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Effectiveness of natural and synthetic blocking reagents and their application for detecting food allergens in enzyme-linked immunosorbent assays

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Abstract

Blocking is an important step before an enzyme-linked immunosorbent assay (ELISA) can be performed. It reduces non-specific binding to the microtiter plate to a minimum. For detecting food allergens by means of ELISA, the problem with protein blocking solutions is obvious. The blocker might interfere with the antibodies of the assay and leads to false positive results. Therefore, other blocking solutions are greatly needed. There are some alternatives like synthetic blockers or carbohydrates. Comparisons of these different blocking agents, namely proteins, carbohydrates, and synthetic blockers, were made at different reaction conditions. The incubation periods and temperatures were varied, as well as the pH. The best combinations were evaluated and compared, in respect of their blocking efficiency. The two best non-proteinaceous blockers, i.e. polyvinylalcohol and Ficoll, were subsequently applied to ELISA tests for the determination of α-casein and peanut. The study showed that Ficoll and PVA did as well as BSA in buffer solution. Therefore, they can be considered as alternative blocking reagents for ELISA, especially for the detection of food allergens.

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Abbreviations

Abs:

Absorbance

BSA:

bovine serum albumin

ELISA:

enzyme-linked immunosorbent assay

HRP:

horse radish peroxidase

PBS:

phosphate buffered saline

PEG:

polyethyleneglycol

PN:

peanut

PVA:

polyvinylalcohol

PVP:

polyvinylpyrrolidone

TMB:

tetramethylbenzidine

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Acknowledgments

The authors thank the Christian Doppler Research Association and RomerLabs for funding the Christian Doppler PilotLaboratory “Rapid Test Systems for Allergenic Food Contaminants”. We also thank Dr. Marcela Hermann, Institute of Medical Biochemistry, Department of Molecular Genetics, University Vienna, for the immunisation of rabbits and chicken.

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Authors and Affiliations

  1. Christian Doppler Pilot Laboratory “Rapid Test Systems for Allergenic Food Contaminants”, Department of Agrobiotechnology, IFA-Tulln, Center for Analytical Chemistry, University of Natural Resources and Applied Life Sciences Vienna, Konrad-Lorenz Strasse 20, 3430, Tulln an der Donau, Austria

    Denise Huber, Judith Rudolf, Parisa Ansari, Brigitte Galler, Manuela Führer, Christoph Hasenhindl & Sabine Baumgartner

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  1. Denise Huber
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  2. Judith Rudolf
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  3. Parisa Ansari
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  4. Brigitte Galler
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  5. Manuela Führer
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  6. Christoph Hasenhindl
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  7. Sabine Baumgartner
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Correspondence to Sabine Baumgartner.

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Huber, D., Rudolf, J., Ansari, P. et al. Effectiveness of natural and synthetic blocking reagents and their application for detecting food allergens in enzyme-linked immunosorbent assays. Anal Bioanal Chem 394, 539–548 (2009). https://doi.org/10.1007/s00216-009-2698-8

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  • DOI: https://doi.org/10.1007/s00216-009-2698-8

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