Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CTNN beta1 in samples. No significant cross-reactivity or interference between Human CTNN beta1 and analogues was observed.

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 CTNN beta1. 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 CTNN beta1 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 CTNN beta1, 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 CTNN beta1. The concentration of Human CTNN beta1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CTNNB1: a regulator of cell adhesion and a key downstream effector in the Wnt signaling pathway. Implicated early embryonic development and tumorigenesis. Phosphorylated and destabilized by CK1 and GSK-3beta. Stabilized cytoplasmic beta-catenin is a hallmark of a variety of cancers. Stabilized beta-catenin translocates to the nucleus, where it acts as a transcriptional activator of T-cell factor (TCF)-regulated genes. Interacts with the PDZ domain of TAX1BP3, inhibiting its transcriptional activity. Two alternatively spliced human isoforms have been described.
UniProt Protein Details:	Protein type:Transcription factor; Motility/polarity/chemotaxis; Cell adhesion; Oncoprotein; Nuclear receptor co-regulator; Actin-binding Chromosomal Location of Human Ortholog: 3p21 Cellular Component: centrosome; basolateral plasma membrane; intercellular junction; fascia adherens; cytosol; beta-catenin destruction complex; transcription factor complex; cell-cell adherens junction; membrane; lamellipodium; perinuclear region of cytoplasm; cytoplasm; synapse; dendritic shaft; lateral plasma membrane; spindle pole; focal adhesion; tight junction; catenin complex; cell cortex; Z disc; nucleoplasm; adherens junction; microvillus membrane; apical part of cell; plasma membrane; nucleus; cell junction Molecular Function:protein C-terminus binding; transcription coactivator activity; transcription factor binding; protein phosphatase binding; ionotropic glutamate receptor binding; protein binding; signal transducer activity; enzyme binding; androgen receptor binding; cadherin binding; double-stranded DNA binding; protein complex binding; estrogen receptor binding; nitric-oxide synthase binding; SMAD binding; transcription factor activity; kinase binding; alpha-catenin binding; nuclear hormone receptor binding Biological Process: regulation of myelination; regulation of centriole-centriole cohesion; positive regulation of apoptosis; protein heterooligomerization; positive regulation of transcription, DNA-dependent; regulation of fibroblast proliferation; cell maturation; negative regulation of chondrocyte differentiation; T cell differentiation in the thymus; positive regulation of fibroblast growth factor receptor signaling pathway; osteoclast differentiation; Wnt receptor signaling pathway through beta-catenin; cell-cell adhesion; positive regulation of endothelial cell differentiation; regulation of cell fate specification; embryonic foregut morphogenesis; positive regulation of mesenchymal cell proliferation; male genitalia development; ectoderm development; synapse organization and biogenesis; cell adhesion; bone resorption; response to drug; positive regulation of neuroblast proliferation; positive regulation of I-kappaB kinase/NF-kappaB cascade; regulation of smooth muscle cell proliferation; transcription, DNA-dependent; hair cell differentiation; negative regulation of protein sumoylation; patterning of blood vessels; genitalia morphogenesis; muscle cell differentiation; midgut development; smooth muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; embryonic digit morphogenesis; negative regulation of transcription, DNA-dependent; oocyte development; embryonic forelimb morphogenesis; negative regulation of osteoclast differentiation; glial cell fate determination; endodermal cell fate commitment; apoptosis; cell-matrix adhesion; neuron migration; dorsal/ventral axis specification; cell fate specification; positive regulation of histone H3-K4 methylation; negative regulation of transcription from RNA polymerase II promoter; embryonic hindlimb morphogenesis; response to estradiol stimulus; negative regulation of cell proliferation; central nervous system vasculogenesis; positive regulation of MAPKKK cascade; pancreas development; positive regulation of interferon type I production; fallopian tube development; proximal/distal pattern formation; layer formation in the cerebral cortex; negative regulation of mitotic cell cycle, embryonic; cell structure disassembly during apoptosis; Wnt receptor signaling pathway; hair follicle morphogenesis; thymus development; in utero embryonic development; regulation of T cell proliferation; neural plate development; stem cell maintenance; embryonic axis specification; synaptic vesicle transport; gastrulation with mouth forming second; liver development; regulation of angiogenesis; odontogenesis of dentine-containing teeth; myoblast differentiation; negative regulation of oligodendrocyte differentiation; Schwann cell proliferation; positive regulation of osteoblast differentiation; response to cadmium ion; ureteric bud branching; response to cytokine stimulus; androgen receptor signaling pathway; epithelial to mesenchymal transition; positive regulation of muscle cell differentiation; embryonic heart tube development; innate immune response; lens morphogenesis in camera-type eye; anterior/posterior axis specification Disease: Pilomatrixoma; Mental Retardation, Autosomal Dominant 19; Ovarian Cancer; Colorectal Cancer; Hepatocellular Carcinoma
NCBI Summary:	The protein encoded by this gene is part of a complex of proteins that constitute adherens junctions (AJs). AJs are necessary for the creation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells. The encoded protein also anchors the actin cytoskeleton and may be responsible for transmitting the contact inhibition signal that causes cells to stop dividing once the epithelial sheet is complete. Finally, this protein binds to the product of the APC gene, which is mutated in adenomatous polyposis of the colon. Mutations in this gene are a cause of colorectal cancer (CRC), pilomatrixoma (PTR), medulloblastoma (MDB), and ovarian cancer. Three transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Oct 2009]
UniProt Code:	P35222
NCBI GenInfo Identifier:	461854
NCBI Gene ID:	1499
NCBI Accession:	P35222. 1
UniProt Secondary Accession:	P35222,Q8NEW9, Q8NI94, Q9H391, A8K1L7,
UniProt Related Accession:	P35222
Molecular Weight:
NCBI Full Name:	Catenin beta-1
NCBI Synonym Full Names:	catenin (cadherin-associated protein), beta 1, 88kDa
NCBI Official Symbol:	CTNNB1
NCBI Official Synonym Symbols:	CTNNB; MRD19; armadillo
NCBI Protein Information:	catenin beta-1
UniProt Protein Name:	Catenin beta-1
UniProt Synonym Protein Names:	Beta-catenin
Protein Family:	Catenin
UniProt Gene Name:	CTNNB1
UniProt Entry Name:	CTNB1_HUMAN

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
2000	51198 60244	55721	55693
1000	23050 24898	23974	23946
500	11388 10676	11032	11004
250	4827 5761	5294	5266
125	2618 2598	2608	2580
62.5	1350 1272	1311	1283
31.25	660 688	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CTNN beta1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CTNN beta1 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	99.73	229.73	837.66	109.03	209.57	774.89
Standard deviation	12.85	23.20	86.87	8.84	23.05	63.39
C V (%)	12.88	10.10	10.37	8.11	11.00	8.18

Recovery

The recovery of Human CTNN beta1 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)	92-105	98
EDTA plasma (n=5)	92-107	99
Cell culture media (n=5)	88-104	95

Linearity

Samples were spiked with high concentrations of Human CTNN beta1 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)
1:2	Range (%)	96-111	87-98	88-101
1:2	Average (%)	104	93	95
1:4	Range (%)	96-110	87-98	100-114
1:4	Average (%)	103	93	107
1:8	Range (%)	84-96	95-112	92-108
1:8	Average (%)	91	103	99
1:16	Range (%)	96-113	95-108	93-103
1:16	Average (%)	103	102	98

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 ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

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.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
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.
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.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
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.
Determine the RLU value of each well immediately.