immunopurified extracellular bartonella henselae antigen for detecting specific antibodies by enzyme...

.AP.IIIS 105: 941-950, 1997 Priiired in Denmark All ri.qlii.7 reserved

C o p y r i E h f C 4 P W I S 1997

Immuno~urified extracellular Bartonella henselae antigen for detecting specific antibodies bv e r n e immunoassav

KRESTEN ENGBEK, LARS OTTO UTTENTHAL and CLAUS KOCH

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

Engbaek, K.. Uttenthal, L. 0. & Koch, C. Immunopurified extracellular Bartonella heizselae antigen for detecting specific antibodies by enzyme immunoassay. APMIS 105; 941-950, 1997.

Protein antigens of Bnrfonella henselae bacterial sonicate supernatant and concentrated cell-free culture filtrate were examined by SDS-PAGE. The sonicatc supernatant gave 38 bands and the culture filtrate at least 21, of which 18 were of bacterial origin. Immunoblotting against 13 monoclonal antibodies obtained from mice infected with live B. henselae showed that 10 of these antibodies reacted with a narrow 225 kDa band and varying smears of bands ranging from 36 to 240 kDa in the sonicate, but only with a single 200 kDa band in the culture filtrate. Testing of pre- and post-infection rabbit sera in immunoblotting against culture filtrate demonstrated that the 200 kDa component gave the most prominent specific reaction with post-infection sera. The 200 kDa antigen was isolated by immunoaffinity chromatography of concentrated culture filtrate. and its molecular size determined by sizc- exclusion chromatography as > 1000 kDa. The immunopurified antigen was compared with bacterial sonicate as coating antigen in EIA for determining humoral immune responses in rabbits inoculated with live B. henselae. The two antigens gave almost identical results for IgM and IgG responses. The specificity of the immunopurified antigen was tested in EIA against hyperimmune rabbit sera and sera of rabbits inoculated with live B. henselae, B. yuintana and Ajpia felis. Only the hyperimmune serum against B. henselae and the sera of the rabbits inoculated with live B. henselae reacted with the immunopurified antigen. whereas the B. henselue sonicatc cross-reacted with hyperimmune and post- infection sera of rabbits inoculated with B. quir7tana and A . felis.

Key words: Burtonella henselae; Rochalimaea henselne; cat-scratch disease; extracellular antigens; monoclonal antibodies.

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

3urtonellu henselae, probably the main causative agent of cat-scratch disease (CSD) and bacillary angiomatosis, has been increasingly rcc- ognized in human disease since it was first described in 1992 (9, 12). However, the diagnosis of CSD is difficult, culture of the organism slow and often unsuccessful, and human sera may contain antibodies against B. henselue even without a history of infection. Hyperimmunc rabbit sera against B. hm.sr/ue contain anti-

Received March 11, 1997 Accepted July 1, 1997.

bodies that cross-react with components of other bacterial species, indicating that similar antigenic determinants are present in other organisms (4).

Afipiu felis, another probable agent of CSD secretes a number of components into the liquid culture medium, of which we have identified 16 by SDS-PAGE. Two of these components form a complex >I000 kDa antigen in the culture medium. Preliminary studies indicate that this antigen has a potential diagnostic use in a serological assay for A . felis infections (6) .

The present study was initiated to identify B.

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henselae components in concentrated culture filtrate, to select monoclonal antibodies that could be used as immunoadsorbants for purify- ing species-specific B. henselae components, and to determine whether one or more of these components could be used as antigen in an enzyme immunoassay for B. henselue infections.

MATERIALS AND METHODS

Bacterial strains and antigen preparation Bartonella henselae ATCC 49882 was cultured at

