A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin
Oxytocin (OT) is a peptide hormone synthesized in the hypothalamus and released into systemic circulation or other areas of the brain. Its physiological roles include action as a hormone with stimulation of uterine contractions and that as a neuromodulator with involvement in social behaviors and re...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-07-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S240558082400030X |
| _version_ | 1827227441905532928 |
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| author | Keenan Gerred Amita Kapoor |
| author_facet | Keenan Gerred Amita Kapoor |
| author_sort | Keenan Gerred |
| collection | DOAJ |
| description | Oxytocin (OT) is a peptide hormone synthesized in the hypothalamus and released into systemic circulation or other areas of the brain. Its physiological roles include action as a hormone with stimulation of uterine contractions and that as a neuromodulator with involvement in social behaviors and regulation of mood. Its small size and low levels within biological matrices make it challenging to accurately measure. The goal of this study was to demonstrate the specificity of the antibody, sensitivity, and reproducibility of the Phoenix Pharmaceuticals (PP) OT radioimmunoassay (RIA) for use in human urine, serum, and saliva. Specificity of the antibody was assessed by high pressure liquid chromatography with ultraviolet (HPLC-UV) separation and assay of the fractions. Immunoreactivity was evaluated using the percent OT bound, and the fraction retention times were compared to the retention time of an intact OT standard to determine which fractions contained OT in the extracted samples. Reproducibility was assessed by running replicates of pools of each biomatrix over several assays. Sensitivity was assessed by repeated measurement of physiologically relevant low-concentration specimens. In all tested specimens the greatest reactivity in assay corresponded to the same fraction(s) as the OT standard. Only minimal reactivity was found in the other fractions, suggesting that in an unfractionated sample the antibody reacts mostly with intact OT. Reproducibility was acceptable for all specimens and the coefficient of variation (CV) ranged from 3.72 to 8.04% and 5.89–12.8%, for intra and inter-assay, respectively. The limits of quantitation (LOQ) were sufficient for measurement of normal values in urine (0.643 & 1.43 pg/mL), serum (1.90 pg/mL), and saliva pools (0.485 & 4.42 pg/mL). In conclusion, the PP OT RIA is specific and sensitive enough for reproducible measurement of intact OT in human peripheral biological matrices. |
| first_indexed | 2024-03-07T20:04:10Z |
| format | Article |
| id | doaj.art-75d535ec535e4401868c8f8c0b99b356 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2025-03-21T17:58:01Z |
| publishDate | 2024-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-75d535ec535e4401868c8f8c0b99b3562024-06-11T04:10:27ZengElsevierBiochemistry and Biophysics Reports2405-58082024-07-0138101666A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocinKeenan Gerred0Amita Kapoor1Corresponding author.; Wisconsin National Primate Research Center, University of Wisconsin-Madison, 1220 Capitol Court, Madison, WI, 53715, USAWisconsin National Primate Research Center, University of Wisconsin-Madison, 1220 Capitol Court, Madison, WI, 53715, USAOxytocin (OT) is a peptide hormone synthesized in the hypothalamus and released into systemic circulation or other areas of the brain. Its physiological roles include action as a hormone with stimulation of uterine contractions and that as a neuromodulator with involvement in social behaviors and regulation of mood. Its small size and low levels within biological matrices make it challenging to accurately measure. The goal of this study was to demonstrate the specificity of the antibody, sensitivity, and reproducibility of the Phoenix Pharmaceuticals (PP) OT radioimmunoassay (RIA) for use in human urine, serum, and saliva. Specificity of the antibody was assessed by high pressure liquid chromatography with ultraviolet (HPLC-UV) separation and assay of the fractions. Immunoreactivity was evaluated using the percent OT bound, and the fraction retention times were compared to the retention time of an intact OT standard to determine which fractions contained OT in the extracted samples. Reproducibility was assessed by running replicates of pools of each biomatrix over several assays. Sensitivity was assessed by repeated measurement of physiologically relevant low-concentration specimens. In all tested specimens the greatest reactivity in assay corresponded to the same fraction(s) as the OT standard. Only minimal reactivity was found in the other fractions, suggesting that in an unfractionated sample the antibody reacts mostly with intact OT. Reproducibility was acceptable for all specimens and the coefficient of variation (CV) ranged from 3.72 to 8.04% and 5.89–12.8%, for intra and inter-assay, respectively. The limits of quantitation (LOQ) were sufficient for measurement of normal values in urine (0.643 & 1.43 pg/mL), serum (1.90 pg/mL), and saliva pools (0.485 & 4.42 pg/mL). In conclusion, the PP OT RIA is specific and sensitive enough for reproducible measurement of intact OT in human peripheral biological matrices.http://www.sciencedirect.com/science/article/pii/S240558082400030XOxytocinUrineSalivaSerumMethod validationRadioimmunoassay |
| spellingShingle | Keenan Gerred Amita Kapoor A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin Biochemistry and Biophysics Reports Oxytocin Urine Saliva Serum Method validation Radioimmunoassay |
| title | A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| title_full | A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| title_fullStr | A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| title_full_unstemmed | A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| title_short | A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| title_sort | fit for purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin |
| topic | Oxytocin Urine Saliva Serum Method validation Radioimmunoassay |
| url | http://www.sciencedirect.com/science/article/pii/S240558082400030X |
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