Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

FEMS MicrobiologyEcology73 (1q90) 299-308 Published by Elsevier

299

FEMSEC 00263

Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil Julie M a s o n a n d R i c h a r d G. Burns Biological Laboratory, University of Kent at Canterbury, Canterbury, Kent, U.K

Received31 October !989 Revision received22 December 1989 Accepted 2 January 1990 Key words: Immunological detection of bacteria; Soil inoculation; Monoclonal antibodies

1. S U M M A R Y Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods (Pseudomonas / Flavobacterium type) isolated from the soil into whicll P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 grown under lownutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism

Correspondence to: R.G. Bums, Biological Laboratory, University of Kent at Canterbury, Kent CT2 7NJ, U.K.

should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the Iocalisation of the antigen in the outer m e m b r a n e / c e l l wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g - i soil can be enumerated.

2. I N T R O D U C T I O N There is growing interest in the release of microorganisms for maintaining or improving soil fertility and for the clean-up of contaminated environments. These microbial inoculants may act to reduce plant pathogens [1,2], solubilise nutrients and stabilise the soil [3,4] or act as degraders of persistent pollutants [5,6]. However, a major constraint to releasing microbes (especially those which have been genetically engineered) into the environment is our lack of knowledge of how microbes survive, proliferate and disperse in soil. Before these questions can be answered, sensitive and accurate methods for detection and enumeration of introduced mic~ ~bial species are needed.

0168-6496/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties

300 Dilution plate methods are commonly used to estimate numbers of bacteria although counts are known to be selective, underestimate bacterial numbers and subject to experimental error [7]. The most recent advances towards accurate and sensitive detection have been the development of serological probes and recombinant DNA techniques [8]. The latter procedure usually involves :he insertion of a gene or genes into an organism which codes for an enzyme(s) which allows the microorganism to be detected by using selective media [9,10]. More direct approaches involve the detection of recombinant DNA sequences using DNA probes [11] and analysis of rRNA profiles. The 16S rRNA is known to be conserved in certain groups of microorganisms and therefore acts as a selective probe [12,13]. Oligonucleotide hybridization probes are also being developed [14] which complement either organism-specific or universally conserved regions of 16S rRNA. However, there are still problems with recovery of rRNA and also rRNA genes may be in several copies per cell and are thus not representative of numbers [7]. Antisera against bacteria are commonly used for the detection of medically important pathogens and since the development of monoclonal antibody technology [15] many papers have been pubfished on the use of monoclonal antibodies in medical diagnosis [16]. There is now increasing interest in the production of monoclonal antibodies against ecologically important microorganisms [17,18] and efforts are being directed towards using monoclonal antibodies to detect plant pathogens [19-21]. We report here the isolation of hybridomas secreting monoclonal antibodies for a common soil organism, Flaoobacterium sp. P25, and describe a powerful method for the detection of P25 which employs monoclonal antibodies which are species or even strain specific. Preliminary data indicate the value of MABS in detecting and enumerating P25 following inoculation of soil.

