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Polyclonal antibody characterization of Babesia caballi antigens
Inremo~ionol
Pergamon
Journalfor Parasrlology, Vol. 24, No. 4, pp. 51 l-517, 1994 Copyright 0 1994 Australian Society for Parasitology Elsevier Science Ltd Prmted in Great Britain. All rights reserved 002%7519/94 $7.00+ 0.00
0020-7519(93)EOOO3-R
POLYCLONAL
ANTIBODY CHARACTERIZATION CABALLI ANTIGENS REINHARD
OF BABESIA
BOSE
Institute of Parasitology, School of Veterinary Medicine, Biinteweg 17, 30559 Hannover, Germany (Received 22 June 1993; accepted
IO October
1993)
Abstract-BOSE R. 1994. Polyclonal antibody characterization of Babesia caballi antigens. international Journal for Parasitology 24: 51 l-517. In a previous study diagnostic B. caballi antigens with apparent molecular mass of 50 and 48 kDa were identified. Another antigen of 141 kDa was recognized by most but not all B. caballi sera tested. Here a further characterization of the three antigens is reported. Rabbits were vaccinated with gel-purified antigens and monospecific antibodies were obtained for the 141 and 48 kDa antigens. Antibodies raised against the 50 kDa antigen cross-reacted with the 48 kDa antigen, suggesting that these two antigens bear unique as well as common epitopes. After two-dimensional electrophoresis the 50 and 48 kDa antigens were present as horizontal bands over a pH range from approximately 5.0-7.0 with focused spots at a pH of 5.5 and 5.9, respectively. The 141 kDa antigen was not present after twodimensional electrophoresis. None of the three antigens could be identified as a glycoprotein. Judging from the immunofluorescence antibody test staining pattern obtained with the rabbit sera the 141 kDa antigen is present on the surface of infected erythrocytes. The 50 and 48 kDa antigens are located in the parasite itself and probably not on the surface of infected erythrocytes. The punctate staining pattern observed with the 48 kDa antiserum suggests that this antigen might be located in or associated with the apical complex of the parasite. INDEX KEY electrophoresis;
WORDS: Babesia caballi; antigen Western blotting; immunofluorescence
characterization; antibody test.
SDS-PAGE;
two-dimensional
other major antigen bands of apparent molecular mass of 50 and 48 kDa were consistently recognized by sera from infected horses. In this study a further characterization of the 141 kDa and the diagnostic antigens of 50 and 48 kDa of B. caballi is reported.
INTRODUCTION
babesioses are widespread throughout most countries in the tropical and subtropical regions of the world (Friedhoff, 1982; Friedhoff, Tenter & Miller, 1990). Other countries like the U.S.A., Australia and Japan are largely or completely free from the diseases ahd, consequently, have implemented quarantine regulations to prevent the introduction of infected equines or ticks. Generally, horses are tested by the complement fixation test (CFT) or the immunofluorescence antibody test (IFAT) prior to importation (Friedhoff et al., 1990). As both tests lack sensitivity and specificity, efforts have been made towards the development of improved serological tests. For Babesia equi diagnostic antigens have been identified by the generation of monoclonal antibodies (Knowles, Perryman, Kappmeyer & Hennager, 1991). For B. caballi the humoral immune response of horses was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western analysis (B&e & Daemen, 1992). Seven major babesial antigens were demonstrated. The 141 kDa was the most abundant antigen and recognized by most but not all sera from infected horses. Two EQUINE
MATERIALS
AND METHODS
Antigenpreparation.Antigen was obtained from microaerophilous stationary phase (MASP) cultures (Levy & Ristic, 1980) as described before (B&e & Daemen, 1992). Briefly, MASP cultures initiated from blood of a donor pony infected with the USDA strain (Tenter & Friedhoff, 1986) were grown to about 4% of parasitized erythrocytes. A preparation of about 100% parasitized cells was obtained using Percoll gradients (Bhushan, Miiller & Friedhoff, 1991). Antigen purifcarion. Antigen was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) using the system devised by Laemmli (1970) as described before (B&e & Daemen, 1992). Infected erythrocytes were solubilized in SDS sample buffer and separated on a preparative lo%, 0.75-mm-thick gel. Gels were stained with colloidal Coomassie Brilliant Blue (Neuhoff. Arold, Taube & Ehrhardt, 1988). The babesial antigen bands of apparent molecular mass of 141, 50 and 48 kDa were excised. Proteins 511
512
R. B&t
were removed from the gel strips by electroelution (50 V, 16 h) using a commercially available apparatus (Centrilutor. Amicon, Witten, Germany). Samples were concentrated to a final volume of approx. 100 ~1 by ccntrifugation in microconcentration units (Centricon 10, Amicon, Witten* Gcrinany) in a fixed angle rotor (5000 g, 60 min, 20°C). Vutrinarion of rubbits. For each vaccination. antigen electroeluted from 2 preparative gels was used. Antigens were emulsified in an equal volume of Freund’s complete adjuvant (Sigma. Deisenhofen, Germany) and injected S.C. Rabbits were boosted 3,6 and 7 weeks after the first vaccination and bled I week after the last vaccination. Wu.cfern
described
htotfh~.
