Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
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 Rat E-Cad in samples. No significant cross-reactivity or interference between Rat E-Cad 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 Rat E-Cad. 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 Rat E-Cad 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 Rat E-Cad, 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 Rat E-Cad. The concentration of Rat E-Cad in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Function: Cadherins are calcium-dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. CDH1 is involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha-E/beta-7. Ref. 25E-Cad/CTF2 promotes non-amyloidogenic degradation of Abeta precursors. Has a strong inhibitory effect on APP C99 and C83 production. Ref. 25
UniProt Protein Details:	Subunit structure: Homodimer; disulfide-linked. Component of an E-cadherin/ catenin adhesion complex composed of at least E-cadherin/CDH1, beta-catenin/CTNNB1 or gamma-catenin/JUP, and potentially alpha-catenin/CTNNA1; the complex is located to adherens junctions. The stable association of CTNNA1 is controversial as CTNNA1 was shown not to bind to F-actin when assembled in the complex. Alternatively, the CTNNA1-containing complex may be linked to F-actin by other proteins such as LIMA1. Interaction with PSEN1, cleaves CDH1 resulting in the disassociation of cadherin-based adherens junctions (CAJs). Interacts with AJAP1, CTNND1 and DLGAP5 By similarity. Interacts with TBC1D2. Interacts with LIMA1. Interacts with CAV1. Interacts with the TRPV4 and CTNNB1 complex By similarity. Interacts with PIP5K1C. Interacts with RAB8B By similarity. Interacts with RAPGEF2 By similarity. Interacts with DDR1; this stabilizes CDH1 at the cell surface and inhibits its internalization. Ref. 10 Ref. 17 Ref. 19 Ref. 20 Ref. 21 Ref. 22 Ref. 23 Ref. 29 Ref. 31 Ref. 32 Subcellular location: Cell junction. Cell membrane; Single-pass type I membrane protein. Endosome. Golgi apparatus trans-Golgi network. Note: Colocalizes with DLGAP5 at sites of cell-cell contact in intestinal epithelial cells. Anchored to actin microfilaments through association with alpha-, beta- and gamma-catenin. Sequential proteolysis induced by apoptosis or calcium influx, results in translocation from sites of cell-cell contact to the cytoplasm. Colocalizes with RAB11A endosomes during its transport from the Golgi apparatus to the plasma membrane. Ref. 4 Ref. 21 Ref. 24 Ref. 32 Tissue specificity: Non-neural epithelial tissues. Induction: Expression is repressed by MACROD1. Ref. 26 Domain: Three calcium ions are usually bound at the interface of each cadherin domain and rigidify the connections, imparting a strong curvature to the full-length ectodomain. Ref. 16 Post-translational modification: During apoptosis or with calcium influx, cleaved by a membrane-bound metalloproteinase (ADAM10), PS1/gamma-secretase and caspase-3 to produce fragments of about 38 kDa (E-CAD/CTF1), 33 kDa (E-CAD/CTF2) and 29 kDa (E-CAD/CTF3), respectively. Processing by the metalloproteinase, induced by calcium influx, causes disruption of cell-cell adhesion and the subsequent release of beta-catenin into the cytoplasm. The residual membrane-tethered cleavage product is rapidly degraded via an intracellular proteolytic pathway. Cleavage by caspase-3 releases the cytoplasmic tail resulting in disintegration of the actin microfilament system. The gamma-secretase-mediated cleavage promotes disassembly of adherens junctions. Ref. 4 Ref. 15 Ref. 18N-glycosylation at Asn-637 is essential for expression, folding and trafficking. Ubiquitinated by a SCF complex containing SKP2, which requires prior phosphorylation by CK1/CSNK1A1. Ubiquitinated by CBLL1/HAKAI, requires prior phosphorylation at Tyr-754. Ref. 34 Ref. 35 Involvement in Disease: Hereditary diffuse gastric cancer (HDGC) [MIM:137215]: A cancer predisposition syndrome with increased susceptibility to diffuse gastric cancer. Diffuse gastric cancer is a malignant disease characterized by poorly differentiated infiltrating lesions resulting in thickening of the stomach. Malignant tumors start in the stomach, can spread to the esophagus or the small intestine, and can extend through the stomach wall to nearby lymph