INFECTION AND IMMUNITY, June 1991, p. 2175-2180 0019-9567/91/062175-06$02.00/0 Copyright X 1991, American Society for Microbiology
Vol. 59, No. 6
Production and Characterization of a Monoclonal Antibody to the Complement Fixation Antigen of Coccidioides immitis MATTHEW J. DOLAN' AND REBECCA A. COX2*
Department of Research Immunology, San Antonio State Chest Hospital, San Antonio, Texas 78223,2 and Department of Infectious Diseases, Wilford Hall, United States Air Force Medical Center, Lackland Air Force Base, Texas 782361 Received 31 January 1991/Accepted 29 March 1991
Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgGl) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Agl4 myeloma cells, and the fusion products were screened for IgG antibody to coccidioidin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgGl MAb was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60°C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, a-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.
The complement-fixing (CF) antibody response to Coccidioides immitis is a valuable aid in establishing the diagnosis and prognosis of coccidioidomycosis. Increasing titers of CF antibody are associated with progressive coccidioidal disease, and conversely, decreasing titers are associated with disease regression or stabilization (15, 16). The CF antibody can be detected by the classical CF test of Smith et al. (15) or an immunodiffusion assay for CF antibody (IDCF) developed by Huppert and Bailey (9). Although the CF test has an acceptable level of sensitivity, it lacks specificity owing to the use of heterogeneous coccidioidin (CDN) preparations which contain cross-reactive antigens (1, 8, 11, 16, 18). In contrast, the IDCF is highly specific but has a low level of sensitivity (18). The availability of a monospecific antibody to the IDCF antigen would allow researchers to purify the antigen by solid-phase immunoadsorption and to screen genomic and cDNA expression libraries from C. immitis for the production of a recombinant antigen. We have previously reported the identification of the IDCF precipitinogen in two-dimensional immunoelectrophoresis (2D-IEP) of CDN and the production of goat antiserum specific for this antigen by immunization with the precipitin arc formed in 2D-IEP (3). Repeated immunizations, however, invariably led to the induction of antibodies directed against other CDN components, thus precluding the use of the antiserum as a biospecific ligand. To circumvent this problem, investigations were undertaken to produce a monoclonal antibody (MAb) against the IDCF antigen.
*
MATERIALS AND METHODS Antigens and antisera. CDN was prepared as a culture filtrate (CDN-F) and as a toluene-induced lysate (CDN-L) of mycelia of the C. immitis strain Silveira as detailed previously (9, 10, 13). Reference IDCF antigen was obtained from Scott Laboratories, Inc. (Carson, Calif.), and was prepared as a concentrated culture filtrate from mycelia. Hyperimmune goat anti-CDN and goat anti-IDCF sera were the same preparations used in earlier studies (2, 3). Blastomycin, histoplasmin, rabbit anti-blastomycin serum, and rabbit antihistoplasmin serum were purchased from Scott Laboratories, Inc. Affinity-purified alkaline phosphatase-conjugated rabbit anti-goat immunoglobulin G (IgG), goat anti-rabbit IgG, and goat anti-mouse IgG were obtained from Kirkegaard and Perry Laboratories (Gaithersburg, Md.). Production of MAb. Six- to eight-week-old female BALB/c mice (Charles Rivers Laboratories, Raleigh, N.C.) were immunized intramuscularly with four monthly injections of CDN (100 ,ug) in incomplete Freund's adjuvant. Mice were bled, and the sera were tested for antibody reactivity by IDCF, enzyme-linked immunosorbent assay (ELISA), and immunoblotting, as described below. Mice demonstrating activity against the IDCF antigen were selected for intrasplenic booster immunization and spleen cell-murine myeloma cell fusion. For intrasplenic injection of CDN, the mice were anesthetized with an intraperitoneal injection of pentabarbitone (45 mg/kg), followed by an intramuscular injection of ketamine hydrochloride (50 mg/kg). An area (2 by 2 cm) in the left midscapular line at the lower costal margin was shaved and swabbed with a 70% ethanol solution. A small incision was made through the skin, the muscles were separated by blunt dissection, and the underlying spleen was visualized through the intact peritoneal membrane. A 30-gauge needle was inserted parallel to the spleen, and two injections of CDN
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(each containing 300 ,ug of CDN in 20 pl of physiologic saline) were delivered into the spleen. The overlying skin edges were approximated, and the incision was closed in a single step with 4-0 silk. The technique described above was adapted from those of other investigators (7, 17) but differs in that it preserves the integrity of the peritoneal barrier yet allows visualization of the spleen so that laceration of the capsule is avoided. Hybridomas were produced by using a previously reported adaptation (4) of the method of Gefter et al. (5). Briefly, 3 days after administration of intrasplenic booster injections, the spleens were harvested, and following lysis of erythrocytes with ammonium chloride buffer, 1.5 x 108 spleen cells were fused with 5.2 x 107 Sp2/0 Agl4 murine myeloma cells by using 1.5 ml of 40% polyethylene glycol 1450 at pH 8.0. Fusion products were plated onto 96-well tissue culture plates of hypoxanthine-amino-pterin-thymidine hybrid selection medium (200 ,lI/well) with a feeder cell layer of 1 x 105 normal BALB/c spleen cells. Wells with growth were screened for antibody against CDN by the ELISA, and positive hybridomas were assayed for IgG antibody to the IDCF antigen in immunoblots of CDN. Of the four hybridomas with activity against CDN, one produced IgG antibody to the IDCF antigen and was selected for further study. This hybridoma was cloned by limiting dilution and then passaged into Pristane-primed BALB/c mice to form ascites. The ascitic cells were cloned twice by limiting dilution, and the MAb was purified by immunoadsorption on an Affi-Gel