3. MATERIALS AND METHODS 3.1. Bacterial strains and culture conditions Fiavobacterium P25 was originally isolated from

a calcareous clay soil (sand 42%, silt 32%, clay 26%; pH 6.9, organic carbon 2.1~) and is currently being used in soil inoculation experiments (Thompson and Burns, personal communication). P25 is a triple antibiotic resistant spontaneous mutant (kanamycin 50/~g m1-1, streptomycin 250 /~g m1-1, rifampicin 100 #g ml -~) which can be enumerated on antibiotic selective media and whose physiological and biochemical properties are identical to those of the wild-type organism. P25 was grown on nutrient broth (No. 2, Oxoid), 25°C and stored on nutrient agar at 4°C. The effect of nutrient limitation on P25 antigen expression was investigated following growth on 1/2 and 1/10 strength nutrient broth. To study the effect of starvation P25 was grown for 16 h in nutrient broth, harvested by centrifugation (25 000 x g, 15 rain, 5°C) and washed twice in 0.1 M phosphate buffer (pH 7.2). The washed suspension was added to 50 ml of the same buffer to give an initial concentration of 1 × 106 cells ml-1 (as determined by dilution plate counts) and stored at 25 °C for 200 days. During this period the culture was sampled and the cells were monitored by indirect immunofluorescence. All other bacteria, namely Pseudomonas aeruginosa (NCIB 8298), P. fluorescens (NCIB 3726), P. syringae (NCIB 1301A), Klebsieila oxytoca (NCIB 5938), Staphylococcus aureus (NCIB 8635), Citrobacter freundii (NCIB 3735), Xanthomonas maltophilia (NCIB 9204), Erwinia caratovora (NCPPB 926), Flavobacterium resinovorum (NCIB 8767), F. capsulatum (NCIB 9890), F. balustinum (NCIB 11409), Erwinia amylovora (UKC culture collection), Arthrobacter sp. (UKC culture collection) and Flavobacterium B I 4 / I and Flavobacterium B2/1 (Dr. K.P. Flint, University of Warwick) were also grown on nutrient broth, 25 ° C. Flavobacterium aquatile (NCIB 8694) was grown on (g/l) tryptone 2.0, beef extract 0.5, yeast extract 0.5, sodium acetate 0.2 (pH adjusted to 7.2). 3.2. Antigen preparation P25 was grown for 16 h in nutrient broth at 25 o C. The cells were harvested in late exponential phase by centrifugation (as described above), washed twice in 30 mM Tris-HCl buffer (pH 7.2)

301 and resuspended in 10 ml of the same buffer. The suspension was passed twice through a French pressure cell at 140 MPa. The homogenate was centrifuged at 2 000 × g for 10 min to remove unbroken cells, and the crude cell wall/membrane pellet obtained by centrifugation at 38 000 × g for 40 rain at 5°C. The pellet was resuspended in 30 mM Tris-HC~ buffer (pH 7.2) and frozen in aliquots for storage at - 2 0 ° C. The protein content of the extract was estimated using a microcolorimetric assay (Bio-rad Laboratories, Munich).

3.3. Monoclonal antibody production Four 6-week-old Balb/c mice were used for immunisation, lntraperitoneal injections of crude cell wall/membrane extract consisting of 30/~g of protein in Freunds incomplete adjuvant were given on days 1, 14 and 28. Mice were tail-bled 8 days after the third injection to assess the response to the antigen. The serum was screened using an enzyme-linked immunosorbant assay described below. The final boost (60/~g protein) was given without Freunds adjuvant on day 41 and the mice were killed on day 46. The spleen was removed and lymphocytes were collected in Rothwell Park Memorial Institute medium (RPMI-1640, Imperial Laboratories, U.K.) using blunt forceps. The spleen cells were fused with SP2/myeloma cells using 50~ polyethylene glycol (Boehringer Mannheim, GMBH) [22]. The fusion mixture was plated out in RPMI supplemented with: glutamine (2mM), non-essential amino acids, foetal calf serum (10%) and hypoxanthine aminopterin thymidine (HAT), all supplied by Gibco. The plates were incubated at 3 7 ° C in a moist 5% CO2 in air atmosphere. The resulting hybridomas were screened for antibody production 2-3 weeks after fusion.

3. 4. Screening procedures Polyclonal serum from the tail bleeds and supernatants obtained from hybridomas were screened using an enzyme-linked immunosorbant assay (ELISA). The antigen was dried (37°C, 12 h) onto a microtiter plate at a concentration of 2.5 /~g protein per well. Non-specific binding sites were blocked with phosphate-buffered saline (PBS; (g/l) NaCI, 8.0; KCI, 0.2; Na2HPO 4 • 7H20, 2.16;