35°C for 14 days in Brucella broth (Difco Labora- tories, Detroit, MI, USA) with 2.5% pepsin-digested horse blood (Statens Serum Institut, Copenhagen, Denmark), 10 mM pyridoxal HCI (Sigma P 6155) and 0.01 mM d-biotin (Sigma B 4639) in tissue-culture flasks (Teknunc, Roskilde, Denmark) in an at- mosphere of 6% C02 and 25% 02. The bacteria were separated from the culture supernatant by centrifuga- tion at 45,000 g for 30 min. The supernatant was fil- tered through a 0.2 pm pore-size Millipore filter and concentrated to lil00 of the original volume by means of Pellicon tangential flow filtration (Milli- pore, Bedford, MA, USA) and Omegacell ultrafil- tration systems (Filtron Technology, Northborough, MA, USA) with a molecular cut-off of approximately 10 kDa. The bacterial pellets were washed three times and finally suspended in 50 mM Tris-HC1 containing 150 mM NaCl, 10 mM EDTA and 0.1% viv Triton X-100, pH 7.6. The suspension was ultrasonicated in a Branson Sonifer model 250 (Branson Ultrasonics, Danbury, CT, USA) for 5 min on ice and centrifuged at 45,000 g for 60 min. The bacterial sonicate supernatant (BSS) and the concentrated culture filtrate (CCF) were divided into 2 and 10 ml aliquots, respectively, and stored at -80°C.

Protein concentration of the BSS was determined by the dye-binding assay of Bradford (2) adapted for microwell plates with bovine serum albumin (BSA) as the standard.

Hybridomu technique Monoclonal antibodies (MAbs) were prepared as

described in (5), female BALB/c mice being inoculated with doses of 5.6X lo4 CFU of live B. henselae ATCC 49882. Hybridoma supernatants were tested for antibody production by enzyme immunoassay (EIA) and immunoblotting against B. henselae sonicate. Thirteen antibodies were selected, coded MAb 198-1 to -13.

Antibody purification Monoclonal antibody was purified from 1000 ml

MAb 198-10 culture supernatant on a column (1.6X20 cm) of Protein A Sepharose 4 Fast Flow (Pharmacia, Uppsala, Sweden) as described in (6).

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Immunoaffinity Chromatography Immunoaffinity chromatography was performed

as described in (6). Protein-A-purified MAb 198-10 (90 mg) was linked to 15 g CNBr-activated Sepharose 4B (Pharmacia) and B. henselae CCF was applied at 1 mlimin to a column (2.6X20 cm) of washed gel. Fractions of the eluted antibody-bound component diluted 1:lOO were tested in EIA against MAb 198- 10, and those giving OD490 values >2.0 were pooled and dialysed against three changes of phosphate-buf- fered saline, pH 7.2. The solution was concentrated with polyethylene glycol 20,000 and the protein concentration determined as previously described.

Molecular mass determination The following methods were used: 1) SDS-PAGE

(1 1) of CCF with standard protein markers (Mark 12, Novex, San Diego, CA, USA), and 2) size-exclusion FPLC (Pharmacia) of CCF or immunoaffinity-purified antigen (IPA) as described in (6). Frac- tions (0.5 ml) from chromatographic runs of antigen were subsequently analysed by EIA for reactivity with MAb 198-10.

200 kDa- 1

116.3 kDa- 97.4 kDa-

66.3 kDa-

55.4 kDa-

36.5 kDa ~

31 kDa-

21.5 kDa- A F

Fig. 1. SDS-PAGE of B. henselae bacterial sonicate supernatant (BBS) and B. henselae concentrated culture filtrate (CCF). (A) molecular mass markers (kDa); (B) B. henselae BBS, stained with Coomassie brilliant blue; (C) B. henselue BBS, silver stained; (D) uninoculated culture medium; (E) B. henselue CCF, stained with Coomassie brilliant blue; (F) B. henselcre CCE silver stained.

SOLUBLE B. HENSELAE ANTIGEN

- Fig. 2. Immunoblots of B. henselue bacterial sonicate swernatant (BBS) incubated with monoclonal antibodies 198-1 to 198-13. Mol- ecular mass markers (kDa) are shown on the left.

1 116.3 kDI- 97.4 kDI -

36.5 kDm-

31 kDm-.

I

21.5 kDI- -

Post-infection und hyperimmune rabbit seru Three types of immunization were used. For the

determination of post-infection IgM and IgG responses, five female rabbits were injected intracutaneously with 0.5X los CFU of live B. henselue and bled weekly. After 6 and 12 weeks, the rabbits were rechallenged with the same organism and dose, and bled weekly for another 5 weeks.