protein A MAPS II column (Bio-Rad Laboratories, Richmond, Calif.). Immunoassays. The procedure for the ELISA was as previously described (4). The target antigens (CDN, 10 ,ug/ml; histoplasmin, 1:1,000; or blastomycin, 1:1,000) were bound to polystyrene plates overnight, and wells were blocked with 1% bovine serum albumin. Serum samples, diluted 1:100 in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-TW), or 50 ,ll of the hybridoma supernatants, diluted with an equal volume of PBS-TW, was added to the wells. After a 2-h incubation at 37°C, the wells were washed and reacted with 100 Rl of alkaline phosphatase-conjugated goat anti-mouse IgG, rabbit anti-goat IgG, or goat anti-rabbit IgG, each diluted 1:1,000. Substrate (p-nitrophenyl phosphate diluted in 10% diethanolamine buffer) was added after a 90-min incubation, and the A410 was read at 30 min. Reducing, denaturing immunoblotting was performed by using a modification of the method previously reported (4). Antigens were diluted in a sample buffer containing 4% sodium dodecyl sulfate (SDS), 4.0% 2-mercaptoethanol, 0.001% bromophenol blue, 0.05 M Tris hydrochloride, and 10% glycerol. The samples were heated for 5 min in a boiling water bath and then electrophoresed through a discontinuous 4 to 20% SDS-polyacrylamide gel under reducing conditions. Following electrophoretic transfer to nitrocellulose acetate membranes at 0.6 A overnight, the membranes were blocked with 4% nonfat powdered milk in PBS and then reacted with reference goat or rabbit antisera or MAb. After overnight incubation, the membranes were washed and individual lanes were incubated for 1 h at 25°C with alkaline phosphatase-conjugated rabbit anti-goat IgG, goat antirabbit IgG, or goat anti-mouse IgG, depending on the primary antibody used, and subsequently developed with 5-bromo-4-chloro-3-indolyl-phosphate-Nitro Blue Tetrazolium (Kirkegaard and Perry). Molecular size standards (Bio-Rad Laboratories) were co-electrophoresed under conditions identical to those described above and stained with
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Coomassie blue. Standards included phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa), and lysozyme (14 kDa). Nonreducing, nondenaturing Western blotting (immunoblotting) was performed as described above, except that the antigens were not preheated and 2-mercaptoethanol and SDS were omitted from the sample and running buffers. The molecular size standards (Bio-Rad) included myosin (200 kDa), P-galactosidase (115 kDa), and the four larger markers indicated above (97, 66, 45, and 31 kDa), all run under nonreducing, nondenaturing conditions. Combined 2D-IEP and autoradiography was performed as detailed elsewhere (4, 6). In brief, 0.9 mg of affinity-purified MAb was radiolabeled with 1 mCi of 1251 (Amersham Corp., Arlington Heights, Ill.) by using 4-N-chlorobenzenesulfonamide-derivatized polystyrene beads (IODO-BEADS; Pierce Chemical Co., Rockford, Ill.). After passage over a Presto desalting column (Pierce), 0.12 mg of the 125I-labeled MAb (specific activity, 0.4 mCi/mg) was admixed with goat antiCDN or goat anti-IDCF serum in the second-dimension gel in 2D-IEP, and after electrophoresis, the gels were washed, dried, and then placed in direct contact with X-ray film at -70°C. Corresponding duplicate gels were stained with Coomassie blue to identify reference precipitin peaks. Enzymatic digestion. The effect of various enzymes on the binding of the IDCF antigen to MAb was examined by using a modified ELISA (12). For this procedure, CDN was immobilized on polystyrene plates, blocked with PBS-TW, and then incubated for 2 h at 37°C with glycolytic or proteolytic enzymes diluted in PBS (pH 6.5). The enzymes were a-mannosidase (0.5 U/ml), glucose oxidase (0.26 U/ml), endoglycosidase H (0.005 U/ml), pronase (45 U/ml), and trypsin (4,500 U/ml), all from Sigma Chemical Co. After incubation, the wells were washed with PBS-TW and assayed for reactivity with the MAb by using the ELISA described above. The degradative effects of pronase and lipase were further examined by using a modification of the immunoblotting procedure (19). After CDN was electrophoresed in SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes, strips were cut out and incubated in 4.5 ml of PBS (pH 6.5) containing pronase (3 U/ml) or lipase (1.5 UIml). Following a 90-min incubation at 24°C, the strips were washed with PBS-TW and assayed for reactivity with the MAb by immunoblotting as described above.
RESULTS Reactivity of the MAb in 2D-IEP. The MAb was not detectable as a precipitin when analyzed either in the IDCF or by 2D-IEP. Its reactivity against the IDCF precipitinogen was demonstrable, however, by using a combination of 2D-IEP and autoradiography, wherein 1251I-labeled MAb was admixed with reference precipitin antisera in the seconddimension gel. The results established that radiolabeled MAb bound to the CDN-F component that was precipitated by monospecific goat anti-IDCF serum (compare Fig. 1A and B). The epitope recognized by the MAb appeared to be specific for the IDCF antigen, as evidenced by the lack of reactivity of the radiolabeled MAb with other CDN-F precipitinogens which were detected by using reference goat anti-CDN serum (compare Fig. 1C and D). Reactivity of the MAb in immunoblots. The specificity of the MAb was further evaluated by Western blotting. Figure
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FIG. 1. Reactivity of the MAb in 2D-IEP of CDN. After first-dimension gel electrophoresis, CDN-F (150 ,ug) was electrophoresed against a second-dimension gel containing goat antiserum (400 jig/cm2 of gel) admixed with 1251I-labeled MAb (0.02 jig/cm2 of gel; specific activity, 0.4 mCi/mg). The IDCF precipitinogen seen on the Coomassie blue-stained gel of CDN-F against goat anti-IDCF (A) was reactive with the 1251I-MAb in the corresponding autoradiogram (B). Second-dimension electrophoresis of CDN-F against goat anti-CDN (300 jig/cm2 of gel) yielded several precipitinogens on the Coomassie blue-stained gel (C), including the IDCF and the tube precipitin (TP) antigens (2, 4). The 251I-MAb was specific for the IDCF antigen (D).
