Journal of Immunological Methods, 144 (1991) 11 - 17

11

© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50

JIM06093

Evaluation of monoclonal antibodies to Listeria monocytogenes flagella by checkerboard ELISA and cluster analysis Eystein Skjerve 1,2, Wilhelm Bos 3 and Bram van der Gaag 3 1Department of Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway, e Department of the Science of Food of Animal Origin, Veterinary Faculty, Universityof Utrecht, The Netherlands, and 3 Department of Microbiology, Section of Molecular Biology, CIVO Institutes TNO, Zeist, The Netherlands (Received 19 February 1991, revised received 8 April 1991, accepted 21 May 1991)

A simple ELISA technique is described which utilizes whole bacteria as solid phase antigens for the evaluation of monoclonal antibodies against flagellar proteins of Listeria monocytogenes. Cluster analysis of the ELISA extinction values gave distinct reaction patterns from 23 tested supernatants. The method offers the possibility of screening supernatants without the necessity for standardization of antibody concentration or the labelling of established monoclonal antibodies. Key words: Monoclonal antibody; Listeria monocytogenes; Cluster analysis

Introduction Listeria monocytogenes has become the subject of renewed interest in public health and food hygiene due to acknowledged epidemics caused by food contaminated with Listeria monocytogenes (Kampelmacher and Mossel, 1989; Mossel, 1989). A number of studies have reported the successful production of monoclonal antibodies (Mab) reacting with different antigen of Listeria monocytogenes. Butman et al. (1988) produced genus-specific antibodies against Listeria spp. whilst Farber and Speirs (1987) produced a Mab against the flagella of Listeria and discussed its use in enzyme immunoassays. More recently

Correspondence to: E. Skjerve, Department of Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 DEP., 0033 Oslo 1, Norway (Tel.: 47.2.964844; Fax: 47.2.964850).

McLauchlin et al. (1989) used Mab in an immunofluorescence technique to detect Listeria in foods. Mab have also been used for the characterization and purification of cell surface antigens from Listeria monocytogenes (McLauchlin and Taylor, 1989). Recently, Skjerve et al. (1990) reported the use of Mab against flagella in an immunomagnetic separation technique to detect Listeria monocytogenes in foods. Thus, the prospects of using Mab for the isolation and identification of Listeria spp. appear to be promising. The characterization of Mab involves techniques of antibody capture, antigen capture, or functional assays, based on different EIA techniques which include ELISA, immunoblotting, dot blots and other techniques (Harlow and Lane, 1988). The identification and characterization of the separate clones is often laborious although methods for screening large numbers of supernatants have been described (Perutelli et al., 1989; Miller et al., 1990). For example Lane et al.

12 (1989) used a grid-blot procedure for screening large numbers of Mab against native and denatured proteins. A similar approach was also used in a checkerboard immunoblotting system reported by Kazemi and Finkelstein (1990). The overall goal of this project was to develop Mabs against flagellar antigens of Listeria monocytogenes which could be used in immunomagnetic separations of live bacteria. Therefore, an assay was needed to evaluate Mabs against different epitopes in situ on the bacteria. One of the first examples of the application of cluster analysis was in the field of bacterial taxonomy (Austin and Priest, 1986). Other examples include the use of biochemical data (Shelly et al., 1987; Faresu and Jones, 1988; Shaw and Latty, 1988; Mugnay et al., 1989), and analysis of SDSPAGE gel protein patterns (Costas et al. 1987). So far these numerical techniques have not been used for screening and characterization of different Mabs. The present paper describes the development of Mab against flagellar antigens of Listeria monocytogenes, and a method to screen and evaluate the optical density data on the antibodies from a simple sandwich ELISA analysed by hierarchical cluster analysis.

Materials and methods

Isolation of flagella A strain of L. monocytogenes (L 10, serotype 4) from the strain collection at the Department of Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway was cultivated at 22°C for 4 days in Tryptic Soy Broth (TSB, Oxoid, Basingstoke, England) supplemented with 0.2% glucose and 0.6% yeast extract. Flagella were isolated following the procedure described by Peel et al. (1988) using mechanical disruption with glass beads followed by centrifugation steps to remove debris before a final ultracentrifugation step. Isolated flagella were resuspended in phosphate-buffered saline, pH 7.4 (PBS). The protein content was measured using the Pierce protein assay (Pierce, Rockford, IL), and the protein content was adjusted to 1 mg flagella/ml. The anti-

gen was then kept frozen in aliquots at - 2 0 °C until use.