previously
Western blotting (B&c & Daemen.
was carried 1992).
out
as
290
It~?nlr~~~c?f~~~ore~~n~~~ cmlihody tot (IFA T). Antigen
was prepared from MASP cultures (Biise & Daemen. 1992) and the IFAT done according to Tenter & Friedhoff (i986). Incubation with horse or rabbit sera was followed by a set of washes and an incubation with rabbit anti-horse IgG (H + L) DTAF or goat anti-rabbit IgG (H + L) DTAF (Dianova, Hamburg, Germany). T~t~o-riinlc~~.s~~~n~~nrr( grf rlectrophowsis. Two-dimensional electrophoresis (O’Farreli. 1975) was carried out in a con~~~erciallya~ailableelectrophoresis unit (Mini Protean II. Bio-Rad Laboratories, Richmond. U.S.A.) essentially according to published procedures (Bollag & Edelstein. 199 I ). Isoelectric focusing (IEF) gels were cast as l-mm-thick 5% polyacrylamide gels containing 9.15 M urea. 2%) (v:v) Nonidet P-40, 4% (v/v) ampholines pH 5 7. 1% (v:v) ampholines pH 3.5.. IO. and 0.4% (v:v) ampholincs pH 7-Y. Gels were allowed to polymerize. the sample slots rinsed with 8 M urea and gels pre-focused for I5 min at 300 V. Subsequently. slots were rinsed with upper buffer and one gel was loaded with IEF sumplc buffer (9.5 M urea. ?‘%I (v/v) Nonidet P-40, SO rnh/l dithiothreitol (DTT). 2% (v/v) ampholines pH 3.5-10) only. These gels were used to determine the pH gradient by cutting horizontal strips from tbc gel and measuring the pH after equilibration in water. The replica gels were loaded with B. cohailiantigen which had been solubilized in IEF sample buffer, centrifuged (lOO,OOOg, 60 min. 20°C) and the supernatant filtcrcd through a hollow tibre filtration unit (Dynagard. 0.2 pm. Microgon. Laguna Hills. U.S.A.). Antigen extracted from 12/1l of lOO”/o infected erythrocytcs \yas applied per gel. Gels wcrc run for 20 min at 100 V followed by 300 V for I6 h. From this gel vertical strips were cut, incubated with equilibration buKer (50 mM Tris pH 6.X. 6 M urea. 2%) (w/v) SDS, 50 mM DTT, 10%) (v:v) glycerol) for 30 min. frozen and stored at --80°C. Gel strips were thawed as needed. equilibrated for a further 30 min and loadedonto thesecond dimension 10% SDS polyacryiamide gel. For SDS-PAGE the system devised by Lacmmli (1970) was used as described before (Biise & Daemen. 1992). Gels wcrc stained with colloidal Coomassic Brilliant Blue (NeuhofT~,/ rrl.. 1988) or blotted as described before (B&e Br Daemen. 1992).
&r<,c,/ir>,z of~!1‘c’ctf,f’ofcirr.r. Glycoproteins
were detected on
1
2
3
Fit;. I. Immunoreactivity of purified Bohrsic~ c~uhrrlli antigens. 5. cnhalli antigens were electroeluted from preparative SDS-PAGE gels. Aliquots of the I41 (lane I). the 50 (lane 2) and the 48 (Iane 3) kDa antigens were re-run on SDS PAGE gels. hiotted and probed with a pool of sera from horses infected with B. &al/i. Molecular mass markers are shown on the left (in kDa).
29-
: 1
3
FK;. 2. Reactivity of rabbit sera with &iih&r (,~~b~~~/i antigens on Western blots after SDS-PAGE. Crude 8. ~ll~~~fli antigen was separated by SDS PAGE and eiectrohlotted. Membrane strips were probed with a pool of sera from horses infected with B. ccrhdi as B control (lane I). with serum from a rabbit vaccinated with the purified B. ctrhtrlli antigen of 141 kDa (lane 2). 50 kDa (lane 3) or 48 kDa (lane 4). Molecular mas? markers arc shown on the left (in kDa).