To obtain hyperimmune antisera, three groups of rabbits were immunized at fortnightly interval with B. henselue, B. quintuna (ATCC VR 358) and A . felis (ATCC 53690) sonicate supernatant, respectively, as described in (3).

To obtain post-infection antisera, three pairs of female rabbits were injected intracutaneously with 0.3X lo9 CFU of live B. henselue, B. quintana (ATCC VR 358) and A . felis (ATCC 53690) organisms, respectively, as described in (4).

Polyucrylumide gel electrophoresis und immunoblotting

SDS-PAGE and immunoblotting were carried out as described in (5) . Reduced samples were electro- phoresed and blotted onto nitrocellulose membranes. Unoccupied binding sites on the membranes were blocked by incubation for 1 h in blocking buffer (PBS containing 5% wiv defatted dried milk and 0.50/0 viv Tween 20). Strips of membranes were incubated overnight with either rabbit antisera diluted 1500 or hy-

bridoma supernatant diluted 1:50 in blocking buffer. Bound antibodies were demonstrated by means of the appropriate peroxidase-labelled second antibodies as described in ref. 3 and 5.

Enzyme immunoussuy (EIAJ In preliminary tests, optimal dilutions of reagents

for use in the EIA were determined by box titration with positive and negative control sera. The highest dilution that gave maximal absorbance in EIA was selected.

Polystyrene 96-well microtitre plates (Maxisorp, Teknunc) were coated with either B. henselae BSS (0.3 pg protein per ml) or immunoaffinity-purified antigen (0.5 pg protein per ml) in 50 mM sodium carbonate buffer, pH 9.6, and incubated at 4°C overnight. After washing the wells, non-specific binding sites were blocked with dilution buffer (PBS containing 1% wiv BSA and 1?/0 viv Tween 20) for 1 h at room tem- perature. Serial two-fold dilutions of rabbit sera were added to the wells and incubated at 4°C overnight. Bound antibodies was demonstrated by means of either HPR-conjugated swine anti-rabbit immuno- globulin (P2 17, Dako, Glostrup, Denmark) diluted 1 : 1000, or HRP-conjugated y-chain-specific anti-rabbit IgG (PP 313, The Binding Site, Birmingham, UK) diluted 1 :2000, or HPR-conjugated p-chain-specific anti-rabbit IgM (PP 315, The Binding Site) diluted 1:250 in dilution buffer. The peroxidase activity was

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ENGBAZK e t a / .

PO0 kDm-

Fig. 3. Immunoblots of B. liensrlue concentrated culture filtrate (CCF) incubated with monoclonal antibodies 198-1 to 198-1 3. Mol-

II II - y*% ecular mass markers (kDa) are shown on the left.

116.3 kDm-

97.4 k D I - ,

66.3 kDm-

55.4 kDI -

36.5 kDm-

31 kDm-

21.5 kDa-

1 2 5 4 5 6 7 6 9 1 0 1 1 1 2 1 3

visualized with 0.04°/;, wlv o-phenylenediamine and 4 mM Hz02 in staining buffer (0.1 M citrate-phosphate buffer, pH 5). The difference in OD490 and OD65o was read with a Thermomax microplate reader (Mol- ecular Devices, Menlo Park, CA, USA).

RESULTS

SDS-PAGE of B.henselae sonicate and culture jiltrute

Analysis of the reduced B. henselae BSS by SDS-PAGE identifled 38 bands stained with either Coomassie brilliant blue or silver in posi- tions corresponding to molecular masses ranging from 23 to 225 kDa. One band was prominent (56.5 kDa), 7 were of intermediate density, and the remainder faint (Fig. 1B & C). Analysis of reduced B. henselae CCF identified at least 2 1 silver-stained bands, 2 heavily stained bands at 51.5 and 53.5 kDa, 7 distinct bands including one at 200 kDa, and at least 12 other faint bands (Fig. 1 E & F). The two heavily stained bands at 51.5 and 53.5 kDa and a distinct band at 67.5 kDa were also found in a concentrate of uninoculated culture medium (Fig. 1 D) and were evidently not of bacterial origin.

