production and characterization of mouse monoclonal antibodies against afipia felis

Copyr igh t 0 A P M I S 1997 A P h f I S 105. 192-198, I997 Priiited in Drnibiurk . All righrs resrrved lwklU8

JSSN 0903-4641

Production and characterization of mouse monoclonal antibodies against Afipia felis

KRRSTEN ENGBBK, LARS OTTO UTTENTHAL and CLAUS KOCH

Department of Immunological Research and Development, Immunology Division, Statens Seruminstitut, Copenhagen, Denmark

Engbzk, K., Uttenthal, L. 0. & Koch, C. Production and characterization of mouse monoclonal antibodies against Afpia felis. APMIS 105: 192-198, 1997.

A series of 10 monoclonal antibodies reacting with Afpia felis antigens were selected from mice immunized with live organisms of the reference strain ATCC 53690. Immunoblotting against SDS- PAGE-separated A. felis sonicate allowed the antibodies to be classified into three groups: 1) 168-4, -6, -7 and -10 reacted with a 53 kDa antigen, 2) 168-1, -3 and -9 reacted with both 53 kDa and 60 kDa antigens, and 3) 168-2, -5 and -9 reacted with other antigens. Antibodies of group 1 did not cross-react with other AJpia species or 36 unrelated bacteria, whereas those of groups 2 and 3 reacted with other Afpia species and some unrelated bacteria. Immunoblots of crossed immunoelectrophoretic patterns of A. felis sonicate against rabbit antiserum showed that antibodies of groups 1 and 2 bound to the same precipitin arcs. Antibodies of group 1 reacted with a species-specific epitope on the 53 kDa antigen, while those of group 2 reacted with other epitopes shared by the 53 kDa and 60 kDa antigens. The binding of antibodies of group 1 to A. felis sonicate was inhibited by post-infection rabbit serum, whereas no inhibition was observed for antibodies of group 2. The species-specific epitope of the 53 kDa antigen and the early appearance of antibodies against this epitope after infection suggest that this antigen can be used in a serodiagnostic test for A . felis infection.

Key words: Afpia jelis; cat-scratch disease; monoclonal antibodies; epitopes.

K. Engbzk, Department of Immunological Research and Development, Immunology Division, Statens Seruminstitut, Artillerivej 5 , DK-2300 Copenhagen S, Denmark.

Afipiu felis has had a chequered career as a rec- ognized pathogen in man. After its first isolation in 1988 from patients with cat-scratch disease (CSD), its pathogenic role was later questioned and attention focused on Bartonellu (formerly Rochulimaea) henselue (2, 4, 7, 14). However, a recent investigation employing dot- blot hybridization of PCR amplification prod- ucts from formalin-fixed lymph-node sections from 12 patients with CSD detected A. felis in 3 patients, B. henselue in 5 patients, and both organisms in 2 patients (1). It seems likely that A . felis is involved in at least some cases of CSD.

Received August 29, 1996. Accepted November 18, 1996.

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We have previously used crossed immunoelectrophoresis to estimate the number and specificity of A . felis antigens, and studied the antibody response in rabbits inoculated with live A. felis. These investigations showed that seven antigens of A . felis were confined to that species, but only three of them provoked a detectable antibody response in the inoculated rabbits ( 5 , 6).

Yu & Ruoult (16) have recently produced a series of monoclonal antibodies (MAbs) against A. felis. All were thought to be directed against lipopolysaccharide antigens and were found to be species-specific for A. felis, reacting with both agar-grown and tissue-culture-propagated organisms.

In this communication we describe the estab-

MABs AGAINST AFIPIA FELIS

lishment and characterization of murine hybridoma cell lines producing MAbs against the reference strain of A . felis. The antibodies were characterized with a view to selecting those that might be useful for purifying an antigen that could be used in a sensitive and specific serodiagnostic test for A . ,fe/is infections.

