Vol. 166, No. 3, 1990 February 14, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1228-1236

BIOCHEMICAL

PREPARATION AND CHARACTERIZATION OF POLYCLONAL AND MONOCLONAL ANTIBODIES AGAINST THE INSECTICIDE DDT Daniel

Biirgisser,

Stefan Frey, Stephan Klauser

Bernd

Gutte

Biochemisches

Institut der Universitat Winterthurerstrasse 190, CH-8057 Ziirich, Switzerland

Received

December

and

Ziirich,

4, 1989

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared antiDDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/l were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site. O1990 Academic Press,Inc. Although diction

is

the still

been reported ratory

design

of

in

infancy,

(l-6).

was the

The proposed

design

B-sheet

by CD measurements, various

peptide

action

between

unpublished designed

24-residue

a naturally

of

structure and the

of

are

a monoclonal

occurring

attempts

have

this

from

labo-

field

peptide

our

polypeptide (2,7)

(2).

was confirmed

of DDT binding

studies

using

the

mode of

inter-

postulated

protein of

pre-

successful

DDT-binding

this

supported

NMR studies of

to

results

polypeptide

based on structure

several

a 24-residue

DDT and binding

results).

proteins

One contribution

analogues

The availability ting

its

novel

(2,7)(S. the

Klauser

solution

et

al.,

structure

of

in progress. anti-DDT

DDT-binding

protein,

antibody, would

represengreatly

Abbreviations: DDE,

DDT, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane; l,l-dichloro-2,2-bis(p-chlorophenyl)ethylene; DDA,

bis(p-chlorophenyl)acetic acid: Kelthan, l,l,l-trichloro-2-hydroxy-2,2-bis(p-chlorophenyl)ethane; N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide melting point. 0006-291x/90 Copyright All rights

EDC, x HCl;

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

1228

the

m.p.,

aid

Vol.

BIOCHEMICAL

166, No. 3, 1990

our work all

on the

artificial

work

in the

future

structure dated

of

the

tion

and compared

reveal

the

proteins.

to

essential

of

that

of

computer of

the

features

design the

tight

and probably

in general.

antibody

modelling,

designed

of

anti-DDT

polypeptide

protein

site

RESEARCH COMMUNICATIONS

could

and specific

antibody

would

be eluci-

and x-ray

polypeptide. also

The diffrac-

This

may

DDT binding

by

be of great

value.

The purpose anti-DDT (bovine

field

analysis,

A monoclonal

analytical

DDT-binding

DDT binding

by sequence

AND BIOPHYSICAL

of

the

antibodies. serum albumin)

polyclonal respectively.

study

required

the

preparation

Here we describe

the

synthesis

conjugate

and monoclonal The isolated

which

anti-DDT

antibodies

antibodies

MATERIALS

elicited

the

of of

a DDT-BSA

formation

in rabbits

were partially

true of

and mice,

characterized.

AND METHODS

Materials. Kelthan (Figure 1) was obtained from Riedel-de Ha& (Seelze, Germany), DDT, DDE and DDA (Figure 1) were from Serva (Heidelberg, Germany). Protein A-Sepharose was received from Pharmacia, Affi-Gel 10 (for coupling of amines) and Affi-Gel 102 (for coupling of carboxylic acids) were from Bio-Rad Laboratories. 125 I-Protein A was kindly provided by Dr. M. Aguet. Synthesis of the DDT derivative (Figure 2, product 4) used for conjugation with BSA. Kelthan (9.5 mmol) and 3-bromopropionitrile (85 mmol) reacted in 96% H2SO4 (23.5 mmol)(8) to give product 1 (Figure 2) which after precipitation with ice water and recrystallizationofrom ethanol was obtained as white needles in 90% yield, m.p. 171 C. Product 1 (4.0 mmol) was converted to product 2 (Figure 2) by reaction with sodium azide (8.0 mmol) and tetrabutylammonium bromide (0.28 mmol)(9,10) in 3 ml of benzene and dimethylformamide each. Product 2 was isolated from ethanolic solution by precipitation with ether in 62% yield, m.p. 155OC. It was reacted with an equimolar amount of triethyl phosphite in benzene (10,ll) and then with HCl gas to give product 3 (Figure 2) which was precipitated by adding ether. The yield of the white powder obtained was 95%, m.p. 103OC. The last step of the derivatization consisted in the reaction of product 3 (1 mmol) with succinic anhydride (5 mmol) in dry pyridine. After evaporation of the pyridine, extraction of excess succinic anhydride with 0.1 M HCl/ methanol (lO:l), and recrystallization from acetone the target DDT derivative (product 4, Figure 2) was obtained in 53% yield, m.p. 153Oc. The course of the synthesis was followed by elementary analysis (Table l), mass spectrometry, infrared and NMR spectroscopy of the products. In the target DDT derivative (product 4, Figure 2), for example, the protons of the two amides could be distinguished of the amide at the a-carbon gave a singuby 'H-NMR: The proton lett at 8.53 ppm whereas the proton of the amide bond in which the 1229

Vol.