2A depicts the results obtained in immunoblots of CDN-L and reference IDCF antigen probed with mouse MAb, goat anti-IDCF serum, or goat anti-CDN serum. Whereas hyperimmune goat anti-CDN serum demonstrated multiple bands in CDN-L and in the IDCF antigen, the mouse MAb and goat anti-IDCF serum were selective for a couplet band having a molecular size distribution of 43 to 45 kDa. The interpretation that this is a couplet band, rather than two distinct components, is based upon the fact that the couplet was detected by the MAb and the reference goat anti-IDCF sera, both of which were specific for the IDCF antigen by 2D-IEP (Fig. 1). Since the couplet band could represent breakdown products of the native IDCF antigen as a result of the denaturing conditions used in SDS-PAGE, immunoblots were also prepared by using nondenaturing, nonreducing conditions for electrophoretic separation of antigen. As shown in Fig. 2B, immunoblots of native antigen yielded a single band having a molecular size of 110 kDa. Heat treatment of the native antigen for 30 min at 60°C ablated its reactivity with MAb (Fig. 2B). Epitope characterization. Characterization of the antigenbinding site was accomplished by in situ enzymatic digestion of CDN-F which had been immobilized on polystyrene plates. This technique allowed small quantities of enzyme and antigen to be used and offered a quantitative assessment of enzymatic degradation. As shown in Table 1, treatment with pronase virtually eliminated reactivity with the MAb;
trypsin effected a 42% reduction in epitope reactivity. In contrast, no degradation was observed after incubation of the immobilized antigen with glucose oxidase, ot-mannosidase, or endoglycosidase H. The degradative effect of pronase on the 43- to 45-kDa couplet band was directly visualized by enzyme treatment of CDN-F which had been electrophoretically separated and immobilized on nitrocellulose strips. This procedure allowed enzymes to be removed from the system, as described above, and ensured that alterations in antigen size by enzymes would not be confused with epitope degradation. As shown in Fig. 3, pronase treatment ablated the reactivity of the 43- to 45-kDa couplet band with MAb; lipase was without effect on epitope recognition. Heat treatment of CDN-F at 60°C for 30 min before application to SDS-PAGE also ablated epitope recognition (Fig. 3). The preceding results, taken together, provide strong evidence that the MAb is directed against a heat-labile peptide epitope. Lack of reactivity of MAb with blastomycin and histoplasmin. The specificity of the epitope recognized by the MAb was assessed by immunoblot assays of blastomycin and histoplasmin. Hyperimmune rabbit antisera revealed the presence of multiple antigens in the two heterologous extracts (Fig. 4). None of these antigens were reactive with the MAb. To exclude the possibility that the denaturing conditions used in immunoblotting might have altered a crossreactive epitope, the specificity of the MAb was further
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FIG. 2. Reactivity of the MAb in immunoblots. (A) Immunoblot of CDN-L (15 p.g) and IDCF antigen (15 jig) prepared under reducing, denaturing conditions. Note that the MAb (ascites diluted 1:25) and goat anti-IDCF serum (30 ,ug/ml) were reactive with a 43- to 45-kDa couplet band. (B) Immunoblot of CDN-L (45 ,ug) prepared under nonreducing, nondenaturing conditions. Note that the MAb recognized a 110-kDa band that was heat labile. Molecular mass markers (in kilodaltons) are shown on the left.
evaluated in the ELISA. The results, shown in Table 2, did any detectable level of cross-reactivity.
not reveal
DISCUSSION We report the production of a murine IgG subclass 1 MAb against a heat-labile peptide epitope of the IDCF antigen. The MAb was not reactive with other CDN antigens or with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis, indicating that the epitope is specific for the IDCF antigen. Our initial attempts to produce an IgG MAb to the IDCF antigen were unsuccessful, even though the mice had serum IgG antibodies to the IDCF antigen. Intrasplenic injection of antigen has been used by other investigators as a route for primary immunization (7, 17) and was adapted in the present study to administer the final antigen boost. The intrasplenic route was found to enhance the production of IgG-secreting hybridomas to the IDCF antigen and other CDN antigens as well. The MAb was not detectable as a precipitin, but its reactivity against the IDCF precipitinogen was demonstrable TABLE 1. Effect of enzyme treatments on epitope recognition as assessed by the ELISA' Enzyme
A410 None Pronase Trypsin Glucose oxidase a-Mannosidase
Endoglycosidase H
1.069 0.063 0.623 1.230 1.175 1.211
by using a combination of 2D-IEP and autoradiography. When the radiolabeled MAb was admixed with goat antiCDN serum in the second-dimension gel, the MAb was observed to specifically bind the IDCF precipitin peak. When used to probe immunoblots of CDN prepared under denaturing, reducing conditions, the MAb recognized a 43to 45-kDa couplet band. In contrast, a 110-kDa band was detected on immunoblots prepared under nondenaturing conditions. These combined results provide evidence that the 43- to 45-kDa couplet band represents degraded compo-
Treatment of CDN untreated
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a CDN-F was immobilized on polystyrene wells and then incubated with buffer alone or with buffer containing enzyme. After a 90-min incubation, the treated and nontreated antigen-coated wells were evaluated for reactivity with the MAb. b Percent reduction as compared with the A410 of nontreated antigen-coated wells.
FIG. 3. Epitope characterization by immunoblotting. CDN-L (15 ,ug) was subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose. Strips containing the immobilized antigen were incubated in buffer alone or in buffer containing enzyme, and after removal of enzyme by washing, the strips were probed with the MAb (ascites diluted 1:25). Also shown is an immunoblot of CDN-L which had been heated to 60°C for 30 min before application to SDS-PAGE. Note that the reactivity of the MAb with the 43- to 45-kDa couplet was abolished by pronase or heat treatment. A molecular mass marker (45 kDa) is shown on the left.
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phase extracts of H. capsulatum or B. dermatitidis. This specificity indicates that the MAb will be useful as a biospecific ligand for the purification of the IDCF antigen from heterogeneous extracts and to screen genomic and cDNA expression libraries prepared from C. immitis (14, 20) for the production of a recombinant product. ACKNOWLEDGMENT This work was supported by Public Health Service grant A121431 from the National Institute of Allergy and Infectious Diseases.
