Preparation of monoclonal antibodies able to discriminate between testosterone and 5α-dihydrotestosterone

453

2946

PREPARATION OF MONOCLONAL ANTIBODIES ABLE TO DISCRIMINATE BETWEEN TESTOSTERONE AND 5a-DIHYDROTESTOSTERONE

F. Kohen. Departments

Received

S. Lichter,

2.

Eshhar*

and H.R. Lindner

of Hormone Research ana *Chemical The Weixmann Institute of Science, Rehovot. 76100, Israel.

Immunology,

3-8-82

ABSTRACT A procedure for proauction of monoclonal antibodies to testosterone The method involves immunization of rats with a bovine is described. serum albumin conjugate of testosterone 3-(O-carboxymethyl)oxime followed by polyethylene glycol induced hybridization of the immune lymphoThe resulting hybridomas were cloned cytes with mouse myeloma cells. and the antibodies produced by each clone were characterized. Ail ,“opg antibodies obtained showed high affinity for testosterone, (K l/mol), but clones differed widely in the degree of cross-rea%ion of such as 5a-dihyarotestosterone the antibodies with other steroids, (range 2-100X) and androstenedione (< 0.1-42). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.

INTRODUCTION Testosterone lar

ana past

and 5a-dihydrotestosterone

attempts

criminate

between

partially

successful.

- one involved to

the

these

variation

macromolecular

Volume 39, Number 4

to

generate

antiboaies

two clinically

the

carrier,

s

of

attachment

through

TDEOXDI

the

very

testosterone steroids

have been adopted

site e.g.

to

important

Two approaches of

are structurally

of steroid

simi-

that

ais-

have only in these

the

steroid carbon

April,

been

studies hapten in posi-

1982

tlon

l(l),

approach

3(2,3),

6(14),

involved

an

5a-dihyarotestosterone gate

of

attempt

5a-dihydrotestosterone with

testosterone

these

methods

yielded

terone,

but

the

still

been

able

ana pregnanediol

nique

Kdhler

monoclonal

to

cross-reaction

generate

glucuroniaes

with with

(11). high

the

animals

A large

acid

in

the

(9,101

immuni(8).

for

100% (2.3).

past

specific

by using

towaras

5a-dihydrotestosterone

antisera

a hybriaoma the

proauction

testosterone

All

testos-

cross-reaction to

to

a conju-

before

affinity

We now aescribe specificity

of

conjugate

showed high

10% (1.8)

alternative tolerant

excess

hemocyanin

substantial

from

An

15(7).

to D-glutamic

that

exhibited

and Milstein

antiboaies

with

limpet

antisera

ano

render

couplea

ranging

estrogen of

to

keyhole

5c-aihyarotestosterone We have

11(6),

by pretreatment

zation

minimal

7(5),

with

to techof and

(2%).

MATERIALS AND METHODS

The steroids used for specificity studies were purchased from Reagents: bovine serum albumin (fraction V)(BSA) was Makor Chemicals. Jerusalem: Louis, MO.: [1,2,6,7-3H(N)l testosterone obtained from -Sigma, St. (90-115 Ci/mmol) from New England Nuclear, Boston, Mass., and Dextran T-70 from Pharmacia, Uppsala, Sweaen. in The assay buffer sodium phosphate, pH 7.4, sodium aziae (1 g/l).

the raaioimmunoassay proceaure was 0.02 M containing 9 g NaCl/l, BSA (100 mg/l) and

Synthesis of testosterone 3-(0-carboxymethyl)oxime bovine serum albumin conjugate: -Testosterone 3_(0-carboxymethyl)oxime(m Goa to an activatea N-succinimide ester derivative by a procedure previously aescribea (9). Bovine serum albumin (20 mg) in 0.5 ml of 0.13 M NaHCO was added to the aimethylformamiae solution (0.5 ml) containing th2 activatea ester. The mixture was stirreo for 15 min, and dialyzed against 0.1 M NaHCO and cistilled water. The proauct was freeze-aried. Ultraviolet aasorpt ? on analysis of the antigenic complex inoicated that the immunogen containea 16 steroia resiaues per mole of BSA.