nodes and organs. It also can metastasize to other parts of the body. Note: The disease is caused by mutations affecting the gene represented in this entry. Heterozygous CDH1 germline mutations are responsible for familial cases of diffuse gastric cancer. Somatic mutations has also been found in patients with sporadic diffuse gastric cancer and lobular breast cancer. Ref. 47 Ref. 52Endometrial cancer (ENDMC) [MIM:608089]: A malignancy of endometrium, the mucous lining of the uterus. Most endometrial cancers are adenocarcinomas, cancers that begin in cells that make and release mucus and other fluids. Note: Disease susceptibility is associated with variations affecting the gene represented in this entry. Ovarian cancer (OC) [MIM:167000]: The term ovarian cancer defines malignancies originating from ovarian tissue. Although many histologic types of ovarian tumors have been described, epithelial ovarian carcinoma is the most common form. Ovarian cancers are often asymptomatic and the recognized signs and symptoms, even of late-stage disease, are vague. Consequently, most patients are diagnosed with advanced disease. Note: Disease susceptibility is associated with variations affecting the gene represented in this entry. Breast cancer, lobular (LBC) [MIM:137215]: A type of breast cancer that begins in the milk-producing glands (lobules) of the breast. Note: The gene represented in this entry may be involved in disease pathogenesis. Ref. 27 Sequence similarities: Contains 5 cadherin domains. Sequence caution: The sequence AAA61259. 1 differs from that shown. Reason: Frameshift at positions 16, 22, 25, 28, 31, 34, 52, 67, 73, 76, 94, 102, 633 and 636.
NCBI Summary:	This gene is a classical cadherin from the cadherin superfamily. The encoded protein is a calcium dependent cell-cell adhesion glycoprotein comprised of five extracellular cadherin repeats, a transmembrane region and a highly conserved cytoplasmic tail. Mutations in this gene are correlated with gastric, breast, colorectal, thyroid and ovarian cancer. Loss of function is thought to contribute to progression in cancer by increasing proliferation, invasion, and/or metastasis. The ectodomain of this protein mediates bacterial adhesion to mammalian cells and the cytoplasmic domain is required for internalization. Identified transcript variants arise from mutation at consensus splice sites. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9R0T4
NCBI GenInfo Identifier:	148921649
NCBI Gene ID:	999
NCBI Accession:	AAI46663. 1
UniProt Secondary Accession:	Q9R0T4,Q13799, Q14216, Q15855, Q16194, Q4PJ14, A8K1U7
UniProt Related Accession:	P12830
Molecular Weight:
NCBI Full Name:	CDH1 protein
NCBI Synonym Full Names:	cadherin 1, type 1, E-cadherin (epithelial)
NCBI Official Symbol:	CDH1
NCBI Official Synonym Symbols:	UVO; CDHE; ECAD; LCAM; Arc-1; CD324
NCBI Protein Information:	cadherin-1; CAM 120/80; E-Cadherin; uvomorulin; cell-CAM 120/80; epithelial cadherin; cadherin 1, E-cadherin (epithelial); calcium-dependent adhesion protein, epithelial
UniProt Protein Name:	Cadherin-1
UniProt Synonym Protein Names:	CAM 120/80; Epithelial cadherin; E-cadherin; Uvomorulin
Protein Family:	Cadherin
UniProt Gene Name:	CDH1
UniProt Entry Name:	CADH1_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	52873 58569	55721	55693
1000	22723 25225	23974	23946
500	11953 10111	11032	11004
250	5135 5453	5294	5266
125	2742 2474	2608	2580
62.5	1399 1223	1311	1283
31.25	664 684	674	646
0	27 29	28	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat E-Cad 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)	108.88	263.46	802.73	102.98	258.06	794.50
Standard deviation	10.08	22.87	57.48	10.97	24.34	65.23
C V (%)	9.26	8.68	7.16	10.65	9.43	8.21

Recovery

The recovery of Rat E-Cad 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)	98-113	105
EDTA plasma (n=5)	97-109	102
Cell culture media (n=5)	96-111	102

Linearity

Samples were spiked with high concentrations of Rat E-Cad 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 (%)	102-119	94-107	85-99
1:2	Average (%)	109	101	92
1:4	Range (%)	103-117	92-104	88-103
1:4	Average (%)	109	97	94
1:8	Range (%)	101-116	102-115	91-103
1:8	Average (%)	109	109	96
1:16	Range (%)	99-116	99-111	96-110
1:16	Average (%)	108	104	102

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.