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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 1997, p. 615–619
1071-412X/97/$04.0010
Copyright © 1997, American Society for Microbiology
Vol. 4, No. 5
Monoclonal Antibody E8-18 Identifies an Integral Membrane
Surface Protein Unique to Mycoplasma capricolum
subsp. capripneumoniae
FRED R. RURANGIRWA,1* PATRICK S. SHOMPOLE,2 ANDERSON N. WAMBUGU,2
STANLEY M. KIHARA,2 AND TRAVIS C. MCGUIRE1
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington
99164-7040,1 and Biotechnology and Immunology Section, National Veterinary Laboratory,
Kenya Agricultural Research Institute, Kabete, Kenya2
Received 6 February 1997/Returned for modification 20 March 1997/Accepted 20 June 1997
Monoclonal antibody (MAb) E8-18 reacted with four isolates of Mycoplasma capricolum subsp. capripneumoniae in Western blots identifying an epitope on a 24 kDa antigen (p24). MAb E8-18 did not react with 11
isolates belonging to four other Mycoplasma species or subspecies closely related to M. capricolum subsp.
capripneumoniae. A combination of trypsin treatment of intact organisms and detergent-phase partitioning
revealed p24 to be an integral M. capricolum subsp. capripneumoniae surface membrane protein.
have not been shown to occur in goats (38). The other tests
have been shown to be specific, but they are difficult to implement
in the diagnostic laboratories in countries where CCPP occurs.
Analysis of antigens by specific antibodies, including MAbs,
has been useful in defining important surface structures of
some Mycoplasma species (3, 15, 21, 22, 32, 34, 40), for example, the initial characterization of M. capricolum subsp. capripneumoniae surface-exposed polysaccharide in our laboratory
(40). Identification of other M. capricolum subsp. capripneumoniae surface antigens is also needed because of the involvement of these antigens in antibody-mediated metabolic and
growth inhibition tests, which are also widely used to classify
Mycoplasma species (3, 16, 29, 43, 48). Such M. capricolum
subsp. capripneumoniae surface antigens could be isolated and
exploited for diagnosis, mycoplasma differentiation, and/or immunoprophylaxis. Therefore, MAbs to membrane proteins of
M. capricolum subsp. capripneumoniae were produced. Here,
we describe MAb E8-18, which reacts with a surface-exposed
integral membrane protein of 24 kDa in only M. capricolum
subsp. capripneumoniae and can be used for the differentiation
of M. capricolum subsp. capripneumoniae from other Mycoplasma species.
Development and identification of MAb E8-18. Five mice
were each injected intraperitoneally with 100 ml containing 50
mg (41) of disrupted M. capricolum subsp. capripneumoniae
mixed with an equal volume of Freund’s complete adjuvant.
Mice were given a booster 4 weeks later with an intraperitoneal
injection with the same amount of antigen in Freund’s incomplete adjuvant. A month later, antigen was given intraperitoneally without adjuvant. The mouse with the highest serum
antibody titer and whose sample reacted with discrete bands as
determined by Western blotting (25) was injected intravenously with 100 ml containing 50 mg of antigen without adjuvant. Three days later, spleen cells were collected, washed in
RPMI 1640 medium, and fused with nonsecreting
P3X63.Ag8.653 myeloma cells in a ratio of 10:1 as previously
described (25, 35). Fused cells were distributed to a 48-well
culture plate, incubated at 37°C with 5% CO2, and fed with
medium containing 5 3 1023 M hypoxanthine, 2 3 1025 M
aminopterin, and 8 3 1024 M thymidine. Supernatants were
screened by enzyme-linked immunosorbent assay (ELISA)
with M. capricolum subsp. capripneumoniae antigen (35), and
Mycoplasma capricolum subsp. capripneumoniae, formerly
designated Mycoplasma strain F38, is the causative agent of
classical contagious caprine pleuropneumonia (CCPP) (31).
The disease is of major economic importance in Africa and
Asia and poses a major constraint to goat production because
of high mortalities. M. capricolum subsp. capripneumoniae was
originally isolated from a goat with pleuropneumonia in Kenya
(30) and produces classical CCPP, which involves transmission
by contact, high mortality, and a fibrinous pleuropneumonia
characterized by massive hepatization and pleurisy (31).