FIG. 4. Specificity of the MAb (ascites diluted 1:25) as assessed in immunoblots of histoplasmin and blastomycin. Histoplasmin (diluted 1:2) and blastomycin (diluted 1:2) contained multiple antigens when assayed against rabbit anti-histoplasmin (diluted 1:1,000) and rabbit anti-blastomycin (diluted 1:1,000) sera, respectively. None of the antigens were reactive with the MAb.
REFERENCES 1. Campbell, C. C., and G. E. Binkley. 1953. Serologic diagnosis with respect to histoplasmosis, coccidioidomycosis, and blastomycosis and the problem of cross reactions. J. Lab. Clin. Med. 42:896-906. 2. Cox, R. A., and L. A. Britt. 1985. Antigenic heterogeneity of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis. Infect. Immun. 50:365-369. 3. Cox, R. A., L. A. Britt, and R. A. Michael. 1987. Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum. Infect. Immun. 55:227-232. 4. Dolan, M. J., R. A. Cox, V. Williams, and S. Wooley. 1989. Development and characterization of a monoclonal antibody
against the tube precipitin antigen of Coccidioides immitis.
nents of the native 110-kDa antigen. The antigen may be a protein multimer with components of various sizes as a result of posttranslational differences, or the effects of heat and SDS may cause a progressive or asymmetric cleavage of a native monomeric molecule. Zimmer and Pappagianis (21) reported earlier that the IDCF activity of CDN was eluted in a fraction having an average molecular size of 110 kDa by molecular-sieve chromatography. Immunoblots of the chromatographically purified IDCF antigen against IDCF-positive sera of coccidioidomycosis patients yielded a 110-kDa band when electrophoresis was performed under nondenaturing conditions, whereas a 48- to 50-kDa band was detected in blots prepared under denaturing conditions (21). The epitope recognized by the MAb was identified as a heat-labile peptide on the basis of the finding that immunologic reactivity was completely abolished after treatment with heat or pronase and partially reduced after enzymatic digestion with trypsin. Zimmer and Pappagianis (21), in analyses of their chromatographically purified IDCF antigen, also observed that heat or pronase treatment ablated the reactivity of the 48-kDa IDCF band in immunoblots against IDCF-positive human sera. They reported a variable effect with trypsin, showing elimination of reactivity on denaturing, reducing blots of a filtrate prepared from strain Silveira but retention of reactivity by using a pooled filtrate of 22 strains of C. immitis. The MAb appears to recognize an epitope that is specific for the IDCF antigen on the basis of the finding that the MAb was not reactive with antigenic components in mycelialTABLE 2. Specificity of MAb as assessed by the ELISA A410 of target antigen Antibody CDN-F Blastomycin Histoplasmin 0.019 0.015 0.615 MAb 0.122 0.651 1.631 Goat anti-CDN 0.727 0.784 0.590 Rabbit anti-histoplasmin 0.613 0.381 0.658 Rabbit anti-blastomycin
Infect. Immun. 57:1035-1039. 5. Gefter, M. L., D. H. Marulies, and M. D. Scharff. 1977. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 3:231-236. 6. Harboe, M., and J. Ivanyi. 1987. Analysis of monoclonal antibodies to Mycobacterium leprae by crossed immunoelectrophoresis. Scand. J. Immunol. 25:133-138. 7. Hong, T. H., S. T. Chen, T. K. Tang, S. C. Wang, and T. H. Chang. 1989. The production of polyclonal and monoclonal antibodies in mice using novel immunization methods. J. Immunol. Methods 120:151-157. 8. Huppert, M., J. P. Adler, E. H. Rice, and S. H. Sun. 1979. Common antigens among systemic disease fungi analyzed by two-dimensional immunoelectrophoresis. Infect. Immun. 23: 479-485. 9. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion in coccidioidomycosis. I. The accuracy and reproducibility of the immunodiffusion test which correlates with complement fixation. Am. J. Clin. Pathol. 44:364-368. 10. Huppert, M., J. W. Bailey, and P. Chitjian. 1967. Immunodiffusion as a substitute for complement fixation and tube precipitin tests in coccidioidomycosis, p. 221-225. In L. Ajello (ed.), Proceedings of the Second Coccidioidomycosis Symposium. University of Arizona Press, Tucson. 11. Kaufman, L., and M. J. Clark. 1974. Value of the concomitant use of complement fixation and immunodiffusion tests in the diagnosis of coccidioidomycosis. Appl. Microbiol. 28:641-43. 12. Mitchell, C. G., R. Smith, and T. Lehner. 1987. Recognition of carbohydrate and protein epitopes by monoclonal antibodies to a cell wall antigen from Streptococcus mutans. Infect. Immun.
55:810-815. 13. Pappagianis, D., C. E. Smith, G. S. Kobayashi, and M. T. Saito. 1961. Studies of antigens from young mycelia of Coccidioides immitis. J. Infect. Dis. 108:35-44. 14. Resnick, S., G. Apodaca, G. Newport, C. Halde, and J. McKerrow. 1987. Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, D-48, p. 80. 15. Smith, C. E., M. T. Saito, R. R. Beard, R. M. Kepp, R. W. Clark, and B. U. Eddie. 1950. Serological tests in the diagnosis and prognosis of coccidioidomycosis. Am. J. Hyg. 52:1-21. 16. Smith, C. E., M. T. Saito, and S. A. Simons. 1956. Pattern of 39,500 serologic tests in coccidioidomycosis. JAMA 160:546552. 17. Spitz, M. 1986. "Single-shot" intrasplenic immunization for the production of monoclonal antibodies. Methods Enzymol. 121: 33-41.
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18. Wood, J. C., G. Friedly, M. Zartarian, S. Aarnaes, and L. M. de la Maza. 1982. Alternatives to the standardized laboratory branch complement fixation test for detection of antibodies to Coccidioides immitis. J. Clin. Microbiol. 16:1030-1033. 19. Woodward, M. P., W. W. Young, and R. A. Bloodgood. 1984. Detection of monoclonal antibodies specific to carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78:
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143-153. 20. Yuan, L., J. H. McKerrow, and G. T. Cole. 1988. Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, F-22, p. 395. 21. Zimmer, B., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion complement fixation antigen. J. Clin. Microbiol. 26:2250-2256.