KH2PO 4, 0.2) with bovine serum albumin (1% w/v). Test antibody was then incubated for 60 rain, followed by anti-mouse lgG peroxidase conjugate (Sigma) (a phosphatase conjugate was not chosen as endogenous phosphatase activity was found). Between additions of blocking agent, primary, and secondary antibodies, the plate was washed using PBS plus 0.05% Tween-20. After addition of o-phenylenediamine dihydrochloride substrate (0.04% w / v ) the colour was allowed to develop for 30 min and the reaction was stopped with 4 M sulphuric acid. Absorbance was read at 490 nm in a Dynatech plate reader. Reactions were generally considered positive if the absorbance value was greater than 0.6 optical density units (ODU), non-specific background readings were never greater than 0 . 1 0 D U . Where the superuatants gave optical densities of 0 . 8 0 D U and above, a second screening procedure (indirect immunofluorescence) was also employed. This method was adapted from Sherwin et al. [23]. Slides were coated with poly-L-lysine (100/~g/ml for 10 min, dried for 16 h at 25°C), and 20/~! of a culture of P25, resuspended to an optical density of 0 . 3 0 D U at 600 nm, was dried onto the slide at 25 o C. The slides were incubated (25 o C, 1 h in a moist atmosphere), with test supernatant as primary antibody or with unrelated antibody (a monoclonal antibody previously shown not to cross-react with P25) as a negative control. After incubation the slides were washed (3 × 5 rain) in PBS. The secondary FITC-labelled anti-mouse serum (Sigma F-0257) was applied at a dilution of 1 : 10 in PBS and the slides were incubated for 1 h at 25 ° C in a moist dark atmosphere. After a final wash (as above) the specimens were mounted in a Mowiol (Hariow Chemical Co. Ltd., Essex) solution (2.4 g Mowiol plus 6 g glycerol added to 6 ml of distilled water plus 8.5 ml 0.2 M Tris buffer, pH 8.5). To reduce fading, 100/~l of p-phenylene diamine solution (100 m g / m l ) was added to 900 ~1 of Mowiol. Immunofluorescence was observed using a Zeiss Axioscop microscope with appropriate filters (Zeiss filter set 48-79-15). Hybridomas producing antibodies with both good ELISA activity and immunofluorescence were cloned three times at one cell per well to ensure cell fine purity. Monoclonal antibody D10 gave a

302 strong immunofluorescence result and was chosen for further study. It was class typed using a mouse monoclonal isotyping kit (Amersham International, Buckinghamsl~re) and found to be class IgG1.

3.5. Cross-reactivity A selection of bacteria commonly found in soil was obtained from culture collections. The indirect immunofluorescence technique was used as the screening procedure and Flavobacterium P25 was used as a positive control. Negative controls included the use of secondary antibody alone, an unrelated antibody, and cell culture medium as a substitute for primary antibody. Screening of isolates obtained from soil samples previously inoculated with P25 was carried out using a modified colony blotting method [24]. The colonies to be tested were streaked onto nutrient agar plates (six isolates plus the positive control (P25) and a negative control, E. coil B). A disc of nitrocellulose was placed on the surface of the plate for 1 min. Once removed, the nitrocellulose was washed in distilled water for 5 min to remove colonies which were adhering to the surface of the paper. The nitrocellulose was transfcred to a wash of phosphate buffered saline (PBS) with 0.3% Twccn-20 (20 min) and washed (3 × 2 rain) in distilled water. The nitrocellulose was then washed for 20 rain in PBS (0.1% Tween-20) and finally in distilled water (3 × 20 min). The undiluted hybridoma supernatant containing MAB D10 was incubated with the nitrocellulose for I h. After three washes in distilled water the nitrocellulose was incubated with secondary antibody: peroxidase-conjugated rabbit anti-mouse immunoglobulin ( 1 : 1 0 0 0 dilution in PBS-0.1% Tween 20; Dakopat*s, Denmark). The colour reagent was prepared by dissolving 18 mg of 4-chloronapthol (Sigma) in 6 ml of methanol at 4 ° C . This solution was added dropwise to 24 ml of PBS with stirring. Immediately before use, 30 /~1 of hydrogen peroxide was added to the reagent. The nitrocellulose was incubated with the colour reagent on a shaker and the blue-grey colour developed in 5-15 min.

