwich enzyme immunoassay and immunohistochemi-

c a l s t a i n i n g t o t h e co r respond ing con juga tes p repared by t h e

non-hinge methods i n which t h e enzyme was con juga ted th rough amino

groups o f Fab" . The use of a f f i n i t y - p u r i f i e d Fab'-enzyme

1

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AFFINITY-PURIFIED FAB'-ENZYME CONJUGATES 2077

conjugates prepared by the hinge methods made i t possible t o

measure macromolecular antigens a t attomole . However, t he hinge method i s not su i t ab le f o r t he conjugation

of a small quantity of a f f in i ty -pur i f i ed o r monoclonal Fab', s ince

Fab' loses i t s t h io l groups and i s l o s t in a l a rge proportion a t a

low concentration during processes of gel f i l t r a t i o n and concent-

r a t ion . The present paper describes a method f o r the prepara-

t ion of a f f in i ty -pur i f i ed Fab'-enzyme conjugates from 0.2-2.0 mg

of 2,4-dini trophenyl F ( ab' ) 2 before a f f in i ty -pur i f ica t ion .

MATERIALS A N D METHODS

Enzymes

Pepsin from porcine g a s t r i c mucosa, 8-D-galactosidase from

Escherichia coli (lyophil ized f o r enzyme immunoassay) and horse-

radish peroxidase (grade I , lyophi l ized , RZ=3.0) were obtained

from Boehringer Mannheim GmbH, Mannheim, West Germany.

Antigens and Antibodies

Human chorionic gonadotropin (hCG) was obtained from

Calbiochem-Behring Corporation, San Diego, Cal i forn ia . Rabbit

anti-hCG serum and r abb i t (anti-ZY4-dinitrophenyl bovine serum

albumin) serum were obtained from Miles Laboratories, Inc. ,

Elkhart , Indiana. Goat ( an t i - r abb i t IgG) IgG was obtained from

Cappel Laboratories Inc., Cochranville, Pennsylvania.

Preparation o f IgG and F (ab ' )2

IgG was prepared by f r ac t iona t ion of serum with Na2S04

followed by passage through a column of diethylaminoethyl ce l lu -

lose (Whatman Chemical Separation L t d . , Kent, U K ) , and F ( a b ' ) 2

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207 a RUAN, HASHIDA, AND ISHIKAWA

I was prepared by pepsin d i g e s t i o n of IgG . F1 uoresce i n -

l a b e l e d normal r a b b i t IgG was prepared by pass ing f l u o r e s c e i n -

labe led r a b b i t (anti-human I g G ) IgG ( N i l e s L a b o r a t o r i e s , I n c . ,

El k h a r t , Ind iana) through a human IgG-Sepharose 48 column.

Assay of Enzyme A c t i v i t y

Peroxidase a c t i v i t y was assayed by f l u o r i m e t r y using 3-(p-

hydroxypheny1)propionic a c i d (Aldr ich Chemical Co. , Inc. ,

Milwaukee, Wisconsin) a s a s u b s t r a t e . 8-0-Galactosidase

a c t i v i t y was assayed by f 1 uorimetry us ing 4-methyl umbel 1 i f e r y l -D-

D-galactoside (Boehringer Mannheim GmbH, Mannheim, West Germany)

a s a s u b s t r a t e . One u n i t o f O-0-ga lac tos idase a c t i v i t y was

def ined a s t h a t which hydrolyzed 1 pmol of t h e s u b s t r a t e per min.

C a l c u l a t i o n of t h e Amount o f P r o t e i n s

4

1

The amounts of IgG and i t s f ragments ( F ( a b ' ) 2 and F a b ' ) were 1 c a l c u l a t e d from t h e measured absorbance a t 280 nm .

The amount of peroxidase was c a l c u l a t e d from the measured

absorbance a t 403 n m , and the amount of B-0-ga lac tos idase was

c a l c u l a t e d from t h e measured absorbance a t 280 nm . 1

Prepara t ion o f Protein-Sepharose 48

I g G (10 mg), bovine serum albumin (10 nig) o r hCG (30,000 U )

was coupled t o CNBr-activated Sepharose 48 (Pharmacia Fine

Chemicals AB, Uppsala, Sweden) ( 1 g ) accord ing t o the i n s t r u c t i o n s

of Pharmacia.

