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Rapid detection of bacterial surface proteins using an enzyme-linked immunosorbent assay system
J. Biochem. Biophys. Methods 34 (1997) 69–71
Short communication
Rapid detection of bacterial surface proteins using an enzyme-linked immunosorbent assay system Andreas Burkovski* ¨ ¨ Biotechnologie 1, Postfach 1913, D-52425 Julich ¨ Forschungszentrum Julich , Institut f ur , Germany Received 13 September 1996; accepted 11 October 1996
Abstract In this communication a rapid and simple enzyme-linked immunosorbent assay is described, suitable for the investigation of bacterial surface proteins. The protocol uses whole cells and avoids any centrifugation or cross-linking steps. Less than 10 6 cells were detected by this procedure. 1997 Elsevier Science B.V. Keywords: Corynebacterium; Cell surface protein; ELISA
Bacterial cell surface proteins are of scientific interest due to various reasons: For example they are (i) receptors for phages; (ii) involved in transport processes; and (iii) important antigens. A widely used technique for the investigation of these proteins is the labelling of antibodies with colloidal gold and the subsequent immunological detection of the protein of interest (for example, see [1]). Alternatively, antibody-decorated proteins are labelled with Staphylococcus aureus gold-protein A particles [2]. However, both methods are time consuming and access to electron microscopy facilities is a prerequisite. An equivalent but rapid and less laborious approach is the use of an enzyme-linked immunosorbent assay (ELISA). A method allowing the investigation of bacterial surface proteins was published recently as an approach for the detection of lactobacilli [3]. The bacteria were coupled to polyvinyl chloride microtiter plates using a *Corresponding author. Tel.: 1 49 2461 61 5556, Fax.: 1 49 2461 61-2710; E-mail: [email protected] 0165-022X / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0165-022X( 96 )00034-6
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A. Burkovski / J. Biochem. Biophys. Methods 34 (1997) 69 – 71
centrifugation step and glutardialdehyde. In this communication a more convenient and faster protocol is described avoiding any treatment of the bacterial cells with chemicals. As a model system the GluB protein of Corynebacterium glutamicum was studied [4]. Peptide-specific antibodies against this protein were generated in rabbits by EUROGENTEC (Seraing, Belgium). Cells were grown in LB medium until the early exponential phase was reached, washed once and resuspended to 5 3 10 8 cell / ml in sodium carbonate buffer (50 mM NaHCO 3 -NaOH, pH 9.6). 200 ml aliquots of various dilution steps were transferred into the wells of microtiter plates. After 1 h of incubation at room temperature, remaining protein binding sites were blocked with 5% powdered skim milk in TBS (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM MgCl 2 ) for 1 h at room temperature. Antibody incubation (200 ml per well) was carried out overnight at 48C or for 2 h at room temperature. Antisera were diluted as in Western blotting experiments (1:1000–1:5000). The wells were washed once with 200 ml TBS containing 0.005% Tween 20 and twice with TBS. The secondary antibody (Anti-Rabbit IgG Alkaline Phosphatase Conjugate; Sigma, Deisenhofen) was diluted 1:10 000 in TBS and incubation was carried out at room temperature for 1 h followed by 3 washing steps (see above). Substrate incubation (1 mg / ml p-nitrophenyl phosphate in 0.1 M glycine-NaOH, pH 10.4, 1 mM ZnCl 2 , 1 mM MgCl 2 , 200 ml per well) was carried out for 30 min at room temperature. The reaction was subsequently stopped by the addition of 50 ml 3 M NaOH. Absorption at 405 nm was measured with a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA). Three different polystyrene microtiter plates were tested, Falcon 3075 tissue culture plates (Becton-Dickinson, Lincoln Park, USA), PolySorp and MaxiSorp immuno plates (Nunc, Roskilde, Denmark). Optimal cell
Fig. 1. Minimal cell number for C. glutamicum bound to MaxiSorp microtiter plates and probed with anti-GluB serum (diluted 1:2000). Experiments were carried out in quadruplicate and standard errors are indicated.
A. Burkovski / J. Biochem. Biophys. Methods 34 (1997) 69 – 71
71
binding was observed with MaxiSorp immuno plates, while approx. 10% less cells were bound to PolySorp plates. Binding to Falcon tissue culture plates was negligible. For MaxiSorp plates the minimal cell number was determined (Fig. 1). Less than 10 6 cells / well were detected with the antiserum against GluB, although this protein is not a major surface protein of C. glutamicum. Using an antiserum against a major cell wall protein or a mixture of antibodies the minimal cell number for detection can be easily improved making the method also suitable for food quality control. If necessary, also quantitation is easily possible. Furthermore, this approach can be applied for the screening of bacteria using species-specific antibodies.
Acknowledgements ¨ The author wishes to thank Prof. R. Kramer and Prof. H. Sahm for continuous support.
References [1] Winkler, M.T., Osorio, F.A., Barahona, H.J. and Taffarel, M. (1995) FEMS Immun. Med. Microbiol., 11, 1–4. ¨ [2] Schulz-Hauser, G., Koster, W., Schwarz, H. and Braun, V. (1992) J. Bacteriol., 174, 2305–2311. [3] Nakamura, J., Minami (Iwata), M., Doi, K. and Hamachi, M. (1995) Biosci. Biotech. Biochem., 59, 2039–2043. ¨ [4] Kronemeyer, W., Peekhaus, N., Kramer, R., Sahm, H. and Eggeling, L. (1995) J. Bacteriol., 177, 1152–1158.
