Human AgRP (Agouti Related Protein) CLIA Kit (HUES00005)

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HUES00005
€599

Description

ELISA Kit Technical ManualMSDS

Human AgRP (Agouti Related Protein) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human AgRP . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human AgRP and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human AgRP, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human AgRP. The concentration of Human AgRP in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range7.81-500 pg/mL
Sensitivity4.69 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human AgRP in samples. No significant cross-reactivity or interference between Human AgRP and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
RLU Average Corrected
50049753
53321
5153751508
25019848
22148
2099820969
1259830
8962
93969367
62.54426
4598
45124483
31.252357
2241
22992270
15.631270
1230
12501221
7.81728
752
740711
028
30
29--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AgRP were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AgRP were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)25.8180.27188.2027.5074.73172.76
Standard deviation3.338.7017.523.556.7214.08
C V (%)12.9010.849.3112.918.998.15

Recovery

The recovery of Human AgRP spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)85-9690
EDTA plasma (n=5)95-113103
Cell culture media (n=5)91-10599

Linearity

Samples were spiked with high concentrations of Human AgRP and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)98-11288-10090-104
Average (%)1049497
1:4Range (%)85-9594-10988-102
Average (%)9010195
1:8Range (%)87-10099-11287-103
Average (%)9310594
1:16Range (%)86-99100-11997-114
Average (%)93108105

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human AgRP (Agouti Related Protein) CLIA Kit (HUES00005) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human AgRP (Agouti Related Protein) CLIA Kit (HUES00005) Protein Information

UniProt Protein Function:AGRP: Plays a role in weight homeostasis. Involved in the control of feeding behavior through the central melanocortin system. Acts as alpha melanocyte-stimulating hormone antagonist by inhibiting cAMP production mediated by stimulation of melanocortin receptors within the hypothalamus and adrenal gland. Has very low activity with MC5R. Is an inverse agonist for MC3R and MC4R being able to suppress their constitutive activity. It promotes MC3R and MC4R endocytosis in an arrestin-dependent manner. Genetic variations in AGRP may be a cause of obesity (OBESITY). It is a condition characterized by an increase of body weight beyond the limitation of skeletal and physical requirements, as the result of excessive accumulation of body fat.
UniProt Protein Details:

Protein type:Secreted; Hormone; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 16q22

Cellular Component: extracellular space; cell soma; Golgi lumen

Molecular Function:neuropeptide hormone activity; receptor binding

Biological Process: maternal process involved in pregnancy; hormone-mediated signaling; eating behavior; neuropeptide signaling pathway; photoperiodism; feeding behavior; adult feeding behavior; response to insulin stimulus

Disease: Obesity

NCBI Summary:This gene encodes an antagonist of the melanocortin-3 and melanocortin-4 receptor. It appears to regulate hypothalamic control of feeding behavior via melanocortin receptor and/or intracellular calcium regulation, and thus plays a role in weight homeostasis. Mutations in this gene have been associated with late on-set obesity. [provided by RefSeq, Dec 2009]
UniProt Code:O00253
NCBI GenInfo Identifier:4501995
NCBI Gene ID:181
NCBI Accession:NP_001129.1
UniProt Secondary Accession:O00253,O15459, Q2TBD9,
UniProt Related Accession:O00253
Molecular Weight:
NCBI Full Name:agouti-related protein
NCBI Synonym Full Names:agouti related neuropeptide
NCBI Official Symbol:AGRP  
NCBI Official Synonym Symbols:ART; AGRT; ASIP2  
NCBI Protein Information:agouti-related protein; agouti related protein homolog
UniProt Protein Name:Agouti-related protein
Protein Family:Agouti-related protein
UniProt Gene Name:AGRP  
UniProt Entry Name:AGRP_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
CLIA
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