Vol.

176,

No.

April

15,

1991

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

262-268

Pages

PRODUCTION OF POLYCLONAL AND MONOCLONAL ANTIBODIES NITROSATION-PROFICIENT N. Dalla International

DENITRIFYING Venezial*

Agency

for

February

14,

Lyon

FOR SPECIFIC

BACTERIA

S. Calmels2

Research

69372

Received

COMMUNICATIONS

IN BIOLOGICAL

FLUIDS

and H. Bartsch

on Cancer, Cedex

DETECTION OF

08,

150 Cours

Albert

Thomas,

France

1991

Polyclonal and monoclonal antibodies were raised against the nitrosating enzyme previously isolated and purified from a denitrifying bacteria Pseudomonas Using the polyclonal antibodies, a preliminary ELISA test was set up aeruginosa. which allowed the detection of the nitrosating enzyme in 2 ml urine samples with The use of such a rapid a minimal total bacteria count of > 105 cells/ml. immunological screening test in cl&ical settings should ascertain whether individual subjects at higher risk for cancer of the stomach or bladder harbour The availability more nitrosation-proficient microorganisms in their microflora. of monoclonal antibodies provides a tool for studying the mechanisms of bacteria-mediated nitrosamine formation from precursor amines. 0 1991 ?+c?.de*~cPress, 1

Endogenous bacteria

formation

has been

carcinogenesis colonized

of

postulated

at

sites (2,3).

pH is

catalysed

enzyme

catalysing

the

from

two denitrifying

mucosae

(8).

antibodies assay

against

for

infected

This

the with

1 Visiting

nitrosation

the

infected

reaction

describes nitrosating

detection

the

bladder

pH 7.25

production and the

protein

been

be addressed.

Ig,

saline; ELISA, irmnunoglobulin;

isolated

an and

and Neisseria and monoclonal

up of urine

from

an immunological subjects

bacteria.

Agency

should

nitrosation

subsequently,

of polyclonal setting

of

and the that

(4-7); has

in human

denitrifying

International

(1)

P. aeruginosa

enzyme of this

to the

2 To whom correspondence

at

presence

in human

demonstrated species

microorganisms,

in the

factor

urinary clearly

bacterial

nitrosation-proficient

scientist

compounds

etiological

results

by various

article

rapid

as the

Previous

purified

g-nitroso

as an important

such

stomach

at neutral

carcinogenic

for

Research

on Cancer.

Abbreviations

PBS, 0.01 M phosphate-buffered BSA, bovin serum albumin: sodium dodecyl sulfate* 0006-291X/91 Copyrighr All rights

$1.50

(!3 1991 by Academic Press. lttc. oj. reproduction in an! form reserved,

262

enzyme-linked immunosorbent assay; DMSO, dimethylsulfoxide: SDS,

Vol.