7’ ZOO-
To
513
‘P
A
w4-68-
43-
2920&-
B . -
9=z468-
43-
29200--
97468-
43-
29FIG. 3. (A-C). Reactivity of rabbit sera on Western blots after two-dime~sio~l cleotrophoresis. crude &&es&zc~~u~~~ant~geo was separated by isoelectric focusing (IEF) and SDS--PAGE and electroblotted. Blots were probed with a pool of sera fram horses infected with B. cub& (A), serum from a rabbit vaccinated with the 141 kDa antigen (B) or with the 50 kDa antigen of B. cubdi (C). Figures on the top indicate the Ph gradient in the first dimension IEF gel, figures on the left indicate position of molecular mass markers (in kDa) in the second dimension SDS gel. A lane ofcrude 3. c~~~~~anti~en soiubilized in SDS sample buffer was also run an the second dimension gel and is shown on the right margin of each blot.
514
R. B&E
Western blots using a commercially available kit (Boehringer Mannheim, Germany) according to the manufacturers instructions. The method is based on the oxidation of the hydroxyl groups of sugar residues to aldehyde groups by periodate treatment. To these, digoxigenin is coupled via a hydrazide group. Digoxigenin is detected with a specific antibody coupled to alkaline phosphatase (Kniep & Miihlradt, 1990).
RESULTS
By electroelution from preparative gels pure antigen fractions were obtained, i.e. single bands were observed on SDS gels after staining with colloidal Coomassie Brilliant Blue (data not illustrated). When blotted these antigen fractions were immunoreactive with sera from horses infected with B. cabal/i (Fig. 1). Upon vaccination with these fractions monospecific antibodies were raised in rabbits against the 141 and 48 kDa antigens; antibodies from the rabbit vaccinated with the 50 kDa antigen cross-reacted with the 48 kDa antigen band (Fig. 2). When analysed by twodimensional electrophoresis and Western blotting the 50 and 48 kDa antigens appeared as horizontal lanes after probing with the pool of equine sera (Fig. 3A). Monospecific antibodies directed against the 141 kDa antigen did not recognize an antigen of this molecular mass after two-dimensional electrophoresis (Fig. 3B). When probed with the rabbit serum against the 50 and 48 kDa antigens focused spots with a pI of 5.5 and 5.9 were observed (Fig. 3C). The serum directed against the 48 kDa antigen was not reactive on Western blots after two-dimensional electrophoresis. The 141, 50 and 48 kDa antigens could not be identified as glycoproteins with the detection method used (Fig. 4). With the equine serum pool a staining of the infected erythrocytes and the parasites was seen (Fig. 5A). Antibodies directed against the 141 kDa antigen showed a staining of infected erythrocytes with no parasite structures visible (Fig. 5B). In contrast, the antiserum directed against the 50 and 48 kDa antigens showed a distinct parasite specific staining (Fig. 5C), whereas the erythrocyte surface was not stained. A pun&ate staining was observed with the anti-48 kDa serum (Fig. 5D).
three antigens. Blotting results with the rabbit sera (Figs. 2 and 3) indicate that epitopes present on the 141,50 and 48 kDa antigens are not found on other B. caballi antigens. However, there appear to be antigenic similarities between the 50 and 48 kDa antigens. The fact that the antibodies raised against the 50 kDa antigen cross-reacted with the 48 kDa antigen, whereas antibodies raised against the 48 kDa antigen did not cross-react, indicates that these two antigens bear unique as well as common epitopes. It is unlikely that the cross-reaction is due to the possibility that the rabbit vaccinated with the 50 kDa antigen received antigen from the 48 kDa antigen band, as pure fractions were used for vaccination. After two-dimensional electrophoresis the 50 and 48 kDa antigens appeared as horizontal bands with focused spots at a p1 of 5.5 and 5.9. This indicates that there is probably a large number of antigens with incremental charge differences but identical molecular mass. Typically a post-translational modification of proteins would cause this. However, with the glycoprotein detection method used this could not be ascertained. It is possible that a post-translational modification other than glycosylation, e.g. phosphor-
DISCUSSION
In a previous study the 50 and 48 kDa antigens were identified as targets for the design of an immunodiagnostic test for B. cabdi infections as those antigens were recognized by all B. caballi sera tested (B&e & Daemen, 1992). The 141 kDa antigen was the most abundant antigen and recognized by most but not all B. caballi sera. For this study rabbits were vaccinated with gel-purified antigens to further characterize these
FIG. 4. Detection ofglycoproteins in a Babesiu caballiandgen preparation. Crude B. caballiantigen was separated by SDSPAGE and electroblotted. Membrane strips were probed with a pool of sera from horses infected with B. caballi as a control (lane 1) or used to demonstrate glycoproteins (lane 2). Molecular mass markers are shown on the left (in kDa).