166, No. 3, 1990 Table

1:

Product Product Product Product

1 2 3 4

Numbers

in

P-NH2

by the

40.74 43.96 43.10 46.40

BIOCHEMICAL Elementary

(40.47) (43.78) (42.80) (46.66)

parentheses

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

analyses in Figure

2.83 2.61 3.25 3.51 are

(2.60) (2.79) (3.36) (3.52)

the

of 2

the

2.71 11.90 5.76 5.00

theoretical

products

shown

(2.78) (12.00) (5.87) (5.18)

-44.70 33.05

(44.60) (32.82)

values.

group participates, produced a triplett protons at the adjacent B-carbon.

at

7.83

ppm caused

Preparation of a DDT-BSA conjugate. The DDT derivative (Figure 2, product 4; 18 umol) was activated with excess EDC (N-ethylN'-(3-dimethylaminopropyl)carbodiimide x HCl; 36 umol) (12) in dimethylformamide (1 ml) and added to a solution of BSA (0.3 umol) in 20% aqueous dimethylformamide (5 ml). The turbid mixture was dialyzed against water, lyophilized, resuspended in 0.05 M NH4HC03, and treated with excess cold acetone to precipitate the DDT-BSA conjugate. The yield of the conjugate was 0.16 umol or 54% based on a DDT/BSA molar ratio of 26 as determined by quantitative p-alanine analysis after acid hydrolysis in 6 M HCl at llO°C for 20 h. The DDT/BSA molar ratio could be largely manipulated by the experimental conditions of the coupling reaction (Table 2). Preparation of a DDT-lysozyme conjugate. Hen egg-white lysowas acetylated using acetic anhydride in a 60-fold molar The yield of this step was excess per primary amino group (13). 62%. The reaction between the acetylated lysozyme (6.8 umol) and the p-alanine amide derivative of DDT (B-alanine amide-DDT, Figwas mediated by EDC (four 2.2-mm01 ure 2 (product 3); 21 umol) portions) in 25% aqueous dimethylformamide (22 ml) at pH 5. After 2 h the mixture was dialyzed against 0.1% trifluoroacetic acid and then lyophilized. The conjugate (yield, 56%) contained equizyme

molar

tative

amounts

of

S-alanine

DDT

derivative

and

lysozyme

as

shown

by

quanti-

analysis.

Antibodies against the DDT-BSA conjugate. To raise pOlyClOna1 antibodies, a rabbit was injected with 100 ug of conjugate II

Table

2:

Variability ratio

of

of the DDT derivative-BSA the conjugates

DDT derivative / BSA / EDC molar ratio employed in the coupling reaction 90 60 90

DDT derivative ratio of the conjugate

: 1 : 90 :l : 120 : 1 : 180

10 : 1 26 : 1 41 : 1 1230

molar

/ BSA molar resulting ( 1) ( 11) (III)