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Production and Characterization of a Monoclonal Antibody to the Complement Fixation Antigen of Coccidioides immitis MATTHEW J. DOLAN' AND REBECCA A. COX2*
Department of Research Immunology, San Antonio State Chest Hospital, San Antonio, Texas 78223,2 and Department of Infectious Diseases, Wilford Hall, United States Air Force Medical Center, Lackland Air Force Base, Texas 782361 Received 31 January 1991/Accepted 29 March 1991
Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgGl) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Agl4 myeloma cells, and the fusion products were screened for IgG antibody to coccidioidin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgGl MAb was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60°C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, a-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.
The complement-fixing (CF) antibody response to Coccidioides immitis is a valuable aid in establishing the diagnosis and prognosis of coccidioidomycosis. Increasing titers of CF antibody are associated with progressive coccidioidal disease, and conversely, decreasing titers are associated with disease regression or stabilization (15, 16). The CF antibody can be detected by the classical CF test of Smith et al. (15) or an immunodiffusion assay for CF antibody (IDCF) developed by Huppert and Bailey (9). Although the CF test has an acceptable level of sensitivity, it lacks specificity owing to the use of heterogeneous coccidioidin (CDN) preparations which contain cross-reactive antigens (1, 8, 11, 16, 18). In contrast, the IDCF is highly specific but has a low level of sensitivity (18). The availability of a monospecific antibody to the IDCF antigen would allow researchers to purify the antigen by solid-phase immunoadsorption and to screen genomic and cDNA expression libraries from C. immitis for the production of a recombinant antigen. We have previously reported the identification of the IDCF precipitinogen in two-dimensional immunoelectrophoresis (2D-IEP) of CDN and the production of goat antiserum specific for this antigen by immunization with the precipitin arc formed in 2D-IEP (3). Repeated immunizations, however, invariably led to the induction of antibodies directed against other CDN components, thus precluding the use of the antiserum as a biospecific ligand. To circumvent this problem, investigations were undertaken to produce a monoclonal antibody (MAb) against the IDCF antigen.
*
MATERIALS AND METHODS Antigens and antisera. CDN was prepared as a culture filtrate (CDN-F) and as a toluene-induced lysate (CDN-L) of mycelia of the C. immitis strain Silveira as detailed previously (9, 10, 13). Reference IDCF antigen was obtained from Scott Laboratories, Inc. (Carson, Calif.), and was prepared as a concentrated culture filtrate from mycelia. Hyperimmune goat anti-CDN and goat anti-IDCF sera were the same preparations used in earlier studies (2, 3). Blastomycin, histoplasmin, rabbit anti-blastomycin serum, and rabbit antihistoplasmin serum were purchased from Scott Laboratories, Inc. Affinity-purified alkaline phosphatase-conjugated rabbit anti-goat immunoglobulin G (IgG), goat anti-rabbit IgG, and goat anti-mouse IgG were obtained from Kirkegaard and Perry Laboratories (Gaithersburg, Md.). Production of MAb. Six- to eight-week-old female BALB/c mice (Charles Rivers Laboratories, Raleigh, N.C.) were immunized intramuscularly with four monthly injections of CDN (100 ,ug) in incomplete Freund's adjuvant. Mice were bled, and the sera were tested for antibody reactivity by IDCF, enzyme-linked immunosorbent assay (ELISA), and immunoblotting, as described below. Mice demonstrating activity against the IDCF antigen were selected for intrasplenic booster immunization and spleen cell-murine myeloma cell fusion. For intrasplenic injection of CDN, the mice were anesthetized with an intraperitoneal injection of pentabarbitone (45 mg/kg), followed by an intramuscular injection of ketamine hydrochloride (50 mg/kg). An area (2 by 2 cm) in the left midscapular line at the lower costal margin was shaved and swabbed with a 70% ethanol solution. A small incision was made through the skin, the muscles were separated by blunt dissection, and the underlying spleen was visualized through the intact peritoneal membrane. A 30-gauge needle was inserted parallel to the spleen, and two injections of CDN
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(each containing 300 ,ug of CDN in 20 pl of physiologic saline) were delivered into the spleen. The overlying skin edges were approximated, and the incision was closed in a single step with 4-0 silk. The technique described above was adapted from those of other investigators (7, 17) but differs in that it preserves the integrity of the peritoneal barrier yet allows visualization of the spleen so that laceration of the capsule is avoided. Hybridomas were produced by using a previously reported adaptation (4) of the method of Gefter et al. (5). Briefly, 3 days after administration of intrasplenic booster injections, the spleens were harvested, and following lysis of erythrocytes with ammonium chloride buffer, 1.5 x 108 spleen cells were fused with 5.2 x 107 Sp2/0 Agl4 murine myeloma cells by using 1.5 ml of 40% polyethylene glycol 1450 at pH 8.0. Fusion products were plated onto 96-well tissue culture plates of hypoxanthine-amino-pterin-thymidine hybrid selection medium (200 ,lI/well) with a feeder cell layer of 1 x 105 normal BALB/c spleen cells. Wells with growth were screened for antibody against CDN by the ELISA, and positive hybridomas were assayed for IgG antibody to the IDCF antigen in immunoblots of CDN. Of the four hybridomas with activity against CDN, one produced IgG antibody to the IDCF antigen and was selected for further study. This hybridoma was cloned by limiting dilution and then passaged into Pristane-primed BALB/c mice to form ascites. The ascitic cells were cloned twice by limiting dilution, and the MAb was purified by immunoadsorption on an Affi-Gel protein A MAPS II column (Bio-Rad