B. caballi antigens
515
FIG. 5. (A-D) Staining pattern of sera from rabbits vaccinated with purified Bubesiu cabdi antigens in the immunofluorescence antibody test. Antigen slides were reacted with a pool of sera from horses infected with B. cabdi as a control (A) or with rabbit sera raised against the 141 (B), 50 (C) or 48 kDa antigens (D) of B. caballi.
516
R. BOSE
ylation, contributed to the pattern observed after twodimensional electrophoresis. Alternatively, it is possible that incomplete solubilization of these antigens has contributed to the behaviour in the two-dimensional electrophoresis. In preliminary studies different detergents were tried for the solubilization of B. caballi antigens. For equine sera as well as for rabbit sera highest ELISA reactions were observed when the crude antigen was solubilized with CHAPS, urea or Nonidet P-40. Consequently urea and Nonidet P-40 were also used for solubilization of antigen for the IEF. It was noted, however,that after solubilization in IEF sample buffer and centrifugation, a clearly visible pellet was remaining. With SDS it was possible to extract babesial antigens from this pellet, the most prominent being the 50 and 48 kDa antigens (data not shown). Thus it appears that these antigens are difficult to solubilize completely or tend to self-aggregate. The diffuse IFAT staining pattern observed with the rabbit sera directed against the 141 kDa antigen indicates that this antigen is present on the surface of infected erythrocytes; it appears to be excreted by the parasite. In contrast to this, the 48 and 50 kDa antigens appear to be located in the parasite itself and are probably not excreted or present on the surface of infected erythrocytes. A punctate staining as seen with the 48 kDa antiserum has been demonstrated for antibodies directed against geographically conserved B. bovis antigens (Gaff, Davis, Palmer, McElwain, Johnson, Bailey & McGuire, 1988; Palmer, McEIwain, Perryman, Davis, Reduker, Jasmer, Shkap, Pipano, Goff & McGuire, 1991). One of these, a 60 kDa protective antigen, is located in the rhoptries of the parasite (Suarez, Palmer, Jasmer, Hines, Perryman & McElwain, 1991; Suarez, McElwain, Stephens, Mishra & Palmer, 1991). In contrast, antibodies with a diffuse staining pattern recognized variable antigens (Palmer et al., 1991). Similarly, for B. caballi, the 141 kDa antigen with a diffuse IFAT staining pattern was not recognized by all sera from infected horses and therefore its diagnostic value is limited. In contrast, the 48 kDa antigen with a punctate pattern was recognized by all B. cub& sera tested so far (Bose & Daemen, 1992; Bose & Peymann, in press; Bose, Peymann & Pfeifer Barbosa, in press). Thus, this and the 50 kDa antigen remain as prime candidates for a diagnostic test based on recombinant antigens.
Ac.knoalrd~m~mts-1 wish to thank Prof. Dr K. T. Friedhoff and Drs B. Peymann and B. Hentrich for review of the manuscript and Mrs K. Daemen and Mr M. Wolfhagen for able technical assistance.
REFERENCES BHUSHAN C., M~JLLER 1. & FRIEDHOFF K. T. 1991. Enrichment of Bahesia c&&i-infected erythrocytes from microaerophilous stationary-phase cultures using Percoll gradients. Parasitology Research 71: 177-l 79. BOLLAG D. M. & EDELSTEIN S. J. 1991. Protein Merhodr.
WileyyLiss, New York. BOSE R. & DAEMEN K. 1992. Demonstration
of the humordl immune response of horses to Babesia cahalli by Western blotting. International Journal .fiw Parasitology 22: 627630. BOSE R. & PEYMANN B. (in press). Diagnosis of Bohrsicr cahalli infections in horses by enzyme-linked immunosorbent assay (ELISA) and Western blot. International Journal for Parasitology. B&E R.. PEYMANN B. & PFEIFER BARBOSA I. (in press). Identification of diagnostic antigens for South American Babesiu cuhalli infections. Internotional Journul,fi,r Parusitology. FRIEDHOF~ K. T. 1982. Die Piroplasmen der Equiden Bedeutung fur den internationalen Pferdeverkehr. Berlitwr und Miinchener Tieriirztliche Wochenschrift 95: 368 374. FRIEDHOF~ K. T., TENT~R A. M. & MCLL~R I. 1990. Haemoparasites of equines: impact on international trade of horses. Revue Scient$que et Technique Cyi-icr International de.7 Epizooties 9: I l87- I 194. GOFF. W. L., DAVIS W. C., PALMER G. H., MCELWAIN T. F.. JOHNSON W. C., BAILEY J. F. & MCGUIRE T. C. 1988. Identification of Bubesiu hovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies. Infection and Immunity 56: 2363-2368. KNIEP B. & M~~HLRADT P. F. 1990. Immunochemtcal detection of glycosphingolipids on thin-layer chromatograms. Analytical Biochemisty 188: S-8. KNOWLES D. P., PERRYMAN L. E., KAPPMEYEK L. S. & HENNAGER S. G. 1991. Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. Journal of’ Clinicul Miwohiology 29: 20562058. LAEMMLI U.K. 1970. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nuturc, London. 227: 680 685. LEVY M. G. & RISTIC M. 1980. Bahesia bovis: continuous cultivation in microaerophilous stationary phase culture. Science 207: 1218-l 220. NE~JHOEF V.. AKOLUN.. TAIJBE D. & EHRHARDT W. 1988 Improved staining of proteins in polyacrylamidc gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G250 and R-250. Electrophoresi.c 9: 255--262. O’FARRELL P. H. 1975. High resolution two-dimensional electrophoresis ofproteins. JournolofBiological Chemlstr,, 250: 4007402 I. PALMER G. H., MCELWAIN T. F., PERRYMAN L. E.. DAVIS W. C., REIXKER D. R., JASMFR D. P.. SHKAP V., PIP,ZNO E., Gott W. L. & MCG~JIRET. C. 1991. Strain variation of Bahrsia hovis merozoite surface-exposed epitopes. //r/cc,/roar und Immunity 59: 3340 3342.