I n t r o d u c t i o n o f 2,4-Dini t rophenyl Groups i n t o F ( a b ' ) 2

2 ,4-Dini t rophenyl groups were introduced i n t o F ( a b ' ) 2 us ing

R a b b i t anti-hCG F ( a b ' ) 2 5 2,4-d in i t robenzenesul fonic a c i d .

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AFFINITY-PURIFIED FAB'-F,NZYME CONJUGATES 2079

(2 .4 mg b e f o r e a f f i n i t y - p u r i f i c a t i o n ) i n 0.8 m l o f 0.15 M NaCl was

mixed w i t h 0.1 m l o f 1 M sodium carbonate b u f f e r , pH 9.5, and

incuba ted w i t h 14 mg (1,000- fo ld mo la r excess o f Fab ' ) o f 2 ,4-d i -

n i t robenzenesu l fon i c a c i d sodium s a l t (Tokyo Kasei Kogyo Co.,

L td . , Tokyo, Japan) i n 0.1 m l o f 0 .1 M sodium carbonate b u f f e r , pH

9.5, a t 37°C. A f t e r 4.5 h i ncuba t ion , t h e r e a c t i o n m i x t u r e was

sub jec ted t o ge l f i l t r a t i o n on a column (1.0 x 45 cm) o f Sephadex

6-25 (Pharmacia F ine Chemicals AB, Uppsala, Sweden) us ing 0.1 M

sodium phosphate b u f f e r , pH 7.0, c o n t a i n i n g 0.2 M NaC1.

The average number o f 2 ,4 -d in i t ropheny l groups i n t roduced pe r

Fab' molecule was c a l c u l a t e d f rom t h e measured absorbance a t 280

nm and 360 nm . Under t h e c o n d i t i o n desc r ibed above, i t was

2.0.

Micro-Scale Method f o r t h e P repara t i on o f A f f i n i t y - P u r i f i e d

2,4-Dini t ropheny l Rabb i t Anti-hCG Fab'-Enzyme Conjugates

1) A f f i n i t y p u r i f i c a t i o n . 2,4-Di n i t ropheny l r a b b i t a n t i - hCG F ( a b ' ) 2 (0.2-2.0 mg b e f o r e a f f i n i t y - p u r i f i c a t i o n ) i n 0.5-5 m l

o f 0.1 M sodium phosphate b u f f e r , pH 7.0, c o n t a i n i n g 0.2 M NaCl

and 0.1 % NaN3 was a p p l i e d t o a column o f hCG-Sepharose 48.

A f t e r washing the column w i t h t h e same b u f f e r , t h e 2 , 4 - d i n i t r o -

phenyl anti-hCG F ( a b ' ) 2 adsorbed was e l u t e d w i t h 0.6 m l o f 0.1 M

glycine-HC1 b u f f e r , pH 2.5, c o n t a i n i n g 1 mg o f normal r a b b i t

F ( a b ' ) 2 as a c a r r i e r . For c o n j u g a t i o n t o B-D-galactosidase,

1 mg o f normal r a b b i t F ( a b ' ) 2 p l u s 0.025 mg o f f l u o r e s c e i n - l a b e l e d

normal r a b b i t F ( a b ' ) 2 was used as a c a r r i e r t o m o n i t o r t h e con ju -

g a t i o n e f f i c i e n c y 1 . pH o f t h e e f f l u e n t (0.6 m l ) was

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2080 RUAN, HASHIDA, AND ISHIKAWA

a d j u s t e d t o 6 .0 with 0.125 ml o f 0.5 M Tris-HC1 b u f f e r , pH 8.0

plus 0.175 ml of 0 .25 M sodium phosphate b u f f e r , pH 6 . 0 .

The hCG-Sepharose 48 column was prepared by packing hCG-

Sepharose 4B i n t o a column of 3 .5 mm i n d iameter . The packed

wet volume of hCG-Sepharose 48 was 5 p l , 25 ~1 and 50 p l f o r t h e

a f f i n i t y - p u r i f i c a t i o n of 0 . 2 mg, 1 rng and 2.0 mg , r e s p e c t i v e l y , o f

anti-hCG F ( ab ' ) 2 . When 5 u 1 of hCG-Sepharose 48 was used,

45 u l in packed wet volume of bovine serum albumin-Sepharose 4B

was over1 a i d .