Whereas M. capricolum subsp. capripneumoniae has been isolated from goats in only seven other countries, including Sudan
(19), Tunisia (33), Ethiopia (45), Chad (28), Oman (23), Turkey (47), and Uganda (4), it is not known whether the pleuropneumonia observed in other parts of the world is due to M.
capricolum subsp. capripneumoniae or other mycoplasmas of
the Mycoplasma mycoides cluster (9, 10, 14). The M. mycoides
cluster includes seven Mycoplasma species: M. mycoides (small
colony), M. mycoides (large colony), Mycoplasma capricolum
subsp. capricolum, M. primatum, M. equigenitalium, Mycoplasma bovine group 7 described by Leach (26a), M. capri, and
M. capricolum subsp. capripneumoniae (9, 10). All these organisms have extensive serological cross-reactivity (12–14, 24, 26)
and biochemical similarities (1, 9, 10, 24, 26). The serological
cross-reactivities have made it difficult to develop simple and
yet specific diagnostic tests for the diseases caused by the
different organisms. Various diagnostic tests have been reported, including latex agglutination (36), tests for monoclonal
antibody (MAb) blocking and/or MAb reactivity with antigen
in body fluids (44), dot blot immunobinding assay (18), and a
combination of PCR and enzyme restriction analysis (5). Latex
agglutination to detect serum antibodies to M. capricolum
subsp. capripneumoniae is simple and can be performed in the
field (36). The specificity of this test for field use is adequate;
however, rabbit antisera to some other Mycoplasma species
cause agglutination of M. capricolum subsp. capripneumoniae
polysaccharide latex-coated beads, although these organisms
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine,
Washington State University, Pullman, WA 99164-7040. Phone: (509)
335-6023. Fax: (509) 335-8529.
615
616
NOTES
CLIN. DIAGN. LAB. IMMUNOL.
FIG. 1. Immunoblot of M. capricolum subsp. capripneumoniae and other
Mycoplasma strains with MAb E8-18 and goat polyclonal serum antibodies.
Lanes 1 and 6 were loaded with Mycoplasma bovine group 7, lanes 2 and 7 were
loaded with whole M. capricolum subsp. capripneumoniae, lanes 3 and 8 were
loaded with Y Goat; lanes 4 and 9 were loaded with M. capri, and lanes 5 and 10
were loaded with M. mycoides. Lanes 1 to 5 were reacted with MAb E8-18, and
lanes 6 to 10 were reacted with goat serum antibodies to whole M. capricolum
subsp. capripneumoniae. Molecular mass standards (in kilodaltons) are shown on
the left.
FIG. 2. Immunoblot of M. mycoides cluster strains (50 mg/lane) with MAb
E8-18. Lanes 1 to 4 contain M. mycoides small-colony strains, lanes 5 to 7 contain
M. mycoides large-colony strains, lane 8 is empty, lanes 9 and 10 contain M.
capricolum strains, lanes 11 and 12 contain Mycoplasma bovine group 7 strains,
lane 13 is empty, and lanes 14 to 17 contain M. capricolum subsp. capripneumoniae strains. All lanes were reacted with MAb E8-18. Molecular mass standards (in kilodaltons) are shown on the left.
20 were identified. Six of these reacted with a 24-kDa antigen
in Western immunoblotting. Two limiting dilutions of one of
these hybridomas was done, the supernatants were screened by
ELISA, and the final supernatant was evaluated by Western
blotting. The MAb continued to identify a 24-kDa antigen and
was designated E8-18. The MAb was precipitated with 50%
saturated ammonium sulfate and dialyzed with phosphatebuffered saline (PBS), and the antibody isotype was determined to be immunoglobulin G 2a (IgG2a) with a commercial
kit (Sigma, St. Louis, Mo.).
Reactivity of MAb E8-18 with other Mycoplasma species.