B. caballi antigens SUAREZ C. E., MCELWAIN T. F., STEPHENS E. B., MISHRA V. S. & PALMER G. H. 1991. Sequence conservation among merozoite apical complex proteins of Bubesiu bovis, Babesia bigemina and other apicomplexa. Molecular and Biochemical Parasitology 49: 329-332. SUAREZ C. E., PALMER G. H.. JASMER D. P., HINES S. A., PERRYMAN L. E. & MCELWAIN T. F. 1991. Characteriza-
517
tion of the gene encoding a 60-kilodalton Babe.& bok merozoite protein with conserved and surface exposed epitopes. Molecular and Biochemical Parasitology 46: 455 52. TENTER A. M. & FRIEDHO~F K. T. 1986. Serodiagnosis of experimental and natural Bubesia equi and B. cab&/i infections. Veterinary Parasitology 20: 49-61.
Pergamon
Journalfor Parasrlology, Vol. 24, No. 4, pp. 51 l-517, 1994 Copyright 0 1994 Australian Society for Parasitology Elsevier Science Ltd Prmted in Great Britain. All rights reserved 002%7519/94 $7.00+ 0.00
0020-7519(93)EOOO3-R
POLYCLONAL
ANTIBODY CHARACTERIZATION CABALLI ANTIGENS REINHARD
OF BABESIA
BOSE
Institute of Parasitology, School of Veterinary Medicine, Biinteweg 17, 30559 Hannover, Germany (Received 22 June 1993; accepted
IO October
1993)
Abstract-BOSE R. 1994. Polyclonal antibody characterization of Babesia caballi antigens. international Journal for Parasitology 24: 51 l-517. In a previous study diagnostic B. caballi antigens with apparent molecular mass of 50 and 48 kDa were identified. Another antigen of 141 kDa was recognized by most but not all B. caballi sera tested. Here a further characterization of the three antigens is reported. Rabbits were vaccinated with gel-purified antigens and monospecific antibodies were obtained for the 141 and 48 kDa antigens. Antibodies raised against the 50 kDa antigen cross-reacted with the 48 kDa antigen, suggesting that these two antigens bear unique as well as common epitopes. After two-dimensional electrophoresis the 50 and 48 kDa antigens were present as horizontal bands over a pH range from approximately 5.0-7.0 with focused spots at a pH of 5.5 and 5.9, respectively. The 141 kDa antigen was not present after twodimensional electrophoresis. None of the three antigens could be identified as a glycoprotein. Judging from the immunofluorescence antibody test staining pattern obtained with the rabbit sera the 141 kDa antigen is present on the surface of infected erythrocytes. The 50 and 48 kDa antigens are located in the parasite itself and probably not on the surface of infected erythrocytes. The punctate staining pattern observed with the 48 kDa antiserum suggests that this antigen might be located in or associated with the apical complex of the parasite. INDEX KEY electrophoresis;
WORDS: Babesia caballi; antigen Western blotting; immunofluorescence
characterization; antibody test.
SDS-PAGE;
two-dimensional
other major antigen bands of apparent molecular mass of 50 and 48 kDa were consistently recognized by sera from infected horses. In this study a further characterization of the 141 kDa and the diagnostic antigens of 50 and 48 kDa of B. caballi is reported.
