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Production of Polyclonal and Monoclonal Antibodies Against the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3Aa16

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Abstract

The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

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Abbreviations

Vip:

Vegetative insecticidal protein

pAbs:

Polyclonal antibodies

mAbs:

Monoclonal antibodies

ELISA:

Enzyme-linked immunosorbent assay

PTA:

Plate-trapped antigen

TAS:

Triple antibody sandwich

PBS:

Phosphate-buffered saline

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Acknowledgments

Special thanks go to the working group Epidemiology and Phathogen Diagnostics at JKI in Quedlinburg Germany under the direction of Dr. Rabenstein, who supported us and allowed us to gain experience in the field of cell culture technology. Here, we would like to thank in particular Katja Thile and Annika Hansmann, for their extraordinary commitment, support, and expertise.

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Authors and Affiliations

  1. Julius Kühn-Institut (JKI), Institute for Biological Control, Heinrichstr. 243, 64287, Darmstadt, Germany

    Dorra Ben Hamadou-Charfi, Annette Juliane Sauer & Dietrich Stephan

  2. Biopesticides Team (LPAP), Centre of Biotechnology of Sfax (CBS), P.O. Box 1177, 3018, Sfax, Tunisia

    Dorra Ben Hamadou-Charfi, Lobna Abdelkafi-Mesrati & Samir Jaoua

  3. Department of Biological & Environmental Sciences, College of Arts and Sciences, Qatar University, P.O. Box 2713, Doha, Qatar

    Samir Jaoua

Authors
  1. Dorra Ben Hamadou-Charfi
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  2. Annette Juliane Sauer
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  3. Lobna Abdelkafi-Mesrati
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  4. Samir Jaoua
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  5. Dietrich Stephan
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Corresponding author

Correspondence to Dorra Ben Hamadou-Charfi.

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Hamadou-Charfi, D.B., Sauer, A.J., Abdelkafi-Mesrati, L. et al. Production of Polyclonal and Monoclonal Antibodies Against the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3Aa16. Appl Biochem Biotechnol 175, 2357–2365 (2015). https://doi.org/10.1007/s12010-014-1426-9

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  • DOI: https://doi.org/10.1007/s12010-014-1426-9

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