Monoclonal antibodies One inbred white mouse (BALB/c) was immunized intraperitoneally with 50 /~g flagellar antigen as described by Spitz et al. (1984). An identical booster was given 14 days later, and the mouse was killed 2 days after the booster. The spleen was aseptically removed for a hybridoma fusion with murine myeloma cells (X63 Ag8.653). Spleen and myeloma cells were fused at a ratio of 2:1 using polyethyleneglycol 4000 (Serva, New York, U.S.A.) after the procedure of Galfr~ et al. (1977). Cells were then washed in Dulbecco's MEM 25 mM Hepes (Gibco, New York, U.S.A.) with 13 ml/500 ml supplement: 0.05 mM /3mercaptoethanol (Sigma Chemical Co., St. Louis, MO), 2 mM glutamine (Flow Laboratories, McLean, VA), 50/~g kanamycin/ml (Flow Laboratories), 10 IU penicillin and 10/~g/ml streptomycin (Gibco, Paisely, Scotland) (fusion medium). Resuspension was in fusion medium with 10% hybridoma growth factor (HECS, Costar, Amsterdam, The Netherlands), 20% bovine calf serum (BCS, Hy Clone, Lotah, UT) 0.1 mM hypoxanthine (Sigma) and 1 /.~g/ml azaserine (Sigma). The cells were distributed into six 96 well microtitre plates (Nunc, Copenhagen, Denmark), 100 ~1 into each well. After 2 weeks the supernatants were screened for specific antibody production in an ELISA using flagella as antigens. Positive supernatants were expanded in fusion medium with 10% BCS to 1 and 2 ml volumes in 24 well plates (Nunc). The supernatants were further subcloned using the limiting dilution method (Campbell, 1986) in fusion medium with 10% BCS and 10% HECS. A new subcloning was undertaken after a second screening for specific antibodies. Established supernatants were then evaluated using an ELISA with whole bacteria as solid phase antigens. After the evaluation of the supernatants, three suitable clones were grown in protein-free hybridoma medium (Gibco), and the antibodies produced were concentrated by filtration of the supernatant using omega (100 K) filters in an Amicon 8200 concentrator (Amicon, Damwers, MA).

13

Bacterial strains Different bacterial strains from the strain collection of the Department of Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway, and from the Department of Food of Animal Origin, Veterinary Faculty, University of Utrecht, the Netherlands were used in the experiments. The strains used were as follows: 14 of Listeria rnonocytogenes, three of Listeria inocua and one each of Staphylococcus aureus, Escherichia coli and Streptococcus faecalis. All strains were passaged twice on 7% ox blood agar (Oxoid), after which one colony was transferred to 10 ml of brain heart infusion broth (BH1B, Oxoid). The BHIB was incubated for 48 h at 25 °C (Listeria spp.), and the remaining colonies at 37°C for 24 h. Bacterial suspensions were centrifuged at 3000 x g for 10 min, washed twice in PBS, and resuspended in 1 ml 50 mM carbonate buffer pH 9.6, giving a concentration of 109 bacteria/ml. ELISA procedures Polyvinylchloride ELISA plates (Costar Europa, Badhoevedorp, the Netherlands) were coated with a 1/100 dilution of bacterial suspension in 50 mM carbonate buffer pH 9.6 using 100 /xl per well. The coating was performed overnight at room temperature. The plates were then washed three times in PBS/0.05% Tween 20 (PBS-Tween) with a plate washer (Flow Laboratories). The same procedure was used in all subsequent washings. Possible free binding sites on the well surfaces were blocked by incubation with 0.1% bovine serum albumin (BSA, Sigma) in carbonate buffer for 1 h at 37 o C. Supernatants from putative clones were diluted 1/10 in PBS-Tween with 1% BCS - sample buffer and 100 tzl assayed in a checkerboard pattern. Plates were incubated at 37°C for 1 h. After washing, peroxidase labelled goat anti-mouse IgG (Tago, Burlingame, CA) was added at a 1/2000 dilution in sample buffer (100/xl per well). After 1 h incubation at 37 o C, the plates were washed and 100 /xl substrate (100 /zl l% TMB (Sigma) in DMSO + 1.5 /xl 30% H 2 0 2 per 10 ml 0.1 M sodium acetate buffer pH 5.5-6) were added. After colour development at room temperature for 10 min, the reaction was stopped by adding 50/xl 1 M sulfu-

ric acid. Optical densities were read by a Multiscan MC photometer (Flow Laboratories) at 450 nm, and results recorded using the Titersoft computer program (Flow Laboratories). Primary screening was performed in a similar ELISA, using the flagellar antigen at a concentration of 10/zg/ml in 50 mM carbonate buffer pH 9.6. IgG subclasses were determined in a similar ELISA procedure using plates coated with subclass specific rat anti-mouse IgG antibodies (Zymed Laboratories, San Francisco, CA). Supernatants were screened at in 1/10 and 1/30 dilutions.

