monoclonal antibodies against spore antigens of bacillus anthracis

FEMS Microbiology Immunology 47 (1988) 169-178 169 Published by Elsevier

FIM00027

Monoclonal antibodies against spore antigens of Bacillus anthracis

A.P. Phillips 1, Ailsa M. Campbe l l 2 and Rosa l ind Q u in n

1 Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, U.K. and 2 Department of Biochemistry, University of Glasgow, Glasgow, U.K.

Received 8 March 1988 Revision received 11 August 1988 Revision accepted 22 August 1988

Key words: Bacillus anthracis; Spore; Monoclonal antibody; Immunofluorescence

1. SUMMARY

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts.

Correspondence to: Dr. A.P. Phillips, Chemical Defence Estab- lishment, Porton Down, Salisbury, Wiltshire SP4 0JQ, U.K.

A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but no reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in West- ern blots of anthrax spore extracts.

2. I N T R O D U C T I O N

In diagnosing the disease anthrax, there are considerable problems in differentiating laboratory isolates of the causative agent, the bacterium Bacillus anthracis, from other members of this genus that are widely distributed in the environ- ment. B. cereus, which can cause food poisoning [1], is arguably the species most closely related to B. anthracis in biochemical and antigenic terms, but some B. thuringiensis and B. mycoides strains may also be confused with B. anthracis in laboratory tests [2-4].

Early attempts to differentiate Bacillus species on the basis of spore antigens involved agglutination or precipitation techniques. Data on B. anthracis are often missing from these studies, perhaps because of the tendency at that time to

0920-8534/88/$03.50 © 1988 Federation of European Microbiological Societies

170

consider B. anthracis and B. thuringiensis as variants of B. cereus [5]. Lamanna and Eisler [6] at tempted to distinguish B. anthracis from B. cereus strains using spore agglutinogens, but were unable to remove the cross reactions between the two species. In view of the difficulty of obtaining spore preparations that are not contaminated with vegetative cell debris [7], an agglutination method is less likely to provide specificity for B. anthracis on the basis of spore antigens than are tests such as immunofluorescence (IF) in which the reaction of antibody with the spore surface is visualised by microscopy, and may thus be assessed without the possibility of confusion by the reactions of anthrax vegetative cell or toxin antigens. Nevertheless, the close relationship between spore surface antigens among Bacillus species evidently causes cross reactions in IF tests based on polyclonal antibodies. Fluck et al. [8] were unable to remove all cross reactions between rabbit anti-anthrax spore fluorescein conjugates and spores of several other Bacillus species, including B. cereus, B. thuringiensis, and B. pumilus. In our hands, fluorescein conjugated rabbit IgG raised against B. anthracis Vollum reacted in IF tests with spores of 11 out of 20 B. cereus strains, but these cross reactions were removed by absorption with two B. cereus strains [9]. The absorbed polyclonal antibody reacted with spores of a range of anthrax strains including Vollum and Sterne, but polyclonal antisera to spores of B. anthracis Sterne reacted with spores of all strains tested except Vollum, suggesting that at least two anthrax spore antigens exist [10,11]. The rabbit anti-Sterne and anti-Vollum reagents displayed cross reactions with spores of a number of the problematic Bacillus strains described by Fluck et al. [8], but we were able to eliminate these reactions by absorption with the two B. cereus strains used previously [11].

Monoclonal antibodies should provide a considerable advantage in the fine analysis of the B. anthracis spore surface antigens, including investigation of antigen structure and the possible role in pathogenesis. In the present communication two mouse monoclonal antibodies raised against B. anthracis spores are examined for their reactivity with B. anthracis strains and with other Bacillus species.

3. MATERIALS A N D M E T H O D S

3.1. Bacterial antigens The sources of Bacillus strains used are given in

Table 1. Strains obtained from the Institut fiir Tiermedizin und Tierhygiene mit Tierklinik der Universit~it Hohenheim, through the courtesy of Dr. R. BiShm, were among those used by Fluck et al. [8]. The penicillin-resistant anthrax strain from the Centre for Applied Microbiology and Re- search was kindly provided by Prof. J. Melling. Strains kindly provided by Dr. J. Ezzell at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) included delta (A) strains of anthrax that had been cured of the pX01 toxin plasmid [12,13]. The Sterne and ST1

Table 1

Bacillus' strains used

Bacillus strains Source a

B. anthracis Vollum, B1, M36A; MRE B. subtilis var niger.

B. anthracis Sterne, 183/78, 187/78. CVL

B. anthracis Penicillin resistant. CAMR

B. anthracis A-Vollum-lB1, USAMRIID Ames, A-Ames-I, M36, New Hampshire, A-New Hamsphire-1, V-770, Colorado, V-770-NPIR, A-V-770-NPIR, A-Sterne, Soviet vaccine strain ST1, Buffalo, M36, Nebraska, Texas, 107, 17TS; B. thuringiensis 4040, 4041.