3.6. Detection of P25 in soil samples Non-stcrilised soil samples inoculated with P25

(1 × 10 ~° g - l ) were analysed using a modified immunofiuorescence technique with MAB D10. A high inocuhim density was used to allow for likely lo~ses due to predation in non-sterilised soil. In addition, preliminary experiments had shown that decay rates of P25 in soil were rapid in some circumstances. Soil dilutions (10 -3 , 10 -4 , 10 -5 , 10 -6) were centrifuged at 4 000 × g in an MSE bench centrifuge for 5 min. Based on dilution plate counts using selective agar extraction efficiency was 96% + 3%. The pellet was resuspended in 1 ml of phosphate buffer (pH 7.2) in an Eppendorf tube, recentrifuged and resuspended in 1 ml of D10 hybridoma supernatant. The contents were left to incubate at 25 ° C for I h, with mixing at 10-min intervals. After centrifugation the pellet was washed and resuspended in secondary antibody (as used in the immunofluoresccnce technique). Incubation was in the dark at 25 ° C for 1 h, with mixing every 10 min. The contents were then harvested and washed in sterile distilled water and 10-/~1 aliquots mounted onto multitest slides (Flow laboratories) for microscopic examination.

3. 7. Electron microscopy and immunogold labelling of e2s Preparation of samples. P25 was harvested after 16 h growth on nutrient broth. The pellets were fixed in 2.5% gluteraldehyde in 30 mM phosphate buffer, pH 7.4, for 1 h. The bacterium was prepared for embedding in Spurrs [25] for observation of sections and also in Lowicryl [26] for immunogold studies. The latter resin preserves antigenicity. For embedding in Spurrs, after fixing, the pellet was washed in phosphate buffer and incubation for 1 h in 1% OsO 4. The pellet was washed again and incubated overnight in 1% uranyl acetate at 4 ° C. The sample was then dehydrated in a series of increasing concentrations of industrial methylated spirits (IMS) (30-100%). After three 1 h incubations with 100% ethanol the sample was incuba:ed overnight in 30% Spurrs/ ethanol. The next stage involved successive incubations with increasing concentrations of the resin. Once 100% Spurts was reached the sample was polymerised overnight at 60 ° C. :~amples for embedding in Lowicryl were washed in phosphate buffer (30 mM, pH 7.4) after

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fixation and then subjected to successive dehydration using IMS as described above except that in this case all incubations were at 4 ° C until 100% IMS was reached when the temperature was reduced to - 2 0 ° C . After two 1 h incubations in ethanol ( - 2 0 ° C) Lowicryl was added to a final concentration of 50% and the samples were incubated overnight at - 2 0 ° C. During the next 12 h three changes of Lowicryl were made and the samples were incubated at - 30 ° C. Polymerisation was carried out at - 3 0 ° C under UV overnight followed by 8 h at room temperature under UV. The blocks obtained were sectioned (Reichert Uitracut E ultramicrotome) and mounted onto copper grids (Spurrs sections) and nickel or gold grids (Lowicryl sections). The Spurrs sections were stained with uranyl acetate (570 in 17o acetic acid) for 30 rain at 60 ° C followed by washing and a final incubation in lead citrate [27] for 10 rain. The grids were rinsed and allowed to air dry before observation. lmmunogold labelling. Gold/nickel grids were placed in a 25-VI drop of a 1070 solution of heat-inactivated normal goat serum in Tris buffer (20 mM Tris, 225 mM NaCI, pH 7.2) containing

0.1% BSA and incubated for 1 h at 25°C. The grids were then transfered to a 25-/~! drop of primary antibody, which in this case was undiluted D10 supernatant. Controls using unrelated antibody and Tris buffer as substitutes for primary antibody D10 were also prepared. The grids were incubated at 25 ° C for I h and then washed for 10 s with buffer (20 mM Tris, 225 mM NaCl with 0.1% BSA at pH 8.2) before being placed in secondary antibody (goat anti-mouse IgG labelled with 10 nm colloidal gold, Biocell, Cardiff) diluted 1 : 10 with Tris buffer at pH 8.2. The grids were incubated at 25 ° C for 1 h and given a final wash in Tris buffer (pH 7.2), allowed to air dry before lightly staining with uranyl acetate (30 min, room temperature) followed by lead citrate (10 min at room temperature). The grids were rinsed and dried before observation.