Short communication
Rapid detection of bacterial surface proteins using an enzyme-linked immunosorbent assay system Andreas Burkovski* ¨ ¨ Biotechnologie 1, Postfach 1913, D-52425 Julich ¨ Forschungszentrum Julich , Institut f ur , Germany Received 13 September 1996; accepted 11 October 1996
Abstract In this communication a rapid and simple enzyme-linked immunosorbent assay is described, suitable for the investigation of bacterial surface proteins. The protocol uses whole cells and avoids any centrifugation or cross-linking steps. Less than 10 6 cells were detected by this procedure. 1997 Elsevier Science B.V. Keywords: Corynebacterium; Cell surface protein; ELISA
Bacterial cell surface proteins are of scientific interest due to various reasons: For example they are (i) receptors for phages; (ii) involved in transport processes; and (iii) important antigens. A widely used technique for the investigation of these proteins is the labelling of antibodies with colloidal gold and the subsequent immunological detection of the protein of interest (for example, see [1]). Alternatively, antibody-decorated proteins are labelled with Staphylococcus aureus gold-protein A particles [2]. However, both methods are time consuming and access to electron microscopy facilities is a prerequisite. An equivalent but rapid and less laborious approach is the use of an enzyme-linked immunosorbent assay (ELISA). A method allowing the investigation of bacterial surface proteins was published recently as an approach for the detection of lactobacilli [3]. The bacteria were coupled to polyvinyl chloride microtiter plates using a *Corresponding author. Tel.: 1 49 2461 61 5556, Fax.: 1 49 2461 61-2710; E-mail: [email protected] 0165-022X / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0165-022X( 96 )00034-6
70
A. Burkovski / J. Biochem. Biophys. Methods 34 (1997) 69 – 71
centrifugation step and glutardialdehyde. In this communication a more convenient and faster protocol is described avoiding any treatment of the bacterial cells with chemicals. As a model system the GluB protein of Corynebacterium glutamicum was studied [4]. Peptide-specific antibodies against this protein were generated in rabbits by EUROGENTEC (Seraing, Belgium). Cells were grown in LB medium until the early exponential phase was reached, washed once and resuspended to 5 3 10 8 cell / ml in sodium carbonate buffer (50 mM NaHCO 3 -NaOH, pH 9.6). 200 ml aliquots of various dilution steps were transferred into the wells of microtiter plates. After 1 h of incubation at room temperature, remaining protein binding sites were blocked with 5% powdered skim milk in TBS (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM MgCl 2 ) for 1 h at room temperature. Antibody incubation (200 ml per well) was carried out overnight at 48C or for 2 h at room temperature. Antisera were diluted as in Western blotting experiments (1:1000–1:5000). The wells were washed once with 200 ml TBS containing 0.005% Tween 20 and twice with TBS. The secondary antibody (Anti-Rabbit IgG Alkaline Phosphatase Conjugate; Sigma, Deisenhofen) was diluted 1:10 000 in TBS and incubation was carried out at room temperature for 1 h followed by 3 washing steps (see above). Substrate incubation (1 mg / ml p-nitrophenyl phosphate in 0.1 M glycine-NaOH, pH 10.4, 1 mM ZnCl 2 , 1 mM MgCl 2 , 200 ml per well) was carried out for 30 min at room temperature. The reaction was subsequently stopped by the addition of 50 ml 3 M NaOH. Absorption at 405 nm was measured with a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA). Three different polystyrene microtiter plates were tested, Falcon 3075 tissue culture plates (Becton-Dickinson, Lincoln Park, USA), PolySorp and MaxiSorp immuno plates (Nunc, Roskilde, Denmark). Optimal cell
Fig. 1. Minimal cell number for C. glutamicum bound to MaxiSorp microtiter plates and probed with anti-GluB serum (diluted 1:2000). Experiments were carried out in quadruplicate and standard errors are indicated.
A. Burkovski / J. Biochem. Biophys. Methods 34 (1997) 69 – 71
71
binding was observed with MaxiSorp immuno plates, while approx. 10% less cells were bound to PolySorp plates. Binding to Falcon tissue culture plates was negligible. For MaxiSorp plates the minimal cell number was determined (Fig. 1). Less than 10 6 cells / well were detected with the antiserum against GluB, although this protein is not a major surface protein of C. glutamicum. Using an antiserum against a major cell wall protein or a mixture of antibodies the minimal cell number for detection can be easily improved making the method also suitable for food quality control. If necessary, also quantitation is easily possible. Furthermore, this approach can be applied for the screening of bacteria using species-specific antibodies.
Acknowledgements ¨ The author wishes to thank Prof. R. Kramer and Prof. H. Sahm for continuous support.
References [1] Winkler, M.T., Osorio, F.A., Barahona, H.J. and Taffarel, M. (1995) FEMS Immun. Med. Microbiol., 11, 1–4. ¨ [2] Schulz-Hauser, G., Koster, W., Schwarz, H. and Braun, V. (1992) J. Bacteriol., 174, 2305–2311. [3] Nakamura, J., Minami (Iwata), M., Doi, K. and Hamachi, M. (1995) Biosci. Biotech. Biochem., 59, 2039–2043. ¨ [4] Kronemeyer, W., Peekhaus, N., Kramer, R., Sahm, H. and Eggeling, L. (1995) J. Bacteriol., 177, 1152–1158.