B. cereus NCTC 8035, NCTC 9946, NCTC NCTC 10320

B~ cereus" BERN 1, LA 925, CCM 88; Iq'TTUH B. subtilis 0453; B. rnegaterium CCM 1711, ATCC 14581; B. polymyxa CCM 1459; B. pumilus CCM 2144; B. coagulans CCM 2056; B. thuringiensis H7BT7/72.

Abbreviations. MRE: the former Microbiological Research Establishment, Porton Down, Salisbury, U.K., that closed in 1979. CVL: the Central Veterinary Laboratory, Weybridge, U.K. CAMR: the Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K. USAMRIID: the US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, U.S.A. NCTC: the National Collection of Type Cultures, London, U.K. ITTTUH: the Institut for Tiermedizin und Tierhygiene mit Tierklinik der Universit~it Hohenheim, F.R.G.

strains of anthrax are not encapsulated and are believed to lack the pX02 plasmid [13].

Spores were grown either on the solid medium described by Williams et al. [14] (WSM) or in Schaeffer's medium (SSM) as modified by Burke [15]; preparations were incubated initially for 48 h at 37°C, then for several days as necessary at room temperature until spore forms were in the majority when visualized by dark ground microscopy. Spores were washed three times in water unless otherwise stated. Spores were either stored live at 4 ° C or were inactivated by one of three methods: incubation with 1% (v /v ) formaldehyde at least overnight at room temperature; gamma irradiation to a dose of 3 megarads; and heating to 90°C for 1 h 45 min. Spores inactivated with formaldehyde were stored in 1% or 0.1% formaldehyde.

Ames spores to be used as the immunogen in the production of monoclonal antibodies were grown in WSM then washed 5-fold in deionised water. Whole spore immunogen was prepared by heating the spores at 90°C for 1 h, storing at 4 ° C until required. Spore extract immunogen was prepared by incubating the spores in 10 mM Tris buffer, pH 9.8, containing 1% sodium dodecyl sulphate (SDS) and 5 mM dithiothreitol, at 70 ° C for 30 min. The extract was removed from the spores by centrifugation, filtered through a low protein binding 0.22 # m membrane filter (Milli- pore Corp.) and stored at - 7 0 ° C . For use in immunoblotting, spores were extracted in 5 mM Tris HCI buffer, pH 9.8, containing 1% (w/v) SDS, 50 mM 2-mercaptoethanol, and 10 mM dis- odium ethylene diamine tetraacetate. Vegetative cell preparations were grown in nutrient broth in shake flasks sealed with cotton plugs or in R medium [16] in screw capped flasks, overnight at 37 ° C. Cells were then washed three times in phos- phate buffered saline (PBS) pH 7.5, and inactivated by addition of formaldehyde to 1% (v /v ) and incubation for at least two days at room temperature.

3.2. Mouse polyclonal and monoclonal antibodies Monoclonal antibodies were generated from

B A L B / C mice by fusion of spleen cells with the P3 X63 Ag8 6.5.3 myeloma cell line [17]. To

171

produce monoclonals against epitopes on the intact spore surface, mice were immunised intra- peritoneally (i.p.) with 10 9 heat inactivated B. anthracis Ames spores five days before fusion [18]. Positive clones were selected by the indirect IF method earlier described [19] but using 10 4 B. anthracis Ames spores on each multispot slide well. The one resulting hybridoma, designated E12, was cloned three times on mouse feeder cells at one cel l / three wells. Hybr idoma supernatants were stored at 4 ° C until required for specificity testing.

To produce monoclonals against the spore extract, mice were inoculated i.p. with spore extract, to provide a protein content of 100 ~g, in complete Freund's adjuvant; this was repeated four weeks later. After a further four weeks, the mice received a similar boost but in saline, and the fusion was performed four days later. The one positive clone, designated A9, was selected by indirect ELISA (enzyme linked immunosorbent assay), using 96-well microtitre plates coated with spore extract. Both monoclonals E12 and A9 were shown to be IgM by ELISA assay using ~ chain specific rabbit anti-mouse IgM, and by double diffusion in agar containing this rabbit antibody.