MATERIALS AND METHODS

Struins A. felis ATCC 53690, A. felis ATCC 49714, A. felis

ATCC 49715, A. hroomeue ATCC 49717, A. broomeue ATCC 4971 8, A. ckvelundensis ATCC 49720, A. genospecies 1 ATCC 49721, A . genospecies 2 ATCC 49722 and A. genospecies 3 ATCC 49723 were procured from the American Type Culture Col- lection, Rockville, MD. The other strains used in the study were obtained from the National Collection at the Department of Clinical Microbiology and Tu- berculin, Statens Seruminstitut, Denmark and were the same as previously (5). Eleven of these strains (Legionellu pneumophila ATCC 33 153, Kingella kin- gur ATCC 23330, Moruxellu oslomsis AB 2422, Bor- detrllu pertussis 1291 6, Branhumella caturrhalis U 101, Pseudomonus (Brevundimonas) diminuta AB 1266, Vibrio cholerae ATCC 14374, Enterobucter ae- rogenes U 23, Klebsiellu oxytoca U 2, Proteus miru- bilis U 27 and Mycohucterium tul>erculosis H37 RV) were examined in greater detail.

Antigen preparution The Afipia strains were cultured on buffered char-

coal-yeast extract (BCYE) agar, the haemophilic species on blood agar base no. 2 (Difco 0696) with 5% horse blood, and the remaining species on brain- heart infusion agar (Difco 0418). The preparation of antigens followed the procedure described previously (3, except that 1.5 mM Pefabloc (Boehringer Mann- heim 1429 868) was added as protease inhibitor. Pro- tein concentrations were determined by the dye-binding method of Bradford (3) with bovine serum albu- min (BSA) as the standard.

Post-injection rabbit sera Live A . felis suspension (McFarland turbidity stan-

dard no. 3, 1.5X lo9 CFU/ml) was injected intracut- aneously at a dose of 0.2 ml into two female rabbits. Blood was drawn before and at weekly intervals after the inoculation. Six weeks after the initial inoculation the rabbits were rechallenged with the same dose, and they were again bled weekly for three weeks.

Hyhridoma technique MAbs were produced essentially as described by

Kohler & Milstein (1 I ) and modified by Reading ( 1 3). BALB/c mice (female, 6-8 weeks old) were inoculated

three times at fortnightly intervals with live A . Jelis ATCC 53690 [lo9 organisms in 0.2 ml 10 mM phosphate buffer, pH 7.2, containing 0.13 M NaCl (PBS) given intraperitoneally]. A booster was given intra- venously 3 days before fusion of the spleen cells. Splenic lymphocytes from inoculated mice were fused with myeloma cells of the P3-X63Ag8.6.5.3 line and polyethylene glycol 1500 as the fusogen. Hybridoma supernatants were tested for antibody production by enzyme immunoassay (EIA) and immunoblotting against A. ,felis sonicate. Cells from 10 positive wells were recloned 3 times by limiting dilution (9). For large-scale production of culture supernatants, cloned hybridoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with penicil- lin, streptomycin and 10% fetal calf serum (Bio- chrom, Germany) in Nunc Cell Factories (Nunc, Roskilde, Denmark). Culture supernatants were har- vested twice weekly.

Immunoglobulin subclasses were determined with a mouse monoclonal antibody isotyping kit (Amers- ham, Buckinghamshire, England) according to the manufacturer’s instructions.

Polyucrylamide gel electrophoresis ( P A G E ) Samples were boiled for 4 min with an equal vol-

ume of reducing sample buffer (62.5 mM Tris-HC1, pH 6.8, containing 2% w/v sodium dodecylsulphate (SDS), 20% v/v glycerol, 5% v/v 2-mercaptoethanol, 0.0125% wlv bromphenol blue) and subjected to electrophoresis on a 10% polyacrylamide gel in the pres- ence of 0.1% wiv SDS, as described by Luemmli (12). Protein bands were stained with Coomassie Brilliant Blue R-250, and molecular weights were determined (1 5 ) by comparison with standard protein markers (Mark 12, Novex, San Diego, CA, USA).

Immunoblot t tins SDS-PAGE gels were equilibrated for 2 min in elec-

trode buffer (50 mM Tris containing 384 mM glycine and 0.1 % w/v SDS), transferred to wetted Immobilon polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Bedford, MA) and electrophoresed at 0.1 mAicm’ overnight in a Semi-dry Electroblotter A (Ancos, H~jby , Denmark). Agarose gels from crossed immunoelectrophoresis (XIE) were electroblotted in PBS containing 20 mM mercaptopropionic acid and 50 mM 3-(cyc1ohexylamino)- 1 - propanesulfonic acid (CAPS). Unoccupied protein binding sites on membranes were blocked by incuba- tion in blocking buffer (PBS containing 5% w/v de- fatted dried milk and 0.5‘56 v/v Tween 20) at room temperature for 30 min with gentle agitation. Mem- branes or 5 mm strips of membranes were incubated overnight with agitation at 5°C either in antisera diluted 1 :50 or in MAb-containing culture supernatant diluted 1:lO in blocking buffer. The membranes were washed three times for 5 min in washing buffer (10 mM sodium phosphate buffer, pH 7.2, containing 0.5

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ENGBEK rr ( I / .

