Microbiol. Immunol. Vol. 36 (8), 791-801, 1992
Production of a Monoclonal Antibody That Recognizes the Lipopolysaccharide of a Campylobacter-Like Organism Steven Kok Chee KUAN,* Peter John COLOE, and Malcolm Raymond ALDERTON Departmentof AppliedBiologyand Biotechnology,Royal MelbourneInstitute of G.P.O.
Box 2476V,
Melbourne,
(Accepted for publication,
Australia
Technology,
3001
May 6, 1992)
Abstract A monoclonal antibody was produced to a Campylobacter-likeorganism (RMIT 32A) which was isolated from the terminal ileum of a pig with proliferative enteritis. Isotyping of the antibody revealed that it was an IgG2a with kappa light chains. Immunoblots using the antibody against proteinase-K-treated whole cell lysates of RMIT 32A, a selection of Campylobacterspecies and other enteric bacteria showed that the antibody was specific for RMIT 32A and was directed against the lipopolysaccharide. This antibody can be used for the specific detection of RMIT 32A.
Porcine proliferative enteritis (PPE) is a disease of pigs characterized by adenomatosis of mucosal epithelial cells in the ileum (18, 23, 43, 45). The disease appears to present in several pathological forms such as necrotic enteritis, proliferative hemorrhagic enteropathy, regional ileitis, intestinal adenomatosis and terminal enteritis. However, these diseases are commonly reported as separate entities (18, 20, 30, 42, 44, 45). The causative agent of the disease has not been fully identified but several Campylobacter species have been reported to be associated with the disease (6, 8, 9, 18, 23, 29, 35, 36, 43). Histological examination of affected ilea has revealed the presence of Campylobacterand Campylobacter-likeorganisms that are not membrane bound but present within the apical cytoplasm of intestinal cells (24-26). Several Campylobacter species have been isolated from intestinal lesions and feces of affected pigs. C. sputorumsubspecies mucosalisoriginally described by Lawson and co-workers (18, 19), has been suggested as a causative agent of PPE. Other researchers have since reported the isolation of C. sputorumsubsp mucosalisfrom intestinal lesions of pigs with PPE (23, 26, 35, 36, 42). Two other Campylobacterspecies, C. hyointestinalisand C. coli, have also been isolated from diseased pigs (8, 9,28-30, 36, 48). Although C. sputorumsubsp mucosalis,C. hyointestinalisand C. coli have been suggested as possible causative agents of PPE, efforts to reproduce the disease by oral inoculation of pigs with pure cultures of these organisms have been mostly unsuccessful. Oral inoculation of pigs with homogenized ilea from pigs with PPE has been the only way to reproduce the disease (24, 25, 29, 30, 42). 791
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KUAN
ET AL
McOrist and co-workers (32, 33) reported the isolation of a non-culturable intracellular Campylobacter-likeorganism from infected pig ileal sections. Protein banding profiles in sodium dodecyl sulfate-polyacrylamide gels and immunoblot analysis
with polyclonal
like organism
and
monoclonal
antibodies
suggested
may be a new Campylobacter species.
