Mouse CD209a ELISA Kit (MOFI00591)

Product Name:	Mouse CD209a ELISA Kit
Product Code:	MOFI00591
Size:	96 Assays
Alias:	CD209a, CD209, CLEC4L, DC-SIGN1, Cire, CD209 antigendendritic cell-specific intracellular adhesion molecules, ICAM-3 grabbingnon-integrin, CD209 molecule, CDSIGNHIV gpl20-binding protein, CLEC4LC-type lectin domain family 4 member L, DC-SIGN1C-type lectin domain family 4, member L, DC-SIGNMGC129965, Dendritic cell-specific ICAM-3-grabbing non-integrin 1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CD209a concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse CD209a and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse CD209a in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-102	96
EDTA plasma(n=5)	85-105	94
UFH plasma(n=5)	92-104	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse CD209a 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)	86-105%	87-95%	95-102%
EDTA plasma(n=5)	85-99%	88-99%	82-97%
UFH plasma(n=5)	83-96%	84-98%	82-100%

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

Q91ZX1

UniProt Protein Function:	CD209: Pathogen-recognition receptor expressed on the surface of immature dendritic cells (DCs) and involved in initiation of primary immune response. Thought to mediate the endocytosis of pathogens which are subsequently degraded in lysosomal compartments. The receptor returns to the cell membrane surface and the pathogen-derived antigens are presented to resting T-cells via MHC class II proteins to initiate the adaptive immune response. Probably recognizes in a calcium-dependent manner high mannose N-linked oligosaccharides in a variety of pathogen antigens, including HIV-1 gp120, HIV-2 gp120, SIV gp120, ebolavirus glycoproteins, cytomegalovirus gB, HCV E2, dengue virus gE, Leishmania pifanoi LPG, Lewis-x antigen in Helicobacter pylori LPS, mannose in Klebsiella pneumonae LPS, di-mannose and tri- mannose in Mycobacterium tuberculosis ManLAM and Lewis-x antigen in Schistosoma mansoni SEA. Homotetramer. Binds to many viral surface glycoproteins such as HIV-1 gp120, HIV-2 gp120, SIV gp120, ebolavirus envelope glycoproteins, cytomegalovirus gB, HCV E2 and dengue virus major envelope protein E. Predominantly expressed in dendritic cells and in DC-residing tissues. Also found in placental macrophages, endothelial cells of placental vascular channels, peripheral blood mononuclear cells, and THP-1 monocytes. 13 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral Chromosomal Location of Human Ortholog: 8\|8 A1.1 Cellular Component: cell surface; integral to membrane Molecular Function:calcium-dependent protein binding; carbohydrate binding; mannose binding; viral receptor activity Biological Process: regulation of blood coagulation; regulation of gene expression; regulation of T cell proliferation
UniProt Code:	Q91ZX1
NCBI GenInfo Identifier:	18875404
NCBI Gene ID:	170786
NCBI Accession:	NP_573501.1
UniProt Secondary Accession:	Q91ZX1,Q3TAT9, Q91ZW5,
UniProt Related Accession:	Q91ZX1
Molecular Weight:	24,055 Da
NCBI Full Name:	CD209 antigen-like protein A
NCBI Synonym Full Names:	CD209a antigen
NCBI Official Symbol:	Cd209a
NCBI Official Synonym Symbols:	CIRE; CD209; CDSIGN; Dcsign; SIGNR5; DC-SIGN; SIGN-R1; DC-SIGN1
NCBI Protein Information:	CD209 antigen-like protein A
UniProt Protein Name:	CD209 antigen-like protein A
UniProt Synonym Protein Names:	Dendritic cell-specific ICAM-3-grabbing non-integrin; DC-SIGN; CD_antigen: CD209
Protein Family:	CD209 antigen
UniProt Gene Name:	Cd209a

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

Step	Procedure
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.