Reactivity was determined by Western blotting after the various strains were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer as described previously (25), separated on a 7.5-to-17% gradient
gel, and transferred to nitrocellulose membranes (46). The
membranes were blocked with Tris-buffered saline (TBS) (50
mM Tris, 150 mM NaCl) containing 5% powdered milk for 1 h,
washed twice in TBS, air dried, and stored at 220°C until used.
For immunoblots, the strips were incubated with either diluted
MAb E8-18 or goat antiserum to M. capricolum subsp. capripneumoniae at room temperature overnight and washed with
TBS; peroxidase-conjugated goat anti-mouse IgG (1:1,000) or
rabbit anti-goat IgG was added as a second antibody. Bound
antibodies were visualized with 3,3-diaminobenzidine and hydrogen peroxide. MAb E8-18 reacted with a 24-kDa antigen of
M. capricolum subsp. capripneumoniae (Fig. 1, lane 2) but did
not react with any antigens of several Mycoplasma organisms,
including bovine group 7 (Fig. 1, lane 1), Y Goat (lane 3), M.
capri (lane 4), and M. mycoides (Gladysdale) (lane 5). To
demonstrate that there were proteins available for reaction in
all the lanes containing the various Mycoplasma organisms,
duplicates of the organisms in lanes 1 to 5 of Fig. 1 were run in
lanes 6 to 10 and reacted with goat antiserum to M. capricolum
subsp. capripneumoniae. MAb E8-18 was further tested against
additional M. mycoides cluster strains to confirm specificity.
The strains were from three sources: the National Veterinary
Research Center, Muguga (NVRC-M), Kenya; the National
Veterinary Research Center, Kabete (NVRC-K), Kenya; and
R. H. Leach, National Collection of Type Cultures (NCTC),
Corrindale, England. The strains (origins in parentheses) included four small-colony strains (T419 and T1M44 [NVRCM], B613/87 [NVRC-K], and Gladysdale [NCTC]), three
large-colony strains (VR1/3172.LB2 [NCTC], 78/441 [NCTC],
and Y-Goat [NCTC]), two M. capricolum strains (74/3220
[NCTC] and ZT [NCTC]), two bovine group 7 strains (L2917
[NCTC] and 4055 [NCTC]), and four M. capricolum subsp.
capripneumoniae strains (G22, G94/83, G108/83, and G280/80
[NVRC-K]). MAb E8-18 reacted strongly with a 24-kDa protein band from all four M. capricolum subsp. capripneumoniae
strains tested (Fig. 2, lanes 14 to 17) but not with any protein
from the other strains tested, including M. mycoides smallcolony strains (Fig. 2, lanes 1 to 4), M. mycoides large-colony
strains (Fig. 2, lanes 5 to 7), M. capricolum (Fig. 2, lanes 8 and
9), and bovine group 7 (Fig. 2, lanes 11 and 12).
Evaluation of growth inhibition of M. capricolum subsp.
capripneumoniae by MAb E8-18. Twenty micrograms of MAb
E8-18 did not inhibit in vitro growth of 104 CFU of M. capricolum subsp. capripneumoniae. Some MAbs to mycoplasmas
may not inhibit in vitro growth and yet may be mycoplasmacidal in the presence of complement. This was demonstrated in the case of M. hyorhinis (34).
Effect of trypsin on MAb E8-18 binding to live M. capricolum
subsp. capripneumoniae organisms. To determine if the peptide bound by MAb E8-18 was exposed on the surfaces of live
M. capricolum subsp. capripneumoniae organisms, a freshly
grown tryptose broth (17) culture of M. capricolum subsp.
