Product Name:	Human PIK3CD ELISA Kit
Product Code:	HUFI01310
Size:	96 Assays
Alias:	PIK3CD, Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit delta isoform, PI3-kinase subunit delta, PI3K-delta, PI3Kdelta, PtdIns-3-kinase subunit delta, Phosphatidylinositol-4, 5-bisphosphate 3-kinase 110 kDa catalytic subunit delta, PtdIns-3-kinase subunit p110-delta, p110delta
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PIK3CD concentrations in serum plasma and other biological fluids.
Sensitivity:	0.469ng/ml
Range:	0.781-50ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PIK3CD and the recovery rates were calculated by comparing the measured value to the expected amount of Human PIK3CD in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-97	92
EDTA plasma(n=5)	89-105	97
UFH plasma(n=5)	86-105	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PIK3CD and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	92-105%	87-101%	93-103%
EDTA plasma(n=5)	87-98%	83-98%	86-100%
UFH plasma(n=5)	86-96%	80-100%	85-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

O00329

UniProt Protein Function:	PIK3CD: Phosphoinositide-3-kinase (PI3K) that phosphorylates PftdIns(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 plays a key role by recruiting PH domain-containing proteins to the membrane, including AKT1 and PDPK1, activating signaling cascades involved in cell growth, survival, proliferation, motility and morphology. Mediates immune responses. Plays a role in B-cell development, proliferation, migration, and function. Required for B-cell receptor (BCR) signaling. Mediates B-cell proliferation response to anti-IgM, anti-CD40 and IL4 stimulation. Promotes cytokine production in response to TLR4 and TLR9. Required for antibody class switch mediated by TLR9. Involved in the antigen presentation function of B-cells. Involved in B-cell chemotaxis in response to CXCL13 and sphingosine 1-phosphate (S1P). Required for proliferation, signaling and cytokine production of naive, effector and memory T-cells. Required for T-cell receptor (TCR) signaling. Mediates TCR signaling events at the immune synapse. Activation by TCR leads to antigen-dependent memory T-cell migration and retention to antigenic tissues. Together with PIK3CG participates in T-cell development. Contributes to T-helper cell expansion and differentiation. Required for T-cell migration mediated by homing receptors SELL/CD62L, CCR7 and S1PR1 and antigen dependent recruitment of T-cells. Together with PIK3CG is involved in natural killer (NK) cell development and migration towards the sites of inflammation. Participates in NK cell receptor activation. Have a role in NK cell maturation and cytokine production. Together with PIK3CG is involved in neutrophil chemotaxis and extravasation. Together with PIK3CG participates in neutrophil respiratory burst. Have important roles in mast-cell development and mast cell mediated allergic response. Involved in stem cell factor (SCF)-mediated proliferation, adhesion and migration. Required for allergen-IgE-induced degranulation and cytokine release. The lipid kinase activity is required for its biological function. Isoform 2 may be involved in stabilizing total RAS levels, resulting in increased ERK phosphorylation and increased PI3K activity. Belongs to the PI3/PI4-kinase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Carbohydrate Metabolism - inositol phosphate; Kinase, lipid; EC 2.7.1.153; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 1p36.2 Cellular Component: mast cell granule; plasma membrane; cytosol; phosphoinositide 3-kinase complex Molecular Function:protein binding; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; 1-phosphatidylinositol-3-kinase activity; ATP binding; phosphoinositide 3-kinase activity; phosphatidylinositol-4-phosphate 3-kinase activity Biological Process: respiratory burst during defense response; phosphoinositide 3-kinase cascade; T cell activation; nerve growth factor receptor signaling pathway; natural killer cell activation; signal transduction; T cell receptor signaling pathway; protein amino acid phosphorylation; B cell receptor signaling pathway; B cell homeostasis; phosphoinositide phosphorylation; natural killer cell differentiation; phospholipid metabolic process; inflammatory response; T cell differentiation; epidermal growth factor receptor signaling pathway; neutrophil chemotaxis; adaptive immune response; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; B cell activation; mast cell chemotaxis; cytokine production; mast cell degranulation; phosphatidylinositol biosynthetic process; innate immune response Disease: Immunodeficiency 14
NCBI Summary:	Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids and are involved in the immune response. The protein encoded by this gene is a class I PI3K found primarily in leukocytes. Like other class I PI3Ks (p110-alpha p110-beta, and p110-gamma), the encoded protein binds p85 adapter proteins and GTP-bound RAS. However, unlike the other class I PI3Ks, this protein phosphorylates itself, not p85 protein.[provided by RefSeq, Jul 2010]
UniProt Code:	O00329
NCBI GenInfo Identifier:	67477424
NCBI Gene ID:	5293
NCBI Accession:	O00329.2
UniProt Secondary Accession:	O00329,O15445, Q5SR49, A6NCG0, G1FFP1,
UniProt Related Accession:	O00329
Molecular Weight:	119,479 Da
NCBI Full Name:	Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform
NCBI Synonym Full Names:	phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta
NCBI Official Symbol:	PIK3CD
NCBI Official Synonym Symbols:	APDS; PI3K; IMD14; p110D; P110DELTA
NCBI Protein Information:	phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform; PI3Kdelta; phosphoinositide-3-kinase C; PI3-kinase p110 subunit delta; ptdIns-3-kinase subunit p110-delta; phosphoinositide-3-kinase, catalytic, delta polypeptide variant p37d
UniProt Protein Name:	Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform
UniProt Synonym Protein Names:	Phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit delta
UniProt Gene Name:	PIK3CD
UniProt Entry Name:	PK3CD_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.