ELISA Kit for Orexin A (OXA)

Hypocretin-1

Specificity

This assay has high sensitivity and excellent specificity for detection of Orexin A (OXA).
No significant cross-reactivity or interference between Orexin A (OXA) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Orexin A (OXA) and the recovery rates were calculated by comparing the measured value to the expected amount of Orexin A (OXA) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 87-101 92
EDTA plasma(n=5) 84-91 88
heparin plasma(n=5) 98-105 102

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Orexin A (OXA) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Orexin A (OXA) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Orexin A (OXA) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 89-101% 88-101% 94-103% 82-103%
EDTA plasma(n=5) 85-95% 79-91% 83-97% 89-104%
heparin plasma(n=5) 78-89% 92-101% 92-105% 89-103%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1 Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
Reagent Diluent 1×300µL Stop Solution 1×6mL
TMB Substrate 1×9mL Instruction manual 1
Wash Buffer (30 × concentrate) 1×20mL

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample to each well.
    And then add 50µL prepared Detection Reagent A immediately.
    Shake and mix. Incubate 1 hour at 37°C;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
7. Add 50µL Stop Solution. Read at 450 nm immediately.

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Magazine Citations
Frontiers in Neurology Neuro-Glial and Systemic Mechanisms of Pathological Responses in Rat Models of Primary Blast Overpressure Compared to “Composite” Blast PubMed: PMC3275793
Neurological Sciences Pentylenetetrazol-induced seizures are exacerbated by sleep deprivation through orexin receptor-mediated hippocampal cell proliferation Pubmed: 23852314
Peptides Orexin-A-induced ERK1/2 activation reverses impaired spatial learning and memory in pentylenetetrazol-kindled rats via OX1R-mediated hippocampal neurogenesis. Pubmed: 24321199
Alcohol Alcohol Orexin in Patients with Alcohol Dependence Treated for Relapse Prevention: A Pilot Study PubMed: 26597795
Indian Journal of Medical Research and Pharmaceutical Sciences Evaluation of Antidiabetic And Insulintropic Potential of Nigella Sativa Seeds Water Extract and /or Alpha Lipoic Acid in Type 2 Diabetic Rats Issues%20Pdf: Vol.2
J Clin Res Pediatr Endocninol Correlation of Brain Neuropeptide (Nesfatin-1 and Orexin-A) Concentrations with Anthropometric and Biochemical Parameters in Malnourished Children PubMed: 26831553
Neural Regen Res Resuscitation therapy for traumatic brain injury-induced coma in rats: mechanisms of median nerve electrical stimulation PubMed: 26170820
Mol Med Rep Wake-promoting actions of median nerve stimulation in TBI-induced coma: An investigation of orexin-A and orexin receptor 1 in the hypothalamic region PubMed: 26059340
Mol Neurobiol Application of the Co-culture Membrane System Pointed to a Protective Role of Catestatin on Hippocampal Plus Hypothalamic Neurons Exposed to Oxygen and Glucose Deprivation article:10.1007
Neural Regeneration Research Wake-promoting effects of vagus nerve stimulation after traumatic brain injury: upregulation of orexin-A and orexin receptor type 1 expression in the prefrontal … Pubmed:29557373
Journal of Translational Medicine Recurrent circadian fasting (RCF) improves blood pressure, biomarkers of cardiometabolic risk and regulates inflammation in men Pubmed: 31426866
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