the CamPPtlobacter-
that
Recently,
a
Campyobacter-like
organism which can be grown in vitro, has been isolated from a pig with PPE (1). This organism, designated as RMIT 32A, has different phenotypic characteristics from the other Campylobacterspecies commonly reported to be associated with PPE and oral inoculation of conventional cross-bred pigs with RMIT 32A initiated symptoms of PPE. In this report, we describe the production of monoclonal antibodies to RMIT 32A. MATERIALS
AND
METHODS
Bacterial strains. Campylobacterspecies (Table 1) and RMIT 32A were grown on 5% defibrinated horse-blood agar (HBA) with or without Skirrow antibiotic supplementation (Oxoid, England) at 37 C in sealed jars (BBL Microbiology Systems, U.S.A.) in a microaerophilic environment induced by gassing the jars for 2 to 3 min with 50% H2 and 50% CO2. Serpulina species (Table 2) were grown on HBA plates at 37 C in an anaerobic environment ("GasPak," BBL Microbiology System, U.S.A.). Other gramnegative bacteria listed in Table 2 were grown aerobically on Columbia base agar (Oxoid) plates at 37 C. Monoclonalantibodyproduction. RMIT 32A was grown for 4 days and harvested with sterile phosphate-buffered saline, pH 7.4 (PBS). After three washes with PBS, 106cells were injected intraperitoneally into an eight-week-old female BALB/c mouse. The mouse received three injections with the final booster injection given 3 days prior to removal of the spleen (11). Dulbecco's Modification of Eagle's Medium (Cytosystems, Australia), supplemented with 10% heat-inactivated newborn bovine serum (iron supplemented; HyClone, U.S.A.), was used as the culture medium. The non-secretory myeloma cell line of Sp2/0-Ag14 (Sp2; 46) and resultant hybridomas were cultured at 37 C in a 5% CO2 environment. Hybridomas were produced by fusion (7, 10) using polyethylene glycol 1500 (Boehringer Mannheim, Germany) and HAT selection procedures (21). Enzyme-linkedimmunosorbent assay (ELISA). Preliminary screening of hybridomas was performed by ELISA in microwell plates (Nunclon, Denmark). Culture supernatants were used to detect sonicated RMIT 32A. After the addition of substrate (2, 2'-Azino-bis[3-ethyl benzthiazoline sulfonate]), wells exhibiting a strong green color were scored as positives. Characterization of immunoglobulinsubclass. The isotyping of monoclonal antibodies were performed using "MISO" mouse monoclonal antibody isotyping kit (Commonwealth Serum Lab., Australia). Neat hybridomas culture supernatants were used.
MONOCLONAL
Ascites
fluid
injections
production.
of 0.5
cal
Co.,
14,
47).
ml
of
U.S.A.).
supernatant
After ascites
was
washed by (0.1
M Tris
for
5 min.
The
samples
lysate
with
5
a
final
min
for
SDS-PAGE volume
were
blocking
Rad
Lab.) ; 20 for
the mm),
gels
four pH
and
one
4,
and
(LKB
the
in
1 ml
was
of
lysate and
of
45% of
10%
SDS
were
then
boiled
(17)
was
performed
System,
(12).
for
Pharmacia, at
(14.4
R
whole
glycerol,
electrophoresed
Blue
Coomassie
lysate
volumes
standards
Brilliant
harvested
cell
6.8,
Midget and
weight
were
volume
samples
loaded
with
skim
mm at
0.5
RT
in
IgG
to
200
94
Banding
brilliant-blue
by
V
kDa;
patterns
or
to
silver
(Bio-
incubation,
and 10
for
(ii) pl
5 min,
modified
the
32A
and
in
a
10
ml
was
pH
sample two
8.3,
at
37
buffer
C
and were
phenol-chloroform-petroleum
every
10 pl
65
of
10%
to
the
the
sec
for
2
per
C SDS
procedure
the 8.0
min
final (50
of
ml
35%
and
the
Mannheim) of
36 ƒÊl).
for
2
hr
were of
was
proteinase-K Proteinase-K
procedures:
wells
in-
diluted
(20 ƒÊl
microliters
hot-phenol ether
pH
H2O2
(i) (27).
60
C
for
Following
added.
And
after
gels.
Lipopolysaccharide of
were in
After Tris
Bio-
Tween-20
water.
protein
then,
cooling.
(0.2 ƒÊm;
(Boehringer
of
performed
membranes
30
distilled
incubation
applied
was
of washings,
and
Four
fig
and
procedures.
modification
methanol)
with
lysates
constant
antibody.
of
cell
containing
containing
replaced
(60
with
periods
solution
different
(50)
hr
The
after
(10 ƒÊg/ƒÊl).
using
whole
membranes
proteinase-K
water
samples
a
2.5
Tween-20).
membranes
lysate
isolation using
in
Lyophilized
sample
10 ƒÊl
Lipopolysaccharide RMIT
mg
overnight
of
V for
peroxidase-conjugated
distilled cell
75
saline,
fluid
solution
performed
and
(31).
nitrocellulose
0.1%
ascites
developed
washing
a whole
was (13),
NaCl,
(30
sterile
al
transfer
at
Tris-buffered
horseradish
treatment. in
added
of
development
Proteinase-K
treatment
incubation
M
sonicates et
Electrophoretic Lab.)
diluted
were
of
Markwell
(Bio-Rad
in
4-chloro-napthol
stopped
of
blotting).
milk
Tris,
1 hr
reconstituted
concentration
procedure
involved
5%
The
reaction
the
(2,
min
phenylmethylsulfonyl)
SDS-PAGE
Remazol
Protein
membranes
solution).
from
g)
mixing
The
were
gels
cells
M Tris-HCl blue)
molecular
the
(Western
anti-mouse
boiling
10
Whole 0.1
by
(0.25
1 ƒÊg/ƒÊl.
samples
with
apparatus
in
(TBS-T
hr
Chemi-
hybridomas
for
7.4.