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Reactivity of MAbs with B. henselae BSS and CCF components

The 13 MAbs derived from mice infected with live B. henselae were screened in immunoblots of B. henselue BSS and CCE In blots against B. henselae BSS (Fig. 2) 10 MAbs (198- 1 to -3, 198-5 to -10 and 198-13) reacted with a narrow 225 kDa band and varying smears of different bands ranging from 36 to 240 kDa, suggesting that these MAbs reacted against epi- topes on a polysaccharide or lipopolysaccharide component of the organism. MAbs 1 9 8 4 and -1 1 reacted with two faint bands (27.5 and 32 kDa) and MAb 198-12 reacted with a broad 34 kDa band and a fainter 32.5 kDa band, as well as with seven other faint bands, including the band at 225 kDa. In blots against B. henselue CCF (Fig. 3) 10 MAbs (198-1, -2, -6 to -10, - 12 and - 13) reacted with a single 200 kDa band, whereas no bands were stained with MAbs 198-3, -4 and - 11.

Immunoblo t t ing with pre- and post- infect ion rabbit sera

Sera from the five rabbits inoculated three times with live B. henselue were tested in im-


Fig. 4. Immunoblots of B. henselue bacterial sonicate supernatant (BBS) with pre- and post-infection rabbit sera. Five rabbits (274278) were inoculated intracutaneously three times at 6-week intervals with live B. henselue and bled weekly. (0) pre-infection serum; (1) 4 weeks after 1st inoculation; (2) 3 weeks after 2nd inoculation; (3) 2 weeks after 3rd inoculation. Molecular mass markers (kDa) are shown on the left.

Fig. 5. Immunoblots of B. henselue concentrated culture filtrate (CCF) with pre- and post-infection rabbit sera. Five rabbits (274278) were inoculated intracutaneously three times at 6- week intervals with live B. lienselue and bled weekly. (0) pre-infection serum; (1) 4 weeks after 1st inoculation; (2) 3 weeks after 2nd inoculation; (3) 2 weeks after 3rd inoculation. Molecu- lar mass markers (kDa) are shown on the left.

200 hD8-

l la.5 LO.- et.4 *Om-

... 2 L D I -

4% hDI -

MI 51 LDm- I

21.8 kD8-

m

0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

Rabbit 274 Rabbit 275 Rabbit 276 Rabbit 277 Rabbit 278

200 YDa-

118.3 YDa-

97.4 YDa-

66.2

!

45 YDa-

31 YDa-

o i t a o i t a o i t a o t t s 0 1 1 1

Rabbit 274 Rabbit 271 Rabbit 278 Rabbit t77 Rabbit 278

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ENGBEK et al.

2.5

2

0 m 2 1.5 0

I

0 m

1 a" 0

0 . 5

0

IgM response

\i

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

IgG response - <

U 0 C -d

a d ru

'4

3

2

1

0 0 1 2 3 4 5 6 7 8 9 1011121314151617 0 1 2 3 4 5 6 7 8 9 1011121314151617

Weeks Weeks

0 > Rabbit 274 Rabbit 275 Rabbit 2 7 6

\ Rabbit 277 ~ *I ~ Rabbit 278

Fig. 6. IgM and IgG antibody responses in five rabbits inoculated with live B. henselae, starting with the preinoculation level and tested weekly thereafter. Six and twelve weeks after the first inoculation the rabbits were re-challenged with the same organism and dose. The antibody level is given as OD490-0Dh50 produced by sera diluted 1: 1600 (IgG response) and 1: 10 (IgM response) in EIA against B. henselae immunoaffinity-purified antigen coated at 0.05 pg/well.

munoblotting against B. henselae BSS and CCF. The pre-infection rabbit sera reacted with 10-12 different bands in the BSS blots (Fig. 4). After inoculation with live B. henselae, the sera stained these bands more intensely, and sera from all five rabbits reacted with five new bands (at 20, 23.5, 29, 30.5 and 83 kDa) which were not stained by pre-infection sera. In immunoblots of the CCF (Fig. 5) one of the pre-infection rabbit sera reacted with bands at 58 and 18 kDa and another serum reacted with the 18 kDa band. After the inoculation, all sera developed reactivity against five new bands at 20,35,54,63 and 200 kDa. The most distinct of these was at 200 kDa; this band could be detected in four of the rabbits after the first inoculation and in the remaining rabbit after the third.

