Monoclonal antibodies, electrophoretically transferred from polyacrylamide gels, retain their ability to bind specific antigens

Vol.

133,

No. 3, 1985

December

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3 1, 1985

XMOCIDN?G ABTIBODIFS, FZECTIWPHORETICALLY TRANSFERRED POLYACRYLAMIDE GELS, RETAIN TBEIR ABILITY To BIND SPECIFIC Stephen

Mathor

Imperial Lincoln's Received

1020-1025

Pages

November

and Joyce

PMM

ABTIGEBS

Taylor-Papadimitriou

Cancer Research Fund, P 0 Box I.23 Inn Fields, London WC2A 3PX. U.K.

11, 1985

s-y: Antibodies subjected to SDS-polyacrylamide gel electrophoresis and trausferred to nitrocellulose paper, have been found to retain the ability to bind specific antigen. This has been demonstrated for two groups of antibodies, directed to a) a large molecular weight glycoprotein of the human milk fat globule and b) human interferon a2. Imaunoreactive antibody fragments produced by protease digestion could also be identified in this way on Western blots, thus permitting the development of optimal conditions for digestion, without the need for extensive purification procedures. @ 1985 Academic Press, Inc.

Separation followed

of protein

by

electrophoretic

characterisation widely

of the

practised

1979 (1).

presence

of sodium

the

an isotope

constituent component

sensitive 0006-291X/85

for

in a biological recognisod (4)

and Towbin

a

(5)

using the

(2).

numerous

technique. $1.50

0 1985 by Academic Press, of reproduction in any form

Inc. reserved.

protein

1020

to the

antibody applications

in cell

in the

bands

are

plane

of

which

can now be incubated

with

proteins

bearing

directly

a conjugated

the

a

to

to

paper

unequivocal

or

or

gels

nitrocellulooe

can be detected

monoclonal list

such as 8era

The separated

bind

becom?

by Towbin

polyacrylaxide

which

example, sample

by

description

The nitrocellulose

which

subsequent

has

at right-angles

or enzyme or indirectly permit,

its

isocratic (SDS).

antibodies

with

probes

samples

them to

determinants

procedures

Gershoni

bound.

or polyclonal

since

electrophoresis

to transfer

strongly

antigenic

Such

Copyright All rights

to a second

become

relevant

sulphate

electrophoresis

nitrocellulose

biological or

gel

by antibody

technique

gradient

dodecyl

to

mixture

complex

on

SDS-PAGE in order

monoclonal

with

run

submitted

they

resolved

Conventionally, are

by polyacrylamide

transfer

laboratory

lysates

then

mixtures

second

detection

characterisation (3).

Recent of this

if

the

labelled antibody.

of a minor of reviews simple

the by but

BIOCHEMICAL

Vol. 133, No. 3, 1985

This protein

communication blotting

themselves

ligand

technique.

submitted

nitrocellulose blotted

describes

paper. antibodies (antigen)

are

a hitherto In

to

this

SDS-PAGE

Subsequently, used

to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

permit

unreported

application

case,

iamunoglobulin

and

electrophoretic

the

antigen

visualisation

of

preparations

are

transfer

binding with

properties labelled

the

to of

the

purified

preparations.