2 ) Conjugat ion. The a f f i n i t y - p u r i f i e d 2 ,4-d in i t rophenyl

anti-hCG F ( a b ' ) 2 mixed wi th normal r a b b i t F ( a b ' ) 2 a s a c a r r i e r was

reduced t o Fab' w i t h 2-mercaptoethylamine, and t h i o l groups i n the

hinge of the Fab' were r e a c t e d with maleimide groups introduced

The f i n a l concent-

r a t i o n s of the maleimide-peroxidase and Fab' i n t h e r e a c t i o n

mixture f o r conjugat ion were 30-50 nmol/ml. The f i n a l concent-

r a t i o n s of the maleimide B-D-galactosidase and Fab' i n t h e

r e a c t i o n mixture f o r conjugat ion were 8 nmol/ml. The Fab ' -

peroxidase conjugate was s e p a r a t e d from both unconjugated per-

ox idase and Fab' by gel f i l t r a t i o n , and t h e Fabl-D-D-galactosidase

conjugate was s e p a r a t e d from unconjugated Fab' by gel f i l t r a t i o n

(1 ) . However, about 30 Yo of the maleimide-D-D-galactosidase

remained unconjugated and could not be s e p a r a t e d from t h e conju-

g a t e by gel f i l t r a t i o n , s i n c e the molecular weight of t h e enzyme

i s much l a r g e r than t h a t of F a b ' .

i n t o peroxidase o r B-D-galactosidase 1 .

3) Removal of normal r a b b i t Fab'-enzyme c o n j u g a t e s and

F r a c t i o n s c o n t a i n i n g the Fab ' - unconjugated D-D-galactosidase.

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enzyme conjugates, which were ob ta ined by ge l f i l t r a t i o n as

desc r ibed above, were pooled and a p p l i e d t o a column (0.55 x 1.1

cm) o f r a b b i t ( a n t i - 2 , 4 - d i n i t r o p h e n y l ) IgG-Sepharose 48 a t a f l o w

r a t e o f 1-2 m l /h . The column was washed w i t h 0.01 M sodium

phosphate b u f f e r , pH 6.5, c o n t a i n i n g 0.1 M NaCl and 0.1 % bov ine

serum albumin f o r perox idase con juga tes and w i t h t h e same b u f f e r

c o n t a i n i n g 1 mM MgC12 and 0.1 % NaN3 f o r 8-D-galactos idase

conjugates. The a f f i n i t y - p u r i f i e d 2 ,4 -d in i t ropheny l anti-hCG

Fabl-enzyme con juga te adsorbed was e l u t e d w i t h 4 m l o f 0.1 mM

2 ,4 -d in i t ropheny l g l y c i n e i n t h e same b u f f e r a t a f l o w r a t e o f 2

m l /h . The e l u a t e was sub jec ted t o ge l f i l t r a t i o n on a column

(1 x 45 cm) o f Sephadex 6-25 u s i n g t h e same b u f f e r , and concent-

r a t e d u s i n g a c o l l o d i o n bag i n t h e c o l d .

A f f i n i t y - p u r i f i c a t i o n o f Rabb i t Anti-hCG F(ab'), i n a Moderate

Scale

L

Rabb i t anti-hCG F ( a b ' ) 2 (52 mg b e f o r e a f f i n i t y - p u r i f i c a t i o n )

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 (0.5 x 3.6 cm) o f

The hCG-Sepharose 4B a t pH 2.5 as desc r ibed p r e v i o u s l y . amount o f a f f i n i t y - p u r i f i e d r a b b i t anti-hCG F ( a b ' ) 2 was 0.9 mg.