Hyper immune mouse serum to whole spore antigens was produced by inoculating mice i.p. with 10 v Ames spores in complete Freund's adjuvant, with a boost in saline at 14 days. The mice received further boosts at week four and week eight. Twelve days after the last immunisation, blood was collected and the serum was separated and stored at - 20 ° C.

Mouse monoclonal against the capsular poly (D-glutamyl peptide) was an ascitic fluid kindly provided by Dr. J. Ezzell, of USAMRIID.

3.3. Other antibodies Guinea pig antiserum to the B. anthracis Sterne

live veterinary vaccine Anvax (anti-Anvax) and antiserum to the EA1 extractable antigen of B. anthracis vegetative cells (anti-EA1) were gifts from Dr. J. Ezzell, of U S A M R I I D [20]. Rabbit IgG preparations against formaldehyde inactivated B. anthracis spores were produced for the Vollum and Sterne strains as previously described [21,22]. Fluorescein-conjugated goat anti-rabbit IgG (H + L chains) and fluorescein-conjugated

172

goat an t i -mouse IgG (H + L chains) were pre- pa red in the D e p a r t m e n t of Viral Pa thogenes is and I mmuno logy at U S A M R I I D .

3.4. I F tests for specificity Bacteria were d i lu ted in de ionised water to

abou t 10 6 o r g a n i s m s / m l . Five ffl of bac te r ia l sus- pens ion were dr ied on the 3 m m wells of mul t i spo t glass microscope slides, at 5 5 ° C , then f ixed in methanol . Each well was incuba ted with 5 ffl of mouse monoc lona l E12 d i lu ted 1 : 5 in bovine serum a lbumin buffer (BSAB: 1% BSA (Sigma), 0.1% Tween-20, in PBS p H 7.5), or 10 ffl of ano ther an t i -bac te r ia l reagent : monoc lona l A9 un- di luted, mouse h y p e r i m m u n e an t i se rum di lu ted 1 : 200, or r abb i t I g G at 100 f fg /ml . Autof luores - cence cont ro l assays were pe r fo rmed by omi t t ing an t ibody in this incuba t ion step. Af te r incuba t ion for 45 rain at r oom tempera ture , sl ides were washed three t imes in PBS conta in ing 0.1% Tween-20, once in deionised water, and were air dried. Each well was then incuba ted for 30 rain with 10 /~1 of f luorescein conjuga ted an t i -mouse or an t i - r abb i t reagent , d i lu ted in BSAB. Slides were then washed, dried, and s tored in the da rk until requi red for microscopy, when they were m o u n t e d in glycerol m o u n t a n t con ta in ing a b leach r e t a rdan t (Cit i f luor: the Ci ty Univers i ty , London) .

F luorescence in tens i ty of bac te r ia was scored subject ively on a scale f rom 0 (autof luorescence) to 4 + , at a magni f ica t ion of X l000 , using an O l ym pus BHS microscope. F luoresc ing bac te r ia

were only scored as spores if they a p p e a r e d highly refract i le under da rk g round i l luminat ion . The I F tests were p e r f o r m e d th roughou t by one of us (APP), and scores for s ta ined bac te r i a were gener- al ly r ep roduc ib le wi th in 0.5 + .

3.5. Immunoblotting (Western blotting) Extrac ts of B. anthracis spores were d i lu ted in

SDS buffer , e l ec t rophoresed on SDS-po lyacry la - mide gels, e l ec t rophore t i ca l ly t r ansb lo t t ed onto ni t rocel lu lose sheets, and visual ised by react ion with the var ious an t i -bac te r ia l an t ibod ies essen- t ial ly as descr ibed by Ezzell and Absh i re for vege- tat ive cell extracts [20]. F o r the monoc lona l antibodies E12 and A9 i m m u n o b l o t t i n g was also per- fo rmed by the m e t h o d of Bat te iger et al. [23].

4. R E S U L T S

4.1. Immunofluorescence reactions with B. anthrac is strains

The resul ts of the first specif ic i ty tests of mono- c lonal E l 2 wi th spores of a range of B. anthracis strains are given in Tab le 2. These spores were grown on W S M and s tored for a year or more in fo rma ldehyde buffer before use. Al l spore pre- pa ra t ions con ta ined spores that s ta ined with E12 to at least 1.5 + under the s t anda rd reac t ion con- di t ions, bu t as shown these p repa ra t i ons could be g rouped under four categor ies in respect of the degree of react ion. A marked degree of hetero-

Table 2

Immunofluorescence reaction of B. anthracis spores stored for more than a year, with monoclonal E12