MA-

AND

MlPmoDS

: KC33 and UC 46 and ST254 are monoclonal antibodies which react with human alpha interferon. They were obtained from hybridomas derived from mice i munisedwithamixtureof human alpha interferons purified from Sendai virus induced Namalwa human lymphoblastoid cells. All are of the IgGl subclass. HC33 and HC% were developed by Lewis and colleagues (6) and ST254 by Shearer et al. (7). The anti-interferon polyclonal antiserum IMP311 was obtained by inmunisation of a calf with Namalwa human interferon (courtesy K. Pa&es). EMFG-1 and IiWFG-2 antibodies were raised against the human milk fat globule (8 ). The determinan ts they react with are carried on a large mlecrilar weight mucin-like material (9) found in milk and on similar components found in some epithelial cells and carcinomas (3,30). Kl7E2 (IgGl) is a monoclonal antibody against Human Placental Alkaline Phosphatase (11). AUA-I (IgGl) is a mnoclonal antibody against an as yet unidentified determinant found in low Jevels in a variety of epithelial cell types and, in greater quantities in a number of adenocarcinomas (1.2). With the exception of 52254, all the monocJonaJ antibodies were used here as fully purified preparations. ST254 was partially purified by amxmium sulphate precipitation from ascites fluid but the antibody was less than 10% pure, the r emaining protein content being primarily albumin. Dre: Suman alpha--2 interferon produced by recombinant DWA tochniquas was obtained from Wellcome Laboratories and labelled with I-125 using the Bolton and Hunter method (13) to a specific activity of lmCi per 100,OOOU of interferon (7). Free iodide and denatured interferon were removed using an anti-interferon affinity column consisting of the monoclonal antibody ST254spled to-Affigel 10. The BWt?Gl antigen was prepared from skimed human milk by affinity chromatography on a column of XiWFG-1 antibody coupled to Sepharose purified protein, (Burchell and Gendler, W in preparation). The labelled with I-125 using the Bolton and Iiunter dissoloved in PBS, was method to a specific activity of lmCi per 5 xicrogrammos of protein. Frecl iodide was removed with a short G-25 sephadex column. m SDS-PAGS was performed according to the : (15) but in the absence of reducing agent to method described by Iaemli preserve interchain disulphide bonds. 10-20 microgranmes of purified iumunoglobulin digest in O.OBN his-EC1 pH 6.8 sample buffer antibody or containing 2% SDS were loaded without boiling onto a 100 x 140 x 7sm 3.0% gel with a 5% stack. A voltage of IOO-15OV was applied giving a isocratic After the dye-front had run to the end of the gel, current of 25OmA. transfer to nitrocellulose was performed in a Riorad 'Trans-blot' cell using 0.025M his / 0.197X glycine buffer pH 8.5 containing 20% methanol 50V producing 15OmA were applied for 5 -15 but in the absence of SDS. hours, the longer time permitting convenient overnight transfer. The blocked with 0.05% 'Jt*een 20 in PRS for one hour nitrocellulose paper was and then incubated with 200,000 cpm of labelled antigen in PBS containing 1% RSA. The nitrocellulose was then dried and bound radioactivity detected by autoradiography on Fuji RX film for 24-48 hours.

1021

Vol.

133,

No. 3, 1985

BIOCHEMICAL

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BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RlWlLTS AND DISCUSSION f a&j.aen demonstrate

the retention

gel elcctrophoresis antibodies

molecular

and blotting

carbohydrate antiserum)

react

specific

which

group

different

each

two

types

epitopos

antibodies

after

groups

of

of

molecules.

found on

containing

recombinant

To

of antibodies

a high

more than 50%

(and

of human interferon by

:

paper,

(3,16) of

.

a polyclonal

alpha including

LWA technology

anti-interferon

antibody

is unigue and represented

in

reacts

only once in

the E.

with the

a 16K

molecule.

case

1 and 2 show the results

to

antibodies, detect

the monoclonal

antibodies

antilxxlios,

3

2

obtained

using

purified

binding

specific

and Western

eloctrophoresis

4

activity

repeating

(a4OOK)

other

that

anti-interferon

control

with

some species

likely

determinant

Figures

each

two widely

HuIFN-tu? produced

is

interferon

the

The with

non-glycosylated it

with

glycoprotein

(9).

binding

to nitrocellulose

HMFGl and 2 react

weight

coli;

of specific

were used reacting

The antibodies

.

to ses

blotting.

with the HMFG antibodies 125 I-labelled antigen by

the

antibodies

H17E2 and AU?-1 (see Methods)

antigen

do not.

in after

It may be seen from Figure

SMFG.1 and 2 bind the specific

and

while

1 that two

Even if the film

1

7

6

5

4

3

2

1 w?!

* 200 - 92.5

.68 - 43

01

AUA-1

Hl7E2

HMFGl

HMFGP

02

AUA-1

1022

H17E2

HMFGP

POLY

254

HC46

HC33

BIOCHEMICAL

Vol. 133, No. 3, 1985

is

for

exposed

non-specific could

be

for

still

be

represent&i

after

In

purified

and

also

effectively

HC33).

.