176,

Materials

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

and Methods

Chemicals: 3,3',5,5'-Tetramethylbenzidine, 3,3'-diaminobenzidine, peroxidase coupled anti-rabbit IgG, peroxidase coupled anti-mouse IgG, dimethylsulfoxide (DMSO), bovine serum albumin (BSA), H202 were supplied by Sigma (St Louis, MO, USA): acrylamide, bis-acrylamide, N,N,E',tJ'-tetramethyl-ethylenediamine, nitrocellulose membrane, ammonium ie;sulfate by Bio-Rad (Richmond, CA,USA); sodium nitrite, sodium nitrate, morpholine hydrochloride and N-methyl-pentylamine by Merck (Darmstadt, Germany). All other chemicals were of aKalytica1 grade and obtained from either Merck or Sigma. Polyclonal antibody production: Two HY/CR rabbits were injected subcutaneously with 200 pg and 400 kg, respectively, of the nitrosating enzyme isolated previously (8), in Freund's complete adjuvant. Fourteen days later, they were boosted with the same amount of antigen. They were bled ten days after the boost injection and titers were determined by ELISA (see below), The highest immune response was obtained with the serum taken from a rabbit immunized with 200 kg antigen. Polyclonal antibodies were purified by chromatography on a 3 ml Avid AL column (BioProbe International Inc., Tustin, CA, USA). IgG were eluted with 0.1 M sodium acetate pH 3.0, and dialysed against phosphate buffered saline (PBS). Protein concentration was measured according to Lowry et al. (9). Titration of polyclonal antibodies by solid-phase ELISA: Falcon 96-well microtest plates were coated with 25 rig/well of nitrosating protein in PBS and incubated at 4'C overnight. The remaining protein-binding sites were blocked by incubation with 100 kl/well of 0.2% BSA in PBS for 30 min at 37'C. The plates were then washed twice with the same solution. Following addition of 100 ~1 of 1:lOOO dilution serum per well, the plates were incubated for 60 rnin at 37OC. The plates were again washed, and 50 ~1 of peroxidase coupled anti-rabbit IgG (1:5000 dilution in PBS) was added and incubated for 60 min at 37'C. After the plates were washed, 50 ~1 of a substrate solution containing 1 mg 3,3'5,5'-tetramethylbenzidine in 0.1 ml DMSO and 2 ~1 H202 (30 wt %) in 10 ml citrate buffer, pH 5.0 was added and incubated for 15 min at room temperature. The reaction was stopped with 50 ~1 1 M HCl and the absorbance was read at 450 run with a Bio-Tek EL 312 plate reader. Immunoblotting: SDS-polyacrylamide gel electrophoresis was carried out on 12.5% acrylamide gel (10). Gels were stained with Coomassie blue or subjected to Western transfer (11). Following Western transfer, the nitrocellulose membrane was blocked in 3% BSA in washing buffer (150 mM NaCl, 50 mM Tris HCl, pH 7.2 plus 0.05% Tween 20), for 1 h at room temperature, washed twice in washing buffer, incubated with antibody (1:5000 diluted rabbit serum) for 1 h at room temperature. The immunoblots were washed four times in washing buffer and developed with a peroxidase-conjugated anti-rabbit IgG reacted with 0.03% H202 in buffer containing 0.2% 3,3'-diaminobenzidine. Sample preparation for non-competitive ELISA: Three types of samples were treated: urine, urine spiked with P. aeruginosa (lo6 bacteria/ml), P. aeruginosa suspension in PBS (lo6 bacteria/ml). Ten ml of each sample was filtered successively through a 5.0 w cut-off filter (Millipore, Millex-SC) and a 0.45 cut-off filter (Millipore, Ultrafree CL). The 0.45 km cut-off filters were resuspended in 1 ml PBS and incubated at 37'C for 15 min. The suspension was then tested by non-competitive ELISA.

em

Falcon 96-well microtest plates were coated Non-competitive ELISA on samples: with 50 ~1 of suspension obtained as above, and incubated at 4OC overnight. After blocking with 100 kl/well of 0.2% BSA in PBS for 30 min at 37'C, the plates were washed and incubated with a 1:lOOO dilution of rabbit serum for 1 h at 37'C. The procedure used was as described above. Monoclonal antibody production: Two female BALB/c mice were injected intraperitoneally with 25 kg of the nitrosating enzyme isolated from 263

5

Vol.

176,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

This was followed by two booster aeruginosa (8) in Freund's complete adjuvant, shots of similar doses of the protein in Freund's incomplete adjuvant and in PBS respectively, at 12-day intervals. Three days before the fusion, each mouse was given 25 pg of protein in PBS. Cell lines producing antibodies that recognize the nitrosating protein were obtained from the fusion of spleen cells of immunized mouse with Sp2 myeloma Bybridoma cells secreting the cells (12), and tested by ELISA (see below). antibody of interest were grown to stationary phase. Cells were removed by centrifugation at 4000 g for 10 min. Immunoglobulins were purified by chromatography on Protein A-Sepharose (13). Screening of clones by solid-phase ELISA: This was carried out under conditions similar to those used for the titration of polyclonal antibodies; the plates were incubated with 100 ~1 of culture supernatant (instead of a 1:lOOO diluted serum): the second antibody solution was a 1:5000 diluted peroxidase-coupled anti-mouse IgG. Culture conditions for the microorganisms and isolation from P. aeruginosa (8) and the procedure for nitrosation nitrosamines (5) have been previously reported.

