Anti-glycosyl antibodies: Resolution of anti-gal isoantibodies into individual components by electrofocusing

Vol. 74, No. 2, 1977

BIOCH.EMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANTI-GLYCOSYL ANTIBODIES: RESOLUTION OF ANTI-GAL ISOANTIBODIES INTO INDIVIDUAL COMPONENTS BY ELECTROFOCUSING John H. Pazur Department

of Biochemistry University

Received

and Kevin

L. Dreher

and Biophysics, Pennsylvania Park, Pennsylvania, 16802

State

University

December 9,1976

Rabbit anti-galactose isoantibodies which are specific for the SUMMARY: terminal galactose units of a streptococcal antigenic diheteroglycan of glucose and galactose have been separated into individual components by a preparative electrofocusing technique: Ultracentrifugation and gel electrophoretic patterns indicate that each component consists of a single protein type of uniform molecular structure and size. Such antibody preparations will be of value for studies on the molecular structure of a natural antibody, on the nature of the bonds in an antigen-antibody complex and on the biological mechanism for the synthesis of antibodies. Antibodies

with

molecular

structural

unit

of a natural

purposes

including

studies

the nature

of the bonds

involved

in

preliminary

in

from

in its

specific

anti-lac

for

be useful

molecular

structure

described

the

sera

wall.

terminal is

specific

for

terminal

of on

and on factors synthesis.

immunized

with

of

anti-

a vaccine

of

of glucose

anti-gal antigen,

lactose

In a

two sets

antigen

of the

a single

a variety

of

of antibodies, units

for

of antibodies,

isolation

a carbohydrate

galactose

for

of antibody the

One set

specificity

complex

of rabbits

containing

cell

antibodies,

would

and regulation

D Streptococcus

galactose

and with

an antigen-antibody

(1) we have

antibodies

a Group

antigen on the

initiation

note

glycosyl

is

the

uniformity

and

antibodies, while

units

the other,

of the

same

antigen. We have now succeeded individual

components

a reconstituted

of the unfractioned analytical Copyright All rights

by the

the individual

electrophoresis, while

in separating

sample anti-gal

and a preparative

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

the anti-gal

electrofocusing

method

components yielded

yielded

a multiple

antibodies. centrifuge

into

(2,3).

On gel

a single

protein

pattern

similar

the anti-gal

band

to that

On ultracentrifugation (1),

818

antibodies

in an antibodies

Vol. 74, No. 2, 1977

yielded

BIOCHEMICAL

symmetrical

Although

patterns

preparations

against

carbohydrate

antibodies

obtained,

a natural

homogeneity

at aromatic

directed

and of electrophoretically haptens

(3,8)

of molecular

has not previously

size.

heterogeneity

(4-9)

of an antibody

antigen

in molecular

of restricted

antigens

directed

the preparation

against

indicating

of antibodies

microbial

homogeneous

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

been

have been

uniformity

directed

achieved.

METHODS AND RESULTS Isolation

of the

isolated

from

viable

cells

procedure (11)

Anti-gal

the sera

and elution

were at

in saline-phosphate

of anti-gal

antibodies column,

acid

gal

were

data

Further

a sample

antibodies

centrifuged shown

Separation

of Anti-gal

antibodies

was performed

using sucrose

a LKB 8100 gradient

pH 6 to 8. duced focusing

into

(110

the

column

was performed

solution

with

From 15

behavior

anti-gal

(1).

ammonium sulfate by centrifugation, against

the

of antiserum

ml

The sedimentation

rate

45

on polyacrylamide

antibodies

gels, and

have been presented

of 0.3 ml of a 0.5% solution E centrifuge

of the

at 60,000

with ml)

- Electrofocusing a LKS instrument The liquid

column.

of 6 mg of the along

with

at a power

antibody

the gradient

of the (Rockville,

column

preparation solution

of 1.5 to 2 watts

819

anti-gal Maryland)

consisted

ampholyte

of a

gradient

of

was introand electro-

for

anti-

RPM yielded

1.