P r e p a r a t i o n o f Fabl-enzyme Conjugates w i t h o u t C a r r i e r

6

F ( a b ' ) 2 (0.9-1.0 mg e i t h e r b e f o r e o r a f t e r a f f i n i t y - p u r i f i -

c a t i o n ) was reduced t o Fab' and conjugated t o perox idase and 0-D-

ga lac tos idase as desc r ibed i n t h e m ic ro -sca le method except f o r

t h e f o l l o w i n g c o n d i t i o n . The f i n a l concen t ra t i ons o f t h e

maleimide-0-D-galactosidase and Fab' i n t h e r e a c t i o n m i x t u r e f o r

c o n j u g a t i o n were 2 and 6 nmol/ml, r e s p e c t i v e l y . The maleimide-

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R-D-ga lac tos idase i n t h e r e a c t i o n m i x t u r e f o r c o n j u g a t i o n was

a l m o s t c o m p l e t e l y c o n v e r t e d t o t h e c o n j u g a t e , s i n c e excess o f Fab '

was added, and t h e ave rage number o f Fab ' m o l e c u l e s c o n j u g a t e d p e r

R - 0 - g a l a c t o s i d a s e m o l e c u l e was 2 . 3 . C a l c u l a t i o n and E x p r e s s i o n of t h e Amount o f C o n j u g a t e s

1

The amount o f F a b ' - p e r o x i d a s e c o n j u g a t e s was c a l c u l a t e d f r o m

e i t h e r absorbance a t 403 nm o r p e r o x i d a s e a c t i v i t y by assuming

t h a t p e r o x i d a s e i n t h e c o n j u g a t e had t h e same s p e c i f i c a c t i v i t y as I

t h a t o f t h e o r i g i n a l enzyme . The amount o f Fabl-B-D-

g a l a c t o s i d a s e c o n j u g a t e s was e i t h e r e x p r e s s e d i n u n i t s o f 8-D-

g a l a c t o s i d a s e a c t i v i t y o r c a l c u l a t e d f r o m R - D - g a l a c t o s i d a s e

a c t i v i t y b y t a k i n g t h e s p e c i f i c a c t i v i t y o f R - D - g a l a c t o s i d a s e t o

be 63 b u n i t s / f n i o l .

T e s t f o r t h e Presence o f Uncon juga ted Enzymes i n t h e C o n j u g a t e

P r e p a r a t i o n s

The p r e p a r a t i o n o f r a b b i t an t i -hCG Fabl-enzyme c o n j u g a t e s was

passed t h r o u g h a co lumn of g o a t ( a n t i - r a b b i t I gG) IgG-Sepharose

4B, and t h e enzyme a c t i v i t y i n t h e e f f l u e n t was compared w i t h t h a t

When normal r a b b i t F a b l - and r a b b i t an t i -hCG a p p l i e d 1,6

Fab'-enzyme c o n j u g a t e s were passed t h r o u g h a normal g o a t IgG-

Sepharose 4B column, 96-104 % o f t h e a p p l i e d a c t i v i t i e s was f o u n d

i n t h e e f f l u e n t .

T e s t f o r t h e A n t i g e n B i n d i n g A b i l i t y o f t h e C o n j u g a t e s

The p r e p a r a t i o n o f an t i -hCG Fab'-enzyme c o n j u g a t e s was passed

t h r o u g h a co lumn o f hCG-Sepharose 4B, and t h e enzyme a c t i v i t y i n

When normal t h e e f f l u e n t was compared w i t h t h a t a p p l i e d 1 6

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rabbit Fab'-enzyme conjugates were applied to the same column,

99-101 % of the applied activities was found in the effluent.

Preparation of Anti-hCG IgG-Coated Polystyrene Balls

Polystyrene balls (Precision Plastic Ball Co., Chicago,

Illinois) (3.2 mm in diameter) were coated with rabbit anti-hCG

IgG (0.1 mg/ml) by physical adsorption

Sandwich Enzyme Immunoassay Technique

1 .

An anti-hCG IgG-coated polystyrene ball was incubated with

hCG and then with 5 ng of anti-hCG Fab'-peroxidase conjugate or

200 punits o f anti-hCG Fab'-B-D-galactosidase conjugate6y7

Peroxidase or 8-D-galactosidase activity bound was assayed by

incubation at 30°C for 90 niin or 30 min as described above.

Calculation o f Fluorescence Intensity for Peroxidase Activity

Specifically Bound

Fluorescence intensity for peroxidase activity specifically

bound was calculated by subtracting fluorescence intensity for

peroxidase activity nonspecifically bound in the absence of hCG

from that bound in the presence of hCG.

