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

Kenneth L Campbell,Matthew Lopresti,William Lukas

doi:10.2174/1874241601407010001

Abstract

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.

Highlights

Immunoassays are among the most commonly employed tools in biomedical and diagnostic research, often used in efficient, highly automated, high-throughput protocols
Most macromolecule assays have taken advantage of enzyme-linked immunosorbent assay (ELISA) formats which often demonstrate broad analytical ranges and reasonable limits of detection and they have been streamlined by employing single incubation protocols in which analyte binds to capture and tracer-labeled antibodies simultaneously prior to a wash step and a detection step
Knowing the identity of any inhibitors will not simplify or correct the urine-based direct analyte assays of interest in most diagnostic contexts. This is so if the inhibition or HRP is a cumulative action of multiple components of the many commonly found in urine. From this brief investigation we conclude: 1) it is apparent that urine in undiluted form is highly unsuited to direct immunoassay protocols that expose the HRP reporter conjugates to that urine; 2) this is true without respect to urine source, assay type, or substrate system employed; and, 3) the inhibition is caused by a component that is only partially removed by charcoal treatment and is probably a water soluble organic compound

Summary

Introduction

Immunoassays are among the most commonly employed tools in biomedical and diagnostic research, often used in efficient, highly automated, high-throughput protocols. Most macromolecule assays have taken advantage of enzyme-linked immunosorbent assay (ELISA) formats which often demonstrate broad analytical ranges and reasonable limits of detection and they have been streamlined by employing single incubation protocols in which analyte binds to capture and tracer-labeled antibodies simultaneously prior to a wash step and a detection step. Small analyte assays still typically employ a competitive enzyme immunoassay (EIA) format with a single capture antibody at limiting dilution along with a tracer-labeled form of the analyte. Competitive assays, which tend to exhibit narrower analytical ranges and good limits of detection, have traditionally used a single incubation approach in which the analyte-tracer conjugate competes directly with the analyte for binding to the capture antibody. The tubes, wells, beads, or strips holding the antibody and bound analyte and/or tracer are washed and developed with enzyme substrate

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