Incorporation of labelled amino-acids in antibodies synthesized in vitro by cells of immunized rabbits

Vol.3,No.5

BIOCHEMJCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INCORPORATION OF LABELLED AMINO-ACIDS

Nov. 1960

IN ANTIBODIES

SYNTHESIZED --IN VITRO BY CELLS OF IMMUNIZED RABBITS. Alain Service

BUSSARD and Vinh de Biochimie

Cellulaire,

Paris

Received

October

incubated

indication

that

released,

if

(1955)

of radioactive

soluble

antibody

immunized

over

a period

later

from

the injected

debris

in avoiding

the animals

through

a nylon

(Ab)

(1960)

fragments

who used were

not

occur.

has been provided

tissue

this

step

from

isolated

studied

cells.

and the yield

the use of a non-

to immunize in the

the left

were

bled

the

culture

sieve.

foot

pad.

extremities. in Eagle’s

The cells 453

and to

fluid.

of 0.1 ml of S.E.,

were

Adult

rabbits

administered

Seven to 21 days after in the same fashion.

and the popliteal

nodes

a particulate

the rabbits

tissue

injections

into

by employing

dose of 0.1 ml was given

teasing

does

as to require

and contralateral

by gently

reaction

antigens

(S.E.),

by 9 intradermal

days

is an

as carrier.

Ab formed

a booster

acid

de -- novo and not merely

with

et al

was so little

of 2 weeks

immunization

amino

of antibody

protein

sheep erythrocytes

were

obtained

labelled

in studies

precipitate

the radioactive

in the medium of a tissue

an exchange

and by Vaughan

We have succeeded

remove

that

and others

homologous

antigen,

Pasteur

Paris

has been synthesized

is believed

experiments

isotopic

an isotopically

protein

rabbits

In these

-

protein

for --de novo synthesis

by Mountain immunized

of radioactive with

it

Such evidence

15e

Institut

14, 1960

The appearance culture

A. HUYNH

lymph nodes

were

Lymph node cells medium and passing centrifuged

Three excised were the

at 600 RPM for

Vol.3,No.5

BIOCHEMICAL

10 minutes

and washed

amount

of Eagle’s

twice.

The cells

medium to give

aliquots

of the cell

suspension

In these

experiments

radioactive

74 mC/mM and in a final four

separate

node,

flasks

L) cells

were

node with

dinitrophenol

rabbits.

In an additional

flasks

were

valine of 1 $/ml

a specific

1) cells lymph

a mixture

of

from the proximal

node,

3) cells

lymph

from the proximal

from non-immunized valine

was added

from the proximal

of 95% air-5%

flasks.

In each experiment

the radioactive cells

and 20 ml

radioactivity

(lOWL M), and 4) cells

to the

cells/ml

in 150 ml Erlenmeyer

was used.

with

control,

with

in a sufficient

of 10’

with

contralateral added

aerated

suspended

a concentration

employed

experiment

were

were distributed

amount

from the

the end of the

Nov. 1960

AND BIOPHYSICAI. RESEARCH COMMUNICATIONS

lymph

at

node.

CO2 and gently

The

shaken

at

370 c. Samples (staining

were

taken

by Trypan

blue),

cipitation)

1 ml of S.E.

and 0.75

represent

This

amount

washed

counter

by cells

experiment

is

synthesis

cells

Ab during

on the

in saline,

cells

we have observed

S.E.

of the

after I.

from the

viable.

samples

in vitro

M),

1 hour

for to

on millipore

filters

were

filters

on a thin

synthesis

stimulation.

can be seen that node)

proceeds

contralateral

the 24 hours

was y-globulin culture

separated

and counted

a secondary

It

(proximal

were

medium

studies,

and the

dried

culture

window

efficiency).

shown in Figure

30% of the

treatment

in 10B2 M valine),

valine

(low2

in preliminary

pre-

was made by

ml of the

valine

soaking

by the cells

synthesized

0.25

non-radioactive

of rabbits

stimulated

with

The S. E. were

such conditions

antibodies

cells)

viability

acid

measurement

of antigen.

with

of cell

(by trichloroacetic

The latter

3 x lo8

(28% counting

Under

determination

of S. E. was found,

by previous

extensively

fixed

protein

containing

an excess

(saturated

only

(with

ml of saline

at 37O C.

locally

total

for

and Ab radioactivities.

shaking

Geiger

at intervals

A

proof

by the

rate

than

However,

both

systems

end of this

the radioactive

was provided

by the

by an excess

of sheep

454

A typical

Ab synthesis

At the

that

S.E.

at a faster

node.

of incubation.

of anti

fact

that

anti-rabbit

Ab

period,

substance previous y-globulin

Vol.3, No.5

followed the

by the

removal

radioactivity

bound

To determine from the

activity

did

the

not

before

on the

to 15% of its

Surprisingly

(Fig.

cipitation

of rabbit

that

antibodies

these

the

culture

Previous

this

node cells

autologous

R.E.

fluid

samples, of the

but it

y-globulin

by sheep

are true

samples

immune serum,

auto-antibodies

taken

value. (R.E.)

were

to fix

C for

used radio-

to a lesser

at 56’

reduced

reduced

erythrocytes

found

although

was greatly

of S.E.,

were

were

Nov. 1960

original

the

lymph

heating

fixation

addition

of Ab attachment,

these

tissue I).

affect

erythrocytes

from which

from the S.E.

of the precipitate,

specificity

same rabbit

as a control.

than

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

degree

30 minutes

by specific

pre-

The possibility

requires

more study.

CPM /ml

lateral nodyhbbit

RBC

ateral node/sheep RBC

246 If the ratio computed

(Fig.

to produce

of radioactive II),

a high

30 to 40%.

the

data

of studying

of their

data

it

et al

the the

system

synthesis

highly protein

output

of antibody 455

of total

protein

provides

cells

are able Ab, i.e.,

are consistent

(1958).

a sensitive

--in vitro.

is

100 to 300 pg of Ab

These values

and of Stavitsky

described

Hours

in specific

that

per 24 hours.

(1960)

24

stimulated

can be calculated

gram of cells,

of Vaughan that

Ab to radioactivity

can be seen that

proportion

per

We believe method

it

From these

are synthesized with

IO

and reliable

Vol.3, No.5

Nov. 1960

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CPM Ab CPM Pfotcinr 50

FIG. 2

Xl00 CONTROLS

40 30 20 IO

24 hrr

-CT =lOhrs

48 hrs

HSA

Antigen

3 hrs

Time

of incubation

REFERENCES Mountain, Stavitsky,

I.M., A.B.,

Vaughan, J.H.,

J. Immunol. and Wolf, Dutton,

J. Immunol. &r

A.H.,

74: 170, 1955 B.

Biochim. Dutton,

Biophys.

Acta.

22:

R.W., George, M., and

258, 1960.

456

4,

1958

Marston,

R. Q.