Results

previous

experiments,

(nitrosating

enzyme)

Subsequently, One of

the

measured

and purified formation

immunized

two rabbits

was found

with

adjuvant to

the

in

protein

P. aeruginosa

nitrosating

and boosted

produce

the

once

significant

(8).

enzyme with

from in

-P. PBS.

titers

as

enzyme

anti-enzyme

by ELISA. of

the

serum was purified

eluted

Ig were

relationship

between

450 run showed

that

the

bacterial

enzyme.

Immunoblot

analysis

purified

the

fractions,

nitrocellulose membrane

produced

of

the

that

responsee that

2) or in different

non-competitive The specificity

enzyme

of

serum

by both

the

infected

crude

serum

with serum

these

(data

not

nitrosating Ig.

with gel

Similar

at towards

different

and on

66 kD band to obtain

(lanes

results

were

(lane

8) on

a specific

band on lanes 2, in a crude bacterial activity

AL

The

specific

reactivity

be used

1).

absorbance

were

of a single could

(Fig

3, 4, 6 and extract

3, obtained

4,

6 and with

shown).

Ig was used

in a suspension

rabbit

SDS-polyacrylamide

by the

test

and the

revealed

The presence

fractions

bound ELISA

by the

on an Avid

ELISA

Ig/enzyme

The presence of this single nitrosating protein present

the

7) was specifically

ratio

rabbit 2).

chromatography

by non-competitive

molar

as assayed

suggested

7 demonstrated

the

(Fig

by affinity

tested

Ig

membrane

immunological

nitrosating

isolated

nitrosamine

were complete

and the

linear

catalysing

in Freund's

An aliquot column,

we have

two rabbits

aeruginosa

(lane

enzymes of

and Discussion

In

the

of nitrosating assay and analysis

to ascertain

of P. aeruginosa 264

the

absence

in which

this

of

the

enzyme

was not

a

Vol.

176,

No.

BIOCHEMICAL

1, 1991

AND

MOLAR Fig.

1.

Binding rabbit

curve serum

induced

by pro-incubation

absence

of

unable

any

band

to nitrosate

nitrosating

enzyme

The serum non-denitrifying

A

RATIO

as determined by direct antibody purified on Avid AL column (Ig:

with

nitrate

or nitrite

3 indicated

that

morpholine

did

contain

inducible

not

in culture

specificity

and did

bacterial

species

(Escherichia

s

P

s

P

P

DEAE

COMMUNICATIONS

3, lanes

bacterial the

not

bind

using

2 and 3).

suspension

66 kD protein;

coli,

CM

binding ELISA immunoglobulin).

(Fig

the

by nitrate

high

MW

RESEARCH

lg/Enzyme

on lane is

showed

BIOPHYSICAL

that

* Fig,

2.

3

4

*

*

5

6

76

*

**

the

from

nitrosating

or nitrite. any

protein

Klebsiella

pneumoniae)

6

SDS-polyacrylamide gel (A) and immunoblot (B) analysis of purified Lanes 2 and 3: sonicated fractions containing the nitrosating enzyme. precipitation; P. aeruginosa suspension; lanes 4 and 5: 40% (NH4)2SO4 lane 6: 75% (NH4)2SO4 precipitation; lane 7: purification after DEAE-cellulose; lane 8: purification after CM-cellulose; P:pellet; (*): fractions containing S:supernatant; MW, molecular weight markers; nitrosating enzyme.

265

The was

thus

8765432 12

a

that

Vol.

