Skip to main content
SpringerLink
Log in

Detection of Clostridium botulinum neurotoxins A and B in milk by ELISA and immuno-PCR at higher sensitivity than mouse bio-assay

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

Using commercially available antibodies and toxoids as templates, an ELISA, immuno-quantitative PCR (iqPCR), and multiplex immuno-PCR (iPCR) were developed for detection of Clostridium botulinum neurotoxins A and B. The obtained sensitivities for ELISA ranged from 1 ng/ml in PBS + 1% BSA to 15 and 10 ng/ml in skimmed milk for botulinum neurotoxins (BoNT)/A and BoNT/B, respectively. In semi-fat milk, the limit of detection (LOD) for both toxoids was 30 ng/ml. Quantitative immuno-PCR (iqPCR) had an LOD of 4.5–9 pg/reaction for BoNT/A in both PBS and semi-fat milk, while this was 18.5–37 pg/reaction for BoNT/B in PBS + 1% BSA and semi-fat milk, respectively. The sensitivities of ELISA and iqPCR were improved to 0.5 ng/ml and 3.75 pg/ml (0.2 pg/reaction) in semi-fat milk, respectively, when toxoid of BoNT/A was substituted with actual toxin. Multiplex iPCR with both toxoids run in the same reaction was able to distinguish presence/absence of tested BoNT/A and BoNT/B at 25 pg/reaction. The tested system offers a realistic alternative with much better sensitivity to the standard mouse assay.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Attree O, Guglielmo-Viret V, Gros V, Thullier P (2007) J Immunol Methods 325:78

    Article  CAS  Google Scholar 

  • Bohnel H, Neufeld B, Gessler F (2005) Vet J 169:124

    Article  CAS  Google Scholar 

  • Cai S, Singh BR, Sharma S (2007) Crit Rev Microbiol 33:109

    Article  CAS  Google Scholar 

  • Caponi L, Migliorini P (1999) ELISA assays. In: Caponi L, Migliorini P (eds) Antibody Usage in the Lab. Springer, Berlin, pp 9–31

    Google Scholar 

  • Cereser ND, Costa FMR, Junior ODR, da Silva DAR, Sperotto VD (2008) Ciencia Rural 38:280

    Article  Google Scholar 

  • Chao HY, Wang YC, Tang SS, Liu HW (2004) Toxicon 43:27

    Article  CAS  Google Scholar 

  • De Medici D, Anniballi F, Wyatt GM, Lindstrom M, Messelhausser U, Aldus CF, Delibato E, Korkeala H, Peck MW, Fenicia L (2009) Appl Environ Microbiol 75:6457

    Article  Google Scholar 

  • Fenicia L, Fach P, van Rotterdam BJ, Anniballi F, Segerman B, Auricchio B, Delibato E, Hamidjaja RA, Wielinga PR, Woudstra C, Agren J, De Medici D, Knutsson R (2011) Int J Food Microbiol 145:S152

    Article  Google Scholar 

  • Franciosa G, Pourshaban M, Gianfranceschi M, Gattuso A, Fenicia L, Ferrini AM, Mannoni V, De Luca G, Aureli P (1999) J Food Prot 62:867

    CAS  Google Scholar 

  • Ganapathy R, Padmanabhan S, Eric YPG, Moochhala S, Lionel LKH, Ponnampalam G (2008) Front Biosci 13:5432

    Article  CAS  Google Scholar 

  • Gofflot S, El Moualij BF, Zorzi D, Melen L, Roels S, Quatpers D, Grassi J, Vanopdenbosch E, Heinen E, Zorzi W (2004) J Immunoass Immunochem 25:241

    CAS  Google Scholar 

  • Hatheway CL (1976) Appl Environ Microbiol 31:234

    CAS  Google Scholar 

  • Hill B, Skerry J, Smith T, Arnon S, Douek D (2010) BMC Microbiol 10:267

    Article  Google Scholar 

  • Keller JE (2008) Clin Vaccine Immunol 15:1374

    Article  CAS  Google Scholar 

  • Lindstrom M, Korkeala H (2006) Clin Microbiol Rev 19:298

    Article  CAS  Google Scholar 

  • Lindstrom M, Myllykoski J, Sivela S, Korkeala H (2010) Crit Rev Food Sci Nutr 50:281