Laboratories, Richmond, Calif.). Immunoassays. The procedure for the ELISA was as previously described (4). The target antigens (CDN, 10 ,ug/ml; histoplasmin, 1:1,000; or blastomycin, 1:1,000) were bound to polystyrene plates overnight, and wells were blocked with 1% bovine serum albumin. Serum samples, diluted 1:100 in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-TW), or 50 ,ll of the hybridoma supernatants, diluted with an equal volume of PBS-TW, was added to the wells. After a 2-h incubation at 37°C, the wells were washed and reacted with 100 Rl of alkaline phosphatase-conjugated goat anti-mouse IgG, rabbit anti-goat IgG, or goat anti-rabbit IgG, each diluted 1:1,000. Substrate (p-nitrophenyl phosphate diluted in 10% diethanolamine buffer) was added after a 90-min incubation, and the A410 was read at 30 min. Reducing, denaturing immunoblotting was performed by using a modification of the method previously reported (4). Antigens were diluted in a sample buffer containing 4% sodium dodecyl sulfate (SDS), 4.0% 2-mercaptoethanol, 0.001% bromophenol blue, 0.05 M Tris hydrochloride, and 10% glycerol. The samples were heated for 5 min in a boiling water bath and then electrophoresed through a discontinuous 4 to 20% SDS-polyacrylamide gel under reducing conditions. Following electrophoretic transfer to nitrocellulose acetate membranes at 0.6 A overnight, the membranes were blocked with 4% nonfat powdered milk in PBS and then reacted with reference goat or rabbit antisera or MAb. After overnight incubation, the membranes were washed and individual lanes were incubated for 1 h at 25°C with alkaline phosphatase-conjugated rabbit anti-goat IgG, goat antirabbit IgG, or goat anti-mouse IgG, depending on the primary antibody used, and subsequently developed with 5-bromo-4-chloro-3-indolyl-phosphate-Nitro Blue Tetrazolium (Kirkegaard and Perry). Molecular size standards (Bio-Rad Laboratories) were co-electrophoresed under conditions identical to those described above and stained with
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Coomassie blue. Standards included phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa), and lysozyme (14 kDa). Nonreducing, nondenaturing Western blotting (immunoblotting) was performed as described above, except that the antigens were not preheated and 2-mercaptoethanol and SDS were omitted from the sample and running buffers. The molecular size standards (Bio-Rad) included myosin (200 kDa), P-galactosidase (115 kDa), and the four larger markers indicated above (97, 66, 45, and 31 kDa), all run under nonreducing, nondenaturing conditions. Combined 2D-IEP and autoradiography was performed as detailed elsewhere (4, 6). In brief, 0.9 mg of affinity-purified MAb was radiolabeled with 1 mCi of 1251 (Amersham Corp., Arlington Heights, Ill.) by using 4-N-chlorobenzenesulfonamide-derivatized polystyrene beads (IODO-BEADS; Pierce Chemical Co., Rockford, Ill.). After passage over a Presto desalting column (Pierce), 0.12 mg of the 125I-labeled MAb (specific activity, 0.4 mCi/mg) was admixed with goat antiCDN or goat anti-IDCF serum in the second-dimension gel in 2D-IEP, and after electrophoresis, the gels were washed, dried, and then placed in direct contact with X-ray film at -70°C. Corresponding duplicate gels were stained with Coomassie blue to identify reference precipitin peaks. Enzymatic digestion. The effect of various enzymes on the binding of the IDCF antigen to MAb was examined by using a modified ELISA (12). For this procedure, CDN was immobilized on polystyrene plates, blocked with PBS-TW, and then incubated for 2 h at 37°C with glycolytic or proteolytic enzymes diluted in PBS (pH 6.5). The enzymes were a-mannosidase (0.5 U/ml), glucose oxidase (0.26 U/ml), endoglycosidase H (0.005 U/ml), pronase (45 U/ml), and trypsin (4,500 U/ml), all from Sigma Chemical Co. After incubation, the wells were washed with PBS-TW and assayed for reactivity with the MAb by using the ELISA described above. The degradative effects of pronase and lipase were further examined by using a modification of the immunoblotting procedure (19). After CDN was electrophoresed in SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes, strips were cut out and incubated in 4.5 ml of PBS (pH 6.5) containing pronase (3 U/ml) or lipase (1.5 UIml). Following a 90-min incubation at 24°C, the strips were washed with PBS-TW and assayed for reactivity with the MAb by immunoblotting as described above.
RESULTS Reactivity of the MAb in 2D-IEP. The MAb was not detectable as a precipitin when analyzed either in the IDCF or by 2D-IEP. Its reactivity against the IDCF precipitinogen was demonstrable, however, by using a combination of 2D-IEP and autoradiography, wherein 1251I-labeled MAb was admixed with reference precipitin antisera in the seconddimension gel. The results established that radiolabeled MAb bound to the CDN-F component that was precipitated by monospecific goat anti-IDCF serum (compare Fig. 1A and B). The epitope recognized by the MAb appeared to be specific for the IDCF antigen, as evidenced by the lack of reactivity of the radiolabeled MAb with other CDN-F precipitinogens which were detected by using reference goat anti-CDN serum (compare Fig. 1C and D). Reactivity of the MAb in immunoblots. The specificity of the MAb was further evaluated by Western blotting. Figure
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FIG. 1. Reactivity of the MAb in 2D-IEP of CDN. After first-dimension gel electrophoresis, CDN-F (150 ,ug) was electrophoresed against a second-dimension gel containing goat antiserum (400 jig/cm2 of gel) admixed with 1251I-labeled MAb (0.02 jig/cm2 of gel; specific activity, 0.4 mCi/mg). The IDCF precipitinogen seen on the Coomassie blue-stained gel of CDN-F against goat anti-IDCF (A) was reactive with the 1251I-MAb in the corresponding autoradiogram (B). Second-dimension electrophoresis of CDN-F against goat anti-CDN (300 jig/cm2 of gel) yielded several precipitinogens on the Coomassie blue-stained gel (C), including the IDCF and the tube precipitin (TP) antigens (2, 4). The 251I-MAb was specific for the IDCF antigen (D).