Western

blots.

antibody

was

initially

in

the

after

several

hours

of

reduced

ST254

illustrated

to

digestion

pepsin molecular

interferon the

antigen

and

was

probably

Western

(ten

against

RC46

only

due

preparation

if

avidity

antibodies

to

the

times

than

interferon

the

light to

in

Figure

3 for

Pepsin

and

separated band

ccrrespcnding digestion

gels

heavy

a position Figure

the

chains

the

ability

on

antibody

HMFG-3.

The

at

various and

I-HNFG

to which

antibody,

moves

corresponding 3b shaws

of

transferred 125

intact

stages

to

a Ccxxuaesie

by blotted anti--interferon the antibodies es described for - I-HUIPN-c2 before processing to the calf plyclonal. antiserum cells.

1023

of

binding

binding to

to

weight.

retain

on

the

Fragments

and

found

with

that

:

J'io. k Specific binding of a radiolebellod milk protein by and 2 aFter gel electrophoresis and electrophoretjc antibodies BMFG-I The antibodies indicated were run transfer to nitroceJ.lulose. gels and blotted as describfd in the Methods. acrylamide p$&rocelIulose blot was processed for autoradiography after jncuhation I--SMFG-1 antigen and washing. r-iiuIPNu2 prepereQf with refers Namalwa

lower

less

digests.

125 ma. 21. Binding of antibodies. Blot2 were 1 and were incubated autoradiography. Poly against a-interferon from

and

partially

Namalwa

fram

of

were

position

is

that

separation

which

even

HuIFN-a)..

also

seen

ST254

antj.bodv

is

gel

the

epitopes

bind

purified

antiserum

regions

be

the

This

radiolabelled

samples

runs

the

also

by

the

multiple

2 shows

for

that

reduced, the

can

antibody

calf

were

can

through

ccnnponent

antibodies,

much

Figure

ascitos.

to

subjected

tt

the

the

polyclonal

This

were

procedure seen

in

variable

nitrccellulose.

of

than from

digestion,

of

be

EIWFG

HWFG

epitopes the

can

molecule

milk

binding

non--repeating

the

the

However,

antiaen

the

of

cbtain

present

of

case

the

determinant

to

EIuIFW-c2

The

nu

immuncqlcbulin

stain

the

2 it

bound

containing

fragment

in

through

antibody

XC46

detected.

precipitation

of

.

to

more

by

is

molecule.

Pigure

bound

level

the

of

binding

specific

carried

blotting.

no

enough

directed being

HC33

the

high

on

antibodies

the

argued

affinity

might

days,

antibodies

It their

several

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on The with

Figure f0r

raised

a Blue

Vol.

133,

No. 3, 1985

4

BIOCHEMICAL

3

AND

BIOPHYSICAL

5

2

RESEARCH

COMMUNICATIONS

4

1 nqo

Q&

92.5 * 68 43

26

A

26

6

-

B -

0

2

26

$-

2

0

pia. _. _ _3; __ Specific binding by blotted antibody fragments of ~~ raalolatelled Hwc-1 antigen. Purified antmody was digested with pepsjn at pE 4 (enzyme:eubstrate ratio 1:50) for the times Sndicated before separating on 5-10% polyacrylamide gradient gels. A) Cocmassie blue stained ge3 Autoradiograph of nitrocellulose blot of gel after reaction with "'1.---l antigen.

We

have

antibodies

reported

SDSpolyacrylamide

gels

(non-reducing)

after

that either

does occur (17) and affects

reversible

after

biologically

configuration

That

there is minimal

The

confirmation scope of

other

proteins

EGF and lipoprotein

that SDS treated proteins can exist

demonstration

after

any unfolding

elcctrophoretic

the protein blotting

is

show a

on Western blots has been shown by the

demonstration of binding of ligand to blotted (18.19).

to

the conformation of the binding site,

removal of the detergent.

active

in

transfer

unfolding of the antibody mo1ecuJ.ein the SDS gel. OK that which

that

electrophoresis

ahd eloctrophoretic

The results indicate

paper.

observation

surprising

rather

retain antigen binding properties

can

nitrocellulose

the

here

transfer

to nitrocellulose

technique.

receptors in a native

fmtenda the

It al80 implies that antibodies

reacting with proteins on Western blots do not

necessarily

react

with

a

linear peptide sequence (20). One direct

application

characterisation

of the technique described here,

of the products of proteolytic

and co-workers (21) and a number of advantages of

antibody

imnunoscintigraphy

and

Fab

fragments and

F(ab')Z

other over

antibody digestions.

