Salivary oxytocin was analyzed with the Oxytocin ELISA kit (ENZO, cat.no ADI‐900‐153A). Salivary samples were purified with solid‐phase extraction strata‐X sorbent and 96‐well plate with 60 mg wells (Phenomenex 8E‐S100‐UGB). SPE columns were first revived with 1 ml of methanol. Methanol residues were washed with 1 ml of water. A total of 300 μl of 1.5% trichloroacetic acid (TFA) in water was added to 500 μl of saliva, stirred, and centrifuged at 6000 × g for 10 min. Samples were loaded into SPE columns, washed with 1.5 ml of 0.1% TFA, and eluted with 1 ml of acetonitrile (0.1% TFA, 80:20). Eluent was evaporated in vacuum centrifuge and samples were stored in a freezer (−20°C) until determination with the ENZO Oxytocin ELISA kit according to the assay procedure. The coefficients of variation percent of intraassay and interassay of the method reported by the manufacturer were 12% and 16%, respectively. For oxytocin, we were able to analyze 64% of the first saliva samples and 63% of the second samples. The rest of the samples were either too low in oxytocin or there was not enough saliva for this assay.
Oxytocin elisa kit
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom, Switzerland
The Oxytocin ELISA kit is a quantitative assay designed to measure the concentration of oxytocin in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) principle to detect and quantify the target analyte.
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Lab products found in correlation
Trifluoroacetic acid, by Fujifilm (2 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Bovine cortisol elisa kit, by MyBioSource (2 mentions)
Acetonitrile, by Kanto Chemical (1 mentions)
Acetonitrile, by Fujifilm (1 mentions)
Monospin c18 column, by GL Sciences (1 mentions)
Cortisol elisa kit, by Enzo Life Sciences (1 mentions)
Melatonin elisa kit, by Abcam (1 mentions)
Serotonin elisa kit, by Enzo Life Sciences (1 mentions)
Dhea elisa kit, by Enzo Life Sciences (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Vacutainer plastic tube, by BD (1 mentions)
16 protocols using oxytocin elisa kit
1
Salivary Oxytocin Analysis Protocol
2
Oxytocin Quantification Protocol
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Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Switzerland). Oxytocin ELISA kits were obtained from Enzo Life Sciences (USA). Trifluoroacetic acid (TFA) was purchased from Wako Pure Chemicals (Japan). Acetonitrile (ACN) and oxytocin were purchased from Kanto Chemical (Japan).
3
Oxytocin Quantification by ELISA
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Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Basel, Switzerland). [3H]-labeled oxytocin was obtained from NEN (USA). Oxytocin ELISA kits were obtained from Enzo Life Sciences (USA). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Wako Pure Chemicals (Japan). Other reagents were of the highest commercially available grade.
4
Comprehensive Metabolic and Hormonal Profiling of Mice
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Blood and urine were collected from isoflurane-anaesthetized mice through cardiac and urinary bladder punctures, respectively. Serum (or urinary) levels of total protein, creatinine, urea nitrogen, AST, and ALT were measured using commercial colorimetric assay kits from Fujifilm-Wako (Osaka, Japan). For the blood oxytocin measurements, blood was collected from the retro-orbital plexus. Serum was prepared and its protein components were denatured by 0.05% trifluoroacetic acid and removed by the MonoSpin C18 column (GL Science, Tokyo, Japan) with acetonitrile elution. After acetonitrile evaporation, oxytocin contents were measured using the Oxytocin ELISA Kit (Enzo Life Sciences, CA, NY, USA) according to the manufacturer’s instruction.
5
Oxytocin Secretion from Paraventricular Nucleus
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Eighteen male Wistar rats were used in this experiment. The measurement of oxytocin from PVN was performed accordingly to previous reports [8 ,25 (link)]. Briefly, Wistar rats aged eight weeks were deeply anesthetized by a mixture of three types of anesthetic agents, decapitated, and their brains were removed. Three 400 μm thick slices were prepared using a vibrating microtome, and the PVN-containing area was punched out.