2 mm
prepared
resolving
of
the
step
cubated
was
g
Bacterial pH
SDS,
bromophenol
Low
using
a Trans-Blot
20
2%
buffer
gradient
estimation.
The
of
106
1,000 •~
(approximately
were
staining
Immunoblotting
wash,
with
at
M Tris-HCl,
glycerol,
of
prestained by
measured
goat
intraperitoneal
U.S.A.).
Protein
using
with
Sigma
injected
Discontinuous
10-20%
visualized
were
were
pellet
of sample
cooling.
Lab.,
793
primed
(SDS-PAGE).
cell
15%
micrograms
constant
Rad
1
the
0.025%
or
Pharmacia)
were
centrifuged
0.1
concentration
Ten
were
mice
sterile
electrophoresis.
12.5%
with
was
6.8,
protein
U.S.A.).
fluid
pH
one
before
using
the
CLO
14-tetramethyl-pentadecane;
electrophoresis
2-mercaptoethanol,
for
mice
10,
days,
with
resuspending
boiling
1%
gel times
buffer
cell
6,
TO
retrieved.
three
obtained
BALB/c
(2,
14
SDS-polyacrylamide and
Male
pristane
Collected
ANTIBODY
(LPS) procedure (mPCP;
was (RIM; 38).
isolated 15)
and
RIM-
794
S.K.C.
KUAN
ET Al,
isolated LPS was treated with RNase prior to analysis. performed by colorimetry (16).
Quantification of LPS was
RESULTS
Preliminary screening using EL1SA identified 50 hybridoma clones which produced antibodies to RMIT 32A. These hybridomas were cloned twice by limiting dilution. A hybridoma clone, C3: 52c1, was found to be secreting IgG2a antibodies (kappa light chains) to RMIT 32A. The specificity of this monoclonal antibody to RMIT 32A was examined by immunoblotting. An immunoblot using whole cell lysate of RMIT 32A and C3: 52cl revealed bands at approximately 19 kDa and immediately behind the dye front (Fig. 1, lane 2). There was also weak recognition of bands at 35 to 80 kDa region. Immunoblots of whole cell lysates of the bacteria listed in Tables 1 and 2 of "MATERIALSAND METHODS," showed some weak cross-reactivity (data not shown). The monoclonal antibody was also used in immunoblotting analysis of proteinase-K-treated (60 C for 1 hr) whole cell lysate of RMIT 32A. No recognition of 35 to 80 kDa was observed but the monoclonal antibody reacted with a broad band
2 3 1 Fig. 1. Immunoblot analysis ofRMIT 32A antigens with the monoclonal antibody, C3: 52cl, after SDS-PAGE using 12.5% gels. Lanes: 1, molecular weight standards (kDa): 2, whole cell lysate of RMIT 32A; 3, proteinase-K-treated (6(1C for 1 hr) whole cell lysate of RMIT 32A.
MONOCLONAL Table
Table
1.
2.
ANTIBODY
TO CLO
795
Campylobacter species used in the study
Gram-negative
bacteria
used
in the
study
located between the dye front and 20 kDa region (Fig. 1, lane 3). When the monoclonal antibody was used in immunoblotting analyses of proteinase-K-treated whole cell lysates of other tested bacteria, no recognition was observed. To study the effectiveness of proteinase-K treatment of whole cell lysate at 60 C for 1 hr, whole cell lysates of RMIT 32A and Campylobacter coli NCTC 11366 (Fig. 2, A and B) were also proteinase-K treated overnight at 37 C and then, 2 hr at 65 C. The products of both proteinase-K treatments were subjected to SDS-PAGE and the gel was Coomassie brilliant-blue stained (Fig. 2A). The 60 C treatment (Fig. 2A, lanes 4 and 5) appears to be as equally effective as the longer overnight treatment (Fig. 2A, lanes 6 and 7) because there are no visible signs of protein bands. However, when a matching gel was silver stained (Fig. 2B), low molecular weight bands could be seen in lanes that were loaded with the products of proteinaseK treatments (Fig. 2B, lanes 4 to 7). Whole cell lysates of RMIT 32A and C.coli NCTC 11366 that were treated with proteinase-K at 60 C for 1 hr have similar banding patterns to their respective counterparts that were treated overnight at 37 C and then, 21 hr at 65 C. The products of proteinase-K treatment of whole cell lysates of gram-negative bacteria have been shown to be of LPS (3, 13, 22, 27, 38-41). To further define the
796
S.K.C.
