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ELISAGenie/Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule) ELISA Kit (MOES01734)/Activated Leukocyte Cell Adhesion Molecule) ELISA Kit (MOES01734/MOES01734

Description
Additional Information

Description

Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule) ELISA Kit

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 Mouse ALCAM . 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 Mouse ALCAM 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 Mouse ALCAM, 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 Mouse ALCAM. The concentration of Mouse ALCAM in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay type	Sandwich
Format	96T
Assay time	4.5h
Reactivity	Mouse
Detection Method	Colormetric
Detection Range	0.16-10 ng/mL
Sensitivity	0.10 ng/mL
Sample Volume Required Per Well	100uL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes Mouse ALCAMin samples. No significant cross-reactivity or interference between Mouse ALCAMand analogues was observed.

Typical Data

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
10	2.4272.453	2.44	2.359
5	1.6771.681	1.679	1.598
2.5	0.9580.954	0.956	0.875
1.25	0.4770.487	0.482	0.401
0.63	0.2630.239	0.251	0.17
0.32	0.1860.178	0.182	0.101
0.16	0.1290.137	0.133	0.052
0	0.0760.086	0.081	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse ALCAMwere 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)	0.46	1.04	3.83	0.44	1.11	3.80
Standard deviation	0.03	0.04	0.12	0.03	0.06	0.12
C V (%)	6.52	3.85	3.13	6.82	5.41	3.16

Recovery

The recovery of Mouse ALCAMspiked 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)	87-102	93
EDTA plasma (n=5)	89-101	96
Cell culture media (n=5)	92-105	98

Linearity

Samples were spiked with high concentrations of Mouse ALCAMand 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 (%)	97-113	92-106	86-99
1:2	Average (%)	105	97	91
1:4	Range (%)	88-102	80-92	82-96
1:4	Average (%)	94	86	88
1:8	Range (%)	95-108	87-99	86-99
1:8	Average (%)	100	93	92
1:16	Range (%)	93-103	85-99	86-100
1:16	Average (%)	98	90	93

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 ELISA Plate(Dismountable)	8 wells X12 strips	-20"C, 6 months
Reference Standard	2 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 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 (25X)	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

Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule) ELISA Kit (MOES01734) 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 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.

11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.

12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule) ELISA Kit (MOES01734) Protein Information

UniProt Protein Function:	ALCAM: Cell adhesion molecule that binds to CD6. Involved in neurite extension by neurons via heterophilic and homophilic interactions. May play a role in the binding of T- and B-cells to activated leukocytes, as well as in interactions between cells of the nervous system. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell adhesion Chromosomal Location of Human Ortholog: 3q13.1 Cellular Component: focal adhesion; cell soma; axon; integral to membrane; external side of plasma membrane Molecular Function:protein binding; receptor binding Biological Process: axon guidance; motor axon guidance; signal transduction; cell adhesion
NCBI Summary:	This gene encodes activated leukocyte cell adhesion molecule (ALCAM), also known as CD166 (cluster of differentiation 166), which is a member of a subfamily of immunoglobulin receptors with five immunoglobulin-like domains (VVC2C2C2) in the extracellular domain. This protein binds to T-cell differentiation antigene CD6, and is implicated in the processes of cell adhesion and migration. Multiple alternatively spliced transcript variants encoding different isoforms have been found. [provided by RefSeq, Aug 2011]
UniProt Code:	Q61490
NCBI GenInfo Identifier:	343168770
NCBI Gene ID:	214
NCBI Accession:	NP_001230209.1
UniProt Secondary Accession:	Q61490,O60892, Q1HGM8, Q1HGM9, Q6PEY4, Q6ZS95, B2RNS3 B4DTU0,
UniProt Related Accession:	Q13740
Molecular Weight:	65102
NCBI Full Name:	CD166 antigen isoform 2
NCBI Synonym Full Names:	activated leukocyte cell adhesion molecule
NCBI Official Symbol:	ALCAM
NCBI Official Synonym Symbols:	MEMD; CD166
NCBI Protein Information:	CD166 antigen
UniProt Protein Name:	CD166 antigen
UniProt Synonym Protein Names:	Activated leukocyte cell adhesion molecule; CD_antigen: CD166
Protein Family:	CD166 antigen
UniProt Gene Name:	ALCAM
UniProt Entry Name:	CD166_HUMAN

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Additional Information

Reactivity:	Mouse
ELISA Type:	Sandwich