M NaCl and 0.05% viv Tween 20), and incubated for 1 h at room temperature in peroxidase-conjugated rabbit anti-mouse immunoglobulin (P260, DAKO, Denmark) diluted 1 : 1000 in blocking buffer. After washing, the peroxidase activity was visualized with 3,3-diaminobenzidine tetrahydrochloride, 1 mgiml in 0.1 M phosphate buffer, pH 7.0 (Kem-En-Tec, Cop- enhagen, Denmark) to which 1 pliml 30% viv hydro- gen peroxide had been added.

Enzyme inimunoassuy (EIA) This was carried out in 96-well polystyrene

microtitre plates (Maxisorp, Nunc) following the gen- eral procedures described (8). The following coating procedures were used: 1) bacterial sonicates were ap- plied at a protein concentration of 0.3 pgiml in 50 mM sodium carbonate buffer, pH 9.6, and incubated at 4°C overnight; 2) whole A. ,felis organisms (50 pli well of a suspension of washed bacteria in PBS, ODGso 1.5) were incubated for 10 min at room temperature in wells precoated with poly-L-lysine (50 pgJ tnl in PBS) for 30 rnin at room temperature, centri- fuged at 1500 rpm for 5 min. and fixed with 50 p1 0.5% viv glutaraldehyde in PBS for 15 min at room temperature. Wells were washed three times with washing buffer, and non-specific binding sites were blocked with PBS containing 1% wtv BSA and 1% vi v Tween 20 for 30 rnin at room temperature. In com- petitive EIA, serial two-fold dilutions of pre- and postinfection rabbit serum were added to alternate rows of wells coated with A. felis sonicate and incubated overnight at 4°C. MAb-containing culture supernatant was added at the appropriate dilution, incubated overnight at 4"C, and bound antibody was demonstrated by means of a peroxidase-conjugated second antibody (P260, DAKO, Glostrup, Denmark).

Itnmunodot tesl This was performed on whole A. jelis sonicate, on

concanavalin A-bound components of the sonicate, and on protease-digested sonicate, following the procedure of Hawks et ul. (10). For concanavalin A chromatography, the sonicate (1 ml of protein, concentration 3.31 mgiml) was diluted in four volumes of binding buffer (20 mM Tris-HC1, pH 7.4, containing 0.5 M NaCl, 1 mM CaC1, and 1 mM MnC1,) and passed through a column (8 ml) of Sepharose- conjugated concanavalin A (Pharmacia, Uppsala, Sweden). The column was washed with binding buffer until OD280 of the eluate reached baseline and then eluted with 20 m10.5 M a-methyl-D-mannopyranoside in PBS. UV-absorbing fractions of eluate were pooled and concentrated by ultrafiltration. Pro- tease digestion was carried out by incubating A . feh sonicate with proteinase K (Sigma; 2 pgiml protein) for 45 rnin at 37°C. The reaction was stopped by boil- ing for 4 min. Antigen solutions (10 kg/ml) were ap- plied to squares of PVDF membrane and incubated with MAb-containing culture supernatant diluted

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1 : 10 in blocking buffer. The remaining procedure was as that described for immunoblotting.

RESULTS

SDS-PAGE of A. felis sonicate Analysis of the reduced A . felis sonicate by

SDS-PAGE (Fig. 1) identified 36 proteins with molecular masses ranging from 16.5 to 182 kDa. Three proteins formed prominent bands (38, 41, and 60 kDa), seven formed bands of intermediate density (43, 47, 53, 75, 80, 82 and 87 kDa) while the remaining bands were faint (Fig. 1, B).

Monoclonal antibody characterization MAbs derived from mice infected with live A .

felis were screened by EIA in wells coated with A . felis sonicate, and their reactivities were sub- sequently confirmed by immunoblotting against reduced sonicate subjected to SDS-PAGE. Ten MAbs were chosen and coded 168-1 to 168-10. Immunoglobulin class and subclass, and some characteristics of their major reactive antigens are shown in Table 1. MAbs 168-4, -6, -7 and -10 reacted with a single 53 kDa band, MAbs 168-1, -3 and -9 with 53 kDa and 60 kDa bands, MAb 168-2 with 59 kDa and 38 kDa bands,

Fig. 1. SDS-PAGE and immunoblots of Afipiajdis sonicate incubated with 10 monoclonal antibodies (168-1 to -10); A: molecular marker proteins; B: sonicate stained with Coomassie Blue; c-L : MAbs 168 -1, -2, -3, -4, -5, -6, -7, -8, -9 and -10.