Definition of the Sensitivity of Does Response Curves by Sandwich

Enzyme Immunoassay Technique

The sensitivity of does response curves for hCG by sandwich

enzyme immunoassay technique was taken as the minimal amount of

hCG which gave peroxidase or 8-D-galactosidase activity bound

significantly above that nonspecifically bound in the absence of

hCG (background). Significant difference from background was

confirmed by the t-test (n=5).

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RESULTS

Absence o f Unconjugated Enzymes i n t h e Conjugate P r e p a r a t i o n s

I n o r a e r t o c o n f i r m t h e absence o f unconjugated enzymes, t h e

a f f i n i t y - p u r i f i e d 2 ,4 -d in i t r o p h e n y l r a b b i t a n t i -hCG Fab'-enzyme

con juga te p r e p a r a t i o n s ob ta ined by t h e m i c r o - s c a l e method were

a p p l i e d t o a goa t ( a n t i - r a b b i t IgG) IgG-Sepharose 48 column.

The a d s o r p t i o n t o t h e column o f enzyme a c t i v i t i e s i n t h e con juga te

p r e p a r a t i o n s were a lmost complete (Tab le I ) . T h i s i n d i c a t e d

t h a t t h e r e was l i t t l e unconjugated enzymes i n t h e con juga te

p r e p a r a t i o n s .

Y i e l d o f t h e Conjugates

Loss o f enzymes and Fab' by g e l f i l t r a t i o n and c o n c e n t r a t i o n

a f t e r t h e i n t r o d u c t i o n o f male imide groups o r a f t e r t h e r e d u c t i o n

The o f F (ab ' ) i b e f o r e c o n j u g a t i o n was l e s s than 10 %

recovery i n t h e con juga tes o f enzymes and Fab' i ncuba ted i n t h e

r e a c t i o n m i x t u r e f o r c o n j u g a t i o n was approx ima te l y 70 %.

1 .

The a d s o r p t i o n o f 2 , 4 - d i n i t r o p h e n y l Fab ' -pe rox idase con ju -

gates t o (anti-2,4-dinitrophenyl) IgG-Sepharose 4B was about 75 %,

when t h e average number of 2 , 4 - d i n i t r o p h e n y l groups i n t r o d u c e d p e r

Fab ' molecule was 2.0. The recove ry o f t h e con juga tes f r o m t h e

Sepharose column by e l u t i o n w i t h 2 , 4 - d i n i t r o p h e n y l g l y c i n e was

about 50 7:.

The amount o f a f f i n i t y - p u r i f i e d anti-hCG F ( a b ' ) 2 was 0.9 mg

(1 .7 :A) from 5 2 mg o f t h e co r respond ing F ( a b ' ) 2 b e f o r e a f f i n i t y -

p u r i f i c a t i o n and was presumably 3.4-34 ug f rom 0.2-2.0 mg o f 2,4-

d i n i t r o p h e n y l anti-hCG F ( a b ' ) 2 f o r t h e m i c r o - s c a l e p r e p a r a t i o n .

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TABL

E I

Pu

rity

and

Ant

igen

-Bin

ding

A

bil

ity

of

Rab

bit

Ant

i-hC

G

Fab'

-Enz

yme

Con

juga

tes

Intr

od

uct

ion

Am

ount

o

f Am

ount

of

of

an

ti -h

CG

Aff

init

y-

norm

al

Ads

orpt

ion

of

the

con

juga

tes

to

Labe

l 2,

4-di

ni t

ro-

F (a

b ' )2

pu

rifi

ca

tio

n

rab

bit

en

zym

e ph

enyl

gro

ups

befo

re

usi

ng

hCG

- F

(ab

')*

goat

(a

nti

- hC

G-

into

ant

i-hC

G

affin

ity-

Seph

aros

e ad

ded

as

rab

bit

IgG

) S

epha

rose

I g

G- S

ep ha

rose

4B

4B

a

ca

rrie

r 4B

F

(ab'

12

pu

rifi

ca

tio

n

mg

mg

70

Per

oxid

ase

No

1.0

No

0 98

3.

