Human PLCgamma2 (Phospholipase C Gamma 2, Phosphatidylinositol Specific) CLIA Kit (HUES00573)

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HUES00573
€599

Description

ELISA Kit Technical ManualMSDS

Human PLCgamma2 (Phospholipase C Gamma 2, Phosphatidylinositol Specific) 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 PLCgamma2 . 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 PLCgamma2 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 PLCgamma2, 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 PLCgamma2. The concentration of Human PLCgamma2 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 range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human PLCgamma2 in samples. No significant cross-reactivity or interference between Human PLCgamma2 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
400056564
61598
5908159046
200025731
29987
2785927824
100014647
12321
1348413449
5006261
6949
66056570
2503252
3234
32433208
1251625
1537
15811546
62.50752
758
755720
034
36
35--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PLCgamma2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PLCgamma2 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)207.91432.591776.47227.12438.811745.09
Standard deviation22.1250.92107.3019.6042.30186.03
C V (%)10.6411.776.048.639.6410.66

Recovery

The recovery of Human PLCgamma2 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)89-10094
EDTA plasma (n=5)85-9990
Cell culture media (n=5)85-9790

Linearity

Samples were spiked with high concentrations of Human PLCgamma2 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 (%)93-110102-11785-98
Average (%)10010992
1:4Range (%)97-11290-10388-102
Average (%)1059693
1:8Range (%)99-11587-9994-110
Average (%)10694101
1:16Range (%)95-11186-9691-106
Average (%)1029199

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 PLCgamma2 (Phospholipase C Gamma 2, Phosphatidylinositol Specific) CLIA Kit (HUES00573) 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 PLCgamma2 (Phospholipase C Gamma 2, Phosphatidylinositol Specific) CLIA Kit (HUES00573) Protein Information

UniProt Protein Function:PLCG2: a calcium dependent phosphatidylinositol-specific phospholipase C. The activated enzyme produces the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate. It is a crucial enzyme in transmembrane signaling. Phosphorylated and activated by tyrosine kinases in response to signaling through a variety of growth factor receptors and immune system receptors.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - inositol phosphate; Phospholipase; EC 3.1.4.11; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 16q24.1

Cellular Component: plasma membrane; cytosol

Molecular Function:signal transducer activity; protein binding; phospholipase C activity; phosphoinositide phospholipase C activity

Biological Process: platelet activation; Wnt receptor signaling pathway; inositol phosphate metabolic process; phospholipid catabolic process; calcium-mediated signaling; response to lipopolysaccharide; T cell receptor signaling pathway; activation of store-operated calcium channel activity; B cell receptor signaling pathway; regulation of gene expression; inositol trisphosphate biosynthetic process; B cell differentiation; release of sequestered calcium ion into cytosol; negative regulation of programmed cell death; phosphatidylinositol biosynthetic process; innate immune response; follicular B cell differentiation; blood coagulation

Disease: Autoinflammation, Antibody Deficiency, And Immune Dysregulation, Plcg2-associated; Familial Cold Autoinflammatory Syndrome 3

NCBI Summary:The protein encoded by this gene is a transmembrane signaling enzyme that catalyzes the conversion of 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate to 1D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) using calcium as a cofactor. IP3 and DAG are second messenger molecules important for transmitting signals from growth factor receptors and immune system receptors across the cell membrane. Mutations in this gene have been found in autoinflammation, antibody deficiency, and immune dysregulation syndrome and familial cold autoinflammatory syndrome 3. [provided by RefSeq, Mar 2014]
UniProt Code:P16885
NCBI GenInfo Identifier:215274231
NCBI Gene ID:5336
NCBI Accession:P16885.4
UniProt Secondary Accession:P16885,Q3ZTS2, Q59H45, Q969T5, D3DUL3,
UniProt Related Accession:P16885
Molecular Weight:1265
NCBI Full Name:1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2
NCBI Synonym Full Names:phospholipase C, gamma 2 (phosphatidylinositol-specific)
NCBI Official Symbol:PLCG2  
NCBI Official Synonym Symbols:FCAS3; APLAID; PLC-IV; PLC-gamma-2  
NCBI Protein Information:1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2; phospholipase C-IV; phosphoinositide phospholipase C-gamma-2
UniProt Protein Name:1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2
UniProt Synonym Protein Names:Phosphoinositide phospholipase C-gamma-2; Phospholipase C-IV; PLC-IV; Phospholipase C-gamma-2; PLC-gamma-2
Protein Family:1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase
UniProt Gene Name:PLCG2  
UniProt Entry Name:PLCG2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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