Immunomagnetic separation Immunomagnetic separations (IMS) of Listeria were performed using the selected Mab in the IMS method described by Skjerve et al. (1990). Data analysis Readings from Titersoft were analysed using the VP-Planner (Paperback Software, Berkeley, CA) and the statistical package SPSS PC + (SPSS, Chicago, IL, ver. 3.0). A correlation matrix for all the supernatants was established. Hierarchical cluster analysis was performed using the average linkage method and the vector cosine as a pattern similarity parameter, giving a dendrogram showing the related clusters with distance estimates.

Results

Primary screening with the flagellar antigens and testing for subclass specificity showed that 23 wells produced monoclonal, flagella specific antibodies. All these supernatants were characterized by the whole-bacteria ELISA. Table I shows the correlation matrix from the ELISA results. Several of the correlations were very high (> 0.99), indicating a strong relationship between these supernatants. The dendrogram in Fig. 1 illustrates graphically the relationship between the different monoclonal antibodies, grouping them into two main clusters (supernatants 3-7 and 8-23 respectively), and one more loosely related clusters (1, 2). Table

ELISA using flagellar antigen

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Mab

0.62 0.18 0.10 0.23 0.20 0.39 0.06 0.12 0.06 0.06 0.18 0.24 0.23 0.21 0.23 0.20 0.23 0.19 0.19 0.21 0.15 0.21

1

0.21 0.23 0.14 0.17 0.05 0.05 0.23 0.00 0.00 0.10 0.19 0.17 0.15 0.19 0.15 0.15 0.12 0.13 0.17 0.10 0.16

2

0.54 0.59 0.67 0.49 0.28 0.20 0.15 0.15 0.28 0.14 0.19 0.23 0.27 0.27 0.22 0.26 0.26 0.23 0.42 0.26

3

0.91 0.89 0.82 0.05 0.14 0.10 0.10 0.04 0.05 0.01 0.02 0.03 0.00 0.02 0.01 0.01 0.01 0.16 0.02

4

0.91 0.87 0.07 0.06 0.05 0.06 0.02 0.06 0.02 0.01 0.00 0.02 0.05 0.02 0.01 0.02 0.11 0.01

5

0.91 0.17 0.02 0.00 0.00 0.14 0.02 0.07 0.12 0.02 0.11 0.07 0.09 0.11 0.10 0.23 0.09

6

ELISA using bacterial suspension

0.05 0.13 0.13 0.10 0.03 0.10 0.07 0.04 0.03 0.06 0.08 0.07 0.05 0.07 0.07 0.04

7

0.90 0.93 0.91 0.93 0.92 0.93 0.95 0.90 0.91 0.91 0.92 0.93 0.92 0.76 0.93

8

0.94 0.88 0.84 0.82 0.82 0.85 0.78 0.81 0.81 0.82 0.84 0.82 0.63 0.88

9

0.98 0.95 0.94 0.94 0.94 0.91 0.93 0.92 0.93 0.94 0.93 0.77 0.94

10

0.95 0.93 0.94 0.93 0.93 0.94 0.92 0.92 0.94 0.93 0.85 0.95

11

0.96 0.97 0.96 0.98 0.98 0.98 0.98 0.98 0.98 0.86 0.97

12

0.99 0.98 0.98 0.98 0.98 0.98 0.98 0.9~ 0.79 0.98

13

0.98 0.99 0.99 0.99 0.98 0.99 0.99 0.82 0.98

14

0.97 0.97 0.97 0.97 0.98 0.98 0.78 0.97

15

0.99 0.99 0.99 0.99 0.99 0.87 0.99

16

0.99 0.99 0.99 0.99 0.87 0.96

17

0.99 0.99 0.99 0.84 0.89

18

0.99 0.99 0.84 0.99

19

0.99 0.84 0.99

20

0.82 0.97

21

0.99

22

23

The matrices were developed based on O D results from an E L I S A procedure using different strains of Listeria monocytogenes (14), Listeria inocua (3), Staphylococcus aureus (1), Enterococcus faecalis (1) and Escherichia coli (1) as solid phase antigens.

