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Endogenous Biotin in Rat Brain

Implications for False-Positive Results With Avidin–Biotin and Streptavidin–Biotin Techniques

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Book cover Avidin-Biotin Interactions

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 418))

summary

The interaction between avidin and biotin or streptavidin and biotin forms the basis of several widely used immunohistochemical techniques. An assumption inherent to these techniques is that endogenous biotin is not present in the tissue in detectable quantities, as neither avidin nor streptavidin can discriminate between endogenous biotin and biotinylated antibodies. However, biotin is a known cofactor for numerous carboxylases required in oxidative metabolism, raising the possibility for potential false-positive results in many tissues. This issue has been appreciated in liver and kidney tissue, but has received very little attention in nervous tissue. To address this concern, we examined the distribution of biotin throughout the rat central nervous system using avidin- and streptavidin-based detection systems, as well as a monoclonal antibody raised against biotin. Significant levels of endogenous biotin were identified within specific neuronal types, particularly in neurons associated with the cerebellar motor system and the brainstem auditory system. Non-specific (non-biotin) interactions of avidin and streptavidin conjugates with rat brain tissue were further identified and were most pronounced in the lower brainstem. The binding of avidin- and streptavidin-conjugated markers to endogenous biotin and other non-specific interactions with neural tissue were overcome by several methods including the use of blocking kits, prolonged post-fixation of the tissue in paraformaldehyde, or omission of Triton X-100 from the working solution. Without these measures, a reliable estimate of immunolabel may only be achieved in certain brain regions with markers conjugated directly to secondary antibodies.

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Acknowledgments

This work was supported by the Canadian Institutes for Health Research and an Alberta Heritage Foundation for Medical Research Scientist Award to RWT. BEM was supported by a Canadian Institutes for Health Research Canada Graduate Scholarship, a Natural Sciences and Engineering Research Council of Canada Postgraduate Fellowship, a Killam Trust Scholarship, and a Steinhauer Doctoral Award. The authors gratefully acknowledge the histological expertise of M. Kruskic and L. McKay.

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Authors and Affiliations

  1. Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada

    Bruce E. McKay & Michael L. Molineux

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  1. Bruce E. McKay
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  2. Michael L. Molineux
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  3. Ray W. Turner
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  1. Mead Johnson Nutritionals, Evansville, IN

    Robert J. McMahon

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McKay, B.E., Molineux, M.L., Turner, R.W. (2008). Endogenous Biotin in Rat Brain. In: McMahon, R.J. (eds) Avidin-Biotin Interactions. Methods In Molecular Biology™, vol 418. Humana Press. https://doi.org/10.1007/978-1-59745-579-4_10

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  • DOI: https://doi.org/10.1007/978-1-59745-579-4_10

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-583-5

  • Online ISBN: 978-1-59745-579-4

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