2

No

52

Yes

0 96

69

Yes

0.2

Yes

1 .o

99

77

Yes

1.0

Yes

1.0

99

74

Yes

1.0

Yes

1.0

99

76

Yes

2.0

Yes

1.0

100

82

B-D

-Gal

acto

- No

si

dase

Ye

s

1.0

No

0

0.2

Yes

1.0

96

95

3.8

76

2 H m

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2.086 RUAN, HASHIDA, AND ISHIKAWA

From t h e above v a l u e s o f t h e y i e l d f o r each s t e p , 1.5-15 u g

o f t h e a f f i n i t y - p u r i f i e d 2 , 4 - d i n i t r o p h e n y l an t i -hCG F a b l - p e r -

o x i d a s e c o n j u g a t e o r 10 ug o f t h e a f f i n i t y - p u r i f i e d 2 , 4 - d i n i t r o -

pheny l an t i -hCG F a b ' - D - D - g a l a c t o s i d a s e c o n j u g a t e s h o u l d have been

o b t a i n e d f r o m 0.2-2.0 mg o r 0.2 mg o f t h e c o r r e s p o n d i n g F ( a b ' ) 2

b e f o r e a f f i n i t y - p u r i f i c a t i o n by t h e m i c r o - s c a l e method. HOW-

e v e r , t h e amount a c t u a l l y o b t a i n e d was 0.81-6.5 v g o f t h e p e r -

oxidase c o n j u g a t e and 5.7 p g o f t h e O-D-ga lac tos idase c o n j u g a t e .

A n t i g e n B i n d i n g A c t i v i t y o f t h e Con juga tes

When t h e a f f i n i t y - p u r i f i e d 2 , 4 - d i n i t r o p h e n y l an t i -hCG F a b l -

enzyme c o n j u g a t e p r e p a r a t i o n s o b t a i n e d by t h e m i c r o - s c a l e method

were a p p l i e d t o a co lumn o f hCG-Sepharose 48, 74-82 % o f enzyme

a c t i v i t i e s a p p l i e d was adso rbed ( T a b l e I ) . T h i s was s i m i l a r t o

t h e a d s o r p t i o n o f t h e c o r r e s p o n d i n g c o n j u g a t e s p r e p a r e d w i t h o u t

c a r r i e r .

U s e f u l n e s s o f t h e C o n j u g a t e P r e p a r a t i o n s

W i t h i n c r e a s i n g numbers o f 2 , 4 - d i n i t r o p h e n y l g roups i n t r o -

duced i n t o an t i -hCG F ( a b ' ) 2 , t h e n o n s p e c i f i c b i n d i n g o f t h e

a f f i n i t y - p u r i f i e d 2 , 4 - d i n i t r o p h e n y l an t i -hCG Fabl-enzyme c o n j u -

g a t e s t o an t i -hCG IgG-coa ted p o l y s t y r e n e b a l l s i n c r e a s e d , and

c o n s e q u e n t l y t h e s e n s i t i v i t y f o r hCG by sandwich enzyme immuno-

assay t e c h n i q u e tended t o decrease.

When t h e ave rage number o f 2 , 4 - d i n i t r o p h e n y l g roups i n t r o -

duced i n t o an t i -hCG F ( a b ' ) 2 was 2.0, t h e n o n s p e c i f i c b i n d i n g o f

t h e c o n j u g a t e s p r e p a r e d by t h e m i c r o - s c a l e method was 1 .7 -2 .2 - fo ld

h i g h e r t h a n t h a t o f t h e c o n j u g a t e s w i t h no 2 , 4 - d i n i t r o p h e n y l

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groups, b u t t h e s e n s i t i v i t i e s ob ta ined u s i n g t h e a f f i n i t y - p u r i f i e d

anti-hCG Fab'-enzyme conjugates w i t h and w i t h o u t Z Y 4 - d i n i t r o p h e n y l

groups were s i m i l a r and about 1 0 - f o l d h i g h e r than t h a t ob ta ined

w i t h t h e con juga te prepared b e f o r e a f f i n i t y - p u r i f i c a t i o n ( F i g . 1).

When t h e conjugates were t e s t e d u s i n g 0.003 mU o f hCG p e r tube by

sandwich enzyme immunoassay technique, t h e - t - t e s t f o r d i f f e r e n c e

between enzyme a c t i v i t i e s bound i n t h e absence and presence o f hCG

gave t h e f o l l o w i n g va lues : p < 0.001 f o r t h e a f f i n i t y - p u r i f i e d

anti-hCG Fab' -perox idase conjugates prepared b o t h w i t h and w i t h o u t

c a r r i e r and 0.01 < p < 0.05 f o r t h e a f f i n i t y - p u r i f i e d anti-hCG

Fab' -8-D-galactos idase conjugate prepared w i t h c a r r i e r .