Wistar-derived female rats (2 to 3 months old) from the Immunization: Departmental Colony were immunized by an intradermal injection of testosterone 3-(O-carboxymethylloxime-BSA (200 ug/rat) inoorporated in comA month later the injection was repeated, and plete Freuna’s adjuvant. booster injections (100 pg/rat) were given three weeks later. All animals were bled from the tail ten aays after the booster injection, and the antisera were tested then for titer ana specificity. The non-Ig producer mouse myeloma Cell lines ana culture conditions: ---cell line NSO/l used in the fusion experiments was kindly supplied by The culture conditions were as Dr. C. Milstein, Cambridge, England. previously described (9). On Days 4 and 3 before the fusion experiment, rats hybridization: Spleen cells given a booster injection (100 ug/ratl in saline. were then removea and fused with the mouse myeloma cells by Using 41% polyethylene glycol (Serva, Type 1550, Heidelberg, West Germanyl(l21. After fusion the cells were distributed in Costar tissue culture plates (Clu ter 24, Costar, Cambridge, Mass.), each well containing less than 5 The hybrias arising in each well were selected in Dulx 10$ cells/ml. becco’s modified Eagle’s medium (DMEM) containing hypoxanthine, aminopterin and thymidine (HAT), as previously described (9). Cell

were

Hybrid cells that secreted antiboaies to testosterCloning -of cells: one-3-BSA were Cloned by limiting dilutions in 96-well microplates in Dulbecco’s modified Eagle’s medium (DflEM) containing hypoxanthine and thymidine (HT). as previously described (9). Two weeks prior to cell inoculation, Growth of hybridomas in mice: -adultmze mice (BALB/c x DBA/z) F, or BALB/c nuae mice receivea an intraperitoneal (i.p.1 injection of 2.6,10,14-tetramethylpentadecane The F (Pristane, Aldrich Chemicals, Wis onsinJ(0.5 ml/mouse). mice were in addition irraaiated with 80 Co at 750 rad for 10 min; cu1 tured hybriaoma cells (2-3~10~ in 5 ml Dulbecco’s modified Eagle’s medium were Ascitic subsequently injected i.p. into the irradiated or nuae mice. fluid (5 - 15 ml/mouse) was harvested 10 to 20 days after hybridoma inoculation. Characterization of antibodies: A dextran-coated charcoal radioimmunoassay method was used to detect specific antibodies to testosterone Antibody titer aenotes the dilution at which 50% of the labelea (1). addea at concentrations of 50 pg/ml, ligana, is bound as determined by radioimmunoassay. Cross reactivity (g) was calculated as proposed by Thorneycroft et al. (131, as F where x is the mass of testoster-one ana y the mass of heterologous compound required to produce 50% inhibition of the binding of the tritiated ligand. Affinity of the antibodies was calculated according to Abraham -et al. (14).

S

456

TDEOXDI RESULTS

Preparation

-of

Fusion cells

of

resultea

of

that

subcloned

retea

high

fluid

of

Table

two

of of

weeks

high

During antiboay

hybria

of

in

agar.

these

of

mice,

the

subsequent

titres

rats

cells

growth,

tissue cell

titers

the

culture culture,

mouse

i.p.

culture

ranging

media

of

30 out lines

ailutions

ana

pristane-treated

grew as tumors

from

wells

3 hybriaoma

into

antiboaies

myeloma

hybridomas

by limiting

hybridomas

anti-testosterone with

the

antibody-secreting

When injected the

with

in 26% of

were cloned

F , mice or nude mice, amounts

-to testosterone

immunized

by a RIA procedure

on semi-solid

irradiated

(Table

cells

147 cultures.

yielaed

antibodies

in the growth

detectea

the

spleen

After

(147/5661. were

monoclonal

into l:lO,OOO

and sec-

the to

ascitic 1:60,000

1).