    Article  Google Scholar 

  • Malakar PK, Barker GC, Peck MW (2011) Food Microbiol 28:321

    Article  CAS  Google Scholar 

  • McLauchlin J, Grant KA, Little CL (2006) J Public Health 28:337

    Article  Google Scholar 

  • McLellan K, Das REG, Ekong TAN, Sesardic D (1996) Toxicon 34:975

    Article  CAS  Google Scholar 

  • Niemeyer CM, Adler M, Wacker R (2007) Nat Protoc 2:1918

    Article  CAS  Google Scholar 

  • Peck M (2010) Clostridium botulinum. In: Juneja VK, Sofos JN (eds) Pathogens and toxins in foods: challenges and interventions. ASM Press, Washington, pp 31–52

    Google Scholar 

  • Poras H, Ouimet T, Orng SV, Fournie-Zaluski MC, Popoff MR, Roques BP (2009) Appl Environ Microbiol 75:4382

    Article  CAS  Google Scholar 

  • Rajkovic A, El Moualij B, Uyttendaele M, Brolet P, Zorzi W, Heinen E, Foubert E, Debevere J (2006) Appl Environ Microbiol 72:6593

    Article  CAS  Google Scholar 

  • Rivera VR, Gamez FJ, Keener WK, White JA, Poli MA (2006) Anal Biochem 353:248

    Article  CAS  Google Scholar 

  • Scarlatos A, Welt BA, Cooper BY, Archer D, DeMarse T, Chau KV (2005) J Food Sci 70:R121

    Article  CAS  Google Scholar 

  • Scarlatos A, Cadotte AJ, DeMarse TB, Welt BA (2008) J Food Sci 73:E129

    Article  CAS  Google Scholar 

  • Scotcher MC, Cheng LW, Stanker LH (2010) Plos One 5

  • Sharma SK, Ferreira JL, Eblen BS, Whiting RC (2006) Appl Environ Microbiol 72:1231

    Article  CAS  Google Scholar 

  • Spengler M, Adler M, Jonas A, Niemeyer CM (2009) Biochem Biophys Res Commun 387:278

    Article  CAS  Google Scholar 

  • Stanker LH, Merrill P, Scotcher MC, Cheng LW (2008) J Immunol Methods 336:1

    Article  CAS  Google Scholar 

  • Taitt CR, Shriver-Lake LC, Ngundi MM, Ligler FS (2008) Sensors 8:8361

    Article  CAS  Google Scholar 

  • Varnum SM, Warner MG, Dockendorff B, Anheier NC, Lou JL, Marks JD, Smith LA, Feldhaus MJ, Grate JW, Bruckner-Lea CJ (2006) Anal Chim Acta 570:137

    Article  CAS  Google Scholar 

  • Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C (2010) Biotechnol Adv 28:232

    Article  CAS  Google Scholar 

  • Wang DX, Baudys J, Kalb SR, Barr JR (2011) Anal Biochem 412:67

    Article  CAS  Google Scholar 

  • Weingart OG, Schreiber T, Mascher C, Pauly D, Dorner MB, Berger TFH, Egger C, Gessler F, Loessner MJ, Avondet MA, Dorner BG (2010) Appl Environ Microbiol 76:3293

    Article  CAS  Google Scholar 

  • Wu HC, Huang YL, Lai SC, Huang YY, Shalo MF (2001) Lett Appl Microbiol 32:321

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The research performed was part of the project RT08/9 (BOTEST) funded by Belgian Federal Public Service Health, Food Chain Safety and Environment. The authors would like to express their gratitude to Mr. Edgard VAN NEROM, as well as Francis TWEEPENNINCKX and Rick NYSSEN for their valuable assistance in the experiments.

Author information

Authors and Affiliations

  1. Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

    Andreja Rajkovic, Ahu Uner & Mieke Uyttendaele

  2. Laboratory of Human Histology, CRPP University of Liege, Liege, Belgium

    Benaissa El Moualij, Willy Zorzi & Ernst Heinen

  3. Scientific Service Food-borne Pathogens, Scientific Institute of Public Health, Brussels, Belgium

    Youssef Fikri & Katelijne Dierick

  4. Department of Food Safety and Food Quality Management, Faculty of Agriculture, Belgrade University, Zemun, Belgrade, Serbia

    Andreja Rajkovic

Authors
  1. Andreja Rajkovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benaissa El Moualij
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Youssef Fikri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katelijne Dierick
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Willy Zorzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ernst Heinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ahu Uner
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mieke Uyttendaele
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreja Rajkovic.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rajkovic, A., El Moualij, B., Fikri, Y. et al. Detection of Clostridium botulinum neurotoxins A and B in milk by ELISA and immuno-PCR at higher sensitivity than mouse bio-assay. Food Anal. Methods 5, 319–326 (2012). https://doi.org/10.1007/s12161-011-9300-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-011-9300-7

Keywords

Search

Navigation