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(Table 2) in complete Freund's adjuvant (Difco) and PBS (phosphate-buffered saline), pH 7.2 (0.5 ml each), followed by four monthly booster injections of conjugate II (50 ug each) in incomplete Freund's adjuvant and PBS (0.25 ml each). Blood samples from the ear vein of the rabbit were kept at4OC overnight and then centrifuged. The supernatant sera were stored frozen. Monoclonal antibodies were obtained by the following procedure: Female BALB/c mice received intraperitoneal injections of 40 ug of conjugate II in complete Freund's adjuvant and PBS (0.25 ml each), followed by three monthly booster doses (40 ug each) in incomplete Freund's adjuvant and PBS (0.25 ml each). After the presence of anti-DDT antibodies had been demonstrated in a competitive solid phase RIA (radioimmunoassay), one mouse received an additional injection of [email protected] ug of conjugate II in PBS and was sacrificed four days later. The washed spleen cells of this mouse were fused with X63Ag8.653 myeloma cells (14,15) and grown on feeder layers of peritoneal cells in 10% FCS (fetal calf serum; Inotech) in Iscove's modified Dulbecco's medium that gave a positive competi(Gibco) (16). Cells from hybridomas tive solid phase RIA were subcloned twice after limited dilution. Positive clones secreting IgG and IgM anti-DDT antibodies were injected into female BALB/c mice primed with 2,6,10,14-tetramethylpentadecane (Sigma). The ascitic fluids obtained from these animals were dilgted with 2 volumes of PBS and dialyzed against 18% Na2S04 at 22 C overnight. The precipitates formed were centrifuged. A monoclonal IgM anti-DDT antibody was purified as follows: The precipitate was redissolved in PBS and the solution applied to a goat anti-mouse IgM antibody agarose column (Sigma; capacity, 0.4 mg/ml gel). Bound IgM was eluted by 0.1 M glycine0.15 M NaCl, pH 2.4, and had a gel electrophoretic purity of -90%. The solution was neutralized with 1 volume of ten-fold concentrated PBS, dialyzed against PBS, and applied to a DDT-lysozyme column (prepared from EDC-activated DDT-lysozyme conjugate and Affi-Gel 102 using 100 mm01 EDC, 11 umol conjugate, and 50 ml Affi-Gel 102 in 200 ml PBS, QH 5; coupling yield, 38 nmol conjugate per ml gel). The IgM was eluted again by the glycine buffer, DH 2.4, and the purity of the monoclonal antibody was demonstrated by gel electrophoresis (Fiqure 3). Competitive solid phase RIA of DDT and DDT derivatives. Each incubation step on the microtitre plates was followed by 3 washes with PBS. Plates were coated with the DDT-lysozyme conjugate (3.5 ug in 50 ul of PBS/well) at 4OC overnight. Additional protein binding sites were saturated with an emulsion of 1% powdered milk in PBS at 4OC for 6 h. Then 50 ul of a 1:l mixture of diluted antiserum and hapten solution were added per well. After 8 h at room temperature the wells were washed and the residues incubated with 1251-protein A in 1% powdered milk in PBS (50 ul per well containing -100'000 cpm) at 4OC overnight. Finally, the radioactivity bound by the washed wells was counted. Diluted antiserum and hapten solutions were prepared as follows: Antiserum (ascitic fluid) was diluted lOOO-fold with 20% FCS in PBS after FCS had been passed through a commercial protein ASepharose column to remove any IqG. Stock solutions (1 mM) of DDT, p-alanine amide-DDT (Figure 2, product 3), DDE, DDA, and p-chlorotoluene in ethanol were also diluted with 20% FCS in PBS to concentrations between 0.02 and 64 uM. The resulting mixtures were sonicated and stored at room temperature at least 12 h before use. 1231

Vol. 166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION The efficiency could

of our design

be assessed

perties

of

the

occurring

best

designed

bodies

protein.

proteins

would

with

great

value

also

studies

with

DDA).

against

DDA-protein

authors

anti-DDT

Synthesis

of

by the

ting

alkylbromide

3) by the

(Figure

azide

present using

analyses (e.g.

have the

work,

and

DDE and

been prepared first

a semisynthetic

ratio

true DDT-BSA

product

a DDT-lysozyme 1) that

the

analysis

reducing

agent

4 and BSA, conjugate

26)

(molar the

solid

i) that

resul(product

and hydra-

showed that

amine was obtained Staudinger

the

amine

phthalimide

were prepared;

was used in

of

corresponding

2) by the

to carrier

3-bromopropionit-

conversion

structural

as the

coupling

with

of potassium

The desired (product

conjugates of

for

Direct

1) to

action

phosphite

DDT-protein

lysozyme,

conjugates

natural Such anti-

environmental

suitable

iH-NMR and x-ray

triethyl

and ii)

accessible

DDT metabolites

the

(8).

(product

was formed.

(molar

a naturally

antibodies.

1) was combined

reaction

consecutive

intermediate ent

In

DDT derivatives

Ritter

failed;

using

major

were obtained

Kelthan

rile

p-lactam

of

pro-

as immunogen.

proteins.

zine

(17-19).

antibodies

conjugate

those

for

polypeptide

and DDT-binding

The most easily

cross-reactivity by several

structure

seemed to be anti-DDT

be of

Antibodies

a DDT-binding

polypeptide

DDT-binding

DDT-binding

of

by comparing

from

reaction (10).

the (11)

Two differ-

a DDT-BSA conjugate was used as immunogen,

ratio

of product

phase RIAs

3 and

and as affini-

DDE

DDT

HO’

C\

‘0

DDA

DDT-OH (KELTHAN)

formulae of the insecticide Figure 1: Structural metabolites DDE and DDA, and Kelthan. 1232

a

DDT,

its

major

Vol.