These processes were performed in a buffer composed of (in mM) 229 mannitol, 3 KCl, 26 NaHCO3, 1H3PO4, 0.5 CaCl2, 7 MgCl2, 7 glucose, and 1 kynurenate at pH 7.4 with 95% O2 and 5% CO2 mixed gas.
Prepared PVN slices were incubated at 37 °C for 30 min in artificial cerebrospinal fluid (aCSF) composed of (in mM) 126 NaCl, 3 KCl, 26 NaHCO3, 1H3PO4, 2 CaCl2 and 1 MgSO4 at pH 7.4, with 95% O2 and 5% CO2 mixed gas for recovery. Next, the PVN slices were incubated in aCSF with or without KKT (500 μg/mL) for 1 h. The concentrations of KKT (500 μg/mL) for Oxt secretion from brain slice were decided based on the concentration of KKT used in brain slice patch clamp experiments (500 μg/mL). These incubations were performed in pairs (control and KKT). Secreted Oxt in the supernatant was determined using an oxytocin ELISA kit (Enzo Life Sciences) [10 (link)].
These processes were performed in a buffer composed of (in mM) 229 mannitol, 3 KCl, 26 NaHCO3, 1H3PO4, 0.5 CaCl2, 7 MgCl2, 7 glucose, and 1 kynurenate at pH 7.4 with 95% O2 and 5% CO2 mixed gas.
Prepared PVN slices were incubated at 37 °C for 30 min in artificial cerebrospinal fluid (aCSF) composed of (in mM) 126 NaCl, 3 KCl, 26 NaHCO3, 1H3PO4, 2 CaCl2 and 1 MgSO4 at pH 7.4, with 95% O2 and 5% CO2 mixed gas for recovery. Next, the PVN slices were incubated in aCSF with or without KKT (500 μg/mL) for 1 h. The concentrations of KKT (500 μg/mL) for Oxt secretion from brain slice were decided based on the concentration of KKT used in brain slice patch clamp experiments (500 μg/mL). These incubations were performed in pairs (control and KKT). Secreted Oxt in the supernatant was determined using an oxytocin ELISA kit (Enzo Life Sciences) [10 (link)].
6
Comprehensive Neuroendocrine Biomarker Analysis
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Plasma cortisol was measured by using a Cortisol ELISA kit (ADI-900-071, Enzo Life Sciences, UK), plasma DHEA was determined by a DHEA ELISA kit (ADI-900-093, Enzo Life Sciences, UK), plasma catecholamines (epinephrine: E, norepinephrine: NE, dopamine: DP) was quantified by 3-CAT ELISA kit (BA E-6600, LDN Labor Diagnostika, Germany). Plasma oxytocin and serotonin were determined by a Oxytocin ELISA kit (ADI-900-153A-0001, Enzo Life Sciences, UK) and Serotonin ELISA kit (ADI-900-175, Enzo Life Sciences, UK), respectively. Also, plasmatic concentration of testosterone and melatonin were measured using a Free Testosterone ELISA Kit (CAN-FTE-260, Diagnostics Biochem Canada Inc) and a Melatonin ELISA kit (E4630-100, BioVision), respectively.
The results were expressed in µg cortisol/mL of plasma, µg DHEA/mL of plasma, pg E/mL of plasma, pg NE/mL of plasma, pg DP/mL of plasma, ng Oxytocin/mL of plasma, ng serotonin/mL of plasma, pg testosterone/mL of plasma and pg melatonin/mL of plasma.
The results were expressed in µg cortisol/mL of plasma, µg DHEA/mL of plasma, pg E/mL of plasma, pg NE/mL of plasma, pg DP/mL of plasma, ng Oxytocin/mL of plasma, ng serotonin/mL of plasma, pg testosterone/mL of plasma and pg melatonin/mL of plasma.