TABLE 1. Characteristics of 10 monoclonal antibodies against Afipia felis Code Isotype Reactive antigen Reactivity against Other bacteria

Ajipia species" Molecular Code in Pro tease- mass (kDa\ XIE resistant

168-1 IgGl 53/60 AF-35AF-26 - 1, 2, 3, 4, 5, 6, 8, 9 Branhamella cutnrrhalis Vibrio cholerae

168-2 IgG2a 38/59 AF-3 3 - 1 , 2 , 3 , 4 , 5 , 6 , 8 , 9 -

168-3 IgM 53/60 AF-35lAF-26 + 1, 2, 3, 4, 5, 6, 8, 9 Legionellupneumophiia Kingella kingae Pseud0rnona.Y diminuta Branhamella catarrhah Moraxella osloensis Bordetella pertussis Klebsiella oxytoca Vibrio cholerae

168-4 IgGl 53 AF-35lAF-26 - 1,2, 3 -

168-6 IgG2b 53 AF-35lAF-26 - 1 , 2, 3 168-7 IgGl 53 AF-35lAF-26 - 1, 2, 3

168-5 IgG2b 38 AF-33 - 1 , 2 , 3 , 4 , 5 , 6 , 8 , 9 - - -

168-8 IgM 43147162182 AF-12 + 1, 2, 3, 4, 5, 6, 7, 8, 9 Kingella kingae Pseudomonas dimin u ta Bordetella pertussis Vibrio cholerae

168-9 IgG2b 53/60 AF-35lAF-26 - 1, 2, 3, 4, 5, 6, 7, 8, 9 Kingella kingae Pseudomonas diminuta Branhamellu catarrhah Bordetella pertussis Pseudomonns aeruginosa

a 1 : A. felis ATCC 53690; 2: A. felis ATCC 49714; 3: A . felis ATCC 49715; 4: A . broomeae ATCC 49717; 5: A. broomeae ATCC 49718; 6: A. clevelandensis ATCC 49720; 7: A. genospecies 1 ATCC 49721; 8: A. genospecies 2 ATCC 49722; 9: A. genospecies 3 ATCC 49723.

168-10IgG2a 53 AF-35lAF-26 - 1, 2, 3 -

MAb 168-5 with a 38 kDa band and MAb 168- 8 with at least four bands ranging from 43 kDa to 82 kDa (Fig. I , C-L).

In electroblots of precipitin patterns from XIE of A. felis sonicate (5), MAbs 168-1, -3 , -4, -6, -7, -9 and -10 reacted with antigen AF- 35 and AF-26, MAbs 168-2 and -5 with AF-33, and MAb 168-8 with AF-12 (Fig. 2).

Species spec@city The reactivities of the MAbs towards blots of

proteins from A. felis strains, AJipia species and unrelated bacteria are given in Table 1. The MAbs were first screened in EIA at 1:lO dilution against sonicates (1.5 pg/ml) of 3 AJpia felis strains, 6 AJipiu species and 36 unrelated bacteria coated on the wells. Sonicates giving an OD490 >0.5 in EIA were tested in immunoblots against the respective MAb. Four MAbs (168- 4, -6, -7 and -10) reacted only with A. felis strains, and not with other Ajipia species or

with unrelated bacteria, while two MAbs (1 68- 2 and -5) reacted with other AJpia species except A. genospecies 1, but not with unrelated bacteria. The remaining four MAbs (168-1, -3, -8 and -9) reacted with unrelated bacteria as well as with Afipia species. Although there is a coincidence of bacterial species cross-reacting with the last group of MAbs, no two antibodies gave the same list of cross-reacting species.

Other antigen characteristics All 10 MAbs reacted with antigens that were

bound to concanavalin A-sepharose and eluted with methyl a-D-mannopyranoside, establish- ing that these antigens were glycoproteins. MAbs 168-3 and -8 reacted with epitopes that were resistant to proteinase, and were thus con- cluded to be located on the carbohydrate moi- ety, whereas the remaining MAbs reacted with epitopes that were sensitive to proteinase (Fig. 3). All 10 antibodies bound to whole A. jdis

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E N G B E K el c r l .