ImmunoafJinity-pur l e d antigen from culture Jiltrate

Immunoaffinity chromatography with MAb 198-10 as adsorbent yielded one component

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from B. henselae CCF which reacted with MAb 198-10 and produced a single band at 200 kDa in SDS-PAGE. The molecular size of the immunoaffinity-purified antigen (IPA), as determined by size-exclusion chromatography, corresponded to a molecular mass >lo00 kDa.

IgM and IgG antibodies in pre- and post-injk- tion rabbit sera

IgM and IgG antibody development (Fig. 6) in rabbits inoculated with live B. henselae was studied by EIA using BSS and IPA as coating antigens. The antibody responses, whether of IgM or IgG, were measured similarly by both antigens. Specific IgM showed a significant response as early as 1-2 weeks after both primary and subsequent inoculations, and maximal responses after the third inoculation. Specific IgG rose slowly and variably after the first inoculation and peaked at 1-2 weeks after the second and third inoculations, the magnitude of the re-


4

3 0 L n W a 0 I 2

0 m

a” 0

1

0 1. b

B

\

Reciprocal serum dilution ( x ~ O - ~ )

-+-- Anti-B. henselae serum

Anti-B. quintana serum

Anti-A. felis serum

......... 0 ........

. _ _ _ _ , ) .-.-

Fig. 7. Titration of hyperimmune rabbit sera produced against B. henselae, B. quintanu and A. felis sonicates in wells coated with (A) B. henselae bacterial sonicate supernatant (0.03 pg/well) or (B) B. henselae immunoaffinity-purified antigen (0.05 yg/well).

sponses varying considerably in individual rabbits.

SpeclJicity of IPA for B. henselae antibody responses

The serological cross-reactivity of B. henselae BBS and IPA was studied by EIA against hyperimmune sera from rabbits immunized with B. henselae, B. quintana and A. felis sonicate or against sera from rabbits inoculated with live organisms of the same species. All three hyperimmune rabbit sera reacted with the B. henselae BBS, whereas only the B. henselae serum reacted significantly with the IPA (Fig. 7). Both rabbits (2904 and 2905) inoculated with live B. quintana and one of the rabbits (2906) inoculated with live A . felis developed cross-reacting antibodies against B. henselae BSS, but none of these rabbits developed antibodies that cross-reacted with the IPA (Fig. 8).

DISCUSSION

After the recognition of Bartonella (previously named Rochalimaea) henselae as probably the leading causative agent of CSD and bacillary angiomatosis, serological assays have become the main diagnostic method for these diseases. The most frequently used assays are the indirect immunofluorescence assay (IFA) and EIA using whole bacterial cells as the antigen (1, lo). How- ever, an obvious disadvantage of using whole cells is that the cell surface presents many different components, some of which may show in- trinsic cross-reactivity with antibodies against other organisms, and none of the previously described assays can distinguish between B. henselae and B. quintana infections. Thus the specificity of all such tests must be verified by com- paring titres before and after absorbing the serum with homologous and heterologous or-

947

ENGBEK PI ul

0 m W a 0 I

0 m a" 0

IA B

i

/

/ 1'

i

/I-' I

- 3

' 2

1

0 0 1 2 3 4 5 6 7 8 9 1 0 0 1 2 3 4 5 6 7 8 9 1 0

Weeks Weeks

Rabbit 2903 inoculated 0 ~ Rabbit 2906 inoculated with B . h e n s e l a e with A . f e l i s

Rabbit 2904 inoculated with B . q u i n t a n a

.........*....-...