7
Measurement of Plasma Oxytocin Levels
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For measuring the plasma OT levels, blood samples were drawn from antecubital veins into 3-ml vacutainer blood vacuettes (Greiner Bio-One International GmbH, Austria) containing Aprotinin (500 KIU/ml of blood) (Sigma-Aldrich, Germany). Vacuettes were stored at −20°C before use. Vacuettes were centrifuged at 4°C at 1.600 g for 15 min. Supernatants were stored at −80°C until analysis. Extraction of samples was undertaken and OT concentrations in the extracts were determined in duplicate by Oxytocin ELISA kit (ADI-900-153A, Enzo Life Sciences, USA), a colorimetric competitive enzyme immunoassay kit at the Center for Medical Research at the Medical University Graz, Austria. The mean intra-assay and inter-assay coefficients of variability were 23.4% and 13.9%, respectively; sensitivity was 15.0pg/ml. All procedures were performed according to the manufacturer’s instructions by authorized personnel.
8
Minimizing Stress in Bovine Blood Sampling
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Although sampling blood from cows may appear a non-invasive procedure, it remains a stressful event potentially affecting the behavioral outcomes during the test. Therefore, we avoided blood sampling before the test. On the other hand, multiple sampling after the test suffers the same problem: we could not exclude a hormonal response due to the sampling resulting in a bias on a second blood sampling. For these reasons, we have chosen to rely on only one sampling performed immediately (less than a minute) after the end of the test. Eight mL of blood were withdrawn from the coccygeal vein into plastic tubes and centrifuged at 4 °C and 1500× g for 15 min to obtain serum. The serum was divided into three aliquots, placed in plastic tubes, and stored at –20 °C until analysis. The average calculated over two dosages was used for the result. The serum concentration of oxytocin [51 (link)] and cortisol [52 ] was measured using competitive enzyme immunoassay (EIA) following the manufacturer’s instructions. For the Oxytocin ELISA kit, (Enzo Life Sciences, Lausen, Switzerland), the wavelength was set at 405 nm and the kit sensitivity ranged from 15.6 to 1000 pg/mL; for the Bovine Cortisol ELISA Kit (MyBioSource, Inc., San Diego, CA, USA), the detection range was from 0.049 up to 200 ng/mL and the wavelength was set at 450 nm.
9
Oxytocin Plasma Levels in Nulliparous and Primiparous Females
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Blood was collected from the tail of 3 months old nulliparous and 3–4 months old primiparous females using kalium-EDTA coated tubes (Sarstedt). Plasma were isolated according to manufacturer's instructions and stored at -80°C. Oxytocin plasma levels were measured using the Oxytocin ELISA kit (Enzo Life Sciences), following the manufacturer's protocol. Optical densities were measured on a FlexStation 3 (Molecular Devices) at 405nm, with correction at 580nm.
10
Plasma Oxytocin and Cortisol Measurement Protocol
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Fasting blood samples were collected between 08:00 and 10:00 in the morning. Blood samples for plasma oxytocin assay were collected from the antecubital vein into pre-chilled 5-ml ethylenediaminetetraacetic acid tubes with 250 KIU of apoprotinin and refrigerated until processing. Plasma was isolated by centrifugation at 1,800 ×g for 15 minutes at 4°C and stored in aliquots at −70°C. Oxytocin immune reactivity levels were quantified in duplicate using a commercial oxytocin ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA; formerly Assays Design, Ann Arbor, MI, USA). The detection range was from 12.35 to 1,000 pg/ml. The sensitivity, i.e., the minimum detectable dose of oxytocin, of our assay was 4.92 pg/ml. No extraction was conducted. The intra-assay precision and inter-assay precision of the assay were lower than 10% and 12%, respectively (coefficient of variance [CV] (%) = standard deviation/mean × 100; intra-assay: CV < 10%; inter-assay: CV < 12%).
Fasting blood sample for cortisol was collected between 08:00 and 10:00 in the morning, too. Cortisol level was assessed using a commercial radioimmunoassay kit (sensitivity, 0.2 ng/dl) (Immulite Cortisol; DPC® Biermann, Bad-Nauheim, Germany). The inter- and intra-assay coefficients of variation were < 7.8% and < 7.7%, respectively.
Fasting blood sample for cortisol was collected between 08:00 and 10:00 in the morning, too. Cortisol level was assessed using a commercial radioimmunoassay kit (sensitivity, 0.2 ng/dl) (Immulite Cortisol; DPC® Biermann, Bad-Nauheim, Germany). The inter- and intra-assay coefficients of variation were < 7.8% and < 7.7%, respectively.
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