Fig. 2. Crossed immunoelectrophoresis gels of 18 pg A . felis sonicate against 40 p1 rabbit antiserum cm-? with blank intermediate gel; (A) gel stained with Coomassie Blue; (B) electroblotted onto a PVDF membrane and incubated with MAb 168-6, and (C) electroblotted onto a PVDF membrane and incubated with MAb 168-8. Electrophoresis was carried out at 10 V cm-' for 18 h in the second dimension with the anode at the top. Precipitin arcs are numbered as in (5).

organisms coated onto microwells, suggesting that their respective antigens were located on the surface of the organisms.

Competition with pre- and post-infection serum The binding of MAbs to A . felis sonicate was

tested for inhibition by post-infection rabbit serum in EIA, with pre-infection serum as con- trol. No inhibition was demonstrated for Mabs 168-1, -3, -8 and -9, whereas MAbs 168-2, -4, -5 , -6, -7 and 10 were inhibited to varying de- gree. Fig. 4 shows the different effects of serial dilutions of pre- and post-infection rabbit serum on the binding of three MAbs (1 68-6, -9 and -10) that all reacted with the 53 kDa protein.

DISCUSSION

The 10 MAbs against A . felis antigens can be classified into three groups: 1) 4 (168-4, -6, -7 and -10) directed against a single 53 kDa antigen, 2) 3 (168-1, -3 and -9) directed against both 53 kDa and 60 kDa antigens, and 3) 3 (168-2, -5 and -8) directed against other antigens. Anti- bodies of group 1 did not react with other A&-

Fig. 3. Reaction of A. ,felis sonicate or derivdtes of sonicate with four MAbs. (1) Whole sonicate, ( 2 ) proteinase-digested sonicate, and (3) methyl a-D- mannopyranoside eluate of concanavalin A-bound sonicate, incubated with (A) MAb 168-1, (B) MAb 168-3, (C) MAb 168-5, and (D) MAb 168-8.

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3

2 2 6 0

1

0

MAb 168-6

1

I

MAb 168-9 MAb 168-10 Fig. 4. Competition EIA between rabbit sera (seri- ally diluted from 1 : l O to 1: 10240) taken before (GO-) and 6 weeks after (-0- infection with lo9 live AJpicr ,fclis organisms and MAbs 168-6, -9 and -10. Binding of MAb was revealed by means of peroxidase- conjugated rabbit anti- mouse immunoglobulin.

Rabbit serum dilution (2-fold from 1:lO)

iu species or with 36 unrelated bacteria. Anti- bodies of group 2 reacted not only with other Ajpiu species but also with certain unrelated bacteria, though the reaction pattern was not the same in each case. MAb 168-1 reacted with four Afipia species and two unrelated bacteria, 168-3 with four Afipitr species and eight unrelated bacteria, and 168-9 with five Afipia species and six unrelated bacteria. In group 3, MAbs 168-2 and -5 reacted with four A$pia species but not with other bacteria, whereas 168-8 reacted with five AJpin species and four unrelated bacteria.

MAbs of groups 1 and 2 reacted with a 53 kDa band, suggesting that this antigen was identical for both groups. To identify this antigen in terms of the previously described XIE reference diagram of A . felis antigens (9, XIE precipitin patterns of A. felis sonicate were tested in immunoblots with the seven MAbs of groups 1 and 2. All seven bound to XIE precipitin arcs AF-26 and AF-35. Antigen AF-35 had been found to share a determinant with an antigen of Agwbacterium tumefuciens, whereas no cross-reaction was found with AF-26. AF-26 was also found to belong to the group of antigens which elicit antibodies at an early stage after experimental infection in rabbits (6). The relation of the 53 kDa and 60 kDa antigens in SDS-PAGE to AF-26 and -35 in XIE requires elucidation by further experiments. The technique of blotting immunoprecipitates from XIE in order to allow other antibodies to react with the antigens is not entirely straightforward. Conditions must be chosen that dissociate the immune complexes during transfer to the mem-

brane, so that some free epitopes are available for reaction with another antibody. This was at- tained with the mercaptopropionic acid-CAPS buffer described.