C O R R E L A T I O N B E T W E E N 23 M O N O C L O N A L A N T I B O D I E S A G A I N S T F L A G E L L A O F LISTERIA M O N O C Y T O G E N E S

TABLE I

15

Rescaled distance 16 17 18 19 20 21 • 13 Q

~

0

5

10

15

20

25

I

I

I

I

J

I

-

14 22 15 23

E 12

© 10 ~11

-

]

4 ~

5 6 7 3 1

2

1

Fig. 1. Dendrogram from cluster analysis of 23 supernatants producing monoclonal antibodies against flagella of Listeria rnonocytogenes, tested in an ELISA using different strains of Listeria monocytogenes (14), Listeria inocua (3), Staphylococcus aureus (1), Enterococcus faecalis (1) and Escherichia coli (1) as solid phase antigens.

21 and 23) were producing several subclasses of I g G and therefore were not true monoclonals. However, they a p p e a r e d to react with the same epitope on the bacteria since they showed a very strong pattern similarity to the neighboring supernatants. The fact that the whole of cluster 1 (8-23) originated from the same primary fusion might explain this result. Cluster 2 (3-7) also arose from one original well whereas cluster 3 (1 and 2) came from two different wells. This method for evaluating Mabs was easy to perform and could readily be standardized. Using the vector cosine as a measure of relationship, the variation in results due to differences in concentrations of antibodies and antigen can be reduced, as the pattern will remain basically the same as long as the optical densities do not approach the upper part of the sigmoid curve typically observed in E L I S A experiments. The method presented offers the advantages of working directly with quantitative measureTABLE II MONOCLONAL ANTIBODIES DEVELOPED AGAINST FLAGELLA OF LISTERIA M O N O C Y T O G E N E S , SHOWING THEIR IgG SUBCLASS, THE ORIGINAL WELLS, AND THE FINAL CLUSTER Mab no.

II shows the different Mab, their I g G subclass, and the relationship between clusters and original subclones. Fig. 2 demonstrates the optical density profile of three of the antibodies. The three selected Mab were also suitable for IMS of Listeria from buffers as well as different food items.

Discussion

In the first main cluster (supernatant no. 8-23), two subgroups were found (8-11 and 12-23) whereas the second cluster contained three subgroups (3, 4 / 5 , 6/7). Mab no. 3 - 7 also reacted with Staphylococcus aureus and to a lesser extent with Enterococcus faecalis and Escherichia coli. The interaction with S. aureus may have been due to the Fc region of the antibody reacting with protein A. Some wells (supernatant nos. 16, 18,

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fusion well

IgG subclass

Main cluster

1

1

3

2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 ~ 3 3 3 3 3 3

2b 1 1 1 1 1 1 1 1 1 1 1 1 1 13 1 1 2a 1 1 1 2a 2b 3 1 2a 2b

3 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

16

A

lO0t:~ "~

806040-

~9.

References

20

B

1/;0 80-

6O E

4O

~.~

20

!1 .... C

100-

80-

2oo b

"d 60.e 40-

L. ino :ua

L. m o n o c y t o g e n e s

Fig. 2. Examples of ELISA optical density (OD) patterns from Mab no. 2 (A), 4 (B) and 16 (C). All OD values are scaled according to their maximal OD value for each of the Listeria strains used as antigen.

ments compared to, e.g., checkerboard immunoblotting. When working with antigens as complicated as whole bacteria, a substantial reduction of workload and waiting time can be achieved. The technique can obviously be applied for the evaluation of Mabs against other bacteria as well as other antigens. A rather large number of different antigens (20) was used in this study but the number of supernatants which can be screened increases when the number of antigens is reduced. The monoclonal antibodies selected using this procedure appeared to be well suited for recognizing surface epitopes when tested in an ELISA system as well as in the applied immunomagnetic separation techniques. A mixture of the three selected Mab could provide a practical method for the separation of this important pathogen from foods or other complex matrices.

