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Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride

Nature Protocols volume 4, pages 452–460 (2009)Cite this article

Abstract

In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to effect coupling of the two proteins. Assessment of the conjugate product was carried out using ELISA and SDS-PAGE electrophoresis where evidence for high antibody activity, high specific activity of the conjugate preparation, coupling of nearly all the antibody and over 90% of the enzyme was shown. The titer of the conjugate exceeded 1/100,000. High molecular weight complexes were observed in the SDS-PAGE results, indicating an efficient conjugation procedure. The presence of high molecular weight complexes indicated an efficient conjugation procedure. The protocol is simple, and the conjugation steps can be completed in 27 h once the preparatory phase has been carried out; the method is entirely generic and may be applied to labeling of any antibody.

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Figure 1: Conjugation steps according to the CC protocol.
Figure 2: Conjugation steps according to the sodium periodate methods.
Figure 3: A flowchart of the conjugation procedure.
Figure 4: Antibody–HRP conjugate dilution response graph.
Figure 5: An image of a Coomassie-stained nonreducing SDS-PAGE gel of a conjugate sample.

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Authors and Affiliations

  1. Department of Pharmacy, Pharmaceutical Science Research Division, King's College London, Analytical Sciences Research Group, School of Biomedical and Health Sciences, Franklin-Wilkins Building, Stamford Street, London, SE1 9NH, UK

    Ramadan A Abuknesha, Fiona Jeganathan, Jocelyn Wu & Zakeya Baalawy

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  1. Ramadan A Abuknesha
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  2. Fiona Jeganathan
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Correspondence to Ramadan A Abuknesha.

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Abuknesha, R., Jeganathan, F., Wu, J. et al. Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride. Nat Protoc 4, 452–460 (2009). https://doi.org/10.1038/nprot.2009.6

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