A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine.

ELISA is a well-established technique used worldwide to quantify analytes present in a diverse milieu of biological samplings. It is especially important to clinicians who rely on the accuracy and precision of the test to administer patient care. Those results are to be held with great scrutiny since the assay is subject to error caused by interfering substances found in the sample matrix. In this chapter, we examine the nature of such interferences and discuss approaches to identify and offer remedies to remove the interference and validate the assay. 

Interference in ELISA.

Non-radiometric assays: Technology and application in polypeptide and steroid hormone detection

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.

/pdf/inhibition-of-horseradish-peroxidase-hrp-by-a-nonhydrophobic-2rfb5g7vdd.pdf

Matthew Lopresti

Papers

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