4. RESULTS A N D DISCUSSION

4.1. Isolation of monoclonal antibodies Hyhridoma supernatants giving good binding to the antigen using both ELISA and indirect

304 Table 1 Cross-reactivityof MAB D10 with some Flavobaeterium species and other bacteria commonly found in soil Microorganism Flavobacterium (P25) Flexibacterium sp. (6) Erwinia caratovora (NCPPB 929) E. amylovora (UKC) Citrobacterfreundii (NICB 3735) Pseudomonas aeruginosa (NCIB 3726) P. putida (NCIB 9494) P. syringae (NCIB 130IA) Xanthomonas maltophilia (NCIB 9204) Klebsiella oxytoca (NCIB 5938) Staphylococcus aure~,s (NCIB 8635) Arthrobacter sp. (U~'.C) Flavobacterium res#,o,Jorum (NCIB 8767) F. capsulatum(NCIB 9890) F aquatile (NCIB 8694) F. balustinum (NCTC 11409) Flavobacterium B14/1 Flavobacterium B2/1

Antibody DI0 +a

a +, fluorescence; - . no fluorescence.

immunofluorescence screens were chosen to clone through to the monoclonal state. Twenty strong positives were obtained from one fusion and three of these positives were selected to clone by the limiting dilution technique. Three consecutive clonings at one cell per well were carried out to obtain monoclonal antibodies, and of the three identified D10 was chosen for detailed study as it gave the strongest immunofiuorescence. The fluorescenee given with D10 and the target bacterium P25 is shown in Figs. l a and l b and indicates that the antigen is associated with the cell wall. We have therefore been able to isolate monoclonal antibodies specific for a surface antigen of P25 using the 'shot-gun' approach whereby a crude antigen is used to raise the antibodies. 4.2. Interaction o f DIO with other bacteria A selection of common soil bacteria did not interact with D10 (Table 1). Several Flavobacterium spp. obtained from culture collections also gave no cross-reactivity with the monoclonal antibody (Table 2). Flavobacterium balustinum, the Flavobacterium sp. most closely related to P25 (as

determined by the NCIB), did not cross-react with D10, indicating the exquisite specificity of the monoclonal antibody for targeting P25. Gramnegative orange/yellow pigmented colonies (i.e. Pseudomonas, Flavobacterium, Flexibacter group) isolated from the original soil were screened using the 'colony blot' procedure and all gave negative responses when probed with D10. A selection o[ the colonies was also screened using the indirect immunofluorescence technique and no binding by D10 was seen. This lack of cross-reactivity confirms tl-~ -¢alue of the monoclonal antibody in specificaliy i~,ontifyin$ P25. The inability of D10 to recognise the strain of Flavobacterium most closely related to P25 indicates that either we have an antibody which is not only species specific but strain specific, or that P25 may be a novel Flavobacterium species. 4.3. Binding o f M A B DIO to P25 grown under low-nutrient and starvation conditions To determine whether nutrient levels affected the expression of the cell surface antigen and therefore the ability of MAB D10 to target P25, immunofluorescence was carried out after the organism was grown on 1 / 2 strength and 1 / 1 0 strength nutrient broth. P25 grown under nutrient limiting conditions was detected by D10. Starvation of P25 was achieved by storing washed cells in phosphate buffer for 12 weeks. The numbers of viable organisms present over time was determined by plate counts and their response to the monoclonal antibody was monitored at each stage.