D I S C U S S I O N

We p r e v i o u s l y r e p o r t e d a method f o r c o n j u g a t i n g a small

amount o f 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' t o ho rse rad ish

perox idase 6 . A small amount (8-40 p g ) o f a f f i n i t y - p u r i f i e d

r a b b i t anti-hCG F ( a b ' ) 2 was mixed w i t h normal goat F ( a b ' ) 2 as

a c a r r i e r and conjugated t o t h e enzyme a f t e r r e d u c t i o n t o Fab'.

The a f f i n i t y - p u r i f i e d r a b b i t Fab ' -perox idase con juga te was

separated f rom normal goat Fab ' -perox idase con juga te by passage

through a column o f r a b b i t ( a n t i - g o a t IgG) IgG-Sepharose 48.

However, w i t h decreas ing amounts (8 u g o r l e s s ) of t h e

a f f i n i t y - p u r i f i e d anti-hCG F ( a b ' ) z y t h e p u r i t y o f t h e

a f f i n i t y - p u r i f i e d anti-hCG Fab' -perox idase con juga te p r e p a r a t i o n

decreased, which was accompanied by loss o f t h e s e n s i t i v i t y o f

sandwich enzyme immunoassay. T h i s d i f f i c u l t y c o u l d n o t be

overcome by p r i o r i n t r o d u c t i o n o f 2 ,4 -d in i t ropheny l groups i n t o

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c 0 m

4 1 4 0.01

hCG I mU ltube 1

FIG. 1. Dose response curves for hCG by sandwich enzyme immunoassay technique using anti-hCG Fab'-conjugates with peroxidase (A) and 6-D-galactoside ( B ) . Open circles: the affinity-purified conjugates were prepared from 0.2 mg of 2,4-dinitrophenyl F(ab')2 before affinity-purification by the micro-scale method with carrier. Open triangles: the affinity-purified conjugate was prepared with- out carrier. Closed circles: the conjugates were not affinity- purified. Each point is the mean of 5 determinations.

normal goat F(ab')2 as a carrier and subsequent removal o f

2,4-dinitrophenyl normal goat Fab'-peroxidase conjugate using

(anti-2,4-dinitrophenyl) IgG-Sepharose 48. This was probably

because a very small amount of dimerized, unconjugated peroxidase

could not be removed from the conjugate. In addition, this

method is applicable to the conjugation only with horseradish

peroxidase but not with other enzymes, since no method is

currently available t o prepare a monomeric conjugate with other

enzymes.

In the present method, very small quantities (presumably

3.4-34 p g ) o f affinity-purified anti-hCG F(ab')* could be

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conver ted t o t h e conjugates w i t h h i g h p u r i t y by p r i o r i n t r o d u c t i o n

o f 2 ,4 -d in i t ropheny l groups i n t o t h e F ( a b ' ) 2 b e f o r e a f f i n i t y -

p u r i f i c a t i o n and subsequent s e p a r a t i o n o f t h e conjugates u s i n g

( a n t i - 2 , 4 - d i n i t r o p h e n y l ) IgG-Sepharose 48. The con juga te p re -

p a r a t i o n s con ta ined l i t t l e unconjugated enzymes, and 74-82 % o f

t h e enzyme a c t i v i t i e s i n t h e con juga te p r e p a r a t i o n s were

assoc ia ted w i t h t h e s p e c i f i c Fab' (Table I ) . The p r o p o r t i o n o f

unconjugated F ( a b ' ) 2 i n t h e con juga te p r e p a r a t i o n s must have

been l e s s than 10 % , a l though n o t est imated, and t h e

conjugates prepared by t h e m ic ro -sca le method w i t h c a r r i e r was as

u s e f u l i n sandwich enzyme immunoassay as t h e conjugates prepared

w i t h o u t c a r r i e r . The c o n j u g a t i o n was h i g h l y r e p r o d u c i b l e , i f

t h e c o n t e n t o f t h i o l groups generated i n Fab' and maleimide groups

i n t r o d u c e d i n t o enzymes was con f i rmed t o be s u f f i c i e n t b e f o r e t h e