1.

Properties

of

various

antibodies

to testosterone.

+__--__-________________________________~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~+ 9 I

I I

Monoclonal Antibodies Polyclonal* I 1 Rat IgG I (Clone b) +__________________________________________~~~_~~~~~~~~~~~~~~~~~~~~~~~~+ I I t 1 F2 Dll H6 +____________-_---_-_-_-_-_-_______________-______------~~~~~~~~~~~~~~~+ I I

I

8

lTitera IKa (l/moljb lHeavy chain

II

a b ’ l

1:60,000

1:50,000

1:5.000 3.7x109

class” 2.1x1010 Yza

1.8z0’o

I@’

Titer as definea in the Methods section. Calculated according to the method in ref. 14. Determined by aouble immunoaiffusion in agar. The polyclonal IgC was from the rats usea for the

fusion

experiments.

1 I 1 I II I1

S Characterization

of

The ascitic D1l was further coatea

Table

monoclonal

fluid

RIA-procedure

2.

antibodies

of mice derived

characterized (1,141.

Specificity of mined by RIA

457

TEEOXDI

for

from three

affinity

ana specificity

The results

to

testosterone

H6, P2 and

by a aextran-

are shown in Table

antiboaies

various

hybridomas,

2.

as

deter-

+___________~~~~~~~_____----~~~~~~______~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~+ 8

I

,

Cross-reaction Monoclonal

I

!Compound (15)

(I)

Polyclonal Rat IRC

I !

I

lTestosterone 15a-Dihydrotestosterone I5 B-Dihydrotestosterone

0 I1

117a-Epitestosterone lAnarosteneaione

I15a-Anarostane-3.17-aione 15j-Anarostane-3,17-aione I3a-Hydroxy-5a-anarostan17-one 13B-Hydroxy-5a-anarostan-

I

I 1 I I

17-one

15a-Anarostane-3a. 17@-aiol 15a-Anarostane-3B, 17B-aiol lDehyaroepiandrosterone 15-Anarostene-3@,17@-diol

I II

lProgesterone

0

I ! Cortisol

100 100

0.5

100 100

0.5

100

2 15

100 10

2

0.2

0.1 4

0.1

0.2

0.1 0.1

0.1 0.1

0.1 0.1

0.1




0.1




0.1

2 2 0.01 2

0.3 0.3 0.01 0.2

0.5 1

0.3

0.01 0.5

0.01 0.1

0.1

0.1

0.1

I I I


0.4


0.01





I )

s

458

TElROXDIll

DISCUSSION

This duce

paper

describes

monoclonal

aerivea

from

immunizea

antiboaies fusion

with

of

ferentially with

myeloma

bound

but the

varied

well

testosterone

clones

also

with

aefined

meet the servea

able

mones.

a given

all

the

minimal

rats

serum

albu-

affinity

antiboaies

that

producea # F2 pre_

H6 ana

l?,,

Table

or

The

clone

clone

immunoassay

with

will

shows A nar-

may be selected clones

antiboay

in ascites

types

three

steroias.

The selectea

preparations

react.ed

2) ana showea min-

hybrid

the

(2%)

cross-reaction

other

each

(see

cross-reaction

of (see

with

were

of

Thus clone

2).

aegree

antiboay of

cells

a high

pro-

lines

bovine

clones

required

culture

that

5a-aihydrotestosterone.

assay.

and whenever in tissue

of

Table

Thus an appropriate of

monoclonal

stanaaraization

the

2 indicate

specificity.

by freezing,

that

of

showea

cross-reaction

requirements

spleen

showea

and androsteneaione

in Table

can be propagated

or

in

to non-significant

rowly

with

BY contrast.