Mach

groups have demonstrated the intact

fragments

1024

been the

has

antibodies have

found

for nuamrous

in-vivo USC8

Vol. 133, No. 3, 1985

in

BIOCHEMICAL

iamunochemistry.

isotypes,

are

easily

others

(including

permits

simple

digestion

to (by

facilitating

the

WS in

digested

with

many

antibodies,

pepsin

useful

IgGl

to antibodies)

of

iaanunoreactive

without

extensive

purification.

optimised

and

non-inaaune

comparison

with

Coomassie

design

of

an

particularly

produce

identification

be

identified

al.,

while

some

systfans

conditions

However,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effective

purification

the are

F(ab')2

Imrmnoblotting

fragments

in

reactive

allows

complex digestion

fragments stained procoduro

IgGl

fragments,

not.

This

Blue

same

to

gels), (Wather

be thUS et

preparation).

ACKNOWLEDGMENTS: The authors are grateful to J. Burchell and s. Gendler for purification and iodination of the BWFG-3 antigen, and to w. Shearer for 1'25 T--HuIPNa2 and antibody ST254. They also thank Drs J. Ivanyi and R. Romford for antjbodies HC46 and EC33 and T. DuhZg for purification of antibodies RMFG-1 and 2.

1. 2. 3. 4. 5. 6. 7. a. 9. 10. 13. 12. 13. 14. 15. 16. 17. la. 19. 20. 21.

Towkin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. x, 4350-4354. Schermar, A. and Sun, T. (1985) Lab. Investigation x, 243-256. Cooper, D., Burchell, J., Durbin, Ii. and Taylor-Papadimitriou, J. (1983) J. Immunol. U, 508-523. Gershoni, J.M. (1985) TIBS 103-106. Towbin, H. and Gordon, J. (3964) J. Inxaunologioal Methods 22.313-340. Lewis, W.G., Pantos, K.N., Allen, G. and Ivanyi, J. (1983) Antiviral Res. 1, 69. (Abstract). Taylor-Papadimitriou, 3.. Griffin, D. and Ralkwi33, P. Shearer. M.. (3984) J. Tmmunol. u, 3096 -3103. Taylor-Papadimitriou, J., Peterson, J.A., Arklia, J., Burchell, J. and Ceriani, R-T,. (1961) Int. J. Cancer a, 17-21. Shimixu, M. and Yamauchi, K. (1982) J. Riochem. z, 515&524. Arklie, J., Taylor-Papadimitriou, J., Sodmer, W.F., Kgan, H. and Millis, R. (1991) Int. J. Cancer a, 23-29. Travers, P. and Rodmer W. (1964) Int. J. Cancer && 633 -641. Arklie, J. (1961) D. Phil Thesis: University of Oxford. Bolton, A.E. and Hunter W.M. (1973) Biochem. J. E& 529-539. H. (3984) J. Interferon Res. Taylor--Papadimitriou. 3. and Shearer, 1, 553-559. Laemlli U.K. (1970) Nature 222, 660-665. Wang, D. and Taylor-Papadimitriou, J. (1984) Jnt. J. Burchell, J., Cancer J& 763-768. Reynolds, J.A. and Tanford, C. (1970) J. Biol. Chem. H, 5361-5165. Fernandes-Pol, J.A. (1992) FEB.9 L&t. J&, 86-92. Daniel, O.T., Schneider, W.J.. Goldstein, J.L. and Rrown, M.S. (1963) J. Biol. Chem. a. 46064611. Burchell, J., Bar-t&, J. and Taylor-Papadimitriou, J. (1965) Rybridoma (in pross). Wach J.-P., Chatal, J.-F., Lumbroso, J.D., Bucheggor, F., Forni, M., Ritschard, J., Bf?rchc, C., Douillard, J.Y., Carrel, S., Helgu, M., Steplewski, 2. and Koprowski, U. (1993) Cancer Ros. =,5593-5600.

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