166, No. 3, 1990

BIOCHEMICAL

R-OHa +

AND BIOPHYSICAL

[H2S041

NC-CH2-CH2-Br

RESEARCH COMMUNICATIONS

R-NH-CO-CH2-CH2-Br Product -1

)

NaN3 1 R-NH-CO-CH2-CH2-N3 Product -2

13 HCl

succinic anhydride ) pyridine

R-NH-CO-CH2-CH2-NH3+ClProduct -3

R-NH-CO-CH2-CH2-NH-CO-CH2-CH2-COOProduct 4 Figure 2: Synthesis scheme of the DDT derivatives used for conjugation with albumin and lysozyme. In product 4, the p-alanine amide moiety is underlined. aR-OH: DDT-OH (Kelthan, Figure 1).

ty matrix.

The distinct

interference,

in

antibodies

solid

formed

Preparation tion

of

pared

against

for

amide-DDT

10 by displacing agarose.

the

agarose

not

bind

conjugate matrix

length

bound partially

purified

M

acid

or

were not

monoclonal

ted

this

from

as eluant

DDT columns

using

First,

of

resulting

the

spacer

was %20 A,

the

activabetween

column

This

antibodies

by 3 M guanidine

x HCl in

5 mM sodium

0.1 M glycine

and were sufficiently

pure

did

affinity

anti-DDT

in

p-

DDT-lysozyme

102.

IgG and IgM anti-DDT

column

were pre-

to Affi-Gel

EDC-activated

glycol

of

purifica-

properties.

polyclonal

eluted

for

portion

the

out

DDT-BSA complex.

matrices

to Affi-Gel

50% ethylene

However,

of

Then,

covalently

they

the

3) was coupled

DDT moiety

antibodies.

that

affinity

ruled

chromatography,

of

N-hydroxysuccinimide the

conjugates

antibody-binding

was linked

acetic

BSA moiety

2, product

and the

anti-DDT

strongly

2.4,

their

Although matrix

these

Two different

(Figure the

of

and affinity

of

antibodies.

and tested

ted

the

and properties

anti-DDT

alanine

utilization phase RIAs

0.25

hydroxide.

antibodies

could

x HCl/O.l5

M NaCl,

(Figure

so

3) for

be elupH

sequence

analysis. Competitive of

solid

the monoclonal

coated tions

plates of

phase radioimmunoassays

IgM anti-DDT-BSA

was followed

DDT, p-alanine

in

the

amide-DDT

antibody presence (Figure

1233

(Figure

4).

Binding

to DDT-lysozymeof

rising

2, product

concentra3),

DDE, DDA,

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

-

97006

-

66200

-

45666

-

29666

-

14366

RESEARCH COMMUNICATIONS

Figure 3: SDS-Polyacrylamide gel electrophoresis of the purified mouse monoclonal IgM anti-DDT antibody. Lane 1, commercial bovine monoclonal IgM (Sigma), reduced with B-mercaptoethanol; lane 2, molecular weight marker proteins; lane 3, purified mouse monoclonal IgM anti-DDT antibody, reduced with B-mercaptoethanol (band near top shows IgM heavy chain, band near bottom IgM light chain). The polyacrylamide gel was 10% cross-linked.

and p-chlorotoluene iS

the

part

alS0

of

binding

of

completely.

the the

Free

DDT and free binding

as

of

a control.

Only

conjugates

with

antibodies

amide-DDT

BSA and lysosyme

to

the

DDT had 70% of

the

activity

of

There

was very

by the

which

repressed

DDT-lvsozyme-coated

DDE was 40% as active. DDA or p-chlorotoluene

P-alanine

elates

P-alanine

amide-

little

monoclonal

if

any

anti-DDT-BSA

antibody. Taking

into

account

the

solubility

of

amide-DDT>DDA>DDT=DDE),

the

the monoclonal

was largely

group

of

the

antibody DDT molecule;

The fact

cross-reactivity part the

of

the

ure

with epitope

derivative.

was applied of

that

To

p-alanine

and the

showed that

p-alanine

the of

DDT-lysozyme p-alanine

alone. DDT with

It

amide

on the

p-alanine dilute

p-alanine

RIA carried amide did

conjugate and

was concluded the

antibody

antibody

anti-DDT-BSA

as described.

not

inhibit

and that

DDT was

that

the

indistinguishable

the

lack

antibody 1234

of

by ethylenic

showed 100% could

mean that

amide portion ascitic

mixt-

micro-

The results binding

of from

to

the mixture that

100% cross-reactivity

was most likely

of

fluid

a 1:l

antibody

behaviour

C13C-

binding

amide and ii)

out

that

the

group

amide-DDT

possibility,

i)

suggested

against

of this

anti-DDT-BSA

this

RIAs

directed

p-alanine

was located with

the

(B-alanine

amide and DDT to a DDT-lysozyme-coated

plate

titre

the

together

haptens

(DDA) abolished only

test

of

replacement

C12C= (DDE) and carboxylate stepwise.

results

the

caused

of

DDT

of by the

Vol.