aifferea

The results

(see

ana

testosterone

Sg-aihydrotestosterone

cells

specificity

wiaely

lines

The hybridoma

antibodies

5a-aihydrotestosterone.

equally

hybriaoma

3-CO-carboxymethyl)oxime

The monoclonal

clones

of

testosterone.

mouse

1) to testosterone,

by aifferent

imal

to

testosterone

min con jugate. Table

the establishment

It

facilitate

proceaures

can be pre-

secreting

fluid.

for

to

cells

seems probvigorous steroid

hor-

ACKNOWLEDGEMENTS This work was supportea by grants from the U.S.A. Binational Scithe Bosch Foundation ana the Rockefeller ence Foundation (Jerusalem), Z.E. is the incumbent of the Recanati Career Development Founaation. H.R.L. is the Adlai E. Stevenson III ProfesChair in Cancer Research. sor of Enaocrinology and Reproauctive Biology at the Weizmann Institute We are grateful to Mrs. M. Kopelowitz for excellent secreof Science. tarial assistance ana to Mrs. J. Ausher, Mr. A. Almoznino ana Ms. T. for the generous assistance and to Dr. C. Milstein Waks for technical of the NSO/l myeloma cells. gift

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13.

14.

15.

In Steroid Immunoassay Kohen, F., Bauminger, S. ana Lindner, H.R. (Cameron, E.H.D., Hillier, S.G. ana Criffiths, K., eas., Alpha Omega Publishing Ltd., Cardiff, Wales (19751, pp. 11-32. D.L. Z. Klin. Chem. u. Klin. Biochem. Nieschlag, E. and Loriaux, lo, 164-168 (19721. Ismail, A., Niswender, G.D. and Midgley, A.R., Jr. J. Clin. Endocrinol. Hetab., 2, 177 (19721. Jones, C.D. ana Mason, N.R. Steroids, 2, 23-32 (1975). Weinstein, A., Lindner, H.R., Friealander, A. ana Bauminger, S. Steroids, 2, 789-812 (19721. Hillier, S.C., Brownsey, C. B. ana Cameron, E.H.D. Steroias, 21, 735-754 (1973). Biochem.. 8. 1165-1169 Condom. R. and Desfosser, A. J. Steroid (19771. Tateishi, K., Hamaoka, T., Takatsu, K. ana Hayashi, C. J. Steroid Biochem., 13, 951-959 (19801. Eshhar, Z., Kim, J.B., Barnard, C., Collins, W.P., Gilad, S., Lindner, H.R. and Kohen, F. Steroids. 3, 89-109 (19811. Eshhar, Z., Kohen. F. ana Linaner. H.R. In XXIXth Colloquium of Protiaes of the Biological Fluids, (Peters, H., ed.1, Pergamon Press, Oxfora, in press. K(lhler. G. and Milstein, C. Nature 256, 495-497 (1975). Eshhar, Z., Ofarim, M. ana Waks, T. J. Immunol. 775-780 2, (1980). Tnorneycroft, I., Tillson, S.A.. Abraham, G.E., Scaramuzzi. R.J. ana Caldwell, B.V. In Immunologic Methods in Steroid Determination Peron, F.G. and Caldwell, B.V. eas.), Appleton-Century-Crofts, New York (19701, pp. 63-86. Abraham, G.E. and Garza. R. In Handbook of Raaioimmunoassay (Abraham, G.E., ed.) Marcel Dekker, Inc., New York. (19771, pp. 591-656. The following trivial names will be used: 5a-aihydrotestosterone = 17g-hyaroxy-5a-androstan-3-one: 5B-aihydrotestosterone = 17@hydroxy-5@-anarostan-3-one: 17c-epitestosterone = 17a-hydroxy-4anarosten-3-one: anarostenedione = 4-androstene-3,17-dione: dehyaroepiandrosterone = 3B-hydroxy-5-androsten-17-one.