2A depicts the results obtained in immunoblots of CDN-L and reference IDCF antigen probed with mouse MAb, goat anti-IDCF serum, or goat anti-CDN serum. Whereas hyperimmune goat anti-CDN serum demonstrated multiple bands in CDN-L and in the IDCF antigen, the mouse MAb and goat anti-IDCF serum were selective for a couplet band having a molecular size distribution of 43 to 45 kDa. The interpretation that this is a couplet band, rather than two distinct components, is based upon the fact that the couplet was detected by the MAb and the reference goat anti-IDCF sera, both of which were specific for the IDCF antigen by 2D-IEP (Fig. 1). Since the couplet band could represent breakdown products of the native IDCF antigen as a result of the denaturing conditions used in SDS-PAGE, immunoblots were also prepared by using nondenaturing, nonreducing conditions for electrophoretic separation of antigen. As shown in Fig. 2B, immunoblots of native antigen yielded a single band having a molecular size of 110 kDa. Heat treatment of the native antigen for 30 min at 60°C ablated its reactivity with MAb (Fig. 2B). Epitope characterization. Characterization of the antigenbinding site was accomplished by in situ enzymatic digestion of CDN-F which had been immobilized on polystyrene plates. This technique allowed small quantities of enzyme and antigen to be used and offered a quantitative assessment of enzymatic degradation. As shown in Table 1, treatment with pronase virtually eliminated reactivity with the MAb;
trypsin effected a 42% reduction in epitope reactivity. In contrast, no degradation was observed after incubation of the immobilized antigen with glucose oxidase, ot-mannosidase, or endoglycosidase H. The degradative effect of pronase on the 43- to 45-kDa couplet band was directly visualized by enzyme treatment of CDN-F which had been electrophoretically separated and immobilized on nitrocellulose strips. This procedure allowed enzymes to be removed from the system, as described above, and ensured that alterations in antigen size by enzymes would not be confused with epitope degradation. As shown in Fig. 3, pronase treatment ablated the reactivity of the 43- to 45-kDa couplet band with MAb; lipase was without effect on epitope recognition. Heat treatment of CDN-F at 60°C for 30 min before application to SDS-PAGE also ablated epitope recognition (Fig. 3). The preceding results, taken together, provide strong evidence that the MAb is directed against a heat-labile peptide epitope. Lack of reactivity of MAb with blastomycin and histoplasmin. The specificity of the epitope recognized by the MAb was assessed by immunoblot assays of blastomycin and histoplasmin. Hyperimmune rabbit antisera revealed the presence of multiple antigens in the two heterologous extracts (Fig. 4). None of these antigens were reactive with the MAb. To exclude the possibility that the denaturing conditions used in immunoblotting might have altered a crossreactive epitope, the specificity of the MAb was further
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FIG. 2. Reactivity of the MAb in immunoblots. (A) Immunoblot of CDN-L (15 p.g) and IDCF antigen (15 jig) prepared under reducing, denaturing conditions. Note that the MAb (ascites diluted 1:25) and goat anti-IDCF serum (30 ,ug/ml) were reactive with a 43- to 45-kDa couplet band. (B) Immunoblot of CDN-L (45 ,ug) prepared under nonreducing, nondenaturing conditions. Note that the MAb recognized a 110-kDa band that was heat labile. Molecular mass markers (in kilodaltons) are shown on the left.
evaluated in the ELISA. The results, shown in Table 2, did any detectable level of cross-reactivity.
not reveal
DISCUSSION We report the production of a murine IgG subclass 1 MAb against a heat-labile peptide epitope of the IDCF antigen. The MAb was not reactive with other CDN antigens or with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis, indicating that the epitope is specific for the IDCF antigen. Our initial attempts to produce an IgG MAb to the IDCF antigen were unsuccessful, even though the mice had serum IgG antibodies to the IDCF antigen. Intrasplenic injection of antigen has been used by other investigators as a route for primary immunization (7, 17) and was adapted in the present study to administer the final antigen boost. The intrasplenic route was found to enhance the production of IgG-secreting hybridomas to the IDCF antigen and other CDN antigens as well. The MAb was not detectable as a precipitin, but its reactivity against the IDCF precipitinogen was demonstrable TABLE 1. Effect of enzyme treatments on epitope recognition as assessed by the ELISA' Enzyme
A410 None Pronase Trypsin Glucose oxidase a-Mannosidase
Endoglycosidase H
1.069 0.063 0.623 1.230 1.175 1.211
by using a combination of 2D-IEP and autoradiography. When the radiolabeled MAb was admixed with goat antiCDN serum in the second-dimension gel, the MAb was observed to specifically bind the IDCF precipitin peak. When used to probe immunoblots of CDN prepared under denaturing, reducing conditions, the MAb recognized a 43to 45-kDa couplet band. In contrast, a 110-kDa band was detected on immunoblots prepared under nondenaturing conditions. These combined results provide evidence that the 43- to 45-kDa couplet band represents degraded compo-
Treatment of CDN untreated
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a CDN-F was immobilized on polystyrene wells and then incubated with buffer alone or with buffer containing enzyme. After a 90-min incubation, the treated and nontreated antigen-coated wells were evaluated for reactivity with the MAb. b Percent reduction as compared with the A410 of nontreated antigen-coated wells.
FIG. 3. Epitope characterization by immunoblotting. CDN-L (15 ,ug) was subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose. Strips containing the immobilized antigen were incubated in buffer alone or in buffer containing enzyme, and after removal of enzyme by washing, the strips were probed with the MAb (ascites diluted 1:25). Also shown is an immunoblot of CDN-L which had been heated to 60°C for 30 min before application to SDS-PAGE. Note that the reactivity of the MAb with the 43- to 45-kDa couplet was abolished by pronase or heat treatment. A molecular mass marker (45 kDa) is shown on the left.
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phase extracts of H. capsulatum or B. dermatitidis. This specificity indicates that the MAb will be useful as a biospecific ligand for the purification of the IDCF antigen from heterogeneous extracts and to screen genomic and cDNA expression libraries prepared from C. immitis (14, 20) for the production of a recombinant product. ACKNOWLEDGMENT This work was supported by Public Health Service grant A121431 from the National Institute of Allergy and Infectious Diseases.
FIG. 4. Specificity of the MAb (ascites diluted 1:25) as assessed in immunoblots of histoplasmin and blastomycin. Histoplasmin (diluted 1:2) and blastomycin (diluted 1:2) contained multiple antigens when assayed against rabbit anti-histoplasmin (diluted 1:1,000) and rabbit anti-blastomycin (diluted 1:1,000) sera, respectively. None of the antigens were reactive with the MAb.