Table 2 Immunofluorescence of Flavobacterium P25 with MAB DI0 when grown under different nutrient conditions or stored in phosphate buffer Growth or storage condition Nutrient broth (25 g/l) Nutrient broth (12.5 g/l) Nutrient broth (2.5 g/l) Stored in phosphate buffer (pH 7.4, 25°C, 200 days) Stored as wet pellet (4 ° C, 14 days)

Antibody D10 + + + + +

Fig. 2. Electron micrographs of P25 sections: a Spurrs section ( × 68 000) b Lowicryl section stained by inununogold labelling using MAB D10 ( × 80 000). (Publisher's magnification 0.97 × ).

Aher 50 days of starvation P25 could be detected using the MAB but cells were smaller and more coccoid in comparison with actively growing cells and the cell surface waz less sharply defined when observed using fluorescence. However, even after 200 days starvation viable P25 cells were present in the buffer and could be detected using D10 (Table 2). Thus the antigen targeted by MAB D10 appears to be a constitutive component of the cell and expressed under a range of nutritional conditions. The immunogold labelling experiments using D10 confirmed that the antigen is predominantly or exclusively located in the cell wall/outer membrane region of P25 (Figs. 2a and 2b).

4.4. Detection of P25 in soil samples We were able to detect P25 grown under nutrient limiting conditions and after prolonged starvation and therefore the ofigotrophic nature of soil should not prove a deterrent to using MAB D10 to monitor P25 released in soil. Soil dilutions from both field (54 days and 76

days after inoculation) and laboratory (24 h after inoculation) experiments were probed u~ing MAB D10 in order to evaluate its applicability for use with environmental samples. P25 was observed in all laboratory soil dilutions although some diffuse background fluorescence was encountered which made quantification difficult. Wheat roots grown in the presence of P'25 were harvested from field experiments, washed and probed with D10. Clusters of P25 were visible on the roots but again some background fluorescence of plant material was observed. The limit of statistically valid enumeration of P25 in soil using dilution plates and selective media approximates to 200 organisms g-1 dry weight of soil. However, in both soil and rhizospbere field samples in which P25 had been detected yet where numbers were below quantifiable limits ( < 200 g - ~ soil), we used D10 to detect significant numbers of the inoculated bacteria which by recording fluorescent cells in a large number of microscope fields suggested a level of sensitivity as low as 20 g-~ soil. The method used

306 to target P25 in soil dilutions is a modified ir~lmunofluorescence technique and this is in need of further modification to reduce the problems o f background fluorescence (using blocking agents) a n d to make the test m o r e quantitative. Probing the bacteria in control (i.e. non-inoculated) soils gave an encouraging result in that n o organisms were targeted. Therefore identification o f the released P25 should not be h a m p e r e d by any crossreactivity from the indigenous population. The sensitivity of b o t h the immunochemicai and recombinant D N A approaches to detect organisms varies from 102-106 organisms per g or per ml o f sample [20,28,29]. If immunochemical probes such as monoclonal antibodies are to be used successfully to detect and enumerate microorganisms certain problems need to b e overcome. T h e most i m p o r t a n t o f these were outlined by Bohool and Schmidt [30] and are: (1) the a n t i b o d y needs to be specific for the organism so as to eliminate problems of cross-reactivity and n o n specific binding; (2) interference from autofluorescence or non-specific binding to background m u s t b e eliminated; (3) the antigen targeted must b e stable and ideally an indicator o f viability in order to distinguish between live, dead or ' d o r m a n t ' cells; and (4) efficiency of recovery of the desired ceils from samples for quantification m u s t be optiraised. M a n y o f these problems have been overcome in the work presented here where we have achieved the isolation o f a specific antibody for P25 which targets a stable antigen. The specificity achieved should allow us to reduce or even eliminate the interference from non-specific binding. Identification and quantification of the released organism could either b e indirect (for example using an enzyme-linked i m m u n o s o r b a n t assay to detect extracted antigen) or direct by targeting the antib o d y with fluorescent markers. Immunofluorescence-based techniques such as image analysis and in particular flow cytometry are currently being investigated. ACKNOWLEDGEMENTS T h e research reported here was s u p p o r t e d by the D e p a r t m e n t of the Environment. Special

thanks go to Paola Quatrini for technical assistance, and Susan Page and Dr. Nigel Jenkins for advice.

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