Score in IF test

no spores less spores mostly spores mostly spores 0.5 +, than 2 + 1-2 + 0.5 2 + with < 10%, 2-3 +

Vollum a A-Ames Sterne Ames B1 187/78 A-Sterne Ames b 183/78 Sterne Pen-resistant M36 A-V770-NPIR A-N. Hampshire-1 M36A

Spores were grown on WSM, inactivated with formaldehyde at 37°C, then washed six times in PBS and stored in 0.1% formaldehyde/PBS for at least a year before use. a Two Vollum preparations were tested; one was the immunogen for rabbit anti-spore IgG. b This preparation, inactivated by heating at 90 °C for 1 h, was the immunogen used in monoclonal production.

geneity was apparent in the fourth group, in which the majority of spores were assessed as 0.5 + but a small number were stained at 2-3 + ; these preparations were a formalised Ames spore preparation [11] and the heated Ames whole spore immunogen. Spore fluorescence intensity 'autofluo- rescence' scores in control assays were 0.25 + at most. In other experiments, spores of the plasmid-cured variant A-Vollum-lB1 reacted with monoclonal E12 only at 0.5-1 + . The B. anthracis Sterne spore preparation No. 52 used previously as the immunogen for preparing rabbit polyclonal antibody [9-11] did not react with monoclonal El2, but still reacted with rabbit anti-spore IgG.

The monoclonal A9 (anti-spore extract) stained about 1% of spores at 1-1.5 + in these preparations of heated Ames and Sterne spores and in the formalised preparations of J-Ames-1 and A-N. Hampshire- l , but did not react at all with spores in the other preparations cited in Table 2. All of the above spore preparations reacted with mouse hyperimmune serum to give IF scores of 2 + or better.

The effect of inactivation of spores and the effect of varying the spore growth medium were investigated. As is clear from Table 3, IF scores

173

for live spores dried immediately after harvesting and for spores inactivated by heating or formalisa- tion then tested within 2 weeks were not substan- tially different. Spores grown in SSM generally contained a far higher proportion of poorly stained spores than the same strain grown on WSM (see also Table 4). SSM preparations were also much more heavily contaminated with cellular debris which was stained by mouse or rabbit hyperimmune reagents thus degrading the resolution of spores, but was not stained by monoclonal El2. As may be seen in Table 3, staining with the antiserum was usually more homogeneous than for monoclonal El2. The single result in the table for the performance of gamma irradiated spores indicates a marked decrease in the degree of reaction with El2 which was typical; however, staining with the mouse hyperimmune serum was not usually reduced by irradiation. Many spores appeared structurally damaged after irradiation.

The substantial staining of those WSM-grown B. anthracis Ames spore suspensions cited in Ta- ble 3 contrasts with the results for the long storage preparations of WSM-grown Ames spores used (Table 2). Because of the possible significance of the time of storage of spore suspensions, Ames

Table 3

Effect of bacterial inactivation on immunofluorescence reactions of B. anthracis spores stored for up to two weeks.

B. anthracis Growth Inactivation IF score ~' strain medium method monoclonal mouse hyper-

E12 immune serum

Ames WSM live 1.5, 2.5, 3 3, 3.5 WSM heat (2) 3 3.5 WSM HCHO (2) 2.5, 3 3 SSM heat 0.25 ( < 5%: 0.5, 1) (2.5) 3

Vollum WSM live 1.5-2.5 2.5, 3 WSM heat 2-3 2-3.5 WSM HCHO 2.5, 3 3 SSM heat 0.25, 0.5-1.5 (2) (1,5) 3

STI WSM HCHO 2.5 3.5 SSM live 0.5-2 (2.5) 3 SSM heat 0.5-2.5 3.5 SSM HCHO 0.5-2 3 SSM ,/-irradiation 0.25 ( < 1%: 1) 1.5-2.5

Multiple IF scores, e.g. 2-3, indicate approximately equal numbers of bacteria of each brightness value within this range. Scores in parentheses indicate that less than 10% of cells stained to this degree, unless other percentage values are given.

