Analytical and physiological validation of an enzyme immunoassay to measure oxytocin in dog, wolf, and human urine samples

AbstractOxytocin (OT) promotes pro-sociality, bonding, and cooperation in a variety of species. Measuring oxytocin metabolite (OTM) concentrations in urine or saliva provides intriguing opportunities to study human and animal behaviour with minimal disturbance. However, a thorough validation of analytical methods and an assessment of the physiological significance of these measures are essential. We conducted an analytical validation of a commercial Enzyme Immunoassay (EIA; Arbor OT assay kit) to measure OTM concentrations in dog, wolf, and human urine samples. To test the assay’s ability to detect changes in OTM concentrations, we administered oxytocin intranasally to 14 dogs. Assay performance with regard to parallelism was acceptable. Assay accuracy and extraction efficiency for dog and wolf samples were comparable to a previously validated assay (Enzo OT assay kit) but variation was smaller for human samples. Binding sensitivity and antibody specificity were better in the Arbor assay. Average OTM concentrations were more than twice as high as in comparable samples measured with the Enzo assay, highlighting a lack of comparability of absolute values between different assays. Changes in OTM concentrations after intranasal treatment were detected reliably. The Arbor assay met requirements of a “fit-for-purpose” validation with improvement of several parameters compared to the Enzo assay.

Download Full-text

Validation of a commercial enzyme immunoassay to assess urinary oxytocin in humans

Endocrine Connections ◽

10.1530/ec-20-0583 ◽

2021 ◽

Vol 10 (3) ◽

pp. 290-301

Author(s):

Franka S Schaebs ◽

Gwen Wirobski ◽

Sarah Marshall-Pescini ◽

Friederike Range ◽

Tobias Deschner

Keyword(s):

Enzyme Immunoassay ◽

Social Relationships ◽

Extraction Efficiency ◽

Quality Criteria ◽

Extraction Methods ◽

Urine Samples ◽

Analytical Validation ◽

Physiological Processes ◽

Commercial Enzyme Immunoassay ◽

Ethanol Extracts

Within the last decade, oxytocin (OT) has attracted a lot of attention in the context of various human social behaviors. Besides its importance in regulating physiological processes in females related to giving birth and lactation, OT is involved in the establishment and maintenance of social relationships, trust and emotion recognition. However, results are not always consistent across studies, which may partly be due to the incomplete validation of methods used to assess OT levels. Carefully validating a method before its use is of crucial importance to ensure that it can be used to accurately, reliably and repeatedly assess OT levels. With this study we evaluated a commercially available Enzyme Immunoassay to assess OT in human urine samples by conducting a careful analytical validation. Results indicate that, with regard to parallelism and immunoreactivity, human urinary OT can be assessed reliably. However, extraction methods need further improvement to optimize measures of accuracy and extraction efficiency, especially in the lower range of the assay system. Tests on OT stability indicate that OT is affected by degradation when stored at 4°C or room temperature. Storing urine samples over longer periods revealed that OT levels are most stable when stored as ethanol extracts at −20°C compared to being stored as samples at −20°C or −80°C. Although some of the validated parameters did not reach the intended quality criteria, this study highlights the importance of such in depth validation procedures and reporting results to make them available to researchers embarking on projects utilizing such methods.

Download Full-text

Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples

Clinical Chemistry ◽

10.1093/clinchem/43.2.363 ◽

1997 ◽

Vol 43 (2) ◽

pp. 363-368 ◽

Cited By ~ 9

Author(s):

Frédéric Taran ◽

Yveline Frobert ◽

Christophe Créminon ◽

Jacques Grassi ◽

Didier Olichon ◽

...

Keyword(s):

Monoclonal Antibody ◽

Enzyme Immunoassay ◽

Human Urine ◽

Homovanillic Acid ◽

Colorimetric Assay ◽

Cross Reactivity ◽

Urine Samples ◽

Vanillylmandelic Acid ◽

Human Urine Samples ◽

Enzyme Detection

Abstract A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 μmol/L, a CV <10% in the 1.25–10 μmol/L range, and intra- and interassay CVs of <10%. Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for other structurally related catecholamine metabolites. Parallelism of the EIA was shown in dilution studies and the correlation with routine HPLC assay in 62 normal and pathologic samples was EIA = 1.492 (HPLC) − 3.46, Sy|x = 47.52, range = 4–1800 μmol/L, r2 = 0.977. Additional data concerning the validity of this assay were provided by HPLC analysis of urinary immunoreactive material.

Download Full-text

Drug screening by enzyme immunoassay with the American Monitor KDA.

Clinical Chemistry ◽

10.1093/clinchem/24.10.1823 ◽

1978 ◽

Vol 24 (10) ◽

pp. 1823-1825 ◽

Cited By ~ 5

Author(s):

J R Pearson

Keyword(s):

Enzyme Immunoassay ◽

Drug Screening ◽

Human Urine ◽

Methadone Treatment ◽

Lysozyme Activity ◽

Urine Samples ◽

Treatment Center ◽

Automatic Correction ◽

Human Urine Samples ◽

Two Systems

Abstract The methods for barbiturate, opiate, methadone, and amphetamine have been modified for use with the American Monitor KDA. The modification, which incorporates automatic correction for endogenous lysozyme activity, was evaluated by comparing results obtained with the KDA for human urine samples containing known amounts of drug(s) with results obtained with the procedure recommended by Syva for the Gilford 3500. There was 98% agreement bewteen the two systems. Six calibrators and 40 samples can be assayed for all four drugs in about 2.5 h. The procedure has proven to be reliable for screening urine samples obtained from clients at a methadone treatment center.

Download Full-text