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

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.

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

Scientific Reports ◽

10.1038/s41598-021-92356-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

G. Wirobski ◽

F. S. Schaebs ◽

F. Range ◽

S. Marshall-Pescini ◽

T. Deschner

Keyword(s):

Enzyme Immunoassay ◽

Human Urine ◽

Extraction Efficiency ◽

Urine Samples ◽

Antibody Specificity ◽

Analytical Validation ◽

Human Urine Samples ◽

Assay Performance ◽

Fit For Purpose ◽

Commercial Enzyme Immunoassay

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.

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Development of an enzyme-linked immunosorbent assay with monoclonal antibody for quantification of homovanillic in human urine samples

Clinical Chemistry ◽

10.1093/clinchem/44.8.1674 ◽

1998 ◽

Vol 44 (8) ◽

pp. 1674-1679 ◽

Cited By ~ 5

Author(s):

Run Zhang Shi ◽

Yee-Ping Ho ◽

John Hok Keung Yeung ◽

Penelope Mei Yu Or ◽

Kenneth Kin Wah To ◽

...

Keyword(s):

Monoclonal Antibody ◽

Human Urine ◽

High Specificity ◽

Urine Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Hybridization Technique ◽

Vanillylmandelic Acid ◽

Coefficients Of Variation ◽

Sensitivity Specificity ◽

Initial Results

Abstract A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and <0.1%, respectively. The assay coefficients of variation were <10% within the working range of 0.5–40 mg/L. Initial results from testing urine samples of patients with neuroblastoma and other diseases were validated by HPLC, suggesting that this ELISA method is a reliable and convenient system for quantification of HVA in urine and can be used in the mass screening of neuroblastoma in infants.

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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.

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An enzyme immunoassay for determining albumin in human urine samples using an ultra-microanalytical system

Journal of Immunoassay and Immunochemistry ◽

10.1080/15321819.2020.1807357 ◽

2020 ◽

Vol 41 (5) ◽

pp. 896-912

Author(s):

Ruben Del Valle ◽

Juliette M. Cazanave Mora ◽

Nancy L. Carrazana San Martín ◽

Liliana Hernández Pérez ◽

Martha E. Legrá Torres ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Human Urine ◽

Urine Samples ◽

Human Urine Samples

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Molecularly Imprinted Nanoparticles Assay (MINA) in Pseudo ELISA: An Alternative to Detect and Quantify Octopamine in Water and Human Urine Samples

Polymers ◽

10.3390/polym11091497 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1497 ◽

Cited By ~ 2

Author(s):

Ewa Moczko ◽

Richard Díaz ◽

Bernabé Rivas ◽

Camilo García ◽

Eduardo Pereira ◽

...

Keyword(s):

Detection Limit ◽

Human Urine ◽

Solid Phase ◽

Cross Reactivity ◽

Urine Samples ◽

Cold Chain ◽

Enzyme Linked Immunosorbent Assay ◽

Molecularly Imprinted ◽

Human Urine Samples ◽

High Affinity

In 2004, octopamine was added to the list of drugs banned by the world anti-doping agency (WADA) and prohibited in any sport competition. This work aims to develop a new analytical method to detect octopamine in water and human urine samples. We proposed a pseudo-enzyme-linked immunosorbent assay (pseudo-ELISA) by replacing traditional monoclonal antibodies with molecularly imprinted polymer nanoparticles (nanoMIPs). NanoMIPs were synthesised by a solid-phase approach using a persulfate initiated polymerisation in water. Their performance was analysed in pseudo competitive ELISA based on the competition between free octopamine and octopamine-HRP conjugated. The final assay was able to detect octopamine in water within the range 1 nmol·L−1–0.1 mol·L−1 with a detection limit of 0.047 ± 0.00231 µg·mL−1 and in human urine samples within the range 1 nmol·L−1–0.0001 mol·L−1 with a detection limit of 0.059 ± 0.00281 µg·mL−1. In all experiments, nanoMIPs presented high affinity to the target molecules and almost no cross-reactivity with analogues of octopamine such as pseudophedrine or l-Tyrosine. Only slight interference was observed from the human urine matrix. The high affinity and specificity of nanoMIPs and no need to maintain a cold chain logistics makes the nanoMIPs a competitive alternative to antibodies. Furthermore, this work is the first attempt to use nanoMIPs in pseudo-ELISA assays to detect octopamine.

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