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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. REFERENCES 1. Andersen, H., G. Christiansen, and C. Christiansen. 1984. 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