BIOCHEMICAL

166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 4: Solid phase radioimmunoassay of the cross-reaction of DDT and DDT derivatives with a monoclonal IgM anti-DDT-BSA antibody on a DDT-lysozyme-coated microtitre plate. Details of the assay procedure are given in MATERIALS AND METHODS. Using an IgM antibody required incubation of the DDT-lysozyme - IgM - coated plate with the IgG fraction of rabbit anti-mouse Ig antiserum (Nordic Immunology) prior to the addition of 1251-protein A. A 1:l mixture of diluted ascitic fluid and 20% FCS in PBS was used as positive control (100% radioactivity), 20% FCS in PBS alone was used as negative control (0% radioactivity). DDT-lysozyme binding of the anti-DDT-BSA antibody was studied in the presence of DDA or p-chlorotoluene m, DDE (a), DDT (O), and p-alanine amide-DDT (0).

limited

solubility

concentrations

above

Competition bodies

or aggregation

solid

similar

0.0035

favourably

with

chromatography

mg/l the

via

its

carboxyl

DDT-BSA antibodies well

DDT in aqueous

polyclonal

limit

nM or 0.035 group

of

mg/l).

method

high

performance

Antibodies

the

specificity

anti-

as low as

by this

DDA was directly lacked

at

anti-DDT-BSA

DDT concentrations

were detectable

in which

buffers

4).

results.

detection

(-100

a DDA-BSA conjugate tein

~1 uM (Figure

phase RIAs using

gave very

%13 nM or

of

comparing liquid

raised

linked

against

to the of our

and bound DDT, DDE, and DDA almost

pro-

anti-

equally

(19). ACKNOWLEDGMENTS

We thank Dr. P. Wipf for advice during the synthesis of the DDT derivatives, Dr. R. Prewo for the x-ray structural analysis of the p-lactam derivative of DDT, Dr. M. Aguet and Martina Metzler for help during the preparation of the monoclonal antibodies, and the Schweizerische Nationalfonds and the Roche Research Foundation for financial support of this work. 1235

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1.

2. 3. 4.

Gutte, B., Daumigen, M., and Wittschieber, E. (1979) Nature 281, 650-655. Moser, R., Thomas, R-M., and Gutte, B. (1983) FEBS Lett. 157, 247-251. Mutter, M. (1988) Trends Biochem. Sci. 13, 260-265. Lear, J.D., Wasserman, Z.R., and DeGrado, W.F. (1988) Science 240,

5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

1177-1181.

Sasaki, T., and Kaiser, E.T. (1989) J.Am.Chem.Soc. 111, 380381. Hecht, M-H., Richardson, D.C., Richardson, J.S., and Ogden, R. (1989) J.Cell.Biochem. 13A, 86. Hehlgans, T., Langen, H., Linden, M., Epprecht, T., Klauser, S ., and Gutte, B. (1989) In Peptides 1988: Proc.20th Eur. Pept.Symp. (G. Jung and E. Bayer, Eds.), pp. 420-422. Walter de Gruyter, Berlin. Ritter, J.J., and Minieri, P.P. (1948) J.Am.Chem.Soc. 70, 4045-4048. Reeves, W.P., and Bahr, M.L. (1976) Synthesis, 823. Koziara, A., Osowska-Pacewicka, K., Zawadzki, S., and Zwierzak, A. (1985) Synthesis, 202-204. Staudinger, H., and Hauser, E. (1921) Helv.Chim.Acta 4, 861886. Hoare, D-G., and Koshland, D-E., Jr. (1967) J.Biol.Chem. 242, 2447-2453. Riordan, J.F., and Vallee, B.L. (1967) Meth.Enzymol. 11, 565570. Kearney, J-F., Radbruch, A., Liesegang, B., and Rajewski, K. (1979) J.Immunol. 123, 1548-1550. Fazekas de St.Groth, S., and Scheidegger, D. (1980) J. Immunol.Meth. 35, l-21. Iscove, N-N., and Melchers, F. (1978) J.Exp.Med. 147, 923933. and Guardia, E.J. (1968) Proc.Soc.Exp.Biol.Med. Haas, G.J., 129, 546-551. Centeno, E.R., Johnson, W.J., and Sehon, A.H. (1970) Int. Arch. Allergy Appl.Immunol. 37, l-13. 18, 95-102. Furuya, K., and Urasawa, S. (1981) Mol.Immunol.

1236

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT.

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized...
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