KUAN
ET AL
A
B
Fig. 2. SDS-polyacrylamide gel (12.5%) after (A) Coomassie brillant-blue, and (B) silver staining. Lanes: 1, molecular weight standards (kDa); 2, whole cell lysate of RMIT 32A; 3, whole cell lysate of C. co/i NCTC 11366; 4 and 6, proteinase-K-treated whole cell lysate of RMIT 32A; 5 and 7, proteinase-K-treated whole cell lysate of C. co/i NCTC 11366. Lanes 4 and 5 were treated at 60 (1 for 1 hr. Lanes 6 and 7 were treated overnight at 37 C and then, 2 hr at 65 C.
nature of the antigen recognized by C3: 52cl, LPS from RM IT 32A was isolated using two different procedures: the mPCP and RIM procedures. SDS-PAGE banding profiles of the isolated LPS are shown in Fig. 3. The immunoblot of the isolated LPS (using mPCP and RIM) and proteinase-K-treated whole cell lysate of RMIT 32A, probed with the monoclonal antibody, is shown in Fig. 4. There arc observable differences in the recognition of the antibody to mPCP-LPS, RIM-LPS and proteinase-K-treated whole cell lysate.
NIONOCLONAL
ANTIBODY
TO CTO
Fig. 3. Silver-stained SDS-polyacrylamide gel (10-20%) of LPS isolated from RMIT Lanes: 1, molecular weight standards (kl)a) 2, mPCP-LPS; 3, RIM-LPS.
797
32A.
Fig. 4. Immunoblot analysis of RMIT 32A LPS with the monoclonal antibody. C3: 52cl, after SDS-PAGE (l0 20% gel). Lanes: 1, molecular weight standards (kDa) ; 2, mPCP-LPS; 3, RI NI-1.PS; 4, proteinase-K-treated whole cell lysate of RMIT 32A.
798
S.K.C.
KUAN
ET AL
DISCUSSION
In this report, we described the production of a monoclonal antibody, C3: 52cl, to a Campylobacter-likeorganism, RMIT 32A. Immunoblots of proteinase-K- and non-proteinase-K-treated whole cell lysates were used to study the specificity of the monoclonal antibody to RMIT 32A. The immunoblots using the monoclonal antibody and whole cell lysates of RMIT 32A, a selection of Campylobacterspecies and other gram-negative bacteria showed some weak cross-reactivities. After proteinase-K treatment of whole cell lysates at 60 C for 1 hr, the weak cross-reactivities have been completely eliminated, resulting in the specific recognition of an antigen of RMIT 32A by the antibody. The recognition (Fig. 1, lane 3) is located between the dye front and the 20 kDa region. Proteinase-K is a potent proteolytic enzyme and this analysis shows that both the 60 C treatment and the overnight treatment at 37 C and then 2 hr at 65 C, are equally effective in digesting proteins as there are no protein bands in the Coomassie brilliant-blue stained gel (Fig. 2A, lanes 4 to 7). However, bands were revealed when a matching gel was silver stained (Fig. 2B, lanes 4 to 7). The banding patterns, on the silver-stained gel of the 60 C treated whole cell lysates of RMIT 32A (Fig. 2B, lane 4) and C. coli NCTC 11366 (Fig. 2B, lane 5) were similar to their respective counterparts which were treated overnight at 37 C and then, 2 hr a t 65 C (Fig. 2B, lanes 6 and 7, respectively). Therefore, the 60 C treatment is the preferred treatment as it involved a shorter incubation duration. To further elucidate the nature of the antigen recognized by C3 : S2c1, LPS was isolated from RMIT 32A using two different procedures. The SDS-PAGE banding patterns of the isolated LPS (Fig. 3) is similar to Salmonellarough LPS chemotypes (13). The immunoblot of the monoclonal antibody against isolated LPS and proteinase-K-treated whole cell lysate of RMIT 32A (Fig. 4) showed recognition bands of low molecular weights (