176,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1.25 1.00 0.75 0.50 0.25

0.00 3

were

5 *

Fig.

&.

Analysis of of unspiked

the

results found

confirm in

that

The sensitivity for

Preliminary

both

intact

bacterial

used

to establish rabbit

cell

serum

of

1:lOOO

concentration

was used.

concentration

revealed

concentration

range

Measurement indicated

of

that

The assay

the

of

(ii) human

urine

(o-o)

and

these

4,

strains

(14,

analysis

S and 6).

differs

from

us to

develop

15).

procedures

enabled

optimize

parameters

color

the

A checkboard development,

containing

absorbance of the

for

versus

protein procedure

for

a 0.3

which

curves

in was

a dilution

- 5 kg/ml

increasing

affinity

the

nitrosating

protein

protein

within

this

protein

4).

amount

was validated

bacteria,

of

portions

of bacterial

a 108 bacteria/ml

unspiked

in

P. aeruginosa. for

The plots

(Fig

(/q/ml)

3, lanes

earlier

to determine

and a suspension

linear

5

samples. to

order

conditions

or

(Fig

and ELISA

biological

in

suspension

optimal

(0-O)

enzyme

performed

ELISA

PBS

as proposed

to were

antibody-based

4

CONCENTRATION

or nitrite

immunoblot

application

experiments

polyclonal

to

nitrosating

strains,

of

an immunoassay

added

of nitrate the

denitrifying

3

of nitrosating enzyme (lane 1) in different Pseudomonas aeruginosa: K.pn, Klebsiella P.aer, Escherichia coli: (*): fractions containing

P. aeruginosa urine (wm).

presence

2

PROTEIN

4

Immunoblot analysis bacterial strains. pneumoniae; E.coIi, nitrosating activity.

in

1

0

3.

These

of

6 *

Fig.

cultured

that

4 *

4 0

human

urine

urine

samples.

protein

suspension

using

(i)

spiked

with

PBS spiked the

PBS and urine 266

according

contained with

known

same quantities were

to

Lowry

a 50 kg/ml

spiked

quantities of

with

et

al.

(9)

protein.

bacteria

of and

(iii)

106 P. aeruginosa

Vol.

176,

No.

I,

1991

BIOCHEMICAL

cells/ml.

Samples

described

in Materials

urine

spiked

bacterial

with

of

all

the

not

urine to

at

(10 ml)

reproducible

a

enzyme

Six

clones purified

of monoclonal bacteria

of bacterial

nitrosation,

were

grown

with

The with the

a 1:lOOO was negative

gastric

test,

(7,

4,

the

requiring

were

produced

from

P. aeruginosa

to

from

stationary

the

enzyme been

are

prevalent

more subjects

remains

enzyme

important

urines.

(Materials

and Methods).

the

cells

were

removed

A-Sepharose. nitrosation

clearly

the

partially

with

volume

mice

catalysing

more

infected

sample

small

phase;

elucidate

from

are

of

two BALB/c

on Protein

has only

nitrosating

4OC (data

detection

samples

a quite

so far

it

showed

24 h at

specific

when urine

to

16),

for

ELISA

be a simple assay to detect fluids such as infected

against

juice

from

be stored

a tool

Enterobacteria

the

can

by chromatography

that

and nasopharynx for

enzyme

antibodies

urines,

immunoassays

4,

obtained

that

Results

allows

would in biological

provides

As non-denitrifying colon

figure

The response

bacteria,

antibodies

positive

infected

in

as

performance.

time,

nitrosating

the

denitrifying

from

to

checked.

therefore,

This

filtration

supernatant

The use

COMMUNICATIONS

ELISA,

was performed

but

in denitrifying

monoclonal

with

stable

and the

assay,

bacteria

addition,

also assay

105 bacteria/ml.

and a short

immunized

shown

rabbit.

be frozen,

immunological

least

were

cannot

nitrosation-proficient In

are

similar

assay

non-injected

a

conditions

allow

nitrosating

with

curve

a

RESEARCH

by non-competitive

The results

A control

from

samples

The present the

taken

storage

shown),

and analysed

exhibited

in PBS.