REFERENCES 1. Campbell, C. C., and G. E. Binkley. 1953. Serologic diagnosis with respect to histoplasmosis, coccidioidomycosis, and blastomycosis and the problem of cross reactions. J. Lab. Clin. Med. 42:896-906. 2. Cox, R. A., and L. A. Britt. 1985. Antigenic heterogeneity of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis. Infect. Immun. 50:365-369. 3. Cox, R. A., L. A. Britt, and R. A. Michael. 1987. Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum. Infect. Immun. 55:227-232. 4. Dolan, M. J., R. A. Cox, V. Williams, and S. Wooley. 1989. Development and characterization of a monoclonal antibody
against the tube precipitin antigen of Coccidioides immitis.
nents of the native 110-kDa antigen. The antigen may be a protein multimer with components of various sizes as a result of posttranslational differences, or the effects of heat and SDS may cause a progressive or asymmetric cleavage of a native monomeric molecule. Zimmer and Pappagianis (21) reported earlier that the IDCF activity of CDN was eluted in a fraction having an average molecular size of 110 kDa by molecular-sieve chromatography. Immunoblots of the chromatographically purified IDCF antigen against IDCF-positive sera of coccidioidomycosis patients yielded a 110-kDa band when electrophoresis was performed under nondenaturing conditions, whereas a 48- to 50-kDa band was detected in blots prepared under denaturing conditions (21). The epitope recognized by the MAb was identified as a heat-labile peptide on the basis of the finding that immunologic reactivity was completely abolished after treatment with heat or pronase and partially reduced after enzymatic digestion with trypsin. Zimmer and Pappagianis (21), in analyses of their chromatographically purified IDCF antigen, also observed that heat or pronase treatment ablated the reactivity of the 48-kDa IDCF band in immunoblots against IDCF-positive human sera. They reported a variable effect with trypsin, showing elimination of reactivity on denaturing, reducing blots of a filtrate prepared from strain Silveira but retention of reactivity by using a pooled filtrate of 22 strains of C. immitis. The MAb appears to recognize an epitope that is specific for the IDCF antigen on the basis of the finding that the MAb was not reactive with antigenic components in mycelialTABLE 2. Specificity of MAb as assessed by the ELISA A410 of target antigen Antibody CDN-F Blastomycin Histoplasmin 0.019 0.015 0.615 MAb 0.122 0.651 1.631 Goat anti-CDN 0.727 0.784 0.590 Rabbit anti-histoplasmin 0.613 0.381 0.658 Rabbit anti-blastomycin
Infect. Immun. 57:1035-1039. 5. Gefter, M. L., D. H. Marulies, and M. D. Scharff. 1977. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 3:231-236. 6. Harboe, M., and J. Ivanyi. 1987. Analysis of monoclonal antibodies to Mycobacterium leprae by crossed immunoelectrophoresis. Scand. J. Immunol. 25:133-138. 7. Hong, T. H., S. T. Chen, T. K. Tang, S. C. Wang, and T. H. Chang. 1989. The production of polyclonal and monoclonal antibodies in mice using novel immunization methods. J. Immunol. Methods 120:151-157. 8. Huppert, M., J. P. Adler, E. H. Rice, and S. H. Sun. 1979. Common antigens among systemic disease fungi analyzed by two-dimensional immunoelectrophoresis. Infect. Immun. 23: 479-485. 9. Huppert, M., and J. W. Bailey. 1965. The use of immunodiffusion in coccidioidomycosis. I. The accuracy and reproducibility of the immunodiffusion test which correlates with complement fixation. Am. J. Clin. Pathol. 44:364-368. 10. Huppert, M., J. W. Bailey, and P. Chitjian. 1967. Immunodiffusion as a substitute for complement fixation and tube precipitin tests in coccidioidomycosis, p. 221-225. In L. Ajello (ed.), Proceedings of the Second Coccidioidomycosis Symposium. University of Arizona Press, Tucson. 11. Kaufman, L., and M. J. Clark. 1974. Value of the concomitant use of complement fixation and immunodiffusion tests in the diagnosis of coccidioidomycosis. Appl. Microbiol. 28:641-43. 12. Mitchell, C. G., R. Smith, and T. Lehner. 1987. Recognition of carbohydrate and protein epitopes by monoclonal antibodies to a cell wall antigen from Streptococcus mutans. Infect. Immun.
55:810-815. 13. Pappagianis, D., C. E. Smith, G. S. Kobayashi, and M. T. Saito. 1961. Studies of antigens from young mycelia of Coccidioides immitis. J. Infect. Dis. 108:35-44. 14. Resnick, S., G. Apodaca, G. Newport, C. Halde, and J. McKerrow. 1987. Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, D-48, p. 80. 15. Smith, C. E., M. T. Saito, R. R. Beard, R. M. Kepp, R. W. Clark, and B. U. Eddie. 1950. Serological tests in the diagnosis and prognosis of coccidioidomycosis. Am. J. Hyg. 52:1-21. 16. Smith, C. E., M. T. Saito, and S. A. Simons. 1956. Pattern of 39,500 serologic tests in coccidioidomycosis. JAMA 160:546552. 17. Spitz, M. 1986. "Single-shot" intrasplenic immunization for the production of monoclonal antibodies. Methods Enzymol. 121: 33-41.
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18. Wood, J. C., G. Friedly, M. Zartarian, S. Aarnaes, and L. M. de la Maza. 1982. Alternatives to the standardized laboratory branch complement fixation test for detection of antibodies to Coccidioides immitis. J. Clin. Microbiol. 16:1030-1033. 19. Woodward, M. P., W. W. Young, and R. A. Bloodgood. 1984. Detection of monoclonal antibodies specific to carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78:
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143-153. 20. Yuan, L., J. H. McKerrow, and G. T. Cole. 1988. Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, F-22, p. 395. 21. Zimmer, B., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion complement fixation antigen. J. Clin. Microbiol. 26:2250-2256.