The reactions of MAbs of groups 1 and 2 demonstrate that the 53 kDa antigen possesses at least three distinct epitopes, one species-specific and at least two shared with other AJipiu species and a limited number of unrelated bacteria. Although it is possible that this antigen consists of two or more proteins that were not resolved in the electrophoretic system, the different epitopes may coexist on a single protein. The species-specific epitope seems to be confined to the 53 kDa protein band, whereas the other epitopes are shared with the 60 kDa band. The reactivities of MAbs 168-1, -3 and -9 would suggest that there are at least two epitopes shared between the 53 kDa and 60 kDa bands: a carbohydrate epitope with which 168-3 reacts, and protein epitopes with which 168-1 and -9 react. That these epitopes are all different in some respects is shown by the different cross- reactivities of 168-1, -3 and -9 with unrelated bacteria. The sharing of epitopes across species boundaries is a common finding and accords with our previous experiments in which hyper- immune rabbit antiserum against A. ,frlis sonicate reacted with antigens from other bacterial species in XIE ( 5 ) . The evidence from the pres- ent work suggests that the 53 kDa and 60 kDa components are closely related surface antigens, sharing sequence homologies with each other and with antigens of other bacteria, but there is a species-specific region of the 53 kDa antigen that is unique to that protein.

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ENGBEK et ul

The finding that all MAbs of group 1, reacting only with the 53 kDa antigen, were inhibited by post-infection rabbit serum, whereas no inhibition was observed for MAbs of group 2 or MAb 168-8, is of interest as it suggests that mice and rabbits react differently against different epitopes during the infection.

The species specificity shown by the group-l- reactive epitope of the 53 kDa antigen and the fact that post-infection rabbit sera contain antibodies directed against it suggest that this antigen, or a fragment of it bearing that epitope, could be used as the basis for a serodiagnostic test for A . felis infection.

The authors acknowledge the excellent technical as- sistance of B. Raasthoj and C. D. Madsen, and many helpful suggestions from G. Houen.

REFERENCES

Alkan, S. , Morgan, M. B., Sandin, R. L., Moscin- ski, L. C. & Ross, C. W.: Dual role for Afipia felis and Rochalimaea henselae in cat-scratch disease. Lancet 345: 385, 1995. Anderson, B., Sims, K., Regnery, R., Robinson, L., Schmidt, M. J., Goral, S., Huger, C. & Ed- wards, K . : Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J. Clin. Microbiol. 32: 942-948, 1994. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. Dolun, M. J . , Wong, M . T., Regnery, R. L., Jorg- ensen, J. H., Garcia, M., Peters, J. & Drehner. D. : Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease. Ann. Intern. Med. 118: 331-336, 1993.

5. Engblek, K. & Koch, C.: Immunoelectrophoretic characterization and cross-reactivity of Rochali- maea henselae, Rochalimaea quintana, and Afipia felis. APMIS 102: 931-942, 1994.

6. Engbak, K. & Koch, C.: Antibody response in rabbits infected with Rochalimaea henselae, Ro- chalimaea quintana and Afipia felis. APMIS 102: 943-949, 1994.

7. English, C. K., Wear, D. J . , Margileth, A. M., Lissner, C. R. & Walsh, G. P.: Cat-scratch disease. Isolation and culture of the bacterial agent. J. Am. Med. Assoc. 259: 1347-1352, 1988.

8. Engvall, E. & Perlmann, P.: Enzyme-linked im- munosorbent assay 111. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen coated tubes. J. Immunol. 109: 129-135, 1972.

9. Goding, J . W.: Monoclonal antibodies: Prin- ciples and Practice. Academic Press, New York 1983.

10. Hawks, R., Niday, E. & Gordon, J.: A dot-immu- nobinding assay for monoclonal and other antibodies. Anal. Biochem. 133: 214-219, 1982.

11. Kohler, G. & Milstein, C.: Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256: 495-497, 1975.

12. Laernmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.

13. Reading, C.-L.: Theory and methods for immu- nization in culture and monoclonal antibody production. J. Immunol. Methods 53: 261-191, 1982.

14. Regnery, R. L., Olson, J . G., Perkins, B. A. & Bibb, W.: Serological response to “Rochalimaea henselae ” antigen in suspected cat-scratch disease. Lancet 339: 1443-1445, 1992.

15. Weber, K. & Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412, 1969.

16. Yu, X. & Raoult, D.: Monoclonal antibodies to Afipia felis - a putative agent of cat scratch disease. Am. J. Clin. Pathol. 101: 603-606, 1994.

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production and characterization of mouse monoclonal antibodies against afipia felis

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