Human CD209 antigen (CD209) ELISA Kit (HUEB1985)

Product Name:	Human CD209 antigen (CD209) ELISA Kit
SKU:	HUEB1985
Size:	96T
Target:	Human CD209 antigen (CD209)
Synonyms:	C-type lectin domain family 4 member L, Dendritic cell-specific ICAM-3-grabbing non-integrin 1, DC-SIGN, CD209, CLEC4L
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/ml
Sensitivity:	40.9pg/mL

Intra CV:

5.3%

Inter CV:

7.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	102-102%	106-118%	104-114%	100-108%
EDTA Plasma(N=5)	82-92%	103-116%	102-112%	113-113%
Heparin Plasma(N=5)	90-103%	107-119%	111-121%	90-103%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	87	81-93
Plasma	89	83-95

Function:

(Microbial infection) Acts as an attachment receptor for HIV-1 and HIV-2 (PubMed:11799126, PubMed:12502850, PubMed:1518869). Acts as an attachment receptor for ebolavirus (PubMed:12502850, PubMed:12504546). Acts as an attachment receptor for cytomegalovirus (PubMed:12433371, PubMed:22496863). Acts as an attachment receptor for HCV (PubMed:15371595, PubMed:16816373). Acts as an attachment receptor for dengue virus (PubMed:12682107). Acts as an attachment receptor for measles virus (PubMed:16537615). Acts as an attachment receptor for herpes simplex virus 1 (PubMed:18796707). Acts as an attachment receptor for Influenzavirus A (PubMed:21191006). Acts as an attachment receptor for SARS coronavirus (PubMed:15140961). Acts as an attachment receptor for Japanese encephalitis virus (PubMed:24623090). Acts as an attachment receptor for Lassa virus (PubMed:23966408). Acts as an attachment receptor for marburg virusn (PubMed:15479853). Acts as an attachment receptor for Respiratory syncytial virus (PubMed:22090124). Acts as an attachment receptor for Rift valley fever virus and uukuniemi virus (PubMed:21767814). Acts as an attachment receptor for west-nile virus (PubMed:16415006). Probably recognizes in a calcium-dependent manner high mannose N-linked oligosaccharides in a variety of bacterial pathogen antigens, including 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 (PubMed:16379498). Recognition of M.tuberculosis by dendritic cells occurs partially via this molecule (PubMed:16092920, PubMed:21203928).

Uniprot:	Q9NNX6
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human CD209 antigen
Sub Unit:	(Microbial infection) Interacts with whole M.bovis cells in a Ca(2+)-dependent and independent manner; in vitro experiments suggest it interacts with CH60.1 (groL1), DnaK, GADPH (gap) and LrpG (PubMed:21203928).
Subcellular Location:	Isoform 12 Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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: 19p13 Cellular Component: cell surface; membrane; cytoplasm; plasma membrane; integral to membrane Molecular Function:mannose binding; protein binding; peptide antigen binding; metal ion binding; virion binding; carbohydrate binding Biological Process: cell-cell recognition; heterophilic cell adhesion; antigen processing and presentation; leukocyte adhesion; virus-host interaction; regulation of T cell proliferation; innate immune response; endocytosis; peptide antigen transport; viral genome replication; virion attachment to host cell surface receptor Disease: Mycobacterium Tuberculosis, Susceptibility To; Human Immunodeficiency Virus Type 1, Susceptibility To
NCBI Summary:	This gene encodes a transmembrane receptor and is often referred to as DC-SIGN because of its expression on the surface of dendritic cells and macrophages. The encoded protein is involved in the innate immune system and recognizes numerous evolutionarily divergent pathogens ranging from parasites to viruses with a large impact on public health. The protein is organized into three distinct domains: an N-terminal transmembrane domain, a tandem-repeat neck domain and C-type lectin carbohydrate recognition domain. The extracellular region consisting of the C-type lectin and neck domains has a dual function as a pathogen recognition receptor and a cell adhesion receptor by binding carbohydrate ligands on the surface of microbes and endogenous cells. The neck region is important for homo-oligomerization which allows the receptor to bind multivalent ligands with high avidity. Variations in the number of 23 amino acid repeats in the neck domain of this protein are rare but have a significant impact on ligand binding ability. This gene is closely related in terms of both sequence and function to a neighboring gene (GeneID 10332; often referred to as L-SIGN). DC-SIGN and L-SIGN differ in their ligand-binding properties and distribution. Alternative splicing results in multiple variants.[provided by RefSeq, Feb 2009]
UniProt Code:	Q9NNX6
NCBI GenInfo Identifier:	46396012
NCBI Gene ID:	30835
NCBI Accession:	Q9NNX6.1
UniProt Secondary Accession:	Q9NNX6,Q2TB19, Q96QP7, Q96QP8, Q96QP9, Q96QQ0, Q96QQ1 Q96QQ2, Q96QQ3, A8KAM4, A8MVQ9, G5E9C4,
UniProt Related Accession:	Q9NNX6
Molecular Weight:	33,874 Da
NCBI Full Name:	CD209 antigen
NCBI Synonym Full Names:	CD209 molecule
NCBI Official Symbol:	CD209
NCBI Official Synonym Symbols:	CDSIGN; CLEC4L; DC-SIGN; DC-SIGN1
NCBI Protein Information:	CD209 antigen; HIV gpl20-binding protein; C-type lectin domain family 4 member L; C-type lectin domain family 4, member L; dendritic cell-specific ICAM-3-grabbing non-integrin 1; dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin
UniProt Protein Name:	CD209 antigen
UniProt Synonym Protein Names:	C-type lectin domain family 4 member L; Dendritic cell-specific ICAM-3-grabbing non-integrin 1; DC-SIGN; DC-SIGN1; CD_antigen: CD209
Protein Family:	CD209 antigen
UniProt Gene Name:	CD209
UniProt Entry Name:	CD209_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.