c o n j u g a t i o n . One p g o f t h e perox idase o r 8 -0 -ga lac tos idase

conjugates was s u f f i c i e n t f o r pe r fo rm ing 200 o r 500 assays by

sandwich enzyme immunoassay technique. Furthermore, t h e

p resen t method i s a p p l i c a b l e t o t h e c o n j u g a t i o n n o t o n l y w i t h

ho rse rad ish perox idase b u t w i t h o t h e r enzymes such as 8-D-galacto-

s idase and a l k a l i n e phosphatase. Therefore, t h e p r e s e n t method

i s usefu l n o t o n l y f o r t e s t i n g t h e c o n d i t i o n o f a f f i n i t y - p u r i f i -

c a t i o n u s i n g a smal l q u a n t i t y o f F ( a b ' ) 2 b u t f o r assay ing a

l i m i t e d number o f specimens, when a n t i b o d i e s a r e expensive.

1

However, t h e r e were seve ra l disadvantages i n h e r e n t t o t h e

p resen t m ic ro -sca le method. F ( a b ' ) 2 had t o be i ncuba ted a t

pH 9.5 f o r i n t r o d u c t i o n o f 2 ,4 -d in i t ropheny l groups. I t was n o t

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easy t o c o n t r o l t h e number o f 2 , 4 - d i n i t r o p h e n y l g r o u p s i n t r o d u c e d

i n t o F(ab ' ) , . W i t h i n c r e a s i n g numbers o f 2 , 4 - d i n i t r o p h e n y l

g roups i n t r o d u c e d i n t o F ( a b ' ) 2 , t h e n o n s p e c i f i c b i n d i n g of t h e

c o n j u g a t e s t o IgG-coa ted p o l y s t y r e n e b a l l s i n c r e a s e d , and t h e

a n t i g e n b i n d i n g z b i l i t y may be i m p a i r e d , a l t h o u g h t h e a d s o r p t i o n

o f 2 , 4 - d i n i t r o p h e n y l Fab ' -enzyme c o n j u g a t e s t o ( a n t i - 2 , 4 - d i n i t r o -

p h e n y l ) IgG-Sepharose 4B became more e f f i c i e n t . The r e c o v e r y

o f 2 , 4 - d i n i t r o p h e n y l Fab ' -enzyme c o n j u g a t e s f rom a co lumn o f

(anti-2,4-dinitrophenyl) IgG-Sepharose 4B was r a t h e r l o w ( a b o u t 50

":).

R E F ERE N C E S

1. E . I s h i k a w a , M. Imagawa, S . Hash ida , S . Y o s h i t a k e , Y . Hamaguchi and T. Ueno, J . Immunoassay 4 209 (1983)

2. E. I s h i k a w a , M. Imagawa and S. Hashid;, Deve lop . Immunol. 18 219 (1983)

3 . S . Hash ida , I ( . Nakagawa, M. Imagawa, S. I noue , S. Y o s h i t a k e , E . I s h i k a w a , Y . Endo, S . O h t a k i , Y . I c h i o k a and K . Naka j ima, C l i n . Chim. A c t a 135 263 (1983)

4. M. Imagawa, S. Hash ida , E. I s h i k a w a , H. M o r i , C . Naka i , Y . I c h i o k a and K. Naka j ima , A n a l . L e t t . 16(B19) 1509 (1983)

5. H. N. E i sen , M. E . C a r s t e n and S. Belman, J . Immunol. 3 296 (1954)

6. S. I noue , M. Imagawa, S. Hash ida , K. H. Ruan and E. I s h i k a w a , Ana l . L e t t . 17(B3) 229 (1984)

7 . M. Imagawa, E. I s h i k a w a , S. Y o s h i t a k e , K. Tanaka, H. Kan, M. I nada , H. Irnura, H. K u r o s a k i , S . Tach ibana, M. Takag i , M . N i s h i u r a , N. Nakazawa, H. Ogawa, Y . T s u n e t o s h i and K. Naka j ima, C l i n . Chim. Ac ta . 126 227 (1982)

-

Rece ived September 10, 1984 Accep ted September 1 2 , 1984

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a micro-scale preparation of affinity-purified fab'-enzyme conjugates with high purity for...

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