ELISA Kit for Obestatin (OB) Rattus norvegicus (Rat) Competition ELISA

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  • ELISA Kit for Obestatin (OB) Packages (Simulation)
  • ELISA Kit for Obestatin (OB) Packages (Simulation)
  • ELISA Kit for Obestatin (OB) Results demonstration
  • CEC039Ra.jpg Typical Standard Curve
  • Certificate ISO9001: 2008, ISO13485: 2003 Registered

Recovery

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

Matrix Recovery range (%) Average(%)
serum(n=5) 89-101 94
EDTA plasma(n=5) 84-94 87
heparin plasma(n=5) 98-105 102

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Obestatin (OB) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Obestatin (OB) 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 Obestatin (OB) 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) 79-91% 79-95% 92-105% 97-104%
EDTA plasma(n=5) 97-105% 78-101% 95-105% 89-103%
heparin plasma(n=5) 80-90% 95-103% 80-89% 92-99%

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 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

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.

ELISA Kit for Obestatin (OB)

Test principle

This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to Obestatin (OB) has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled Obestatin (OB) and unlabeled Obestatin (OB) (Standards or samples) with the pre-coated antibody specific to Obestatin (OB). After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of Obestatin (OB) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of Obestatin (OB) in the sample.

Citations

  • Evaluation of effects of diet on serum obestatin levels in over-weight patients with polycystic ovary syndromeOrg:Source
  • Significance of appetite hormone ghrelin and obestatin levels in the assessment of the severity of acute pancreatitisPubmed:25141321
  • Serum complement factor 5a levels are associated with nonalcoholic fatty liver disease in obese childrenDOI: 10.1111/apa.14106
  • The role of the adipocytokines vaspin and visfatin in vascular endothelial function and insulin resistance in obese childrenPubmed: 31771561

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