174

Table 4

Immunofluorescence reactions of B. anthracis spores tested within two days of harvesting

B. anthracis Growth Inactivation IF score a strain medium method monoclonal

El2 mouse hyperimmune serum

Ames WSM HCHO (2, 2,5) 3 (3.5) A-Ames-1 WSM HCHO 0.25, 1.5 (2, 2.5)

SSM heat 0.25 ( < 5%: 1, 1.5) Colorado WSM HCHO (l.5, 2) 2.5, 3 Buffalo WSM HCHO (0.5) 2.5, 3 N. Hampshire WSM HCHO 2.5, 3

SSM heat 0.5, 1.5, 2 A-N. Hampshire-I WSM HCHO 2.5, 3

SSM heat (0.5) 1.5-2.5 Nebraska WSM HCHO (0.25, 1) 2, 2.5 (3) M36 WSM HCHO (1, 1.5) 2, 2.5 V-770 WSM HCHO (0.25) 0.5 (1.5 2.5) V-770-NPIR WSM HCHO 1.5, 2 107 WSM HCHO (1.5) 2.5, 3 17TS WSM HCHO (2.5) 3, 3.5

4

3.5 3 3.5 3.5 4

3.5 3.5 (2) 3.5 3.5 3 3.5 3.5 3.5 3.5

IF scores are explained in the caption to Table 3.

a n d a n u m b e r of o t h e r s t r a i n s o f B. a n t h r a c i s w e r e

g r o w n o n W S M a n d t e s t e d a f t e r i n c u b a t i o n ove r -

n i g h t w i t h f o r m a l d e h y d e . R e s u l t s of I F t e s t s of

t h e s e s p o r e s w i t h m o n o c l o n a l E l 2 a r e g i v e n in

T a b l e 4, in s o m e cases t o g e t h e r w i t h t h e r e s u l t for

s p o r e s g r o w n in S S M , t h e n h e a t e d a n d u s e d o n t h e

n e x t day . Al l s p o r e s in t he W S M - g r o w n A m e s

p r e p a r a t i o n r e a c t e d wel l w i t h E l 2 . F o r t h e o t h e r

s t r a i n s g r o w n o n th i s m e d i u m , a s u b s t a n t i a l p ro -

p o r t i o n o f u n s t a i n e d (0.25 + ) s p o r e s was r e c o r d e d

o n l y in t he A - A m e s - 1 a n d V - 7 7 0 p r e p a r a t i o n s ; for

t h e r e m a i n i n g n i n e s t r a i n s s p o r e s we re for the

m o s t p a r t s t a i n e d a t 1.5 + o r be t t e r . JSpores of

a n o t h e r c u r e d s t r a i n , A - V o l l u m - l B 1 , r e a c t e d o n l y

a t 1.5 + w i t h h y p e r i m m u n e a n t i s e r u m a n d n o t at

al l w i t h E l 2 ( r e su l t s n o t s h o w n ) .

Table 5

Immunofluorescence reactions of spores of B. anthracis Sterne and A-Sterne, tested within two weeks of harvesting

B. anthracis Growth Inactivation IF score a strain medium method monoclonal mouse hyper-

E12 immune serum

Sterne WSM live WSM heat WSM HCHO SSM heat

A-Sterne WSM live WSM heat WSM HCHO SSM heat

Sterne WSM, 25 h HCHO WSM, 48 h HCHO WSM, 71 h HCHO

0.25, 0.5 ( <1%: 1.5) (2) 3.5 0.25, 0.5, 1 3.5 0.25, 0.5, 1 3, 3.5 0.25 ( < 5%; 1.5, 2) 1.5, 2, 3.5

0.25, 0.5-1,5, (2) 3, 3.5 0.25, 0.5-1,5 (2) 3.5 0.25, 0.5-1,5 (2) (3) 3.5 0.25, 0.5, 1.5, 2 (2.5) 3

0.25,0.5 ( <1%: 1.5) 3.5 0.5-2 (2.5) 3.5 0.25, 0.5, 1.5 (2) 3.5

a IF scores are explained in the caption to Table 3.

Table 5 shows IF results for spores of B. anthracis Sterne stored only for short periods before use. Live suspensions were dried on slides on the day of harvesting, and the aliquots that were inactivated by heat or formaldehyde were im- mobilised 2 weeks later. Sterne spores grown on WSM or SSM were for the most part only stained to 1 + or less; the toxin cured strain A-Sterne was stained slightly more strongly, up to 1.5 + . To investigate the effect of the time in growth medium on the IF performance of Sterne spores, cultures on WSM were harvested after 25 h, 48 h, and 71 h, treated with formaldehyde, and used on the next day. As may be seen in the table, spores in the earliest harvested preparations stained less well with E12. Similar time resolved experiments with B. anthracis Ames and Vollum also indicated that spores harvested at 25 h gave IF scores with E12 on average about 1 + lower than spores harvested later. Overall, the several Sterne spore preparations tested with monoclonal E12 gave lower IF scores than other anthrax strains, including another non-encapsulated strain ST1.

