Human CD44 ELISA Kit

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SKU:
HUFI00472
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human CD44 ELISA Kit - Information

The ELISA Genie CD44 ELISA Kit can assay for CD44 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our CD44 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound CD44 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human CD44 ELISA Kit - Data

Description

Receptor for hyaluronic acid (HA). Mediates cell-cell and cell-matrix interactions through its affinity for HA, and possibly also through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Adhesion with HA plays an important role in cell migration, tumor growth and progression. In cancer cells, may play an important role in invadopodia formation. Also involved in lymphocyte activation, recirculation and homing, and in hematopoiesis. Altered expression or dysfunction causes numerous pathogenic phenotypes. Great protein heterogeneity due to numerous alternative splicing and post-translational modification events. Receptor for LGALS9; the interaction enhances binding of SMAD3 to the FOXP3 promoter, leading to up-regulation of FOXP3 expression and increased induced regulatory T (iTreg) cell stability and suppressive function.

Post-Translational Modification

Proteolytically cleaved in the extracellular matrix by specific proteinases (possibly MMPs) in several cell lines and tumors. N- and O-glycosylated. O-glycosylation contains more-or-less-sulfated chondroitin sulfate glycans, whose number may affect the accessibility of specific proteinases to their cleavage site(s). It is uncertain if O-glycosylation occurs on Thr-637 or Thr-638. Phosphorylated; activation of PKC results in the dephosphorylation of Ser-706 (constitutive phosphorylation site), and the phosphorylation of Ser-672.

Uniprot ID P16070
Alias

CD44/ECMR-III/HCAM/HCELL/LHR/MDU2/MDU3/MIC4/MUTCH-I/Pgp1/CD44R/CDw44/CSPG8/Epican/HUTCH-I/Hyaluronate receptor/IN/LHR/MC56/PGP-1/PGP-I/Phagocytic glycoprotein 1/Phagocytic glycoprotein I/IN

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of CD44 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.156-10ng/ml

Sensitivity

< 0.094ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of CD44 and the recovery rates were calculated by comparing the measured value to the expected amount of CD44 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 88-95 91
EDTA plasma(n=5) 86-103 92
UFH plasma(n=5) 85-104 96
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CD44 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 86-100% 88-96% 92-105% 92-101%
EDTA plasma(n=5) 83-99% 82-91% 89-98% 84-101%
UFH plasma(n=5) 80-99% 81-100% 84-93% 84-97%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human CD44 ELISA Kit Protocol

The below protocol is a sample protocol for Human CD44 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human CD44 present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

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.

Human CD44 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human CD44 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Human CD44 ELISA Kit Protein Information

UniProt Protein Function:CD44: Receptor for hyaluronic acid (HA). Mediates cell-cell and cell-matrix interactions through its affinity for HA, and possibly also through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Adhesion with HA plays an important role in cell migration, tumor growth and progression. Also involved in lymphocyte activation, recirculation and homing, and in hematopoiesis. Altered expression or dysfunction causes numerous pathogenic phenotypes. Great protein heterogeneity due to numerous alternative splicing and post-translational modification events. Interacts with PKN2. Interacts with HA, as well as other glycosaminoglycans, collagen, laminin, and fibronectin via its N-terminal segment. Interacts with ANK, the ERM proteins (VIL2, RDX and MSN), and NF2 via its C-terminal segment. Isoform 10 (epithelial isoform) is expressed by cells of epithelium and highly expressed by carcinomas. Expression is repressed in neuroblastoma cells. 19 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell adhesion; Motility/polarity/chemotaxis; Membrane protein, integral; Receptor, misc.

Chromosomal Location of Human Ortholog: 11p13

Cellular Component: Golgi apparatus; focal adhesion; cell surface; basolateral plasma membrane; integral to plasma membrane; cytoplasm; plasma membrane; external side of plasma membrane

Molecular Function:collagen binding; hematopoietin/interferon-class (D200-domain) cytokine receptor activity; protein binding; hyaluronic acid binding; hyalurononglucosaminidase activity

Biological Process: extracellular matrix organization and biogenesis; Wnt receptor signaling pathway; positive regulation of heterotypic cell-cell adhesion; glycosaminoglycan metabolic process; cytokine and chemokine mediated signaling pathway; cell-matrix adhesion; pathogenesis; negative regulation of caspase activity; positive regulation of peptidyl-serine phosphorylation; hyaluronan catabolic process; extracellular matrix disassembly; negative regulation of DNA damage response, signal transduction by p53 class mediator; cell-cell adhesion; positive regulation of peptidyl-tyrosine phosphorylation; ureteric bud branching; cartilage development; carbohydrate metabolic process; blood coagulation; leukocyte migration; healing during inflammatory response; hyaluronan metabolic process; negative regulation of apoptosis

Disease: Blood Group, Indian System

NCBI Summary:The protein encoded by this gene is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid (HA) and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs). This protein participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. Transcripts for this gene undergo complex alternative splicing that results in many functionally distinct isoforms, however, the full length nature of some of these variants has not been determined. Alternative splicing is the basis for the structural and functional diversity of this protein, and may be related to tumor metastasis. [provided by RefSeq, Jul 2008]
UniProt Code:P16070
NCBI GenInfo Identifier:308153615
NCBI Gene ID:960
NCBI Accession:P16070.3
UniProt Secondary Accession:P16070,O95370, P22511, Q04858, Q13419, Q13957, Q13958 Q13959, A5YRN9, B6EAT9, D3DR12, D3DR13,
UniProt Related Accession:P16070
Molecular Weight:81,538 Da
NCBI Full Name:CD44 antigen
NCBI Synonym Full Names:CD44 molecule (Indian blood group)
NCBI Official Symbol:CD44  
NCBI Official Synonym Symbols:IN; LHR; MC56; MDU2; MDU3; MIC4; Pgp1; CDW44; CSPG8; HCELL; HUTCH-I; ECMR-III  
NCBI Protein Information:CD44 antigen; epican; Hermes antigen; hyaluronate receptor; phagocytic glycoprotein 1; heparan sulfate proteoglycan; cell surface glycoprotein CD44; extracellular matrix receptor III; chondroitin sulfate proteoglycan 8; GP90 lymphocyte homing/adhesion receptor; hematopoietic cell E- and L-selectin ligand; homing function and Indian blood group system
UniProt Protein Name:CD44 antigen
UniProt Synonym Protein Names:CDw44; Epican; Extracellular matrix receptor III; ECMR-III; GP90 lymphocyte homing/adhesion receptor; HUTCH-I; Heparan sulfate proteoglycan; Hermes antigen; Hyaluronate receptor; Phagocytic glycoprotein 1; PGP-1; Phagocytic glycoprotein I
Protein Family:CD44 antigen
UniProt Gene Name:CD44  
UniProt Entry Name:CD44_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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