Fig. 8. Development of antibodies to B. henselue in rabbits inoculated with live B. henselue, B. yuintuna and A . felis, respectively, starting with the preinoculation level and tested weekly thereafter. Six weeks after the first inoculation the rabbits were rechallenged with the same organism and dose. The antibody level is given as OD490-0D650 produced by sera diluted 1:800 and tested in microtitre plates coated with (A) B. henselue sonicate supernatant or ( B ) B. henselue immunoaffinity-purified antigen.

ganisms. However, in our hands most sera show a fall in titre after absorption with B. henselue cells and comparison of pre- and post-absorption titres has not solved the problem of specificity. Similar problems are encountered when supernatants of sonicated or lysed bacteria are used as antigens in EIA. Furthermore, the IFA is subjective and not easily adapted for testing a large number of specimens, while whole-cell EIAs have not proved reliable, mainly because of instability of cell attachment or because the chemistry of attachment interferes with the assay. The identification and purification of one or more immunoreactive antigens would greatly improve the reliability of the assay system.

We have attempted to identify such antigen(s) by means of the inoculated rabbit model previously described (4). In immunoblots, all pre- infection rabbit sera contained antibodies re-

acting with 10-12 components in the BSS and 2 of the sera also contained antibodies against components in the CCE After inoculation of the rabbits with live organisms, antibodies appeared against five different bands in either BSS or CCF which did not react with pre-infection sera. The most notable of these components was the 200 kDa band in CCF, as antibodies against it appeared in four of five rabbits after the first inoculation and in the remaining rabbit after the third injection.

Thirteen MAbs were produced from mice injected with live B. henselue organisms and selected on the basis of their binding to BSS in EIA and immunoblotting. Ten of these MAbs reacted with a narrow 225 kDa band and varying smears of bands in BSS, whereas the same antibodies reacted with a single 200 kDA band in the CCF. It could be argued that these mono-

948


clonal antibodies were in fact produced against the soluble 200 kDa component subsequently isolated from CCF rather than against cellular components. A comparison with polyclonal antibodies produced in experimental infection in rabbits showed that these were directed against the same 200 kDa band in the CCF as the 10 MAbs, providing further evidence that this component plays a leading role in the serological response. Consequently, one of the MAbs (198-10) was used as the adsorbent in immunoaffinity chromatography to purify this component (IPA) from CCE Size-exclusion chromatography showed its molecular size to be > 1000 kDa, indicating that the 200 kDa mono- mers seen on SDS-PAGE after reduction are ag- gregated in the culture medium.

The IPA was compared with B. henselue BSS in indirect EIA for IgM and IgG responses in the rabbits. All pre-infection sera showed low titres against B. henselue with both IPA and BSS antigens. After injection with live organisms some of the rabbits responded with a slow and modest IgG increase, whereas others were unre- sponsive. However, a significant increase in the IgM response was observed in all rabbits, though of short duration. The low pre-infection titres contrast with the rather high background titres found in humans with no history of infection, when their sera are tested against BSS. This background is probably a consequence of B. henselue sharing antigenic determinants with other bacteria commonly encountered in humans, but to which rabbits are not exposed. In some humans with clinically and histopatho- logically evident cat-scratch disease, the IgG titre fails to rise much above the background, whereas other individuals respond with a high titre (1, 10). Except for a genetic variation in the immunological response, the cause of this variation is not known. Purification of a more specific antigen may reduce the background and increase the diagnostic sensitivity of the EIA. This has been shown for Borrelia burgdorferi when flagella were substituted for sonic extracts as antigens in EIA (7, 8).

To assess the specificity of the B. henselue IPA, we tested the B. henselue BSS and IPA against hyperimmune B. henselue, B. quintuna or A. felis rabbit sera and against sera from rabbits inoculated with the corresponding live organisms. Whereas the BSS reacted with all three

hyperimmune sera, the IPA reacted only with the B. henselue hyperimmune serum. The specificity of the IPA was confirmed by the sera from rabbits inoculated with live organisms: IPA reacted only with sera from rabbits inoculated with B. henselue, whereas BSS also reacted with sera from rabbits inoculated with B. quintunu and A. felis. The close evolutionary relationship between the three organisms means that they share many antigenic determinants, so that heterologous antisera cross-react with B. henselue BSS. However, the B. henselue IPA appears to present an immunodominant epitope that is specific to this organism. Further investigation of the use of the IPA in EIA will assess its use- fulness for the serodiagnosis of B. henselue infections in humans.

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

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Bibb, W.: Serological response to “Rochulimaea henselae” antigen in suspected cat-scratch disease. Lancet 339: 1443-1445, 1992.

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