BIOPHYSICAL

samples.

Different that

bacteria

serum

three

prepared and Methods.

suspension

dilution for

were

AND

characterized in

the

by

mechanism(s) (14).

human microflora

achlorhydric to develop

stomach, similar

in E. coli.

Acknowledgments

We thank generous This

work

Dr C. Trepo

help

during

was supported

and Mr J.

this

study in

part

Grange

(INSERM Unit

and Dr J.

Cheney

by NIH US Grant

for No.

271,

Lyon)

editing 1 ROl

the CA

for

their

manuscript.

47591-OlAl.

References 1.

2. 3.

Hicks, R.M., Walters, C.L., Elseboi, I., El Aasser, A.B., Proc. Roy. Sot. Med., 2, 413-416. and Gouch, T.A. (1977) J. Natl. Cancer Inst., 1, 631-647. Mirvish, S.S. (1983) Preston-Martin, S., and Correa, P. (1989) Cancer Surveys, 267

El Merzabani, g,

459-473.

M.,

Vol.

176,

No.

1, 1991

BIOCHEMICAL

AND

4.

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Suzuki, K., and Mitsuoka, T. (1984) In: N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer (O'Neill, I.K., Von Borstel, R.C., Miller, C.T., Long, J* and Bartsch, H.), IARC Scientific Publications, No. 57, pp 275-281, International Agency for Research on Cancer, Lyon, France. 5. Calmels, S., Ohshima, H., Vincent, P., Gounot, A.-M. and Bartsch, H. (1985) Carcinogenesis, 6, 911-915. 6. Leach, S., Challys, B., Cook* A., Hill, M. and Thompson, M. (1985) Biochem. Sot. Trans., 2, 380-381. 7. Calmels, S., Ohshima, H., Crespi, M., Leclerc, H., Cattoen, C. and Bartsch, H. (1987) In: The Relevance of g-Nitroso Compounds to Human Cancer: Exposure and Mechanisms (Bartsch, H., O'Neill, I.K. and Schulte-Hermann, R.), IARC Scientific Publications No* 84, pp 391-395, International Agency for Research on Cancer, Lyon, France. 8. Venezia, N. and Bartsch, H. (1990) Biochem. Biophys. Res. Calmels, S., Dalla *, I&, 655-660. A.L. and Randall, R.J. (1951) J. Biol. 9. Lowry, O.H., Rosebrough, N.J., Farr, Chem ., E, 265-272. 10. Laemmli, U.K. (1970) Nature, E, 680-685. 11. Towbin, H., Staehelim, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci., E, 4350-4354* 12. Galfre, G., Howe, S.C., Milstein, C., Butcher, G.W. and Howard, J.C. (1977) Nature, @, 550-552. 13. Ey, P.L., Prowse, S.J. and Jenkin, C.R. (1978) Immunochemistry, g, 429-436. 14. Calmels, S., Ohshima, H. and Bartsch, H. (1988) J. Gen. Microbial., e, 221-226. 15. Calmels, S., Ohshima, H., Rosenkranz, H., McCoy, E. and Bartsch, H. (1987) Carcinogenesis, g, 1085-1088. 16. Charriere, M., Poirier, S., Calmels, S., De Montclos, H., Dubreuil, C., Poizat, R., Hamdi Cherif, M., and de The, G* (1991) In: Relevance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins (O'Neill, I.K., Chen, H., &d Bartsch, H.), IARC Scientific Publications, No. 105, pp 158-161, International Agency for Research on Cancer, Lyon, France.

268

Production of polyclonal and monoclonal antibodies for specific detection of nitrosation-proficient denitrifying bacteria in biological fluids.

Polyclonal and monoclonal antibodies were raised against the nitrosating enzyme previously isolated and purified from a denitrifying bacteria Pseudomo...
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