Antibodies

mg

in a density

of the carbohydrate

(5% to 50%) and a 2% carrier

A sample

Sepharose

of pH 7 and dialyzed

72 hrs.

in a Model

in Fig.

of non-

The isolation

was collected

and the identification of the

were

on lactosyl

the solution

obtained.

constituents

(1).

the patterns

at 4" for

N (9).

0.5 M galactose

buffer

the electrophoretic

inhibition

earlier

from

a vaccine

(10)

The precipitate

4O.

solution

strain

with

antibodies

with

chromatography

precipitated

saline-buffer

amino

faecalis,

affinity

at 50% saturation

hapten

innnunized

of the antibodies

The antibodies

gradient

- The anti-gal

of rabbits

of Streptococcus

involved

redissolved

Antibodies

60 hours

at

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I 4

40

72

TJME (MIN.) Fig.

1

Sedimentation the analytical

velocity patterns ultracentrifuge.

of the

anti-gal

antibodies

8

E’ c s N z a

0. 3-I

5

0.2

. 0.1

II 30

40

50

60

70

FRACTION

Fig.

2

Electrofocusing volume for the

40.

The column

was then

of 1

fractions

and the pH were

and a Corning

ml

pH

were

meter.

100

and plot of pH values isoantibodies.

at

collected.

a

flow

of 60

rate

The absorbance

measured A plot

90

NUMBER

pattern anti-gal

emptied

fractions

80

820

ml/hr

effluent

and

at 280 mu of all

in a Beckman

of the results

versus

DU spectrophotometer is

shown

in Fig.

2.

in

Vol. 74, No. 2, 1977

BIOCHEMICAL

M Fig.

3

preparation

acrylamide

blue

G-250

gels

(fractions

galactose

for

49,

solution

a trough

with

of antigen-antibody appeared

1% agarose

to the protein

formation

in a moist were

placed After

in the for

A broad,

preparation

because

of the antigen-antibody

trough.

several

in

antiun-

had

and a 0.15%

days at room the appearance

elongated 48 hrs

petri

Diffusion

for

precipitin

corresponding

Bands were

the ampholene complex

821

of 6 of the

the agarose gel

periodically

6

Goomassie

in separate

the polyacrylamide

bands.

with

The duplicate

3.

in the preparation.

components

stained

and the original

were

chamber

8.5 for

A photograph

solution.

examined

the anti-gal

components

the individual

along

in 5% polyatpH

were

in Fig.

was placed

precipitin

with

73, 85 and 90)

reproduced

was cut

The plates

electrophoresis

components.

200 ul samples

to proceed

temperature.

the

56, 66, is

gel

50 u1 samples

of the diheteroglycan

was allowed

with

VI

and the unfractionated

of 30 milliamps

protein

containing

and covered

solidified

current

locating

fractions

used for

containing

preparation gels

dishes

were

at a constant The gels

stained

111 IV v

of 50 1~1 and 200 1-11 of all

anti-gal

hours.

If

Gel electrophoretic patterns for the mixture of anti-gal isoantibodies (M) and the individual components (I to VI).

Samples

band

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

not

interfered

as revealed

obtained with

in separate

BIOCHEMICAL

Vol. 74, No. 2, 1977

diffusion

tests.

Additional

electrophoresis

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

agar

diffusion

tests

coupled

with

immuno-

in progress. DISCUSSION

Anti-galactose rabbits

antibodies

immunized

by affinity

with

solution

aration

showed

proteins,

preparation

galactose

in the

test.

proteins the

terminal

the

individual

of

of 6 distinct This

diheteroglycan

of glucose

and

and the double

diffusion

was inhibited

B-galactosyl

anti-gal

behavior (Fig.

molecular

weight

centrifugation

the

units unit

term

in

by galactose

(1).

Since

the

of the antigen,

isoantibody

is

of the

various

namely

suggested

for

that

as shown

into

in Fig.