INTRODUCTION
babesioses are widespread throughout most countries in the tropical and subtropical regions of the world (Friedhoff, 1982; Friedhoff, Tenter & Miller, 1990). Other countries like the U.S.A., Australia and Japan are largely or completely free from the diseases ahd, consequently, have implemented quarantine regulations to prevent the introduction of infected equines or ticks. Generally, horses are tested by the complement fixation test (CFT) or the immunofluorescence antibody test (IFAT) prior to importation (Friedhoff et al., 1990). As both tests lack sensitivity and specificity, efforts have been made towards the development of improved serological tests. For Babesia equi diagnostic antigens have been identified by the generation of monoclonal antibodies (Knowles, Perryman, Kappmeyer & Hennager, 1991). For B. caballi the humoral immune response of horses was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western analysis (B&e & Daemen, 1992). Seven major babesial antigens were demonstrated. The 141 kDa was the most abundant antigen and recognized by most but not all sera from infected horses. Two EQUINE
MATERIALS
AND METHODS
Antigenpreparation.Antigen was obtained from microaerophilous stationary phase (MASP) cultures (Levy & Ristic, 1980) as described before (B&e & Daemen, 1992). Briefly, MASP cultures initiated from blood of a donor pony infected with the USDA strain (Tenter & Friedhoff, 1986) were grown to about 4% of parasitized erythrocytes. A preparation of about 100% parasitized cells was obtained using Percoll gradients (Bhushan, Miiller & Friedhoff, 1991). Antigen purifcarion. Antigen was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) using the system devised by Laemmli (1970) as described before (B&e & Daemen, 1992). Infected erythrocytes were solubilized in SDS sample buffer and separated on a preparative lo%, 0.75-mm-thick gel. Gels were stained with colloidal Coomassie Brilliant Blue (Neuhoff. Arold, Taube & Ehrhardt, 1988). The babesial antigen bands of apparent molecular mass of 141, 50 and 48 kDa were excised. Proteins 511
512
R. B&t
were removed from the gel strips by electroelution (50 V, 16 h) using a commercially available apparatus (Centrilutor. Amicon, Witten, Germany). Samples were concentrated to a final volume of approx. 100 ~1 by ccntrifugation in microconcentration units (Centricon 10, Amicon, Witten* Gcrinany) in a fixed angle rotor (5000 g, 60 min, 20°C). Vutrinarion of rubbits. For each vaccination. antigen electroeluted from 2 preparative gels was used. Antigens were emulsified in an equal volume of Freund’s complete adjuvant (Sigma. Deisenhofen, Germany) and injected S.C. Rabbits were boosted 3,6 and 7 weeks after the first vaccination and bled I week after the last vaccination. Wu.cfern
described
htotfh~.
previously
Western blotting (B&c & Daemen.
was carried 1992).
out
as
290
It~?nlr~~~c?f~~~ore~~n~~~ cmlihody tot (IFA T). Antigen
was prepared from MASP cultures (Biise & Daemen. 1992) and the IFAT done according to Tenter & Friedhoff (i986). Incubation with horse or rabbit sera was followed by a set of washes and an incubation with rabbit anti-horse IgG (H + L) DTAF or goat anti-rabbit IgG (H + L) DTAF (Dianova, Hamburg, Germany). T~t~o-riinlc~~.s~~~n~~nrr( grf rlectrophowsis. Two-dimensional electrophoresis (O’Farreli. 1975) was carried out in a con~~~erciallya~ailableelectrophoresis unit (Mini Protean II. Bio-Rad Laboratories, Richmond. U.S.A.) essentially according to published procedures (Bollag & Edelstein. 199 I ). Isoelectric focusing (IEF) gels were cast as l-mm-thick 5% polyacrylamide gels containing 9.15 M urea. 2%) (v:v) Nonidet P-40, 4% (v/v) ampholines pH 5 7. 1% (v:v) ampholines pH 3.5.. IO. and 0.4% (v:v) ampholincs pH 7-Y. Gels were allowed to polymerize. the sample slots rinsed with 8 M urea and gels pre-focused for I5 min at 300 V. Subsequently. slots were rinsed with upper buffer and one gel was loaded with IEF sumplc buffer (9.5 M urea. ?‘%I (v/v) Nonidet P-40, SO rnh/l dithiothreitol (DTT). 2% (v/v) ampholines pH 3.5-10) only. These gels were used to determine the pH gradient by cutting horizontal strips from tbc gel and measuring the pH after equilibration in water. The replica gels were loaded with B. cohailiantigen which had been solubilized in IEF sample buffer, centrifuged (lOO,OOOg, 60 min. 20°C) and the supernatant filtcrcd through a hollow tibre filtration unit (Dynagard. 0.2 pm. Microgon. Laguna Hills. U.S.A.). Antigen extracted from 12/1l of lOO”/o infected erythrocytcs \yas applied per gel. Gels wcrc run for 20 min at 100 V followed by 300 V for I6 h. From this gel vertical strips were cut, incubated with equilibration buKer (50 mM Tris pH 6.X. 6 M urea. 2%) (w/v) SDS, 50 mM DTT, 10%) (v:v) glycerol) for 30 min. frozen and stored at --80°C. Gel strips were thawed as needed. equilibrated for a further 30 min and loadedonto thesecond dimension 10% SDS polyacryiamide gel. For SDS-PAGE the system devised by Lacmmli (1970) was used as described before (Biise & Daemen. 1992). Gels wcrc stained with colloidal Coomassic Brilliant Blue (NeuhofT~,/ rrl.. 1988) or blotted as described before (B&e Br Daemen. 1992).
