Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.78-50 ng/mL
Sensitivity:	0.47 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human CUBN in samples. No significant cross-reactivity or interference between Human CUBN and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human CUBN. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CUBN 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 CUBN, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human CUBN. The concentration of Human CUBN in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	CUBN: Cotransporter which plays a role in lipoprotein, vitamin and iron metabolism, by facilitating their uptake. Binds to ALB, MB, Kappa and lambda-light chains, TF, hemoglobin, GC, SCGB1A1, APOA1, high density lipoprotein, and the GIF-cobalamin complex. The binding of all ligands requires calcium. Serves as important transporter in several absorptive epithelia, including intestine, renal proximal tubules and embryonic yolk sac. Interaction with LRP2 mediates its trafficking throughout vesicles and facilitates the uptake of specific ligands like GC, hemoglobin, ALB, TF and SCGB1A1. Interaction with AMN controls its trafficking to the plasma membrane and facilitates endocytosis of ligands. May play an important role in the development of the peri-implantation embryo through internalization of APOA1 and cholesterol. Binds to LGALS3 at the maternal-fetal interface. Defects in CUBN are a cause of recessive hereditary megaloblastic anemia 1 (RH-MGA1); also known as MGA1 Norwegian type or Imerslund-Grasbeck syndrome (I-GS). RH-MGA1 is due to selective malabsorption of vitamin B12. Defects in vitamin B12 absorption lead to impaired function of thymidine synthase. As a consequence DNA synthesis is interrupted. Rapidly dividing cells involved in erythropoiesis are particularly affected.
UniProt Protein Details:	Chromosomal Location of Human Ortholog: 10p12. 31 Cellular Component: Golgi apparatus; lysosomal lumen; lysosomal membrane; endoplasmic reticulum; brush border membrane; coated pit; cytosol; extrinsic to external side of plasma membrane; membrane; endocytic vesicle; apical plasma membrane; plasma membrane; endosome membrane Molecular Function:protein binding; protein homodimerization activity; transporter activity; receptor activity; calcium ion binding; cobalamin binding Biological Process: steroid metabolic process; receptor-mediated endocytosis; cholesterol metabolic process; vitamin metabolic process; cobalamin metabolic process; lipoprotein transport; cobalamin transport; tissue homeostasis; lipoprotein metabolic process; water-soluble vitamin metabolic process; vitamin D metabolic process Disease: Megaloblastic Anemia 1
NCBI Summary:	Cubilin (CUBN) acts as a receptor for intrinsic factor-vitamin B12 complexes. The role of receptor is supported by the presence of 27 CUB domains. Cubulin is located within the epithelium of intestine and kidney. Mutations in CUBN may play a role in autosomal recessive megaloblastic anemia. [provided by RefSeq, Jul 2008]
UniProt Code:	O60494
NCBI GenInfo Identifier:	126091152
NCBI Gene ID:	8029
NCBI Accession:	NP_001072. 2
UniProt Secondary Accession:	O60494,Q5VTA6, Q96RU9, B0YIZ4,
UniProt Related Accession:	O60494
Molecular Weight:	398,736 Da
NCBI Full Name:	cubilin
NCBI Synonym Full Names:	cubilin (intrinsic factor-cobalamin receptor)
NCBI Official Symbol:	CUBN
NCBI Official Synonym Symbols:	IFCR; MGA1; gp280
NCBI Protein Information:	cubilin; 460 kDa receptor; cubilin precursor variant 1; cubilin precursor variant 2; intestinal intrinsic factor receptor; intrinsic factor-vitamin B12 receptor
UniProt Protein Name:	Cubilin
UniProt Synonym Protein Names:	460 kDa receptor; Intestinal intrinsic factor receptor; Intrinsic factor-cobalamin receptor; Intrinsic factor-vitamin B12 receptor
Protein Family:	Cubilin
UniProt Gene Name:	CUBN
UniProt Entry Name:	CUBN_HUMAN

As the OD 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 (ng/mL)	O.D	Average	Corrected
50	2.483 2.539	2.511	2.442
25	1.588 1.596	1.592	1.523
12.5	0.968 0.936	0.952	0.883
6.25	0.451 0.465	0.458	0.389
3.13	0.284 0.282	0.283	0.214
1.57	0.188 0.16	0.174	0.105
0.78	0.113 0.131	0.122	0.053
0	0.064 0.074	0.069	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CUBN 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 (ng/mL)	2.35	5.20	17.41	2.41	4.70	18.84
Standard deviation	0.16	0.28	0.87	0.16	0.27	0.69
C V (%)	6.81	5.38	5.00	6.64	5.74	3.66

Recovery

The recovery of Human CUBN 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)	93-106	101
EDTA plasma (n=5)	88-100	93
Cell culture media (n=5)	95-108	100

Linearity

Samples were spiked with high concentrations of Human CUBN 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 (%)	87-101	85-97	92-109
1:2	Average (%)	92	92	100
1:4	Range (%)	90-103	79-93	88-102
1:4	Average (%)	97	85	95
1:8	Range (%)	92-104	86-101	83-97
1:8	Average (%)	97	93	90
1:16	Range (%)	93-105	84-97	83-97
1:16	Average (%)	98	90	88

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.