Melatonin was quantified using a commercially available melatonin ELISA kit (Abcam, Cambridge, UK). Melatonin was extracted from the MelaNVs using the ethyl acetate extraction method before performing ELISA. Briefly, 50 μg of MelaNVs in protein amount was mixed with the same volume of cold ethyl acetate, and the sample was incubated on ice for 2 min. After centrifugation at 1000× g for 10 min at 4 °C, the upper organic layer containing melatonin was obtained. The sample was dried overnight under a stream of inert gas. The resulting pellet was reconstituted with a stabilizer solution provided with the kit, and the ELISA assay was conducted according to the manufacturer’s instructions.
Melatonin elisa kit
Manufactured by Abcam
Sourced in United Kingdom
The Melatonin ELISA Kit is a quantitative in vitro diagnostic assay designed for the measurement of melatonin levels in various sample types, including serum, plasma, and urine. The kit utilizes the competitive ELISA principle to determine the concentration of melatonin present in the samples.
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Lab products found in correlation
7 protocols using melatonin elisa kit
1
Melatonin Quantification in Nanoparticles
2
Maternal and Fetal Melatonin Levels
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The concentration of maternal and fetal melatonin levels in plasma was determined in all samples within one assay/standard curve. Melatonin levels were determined using a colorimetric melatonin ELISA kit (code ‐ ab283259; Abcam). The sensitivity of the assay was 2.5 pg/ml.
3
Melatonin Quantification by ELISA
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The concentration of melatonin was measured by Melatonin ELISA kit (abcam,
ab213978). All the materials were equilibrated and prepared reagents to room
temperature prior to use. The assay procedure was below. 100 μL of standards and
100 μL of the samples were added into the appropriate wells. Then to the above
wells, 50 μL of the melatonin tracer was added to all wells except for the
blank. Later, 50 μL of the melatonin antibody was added to all wells except for
the NSB and blank. Then sealed and incubated at room temperature on a plate
shaker for 1 h at ∼500 r/min. The wells were washed by adding 400 μL of wash
buffer thrice. Then, 200 μL of the melatonin conjugate solution was added to
each well except the blank. Sealed the plate and incubated at room temperature
on a plate shaker for 30 min at ∼500 r/min. The wells were washed by adding
400 μL of wash buffer thrice. Then, 200 μL of TMB substrate solution was added
into each well. Sealed the plate and incubated for 30 min at room temperature on
a plate shaker at ∼500 r/min. To this, 50 μL of the stop solution was added into
each well and read the OD at 450 nm.
ab213978). All the materials were equilibrated and prepared reagents to room
temperature prior to use. The assay procedure was below. 100 μL of standards and
100 μL of the samples were added into the appropriate wells. Then to the above
wells, 50 μL of the melatonin tracer was added to all wells except for the
blank. Later, 50 μL of the melatonin antibody was added to all wells except for
the NSB and blank. Then sealed and incubated at room temperature on a plate
shaker for 1 h at ∼500 r/min. The wells were washed by adding 400 μL of wash
buffer thrice. Then, 200 μL of the melatonin conjugate solution was added to
each well except the blank. Sealed the plate and incubated at room temperature
on a plate shaker for 30 min at ∼500 r/min. The wells were washed by adding
400 μL of wash buffer thrice. Then, 200 μL of TMB substrate solution was added
into each well. Sealed the plate and incubated for 30 min at room temperature on
a plate shaker at ∼500 r/min. To this, 50 μL of the stop solution was added into
each well and read the OD at 450 nm.
4
Hormonal Biomarker Profiling Protocol
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Serum prolactin was measured by prolactin Human ELISA kit (Cat. No. ab108655, Abcam, UK), melatonin was measured by melatonin ELISA kit (Cat. No. ab213978, Abcam, UK), TSH was measured by TSH Human ELISA kit (Cat. No. ab108660, Abcam, UK), fT3 was measured by fT3 Human ELISA kit (Cat. No. ab108663, Abcam, UK), fT4 was measured by fT4 Human ELISA kit (Cat. No. ab108686, Abcam, UK), Estradiol was measured by Estradiol E2 Human ELISA kit (Cat. No. ab108640, Abcam, UK), testosterone was measured by testosterone Human ELISA kit (Cat. No. ab174569, Abcam, UK), Cortisol was measured by Cortisol Human ELISA kit (Cat. No. ab108665, Abcam, UK), and ACTH was measured by Human ACTH ELISA kit (Cat. No. MBS2511227, MyBioSource. Inc., San Diego, CA, USA).
5
Circadian Rhythm Assessment via Melatonin and Cortisol
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Since direct measurement of circadian rhythm is not generally feasible, the secretion of melatonin and cortisol is commonly used as a surrogate for circadian rhythmicity [6 ]. Normally, melatonin exhibits a peak during the rest period, while cortisol demonstrates a peak during the inactivity to activity transition period [18 (link)]. Both were measured three times a day on the first three days after admission to the ICU. Samples were drawn at the specified time according to human physiology. Melatonin samples were drawn at 3:00, 8:00 and 16:00, while cortisol samples were drawn at 0:00, 8:00 and 16:00. We assessed cortisol levels in a subgroup of patients who had not used corticosteroids during the experimental period. The melatonin blood sample was immediately centrifuged and stored at -80 °C for later analysis. Serum melatonin was measured by a melatonin ELISA® kit (Abcam, USA). Plasma cortisol was tested at the central laboratory in Zhongshan Hospital, Fudan University.
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
Quantifying Melatonin and FGF19 in HNSCC
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The present studies in human biological specimens were reviewed and approved by the Institutional Review Board of the First Affiliated Hospital of Nanchang University, China. All samples were obtained with informed consent. The blood samples from thirty-five adult patients who have HNSCC with median age 58 years old were collected from the antecubital vein between 6:00 am and 8:00 am 1 day before surgery. The blood samples in the control group were from ten healthy age-matched people. Within 1 hour after collection, the blood samples were placed immediately on ice until centrifugal sera separation and storage at − 80 °C. The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples. Tumor tissues from the same cohort of HNSCC patients were collected for IHC to assess FGF19 levels. The clinicopathologic characteristics of sample sets was shown in Supplementary Table S1 .
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