&r<,c,/ir>,z of~!1‘c’ctf,f’ofcirr.r. Glycoproteins
were detected on
1
2
3
Fit;. I. Immunoreactivity of purified Bohrsic~ c~uhrrlli antigens. 5. cnhalli antigens were electroeluted from preparative SDS-PAGE gels. Aliquots of the I41 (lane I). the 50 (lane 2) and the 48 (Iane 3) kDa antigens were re-run on SDS PAGE gels. hiotted and probed with a pool of sera from horses infected with B. &al/i. Molecular mass markers are shown on the left (in kDa).
29-
: 1
3
FK;. 2. Reactivity of rabbit sera with &iih&r (,~~b~~~/i antigens on Western blots after SDS-PAGE. Crude 8. ~ll~~~fli antigen was separated by SDS PAGE and eiectrohlotted. Membrane strips were probed with a pool of sera from horses infected with B. ccrhdi as B control (lane I). with serum from a rabbit vaccinated with the purified B. ctrhtrlli antigen of 141 kDa (lane 2). 50 kDa (lane 3) or 48 kDa (lane 4). Molecular mas? markers arc shown on the left (in kDa).
7’ ZOO-
To
513
‘P
A
w4-68-
43-
2920&-
B . -
9=z468-
43-
29200--
97468-
43-
29FIG. 3. (A-C). Reactivity of rabbit sera on Western blots after two-dime~sio~l cleotrophoresis. crude &&es&zc~~u~~~ant~geo was separated by isoelectric focusing (IEF) and SDS--PAGE and electroblotted. Blots were probed with a pool of sera fram horses infected with B. cub& (A), serum from a rabbit vaccinated with the 141 kDa antigen (B) or with the 50 kDa antigen of B. cubdi (C). Figures on the top indicate the Ph gradient in the first dimension IEF gel, figures on the left indicate position of molecular mass markers (in kDa) in the second dimension SDS gel. A lane ofcrude 3. c~~~~~anti~en soiubilized in SDS sample buffer was also run an the second dimension gel and is shown on the right margin of each blot.
514
R. B&E
Western blots using a commercially available kit (Boehringer Mannheim, Germany) according to the manufacturers instructions. The method is based on the oxidation of the hydroxyl groups of sugar residues to aldehyde groups by periodate treatment. To these, digoxigenin is coupled via a hydrazide group. Digoxigenin is detected with a specific antibody coupled to alkaline phosphatase (Kniep & Miihlradt, 1990).
RESULTS
By electroelution from preparative gels pure antigen fractions were obtained, i.e. single bands were observed on SDS gels after staining with colloidal Coomassie Brilliant Blue (data not illustrated). When blotted these antigen fractions were immunoreactive with sera from horses infected with B. cabal/i (Fig. 1). Upon vaccination with these fractions monospecific antibodies were raised in rabbits against the 141 and 48 kDa antigens; antibodies from the rabbit vaccinated with the 50 kDa antigen cross-reacted with the 48 kDa antigen band (Fig. 2). When analysed by twodimensional electrophoresis and Western blotting the 50 and 48 kDa antigens appeared as horizontal lanes after probing with the pool of equine sera (Fig. 3A). Monospecific antibodies directed against the 141 kDa antigen did not recognize an antigen of this molecular mass after two-dimensional electrophoresis (Fig. 3B). When probed with the rabbit serum against the 50 and 48 kDa antigens focused spots with a pI of 5.5 and 5.9 were observed (Fig. 3C). The serum directed against the 48 kDa antigen was not reactive on Western blots after two-dimensional electrophoresis. The 141, 50 and 48 kDa antigens could not be identified as glycoproteins with the detection method used (Fig. 4). With the equine serum pool a staining of the infected erythrocytes and the parasites was seen (Fig. 5A). Antibodies directed against the 141 kDa antigen showed a staining of infected erythrocytes with no parasite structures visible (Fig. 5B). In contrast, the antiserum directed against the 50 and 48 kDa antigens showed a distinct parasite specific staining (Fig. 5C), whereas the erythrocyte surface was not stained. A pun&ate staining was observed with the anti-48 kDa serum (Fig. 5D).
