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