One isolate of B. anthracis, Texas, had to be incubated for at least 70 h on WSM before spores were detected, and spore yields were 2 orders less than for the Vollum strain. Texas spores were scored as 3.5 + in IF tests with mouse hyperimmune serum and 0.25 + or 2 + with monoclonal El2.

Some of the B. anthracis spore preparations that were stored for 2 weeks or less before im- mobilisation were screened in IF tests with monoclonal A9. The only strain to react was Ames, grown on either medium. However, the majority of the Ames spores were unstained, and the few 1.5-2 + stained cells often appeared incomplete. No reactions were observed in preparations of Sterne, A-Ames-l, or A-N. Hampshire-1 (contrast the older preparations of these strains, above).

Non-encapsulated vegetative cells were seen oc- casionally in B. anthracis spore preparations; vegetative cells of most strains were stained at 2 4 + by mouse hyperimmune serum, but none reacted with monoclonals E12 or A9. Encapsu- lated vegetative cells of strains Sterne, Ames, and Vollum, grown in R medium, reacted with mouse hyperimmune serum but not with monoclonal E12.

175

4.2. Immunofluorescence cross-reactions with non- anthrax Bacillus species

Monoclonals El2 and A9 were evaluated in IF tests with spores of the non-anthrax Bacillus strains listed in Table 1, grown on solid (WSM) or liquid (SSM) medium and used either live or after inactivation, generally after storage at 4 ° C for several months. Live preparations of Bacillus spores grown on either medium reacted with monoclonal E12 up to 0.5 + , but inactivated spores were only scored as 0.25 + . None of the spore preparations reacted with monoclonal A9. Spores of B. subtilis var niger did not react with mouse hyperimmune serum, but all other spore preparations stained at 2-3.5 + .

In many of the non-anthrax preparations, especially those grown in SSM, intact vegetative cells and considerable debris were stained by mouse hyperimmune serum, but neither cells nor debris were stained by monoclonals El2 or A9. For- malised vegetative preparations of B. cereus NCTC 8035 and 9946 and B. thuringiensis 4041, grown in nutrient broth or R medium, and preparations of B. thuringiensis 4040 and B. subtilis vat niger grown in nutrient broth, also failed to react with monoclonal E12.

To ensure that the lack of reaction with the monoclonal El2 was not due to degradation of the spore antigen during storage, spores of B. cereus NCTC 8035, 9946, and 10320 were grown on WSM, then dried on slides immediately after harvesting, and tested; again, no spores were stained more strongly than 0.5 + .

4.3. Western blotting of B. anthracis spore extracts Western blots performed by the enzyme

visualisation method of Ezzell and Abshire [20] are shown in Fig. la for spore extracts of B. anthracis Sterne and Vollum. After incubation with monoclonal A9 only three bands were visible. One of these bands occurred just over half way through the blot; the other two were lower in molecular weight, with the predominant band equivalent in mobility to a 14 kDa protein. Bands at these three positions were also seen in the lanes developed against the rabbit anti-spore IgG preparations, but not in lanes developed with either guinea pig antiserum. No bands were visible in

176

lb C D

. . . . . ~E~ E ~ E ¸

Fig. 1. Western blotting of spore extracts of B. anthracis. (a) blotting of polyclonal and monoclonal antibodies against spore extract of B. anthracis Sterne (A) and Vollum (B), by the method of Ezzell and Abshire [20]. Lanes: 1, guinea pig anti-Anvax antiserum, diluted 1 : 100; 2, rabbit anti-Vollum spore IgG, at 100 ~ g / m l ; 3, rabbit anti-Sterne spore IgG, at 100 ~ g / m l ; 4, mouse, monoclonal El2, undiluted; 5, mouse monoclonal A9, undiluted; 6, mouse hyper immune serum, diluted 1:100; 7, mouse monoclonal anti-capsule, diluted 1 : 100; 8, guinea pig aEA1 antiserum, diluted 1 : 100. The position of the 14 kDa antigen band is indicated. (b) blotting of spore extract of B. anthracis Ames against mouse monoclonal E12 (lane C) and monoclonal A9 (lane D), using the method of Batteiger et al. [23].

lanes incubated with monoclonal E12 or with the U S A M R I I D anti-capsule monoclonal. The mouse hyperimmune antiserum raised against whole spores recognised a number of antigen bands in common with the rabbit anti-spore IgGs and the two guinea pig antisera, but did not react with the three bands visualised by the anti-spore monoclonal A9. Blots for the other B. anthracis strains tested, Ames and Texas, gave broadly similar results (not shown), except that Texas strain gave far fewer bands with polyclonal reagents.