three antigens. Blotting results with the rabbit sera (Figs. 2 and 3) indicate that epitopes present on the 141,50 and 48 kDa antigens are not found on other B. caballi antigens. However, there appear to be antigenic similarities between the 50 and 48 kDa antigens. The fact that the antibodies raised against the 50 kDa antigen cross-reacted with the 48 kDa antigen, whereas antibodies raised against the 48 kDa antigen did not cross-react, indicates that these two antigens bear unique as well as common epitopes. It is unlikely that the cross-reaction is due to the possibility that the rabbit vaccinated with the 50 kDa antigen received antigen from the 48 kDa antigen band, as pure fractions were used for vaccination. After two-dimensional electrophoresis the 50 and 48 kDa antigens appeared as horizontal bands with focused spots at a p1 of 5.5 and 5.9. This indicates that there is probably a large number of antigens with incremental charge differences but identical molecular mass. Typically a post-translational modification of proteins would cause this. However, with the glycoprotein detection method used this could not be ascertained. It is possible that a post-translational modification other than glycosylation, e.g. phosphor-
DISCUSSION
In a previous study the 50 and 48 kDa antigens were identified as targets for the design of an immunodiagnostic test for B. cabdi infections as those antigens were recognized by all B. caballi sera tested (B&e & Daemen, 1992). The 141 kDa antigen was the most abundant antigen and recognized by most but not all B. caballi sera. For this study rabbits were vaccinated with gel-purified antigens to further characterize these
FIG. 4. Detection ofglycoproteins in a Babesiu caballiandgen preparation. Crude B. caballiantigen was separated by SDSPAGE and electroblotted. Membrane strips were probed with a pool of sera from horses infected with B. caballi as a control (lane 1) or used to demonstrate glycoproteins (lane 2). Molecular mass markers are shown on the left (in kDa).
B. caballi antigens
515
FIG. 5. (A-D) Staining pattern of sera from rabbits vaccinated with purified Bubesiu cabdi antigens in the immunofluorescence antibody test. Antigen slides were reacted with a pool of sera from horses infected with B. cabdi as a control (A) or with rabbit sera raised against the 141 (B), 50 (C) or 48 kDa antigens (D) of B. caballi.
516
R. BOSE
ylation, contributed to the pattern observed after twodimensional electrophoresis. Alternatively, it is possible that incomplete solubilization of these antigens has contributed to the behaviour in the two-dimensional electrophoresis. In preliminary studies different detergents were tried for the solubilization of B. caballi antigens. For equine sera as well as for rabbit sera highest ELISA reactions were observed when the crude antigen was solubilized with CHAPS, urea or Nonidet P-40. Consequently urea and Nonidet P-40 were also used for solubilization of antigen for the IEF. It was noted, however,that after solubilization in IEF sample buffer and centrifugation, a clearly visible pellet was remaining. With SDS it was possible to extract babesial antigens from this pellet, the most prominent being the 50 and 48 kDa antigens (data not shown). Thus it appears that these antigens are difficult to solubilize completely or tend to self-aggregate. The diffuse IFAT staining pattern observed with the rabbit sera directed against the 141 kDa antigen indicates that this antigen is present on the surface of infected erythrocytes; it appears to be excreted by the parasite. In contrast to this, the 48 and 50 kDa antigens appear to be located in the parasite itself and are probably not excreted or present on the surface of infected erythrocytes. A punctate staining as seen with the 48 kDa antiserum has been demonstrated for antibodies directed against geographically conserved B. bovis antigens (Gaff, Davis, Palmer, McElwain, Johnson, Bailey & McGuire, 1988; Palmer, McEIwain, Perryman, Davis, Reduker, Jasmer, Shkap, Pipano, Goff & McGuire, 1991). One of these, a 60 kDa protective antigen, is located in the rhoptries of the parasite (Suarez, Palmer, Jasmer, Hines, Perryman & McElwain, 1991; Suarez, McElwain, Stephens, Mishra & Palmer, 1991). In contrast, antibodies with a diffuse staining pattern recognized variable antigens (Palmer et al., 1991). Similarly, for B. caballi, the 141 kDa antigen with a diffuse IFAT staining pattern was not recognized by all sera from infected horses and therefore its diagnostic value is limited. In contrast, the 48 kDa antigen with a punctate pattern was recognized by all B. cub& sera tested so far (Bose & Daemen, 1992; Bose & Peymann, in press; Bose, Peymann & Pfeifer Barbosa, in press). Thus, this and the 50 kDa antigen remain as prime candidates for a diagnostic test based on recombinant antigens.
Ac.knoalrd~m~mts-1 wish to thank Prof. Dr K. T. Friedhoff and Drs B. Peymann and B. Hentrich for review of the manuscript and Mrs K. Daemen and Mr M. Wolfhagen for able technical assistance.
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