Austin, B. and Priest, F. (1986) Modern bacterial taxonomy. Van Nostrand Reinhold, Edinburgh, pp. 27-31. Butman, B.T., Plank, M.C., Durham, R.J. and Mattingly, J.A. (1988) Monoclonal antibodies which identify a genusspecific Listeria antigen. Appl. Environ. Microbiol. 54, 1564-1569. Campbell, A.M. (1986) Monoclonal antibody technology, 4th edn. Elsevier, Amsterdam, pp. 158-159. Costas, M., Leach, R.H. and Mitchelmore, D.L. (1987) Numerical analysis of PAGE protein patterns and the taxonomic relations within the 'Mycoplasma mycoides cluster'. J. Gen. Microbiol. 133, 3319-3329. Farber, J.M. at~d Speirs, J.I. (1987) Monoclonal antibodies directed against the flagellar antigens of Listeria species and their potential in EIA-based methods. J. Food Protect. 50, 479-484. Faresu, S.B. and Jones, D. (1988) Taxonomic studies on Brochotrix, Erysopelothrix, Listeria and atypical lactobacilli. J. Gen. Microbiol. 134, 1165-1183. Galfr&, G., Howe, S.C., Milstein, C., Butcher, G.W. and Howard, J.C. (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550-552. Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbour Laboratory, New York. Kampelmacher, E.H. and Mossel, D.A.A. (1989) Evaluation and management of the risk of transmission of Listeria monocytogenes by foods. Culture 2, 1-6. Kazemi, M. and Finkelstein, R.A. (1990) Checkerboard immunoblotting (CBIB): an efficient, rapid and sensitive method of assaying multiple antigen/antibody cross-reactivities. J. Immunol. Methods 128, 143-146. Lane, R.D., Mellgren, R.L., Hegazy, M.G., Gonzalez, S.R., Nepomuceno, V., Reimann, E.M. and Schlender, K.K. (1989) The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins. Hybridoma 8, 661-669. McLauchlin, J. and Taylor, A.G. (1989) The use of monoclonal antibodies in the characterization and purification of cell surface antigens of Listeria monocytogenes serogroup 4. Acta Microbiol. Hung. 36, 459-465. McLauchlin, J., Ridley, A.M. and Taylor, A.G. (1989) The use of monoclonal antibodies against Listeria monocytogenes in a direct immunofluorescence technique for the rapid presumptive identification and direct demonstration of Listeria in foods. Acta Microbiol. Hung. 36, 467-471. Miller, K.M., Hotze, C., Brotman, B. and Prince, A.M. (1990) An economic procedure for screening of hybridoma supernatants for surface reactive antibodies to filarial larvae. Trop. Med. Parasitol. 41,221-222. Mossel, D.A.A. (1989) Listeria monocytogenes in foods. Isolation, characterization and control. Int. J. Food Microbiol. 8, 183-195. Mugnai, L., Bridge, P.D. and Evans, H.C. (1989) A chemotaxonomic evaluation of the genus Beauveria. Mycol. Res. 92, 199-209.

17 Peel, M., Donachie, W. and Shaw, A. (1988) Temperature dependent expression of flagella of Listeria monocytogenes studied by electron microscopy, SDS-PAGE and Western Blotting. J. Gen. Microbiol. 134, 2171-2178. Perutelli, P., Marchese, P., Mori, P.G. and Damiani, G. (1989) A simple, rapid method for screening hybridomas producing monoclonal antibodies to platelet proteins. J. Immunol. Methods 123, 227-232. Shaw, B.G., Latty, J.B. (1988) A numerical taxonomy study of non-motile non-feremtative Gram-negative bacteria from food. J. Appl. Bacteriol. 65, 7-21.

Shelley, W.A., Deeth, H.C. and MacRae, I.C. (1987) A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk. J. Appl. Bacteriol. 62, 197-207. Skjerve, E., R~rvik, L.M. and Olsvik, ~. (1990) Detection of Listeria monocytogenes in foods by Immunomagnetic separation. Appl. Environ. Microbiol. 56, 3478-3481. Spitz, M., Spitz, R., Thorpe, R. and Eugui, E. (1984) Intrasplenic primary immunization for the production of monoclonal antibodies. J. Immunol. Methods 70, 39-43.

Evaluation of monoclonal antibodies to Listeria monocytogenes flagella by checkerboard ELISA and cluster analysis.

A simple ELISA technique is described which utilizes whole bacteria as solid phase antigens for the evaluation of monoclonal antibodies against flagel...
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