When Western blotting of B. anthracis Ames spore extract was performed by the arguably more sensitive method of Batteiger et al. [23] in which radioactive Protein A is visualised by autoradi- ography, similar results were obtained (Fig. lb ) in that no reactions of monoclonal E12 were de-

tected but monoclonal A9 reacted predominantly with an antigen of 14 kDa.

5. DISCUSSION

The two mouse monoclonal antibodies raised against spores of B. anthracis Ames appear to react with different epitopes. The monoclonal E12, produced after immunisation with whole spores, reacted well with the surface of spores of several B. anthracis strains in IF tests, but did not react in Western blots of spore extracts. The simplest ex- planation would be that the epitope for El2 either was not extracted or was altered during extraction. Monoclonal A9, produced after immunisation with spore extracts, in IF tests only reacted consistently

with the immunogen strain Ames, and then with a very small proportion of the spores. Such IF results would be explained if the antigen involved were not a structural surface antigen but cell debris non-specifically absorbed to the spore surface. Monoclonal A9 also differed from El2 in that it reacted in Western blots of the four B. anthracis strains tested. Thus, it seems likely that the extractable epitope for A9 is not normally exposed on the spore surface.

The identity of the surface antigen specific for monoclonal El2 will be difficult to determine. The present results indicate that this antigen retains its immunological activity during heating or formali- sation treatments sufficient to render the spores non-viable in laboratory culture, but there are some indications that antigenic activity is not stable during prolonged storage of spore suspensions. Spores stained with E12 usually displayed a greater heterogeneity of staining than when mouse hyperimmune serum was used. The effect of spore growth method on the heterogeneity of staining with E12, together with the evidence of increased antigenicity towards monoclonal El2 with increas- ing incubation time during spore preparation on the solid medium, suggests that the maturation of this antigen on the spore surface may continue late in spore development. The flow immunofluorescence techniques we have developed for bacteria would provide an ideal vehicle for more precise population studies of the staining of spores with monoclonal El2, especially if cell sorting could be used to investigate the genetic basis of variation in antibody uptake [24,25]. Cell sorting would also facilitate investigation of the non-refracti le bacteria, presumably spores, sometimes seen in B. anthracis spore preparations, which were similar in size to the refractile spores and were often stained by monoclonal E12.

Even after growth on WSM, preparations of the B. anthracis strains A-Vollum-lB1 and a_ Ames-1 that had been cured to remove the pX01 plasmid contained a low proportion of spores that reacted with El2. This may be considered further evidence that curing for this plasmid also affects the spore surface [11].

The absence of IF cross reactions between monoclonal E12 and spores of a number of non-

177

anthrax Bacillus strains whose antigenic relationship with B. anthracis has been established using rabbit antisera [8,9,11] and now using mouse antiserum, is good evidence that the epitope for this monoclonal is restricted to B. anthracis. The epitope also appears to be specific for spores as opposed to vegetative cells. The absence of stained cell debris in IF tests based on monoclonal El2 is a major advantage over assays using anti-spore antisera, especially beneficial to the inexperienced microscopist. We reported earlier that a rabbit polyclonal antiserum against B. anthracis Sterne spores reacted in IF tests with spores of Sterne and several encapsulated, virulent strains, but not with the encapsulated strain Vollum [9,11]. Poly- clonal antisera against Vollum spores reacted widely with anthrax strains, including Sterne, evidence for a second spore antigen with a different distribution among anthrax strains. The present IF results indicate that the epitope for monoclonal El2 occurs on the surface of Vollum spores and at a lower density on Sterne spores, and thus the specificity of El2 reflects that of the polyclonal anti-Vollum reagent. Monoc[onals against Sterne spores are now needed to confirm the existence of the second antigen type.

A C K N O W L E D G E M E N T

The specificity studies were performed while one of us (APP) was seconded to the US Army Medical Research Institute of Infectious Diseases, by kind permission of the Commander. Among the U S A M R I I D staff, we particularly wish to acknowledge the advice and help given by Dr. John W. Ezzell, Jr., and the technical support of Mrs. Teresa G. Abshire.

REFERENCES

[1] Doyle, R.J., Keller, K.F. and Ezzell, J.W. (1985) Bacillus, in Manual of Clinical Microbiology, (Lennette, E.H, Bal- ows, A., Hausler, W.J. and Shadomy, H.J., eds.), 4th edn., pp. 211-215, Am. Soc. Microbiol., Washington D.C.

[2] Pederson, C.S. (1956) Symposium on problems in taxon- omy. Bact. Rev. 20. 274-276.

