Inhibition of Horseradish Peroxidase (HRP) by a Nonhydrophobic Component of Urine: A Caution for Immunoassays

Kenneth L. Campbell*, 1, Matthew Lopresti2, William Lukas3
1 Department of Biology, University of Massachusetts at Boston, Boston, MA 02125- 3393, USA
2 Spinal Injury Medicine, Boston Medical Center, Boston, MA 02118, USA
3 Albuquerque NM, 87106, USA

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© 2014 Campbell et al;

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125-3393, USA; Tel: 617-287-6676; Fax: 617-287-6650; E-mail:


Undiluted urine mixed directly with HRP suppresses generation of enzyme product to less than 80% of that seen in buffer controls. Incubating dilutions of various urine preparations with HRP immobilized on concanavalin A coated microtiter plates reveals that the source of urine or HRP, and the type of HRP substrate used have minimal effect on the degree of HRP suppression; only dilutions of urine greater than 8-fold with buffer produce HRP activities equivalent to those in buffer. Treating urine with charcoal or C18 silica only partially reverses the HRP suppression. Inhibition of HRP in competitive assays biases results to the high side and in noncompetive assays biases results to the low side. The present findings suggest analysts should avoid immunoassay protocols that allow direct contact between undiluted urine and HRP reporter conjugates and should be cautious with quantitative results previously reported from assays that used undiluted urine with HRP reporters.

Keywords: Direct Immunoassays, HRP Inhibition, Horseradish Peroxidase Inhibition, Immunoassay Interferences, Urinary Assays, Urine Interference.