Mouse PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) CLIA Kit (MOES00476)

SKU:
MOES00476
€649

Description

ELISA Kit Technical ManualMSDS

Mouse PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) 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 Mouse PACAP . 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 Mouse PACAP 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 Mouse PACAP, 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 Mouse PACAP. The concentration of Mouse PACAP in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
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 Mouse PACAP in samples. No significant cross-reactivity or interference between Mouse PACAP 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
50049266
53808
5153751508
25019626
22370
2099820969
12510179
8613
93969367
62.54394
4630
45124483
31.252350
2248
22992270
15.631256
1244
12501221
7.81706
774
740711
028
30
29--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)25.0170.93219.8224.4067.84226.51
Standard deviation2.207.4913.632.297.5118.17
C V (%)8.8010.566.209.3911.078.02

Recovery

The recovery of Mouse PACAP 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)92-106100
EDTA plasma (n=5)84-9890
Cell culture media (n=5)94-107101

Linearity

Samples were spiked with high concentrations of Mouse PACAP 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 (%)100-11690-10494-109
Average (%)10897100
1:4Range (%)94-106100-11295-108
Average (%)100106102
1:8Range (%)94-10599-11187-99
Average (%)9910592
1:16Range (%)84-99100-11489-105
Average (%)9110796

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

Mouse PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) CLIA Kit (MOES00476) 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.

Mouse PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) CLIA Kit (MOES00476) Protein Information

UniProt Protein Function:Binding to its receptor activates G proteins and stimulates adenylate cyclase in pituitary cells. Promotes neuron projection development through the RAPGEF2/Rap1/B-Raf/ERK pathway.
NCBI Summary:This gene encodes a member of the glucagon superfamily of hormones that have important roles in growth and metabolism. The encoded protein undergoes proteolytic processing to generate mature peptides that function as hypophysiotropic hormones, neurotransmitters, neuromodulators and vasoregulators. Mice lacking the encoded protein exhibit a high rate of early mortality. The surviving adult animals lacking the encoded protein exhibit decreased anxiety, hyperactive behavior and impaired steroidogenesis. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Oct 2015]
UniProt Code:O70176
NCBI GenInfo Identifier:937500806
NCBI Gene ID:11516
NCBI Accession:NP_001302432.1
UniProt Related Accession:O70176
Molecular Weight:19,381 Da
NCBI Full Name:pituitary adenylate cyclase-activating polypeptide isoform 1 preproprotein
NCBI Synonym Full Names:adenylate cyclase activating polypeptide 1
NCBI Official Symbol:Adcyap1  
NCBI Official Synonym Symbols:PACAP  
NCBI Protein Information:pituitary adenylate cyclase-activating polypeptide
UniProt Protein Name:Pituitary adenylate cyclase-activating polypeptide
UniProt Synonym Protein Names:PACAP-related peptideAlternative name(s):PRP-48
Protein Family:Glucagon family neuropeptides
UniProt Gene Name:Adcyap1  
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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
CLIA
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