capripneumoniae was centrifuged (12,000 3 g, 4°C, 10 min),
washed three times in PBS, and resuspended in PBS. Trypsin
was added to a final concentration of 0.2 mg/ml and the suspension was incubated at 37°C for 30 min. Organisms were
pelleted by centrifugation (12,000 3 g, 4°C, 10 min), and the
supernatant was collected. The pelleted organisms were
washed twice with PBS and resuspended to the volume of the
collected supernatant. Treated organisms, the supernatant,
and untreated organisms were immediately boiled in SDSPAGE sample buffer including a reducing agent (25), and
Western blotting was done as described above. There was
binding of MAb E8-18 to a 24-kDa protein in the pellet of the
untreated organisms (Fig. 3, lane 1), while binding was completely abolished by treatment with 0.2 mg of trypsin per ml
(Fig. 3, lane 3). MAb E8-18 did not bind to proteins in the
supernatants, indicating either that p24 was digested to peptides too small to be retained in the gradient gel or that the
epitope recognized by E8-18 was destroyed by trypsin. Neither
a MAb isotype control (Fig. 3, lanes 5 to 8) nor uninfected goat
serum (Fig. 3, lanes 13 to 16) bound proteins in the pellets or
supernatants. However, proteins were bound in both the pellets and supernatants when reacted with serum from an M.
capricolum subsp. capripneumoniae-infected goat (Fig. 3, lanes
VOL. 4, 1997
FIG. 3. Immunoblot of M. capricolum subsp. capripneumoniae following
treatment of intact organisms with trypsin. Freshly harvested M. capricolum
subsp. capripneumoniae organisms were incubated with or without trypsin and
then centrifuged to obtain supernatant and pellet fractions, as described in
Materials and Methods. Lanes 1, 5, 9, and 13 were loaded with untreated pellet;
lanes 2, 6, 8, and 14 were loaded with untreated supernatant; lanes 3, 7, 11, and
15 were loaded with treated pellet; and lanes 4, 8, 12, and 16 were loaded with
treated supernatant. Lanes 1 to 4 were reacted with MAb E8-18, lanes 5 to 8
were reacted with an unrelated MAb of the same isotype, lanes 9 to 12 were
reacted with goat serum antibodies to M. capricolum subsp. capripneumoniae,
and lanes 13 to 16 were reacted with negative goat serum. Molecular mass
standards (in kilodaltons) are shown on the left.
9 to 12). In a separate experiment, cell suspensions of washed
M. capricolum subsp. capripneumoniae were treated with trypsin in the presence of soybean trypsin inhibitor while others
were treated with periodate. The trypsin inhibitor abrogated
the effect of trypsin on p24 and MAb E8-18 reacted normally
with p24, while periodate had no effect on the activity of MAb
E8-18 on p24 (results not shown). Portions of treated and
untreated M. capricolum subsp. capripneumoniae pellets were
plated on tryptose agar plates to assay viability. Trypsin treatment did not alter the viability of the organisms, allowing the
conclusion that the epitope recognized by MAb E8-18 was on
the surfaces of live M. capricolum subsp. capripneumoniae organisms.
Determination of whether p24 was an integral M. capricolum
subsp. capripneumoniae membrane protein. M. capricolum
subsp. capripneumoniae components were separated into hydrophobic and hydrophilic fractions by a Triton X-114 partitioning method (7) with some described modifications (25).
Both the detergent and aqueous phases along with M. capricolum subsp. capripneumoniae organisms were evaluated for
binding to MAb E8-18 in Western immunoblots as described
above. p24 partitioned primarily in the Triton X-114 phase
(Fig. 4, lane 2). However, there was residual p24 detected in
the aqueous phase (Fig. 4, lane 1), possibly due to inefficient
phase separation. The selective segregation of hydrophobic
p24 by its ability to form Triton X-114 micelles enabled identification of the p24 protein as an integral membrane protein.
Similar detergent partitioning has been used to define integral
membrane proteins in other Mycoplasma species (8, 20, 25, 34,
42).
Evaluation of MAb E8-18 in competitive ELISA. Since MAb
E8-18 was specific for M. capricolum subsp. capripneumoniae,
competitive ELISA was used to assess the possibility of using
the MAb in CCPP diagnosis. Microtiter plates were coated
with 100 ml/well containing 10 mg of solubilized M. capricolum
subsp. capripneumoniae in carbonate buffer (15 mM Na2CO3,
35 mM NaHCO3 [pH 9.0]) overnight at 4°C with gentle shak-
NOTES
617
FIG. 4. Immunoblot of M. capricolum subsp. capripneumoniae Triton X-114phase and aqueous-phase proteins with MAb E8-18. Lanes 1 and 4 were loaded
with aqueous-phase proteins, lanes 2 and 5 were loaded with detergent-phase
proteins, and lanes 3 and 6 were loaded with whole M. capricolum subsp. capripneumoniae. Lanes 1 to 3 were reacted with MAb E8-18, and lanes 4 and 6 were
reacted with goat serum antibodies to M. capricolum subsp. capripneumoniae.