178

[3] Kaneko, T., Nozaki, R. and Aizawa, K. (1978) De- oxyribonucleic acid relatedness between Bacillus anthra-

cis, Bacillus cereus, and Bacillus thuringiensis. Microbiol. Immunol. 22, 639-641.

[4] Cole, H.B., Ezzell, J.W., Keller, K.F. and Doyle, R.J. (1984) Differentiation of Bacillus anthracis and other Bacillus species by lectins. J. Clin. Microbiol. 19, 48-53.

[5] Smith, N.R., Gordon, R.E. and Clark, F.E. (1952) Aerobic spore-forming bacteria. US Department of Agriculture Monograph no. 16, Washington, DC.

[6] Lamanna, C. and Eisler, D. (1960) Comparative study of the agglutinogens of the endospores of Bacillus anthracis

and Bacillus cereus. J. Bacteriol. 79, 435-441. [7] Norris, J.R. (1962) Bacterial spore antigens: a review. J.

Gen. Microbiol. 28, 393-408. [8] Fluck, R., Bohm, R. and Strauch, D. (1977) Fluo-

reszenzserologische Untersuchungen von Kreuzreaktionen zwischen Sporen yon Bacillus anthracis und Sporen anderer aerober Sporenbildner. Zbl. Vet. Med. B 24, 497-507.

[9] Phillips, A.P., Martin, K.L. and Broster, M.G. (1983) Differentiation between spores of Bacillus anthracis and Bacillus cereus by a quantitative immunofluorescence technique. J. Clin. Microbiol. 17, 41-47,

[10] Phillips, A.P. and Martin, K.L. (1983) Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores. Appl. Environm. Microbiol. 46, 1430-1432.

[11] Phillips, A.P. and Martin, K.L, (1988) Investigation of spore surface antigens in the genus Bacillus by the use of polyclonal antibodies in immunofluorescence tests. J. Appl. Bacteriol. 64, 47-55.

[12] Ezzell, J.W., lvins, B.E. and Leppla, S.H. (1984) Im- munoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal fac- tor components of Bacillus anthracis toxin. Infect. Im- mun. 45, 761-767.

[13] Ivins, B.E., Ezzell, J.W., Jemski, J., Hedlund, K.W., Ristroph, J.D. and Leppla, S.H. (1986) Immunisation studies with attenuated strains of Bacillus anthracis. In- fect. Immun. 52, 454-458.

[14] Williams, D.J., Franklin, J.G., Chapman, H.R. and Clegg, L.F.L. (1957) Methods of assessing the sporocidal el-

ficiency of an ultra high temperature milk sterilising plant. Part 1. Experiments with suspensions of spores in water. J. Appl. Bacteriol. 20, 43-49.

[15] Burke, W.F. (1982) Bacillus subtilis extracellular nuclease production associated with the spoOH sporulation locus. J. Gen. Microbiol. 128, 1591-1597.

[16] Ristroph, J.D. and Ivins, B.E. (1983) Elaboration of Batil- lus anthracis antigens in a new, defined culture medium. Infect. Immun. 39, 483-486.

[17] Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewsky, K. (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. lmmunol. 123, 1548-1550.

[18] Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 296, 495-497.

[19] Quinn, R., Campbell, A.M. and Phillips, A.P. (1984) A monoclonal antibody specific for the A antigen of Brucella

spp. J. Gen. Microbiol. 130, 2285-2289. [20] Ezzell, J.W. and Abshire, T.G. (1988) Immunological

analysis of cell-associated antigens of Bacillus anthracis. Infect. Immun. 56, 349-356.

[21] Phillips, A.P. and Martin, K.L. (1982) Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus. J. Immunol. Methods 49, 271-282.

[22] Phillips, A.P. and Martin, K.L. (1982) Assessment of immunofluorescence measurements of individual bacteria in direct and indirect assays for Bacillus anthracis and Bacillus cereus spores. J. Appl. Bacteriol. 53, 223-231.

[23] Batteiger, B., Newhall, W.J. and Jones, R.B. (1982) The use of Tween-20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose mem- branes. J. Immunol. Methods 55, 297-307.

[24] Phillips, A.P. and Martin, K.L. (1985) Dual-parameter scatter-flow analysis of Bacillus spores. Cytometry 6, 124-129.

[25] Phillips, A.P., Martin, K.L. and Capey, A.J. (1987) Direct and indirect immunofluorescence analysis of bacterial populations by flow cytometry. J. lmmunoL Methods 101, 219-228.

monoclonal antibodies against spore antigens of bacillus anthracis

Documents