W

electrofocusing

822

the ultraX 105. components

absorbance

the

were

into

composed

fractions

49,

and are absorbing

W

on 50 ~1 samples

of the isoantibodies

3 for

1.5

The

The

apparatus

of 1 ml contained

Many fractions

size.

individual

The

electrophoresis

a resolution

is

(1). ultra-

from

(12,131

method.

fractions gel

analytical

calculated

isoantibodies

gradient

UV scan

in molecular

formulae

from the

Forty-five

had occurred.

component

uniformity antibodies

anti-gal

fractions

Polyacrylamide showed

in the

by an electrofocusing

of the 2.

isoantibodies

and appropriate of the

on density

a symmetrical

anti-gal

the

sedimented

yielding

for

achieved

in Fig.

band

indicates

data

pH gradient

isoantibodies

1) also

A separation

components

3) of the prep-

mobility.

same structural

as a single

centrifuge

fractions

with

consisted

reaction

unit,

and elution

N

components.

sedimentation

protein

the

8-galactosyl

centrifugation

materials.

precipitin

terminal

strain

electrophoretic the

of

faecalis,

(Fig.

preparation

with

capillary

with

The mixture

shown

Sepharose

a characteristic

with

react

has been

antibody

reacted

the antisera

of Streptococcus

The antigen-antibody

and compounds

from

Gel electrophoresis

the

each with

isolated

on lactosyl

(1).

that

antibody

agar

a vaccine

chromatography

galactose

were

of these

individual

of a single 56, 66,

73, 85

BIOCHEMICAL

Vol. 74, No. 2, 1977

and 90.

However,

fractions

some overlapping

consisted

fastest

A second

of

the diheteroglycan column

on the resolution

components

lations

specific

on the genetic

are highly are also individual

size

for

been observed

Studies

vidual

are

desirable underway

and the relationship

Acknowledgement department for

antigens

of synthesis

which

exists

of the

antigen

of the

of the amino

homogenous

indipopu-

in our

will

into

(1).

of antibody

of such sets

initiated

of

moiety

solution

isoantibodies

Such information are indeed

being

Sepharose

lactose

moieties

carbohydrate

control

component

the lactose

The occurrence

structural

with

and a few isoantibodies

moving

with

of the latter

different

occur

of the lactosyl

absorbed

on the determination

isoantibodies

for

by elution

in progress.

did

VI.

specific

and have been

isoantibodies.

of an isoantibody

the slowest

were

RESEARCH COMMUNICATIONS

The individual

being

isoantibodies

the antibodies

vidual

has also

component

was obtained

on which

Experiments

from I to VI with moving

set

of components

of 2 or 3 components.

have been numbered I and the

AND BIOPHYSICAL

same

dextran

type

of isoantibodies

laboratory. acid

among the individual

Experiments

sequence

show whether

in structure

2. 3. 4. 5. 6. 7.

8.

of the

the indi-

and molecular components

set.

- The authors wish to thank R. C. Bolts, Jr. of this assistance with the electrofocusing methodology.

REFERENCES 1.

(14).

Pazur, J. H., Miller, K. B., Dreher, K. L. and Forsberg, L. S. (1976) Biochem. Biophys. Res. Commun., 70, 545-550. Valmet, E. (1968) Science Tools, 15, 8-11. Freedman, M. H. and Painter, R. H. (1971) J. Biol. Chem., 246, 4340-4349. 29, 59-65. Krause, R. M. (1970) Fed. Proceed., Kim, Y. D. and Karush, F. (1973) Immunochem., 10, 365-371. Chen, F. W., Strosberg, A. D. and Haber, E. (1973) J. Immunol., 110, 98-106. Freedman, M., Yeger, H., Milandre, J. and Slaughter, M. (1975) Immunochem., 12, 137-147. Freedman, M. H., Guyer, R. B. and Terry, W. D. (1972) J. Biol. Chem., 247, 7051-7061.

Vol. 74, No. 2, 1977

9.

Pazur,

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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