Molecular mass standards (in kilodaltons) are shown on the left.
ing. Coated plates were blocked with 5% fraction V bovine
serum albumin in PBS for 1 h at room temperature, the
blocker was poured off, and 100 ml of test serum (goat serum)
diluted 1:10 in blocking buffer was added to duplicate wells.
After 30 min of incubation at room temperature with gentle
shaking, the plates were washed three times with PBS containing 0.1% Tween 20. Then, 0.2 mg of MAb E8-18 per well was
added, the mixture was incubated for 30 min, and the plates
were washed. Alkaline phosphatase-conjugated goat antimouse IgG was added at a dilution of 1:1,000, the mixture was
incubated at room temperature for another 30 min, and the
plates were washed. Bound MAb was detected by the addition
of 100 ml of a substrate (p-nitrophenyl phosphate disodium
[one tablet dissolved in 10 ml of substrate buffer]), a 15-min
incubation, and determination of the optical density at 414 nm.
Duplicate serum samples from five goats from Washington
State University, Pullman, known to be negative were included
on each plate and used to determine a mean optical density
and standard deviation. A test serum sample was scored as
positive for antibody to the epitope recognized by MAb E8-18
if the optical density was 3 standard deviations or more below
the mean optical density of the known negative samples.
The binding of the MAb to M. capricolum subsp. capripneumoniae was inhibited by 22 of 30 sera from goats vaccinated
against CCPP at the Small Ruminant Collaboration Research
Support Program farm at Naivasha, Kenya. There was no inhibition of binding of the MAb to M. capricolum subsp. capripneumoniae by 24 sera from nonvaccinated goats from the
same farm and no inhibition by 12 sera from nonvaccinated
goats from the Baringo District, Kenya. Sera from serial bleedings of five goats experimentally infected with M. capricolum
subsp. capripneumoniae by contact exposure, and which eventually died of CCPP (40), did not inhibit the binding of the
MAb to M. capricolum subsp. capripneumoniae even though
these goats had antibody to M. capricolum subsp. capripneumoniae antigen detected by the latex agglutination test (40).
Conclusions. A 24-kDa M. capricolum subsp. capripneumoniae protein was identified with MAb E8-18 and found to be
an integral membrane protein by partitioning with Triton X114; it was found on the surface of live M. capricolum subsp.
capripneumoniae by trypsin treatment. The reactivity of MAb
E8-18 with M. capricolum subsp. capripneumoniae in a competitive ELISA could not be blocked by antibodies in sequential sera from experimentally infected goats with clinical CCPP.
618
NOTES
CLIN. DIAGN. LAB. IMMUNOL.
This observation prevents the use of the competitive ELISA
based on MAb E8-18 in the diagnosis of CCPP. However, p24
may be useful in inducing protective immunity because the
epitope recognized by MAb E8-18 was bound by sera from
most vaccinated goats and other studies have demonstrated
that .90% of goats vaccinated by the same method are resistant to M. capricolum subsp. capripneumoniae challenge (39).
The vaccine against CCPP consists of freeze-dried whole M.
capricolum subsp. capripneumoniae inactivated with saponin
(37), and a Western blot of the vaccine probed with MAb
E8-18 and/or sera from vaccinated goats revealed the presence
of p24 (results not shown). In addition, since MAb E8-18
reacted only with M. capricolum subsp. capripneumoniae in
Western blots, it can be used to help differentiate M. capricolum subsp. capripneumoniae from closely related and difficult-to-distinguish Mycoplasma species (2, 6, 11, 27).
This research was conducted as part of the United States Agency for
International Development Title XII Small Ruminant-Collaborative
Research Support Program under grant AID/DSAN/XII-G-0049 in
collaboration between the Kenya Agricultural Research Institute and
Washington State University.
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