Human AQP-4 (Aquaporin 4) CLIA Kit (HUES00352)

SKU:
HUES00352
Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
€599

Description

ELISA Kit Technical ManualMSDS

Human AQP-4 (Aquaporin 4) 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 AQP-4 . 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 AQP-4 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 AQP-4, 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 AQP-4. The concentration of Human AQP-4 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 range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human AQP-4 in samples. No significant cross-reactivity or interference between Human AQP-4 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
500056035
66477
6125661233
250033891
34817
3435434331
125018835
17503
1816918146
6258968
9816
93929369
312.55206
4460
48334810
156.252655
2367
25112488
78.131321
1357
13391316
022
24
23--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AQP-4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AQP-4 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)275.58600.412436.64289.73552.472533.49
Standard deviation29.0557.58224.6632.6248.62250.82
C V (%)10.549.599.2211.268.809.90

Recovery

The recovery of Human AQP-4 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)93-107100
EDTA plasma (n=5)98-117107
Cell culture media (n=5)87-10193

Linearity

Samples were spiked with high concentrations of Human AQP-4 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 (%)95-109101-116100-113
Average (%)102108107
1:4Range (%)100-11591-10786-97
Average (%)1089892
1:8Range (%)104-12198-11494-108
Average (%)11010699
1:16Range (%)91-10789-10288-101
Average (%)979595

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 AQP-4 (Aquaporin 4) CLIA Kit (HUES00352) 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 AQP-4 (Aquaporin 4) CLIA Kit (HUES00352) Protein Information

UniProt Protein Function:AQP4: a member of the aquaporin family of intrinsic membrane proteins that function as water-selective channels in the plasma membranes of many cells. The predominant aquaporin found in brain. Two alternatively spliced transcript variants have been described
UniProt Protein Details:

Protein type:Transporter, aquaporin family; Membrane protein, integral; Channel, misc.; Membrane protein, multi-pass; Transporter

Chromosomal Location of Human Ortholog: 18q11.2-q12.1

Cellular Component: basolateral plasma membrane; cytoplasm; external side of plasma membrane; integral to membrane; plasma membrane

Molecular Function:glycerol channel activity; water channel activity; water transporter activity

Biological Process: glycerol transport; multicellular organismal water homeostasis; renal water homeostasis; transmembrane transport; transport; water transport

NCBI Summary:This gene encodes a member of the aquaporin family of intrinsic membrane proteins that function as water-selective channels in the plasma membranes of many cells. This protein is the predominant aquaporin found in brain and has an important role in brain water homeostasis. Alternatively spliced transcript variants encoding different isoforms have been described for this gene. A recent study provided evidence for translational readthrough in this gene and expression of an additional C-terminally extended isoform via the use of an alternative in-frame translation termination codon. [provided by RefSeq, Dec 2015]
UniProt Code:P55087
NCBI GenInfo Identifier:2506859
NCBI Gene ID:361
NCBI Accession:P55087.2
UniProt Secondary Accession:P55087,P78564,
UniProt Related Accession:P55087
Molecular Weight:32,300 Da
NCBI Full Name:Aquaporin-4
NCBI Synonym Full Names:aquaporin 4
NCBI Official Symbol:AQP4  
NCBI Official Synonym Symbols:MIWC; WCH4  
NCBI Protein Information:aquaporin-4
UniProt Protein Name:Aquaporin-4
UniProt Synonym Protein Names:Mercurial-insensitive water channel; MIWC; WCH4
Protein Family:Aquaporin
UniProt Gene Name:AQP4  
UniProt Entry Name:AQP4_HUMAN
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