Human cluster of differentiation 63,CD63 ELISA Kit

Instructions
Code CSB-E14107h
Size 96T,5×96T,10×96T
Trial Size 24T ELISA kits trial application
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Product Details

Description

The Human cluster of differentiation 63 (CD63) ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of human CD63 in many biological fluids, including serum, plasma, and tissue homogenates. This assay employs the Sandwich-ELISA technique, in which CD63 in the samples or standards is sandwiched between pre-coated an anti-human CD63 and Biotin-labeled CD63 antibody. The Ag-Ab-Ag immune complex is labeled with HRP-avidin and then develops a color reaction after the addition of the TMB substrate. The intensity of the color is positively correlated to the amount of CD63 in the sample. The kit has high sensitivity, excellent specificity, good linearity, precision low than 10%, high recovery, good lot-to-lot consistency. Get access to more details from the product instructions.

CD63 is a type II cellular membrane protein belonging to the tetraspanin superfamily. Mounting studies have revealed that CD63 incorporates into HIV-1 virions and colocalizes with HIV-1 Env and Gag proteins in HIV-1 producing cells. CD63 associates with tetraspanin-enriched microdomains (TEMs), which have been demonstrated to serve as gateways for HIV-1 budding at the plasma membrane. A recent study has shown that CD63 inhibits trafficking of the chemokine receptor CXCR4 to the cell surface, which affects HIV-1 entry into T lymphocytes. It also plays a positive role in the HIV-1 replication cycle.

Target Name CD63 molecule
Alternative Names Lysosomal associated membrane protein 3 ELISA Kit; CD 63 ELISA Kit; CD63 ELISA Kit; CD63 antigen (melanoma 1 antigen) ELISA Kit; CD63 antigen ELISA Kit; CD63 antigen melanoma 1 antigen ELISA Kit; CD63 molecule ELISA Kit; CD63_HUMAN ELISA Kit; gp55 ELISA Kit; Granulophysin ELISA Kit; LAMP 3 ELISA Kit; LAMP-3 ELISA Kit; LAMP3 ELISA Kit; LIMP ELISA Kit; Lysosomal-associated membrane protein 3 ELISA Kit; Lysosome associated membrane glycoprotein 3 ELISA Kit; Lysosome-associated membrane glycoprotein 3 ELISA Kit; Mast cell antigen AD1 ELISA Kit; ME491 ELISA Kit; Melanoma 1 antigen ELISA Kit; Melanoma associated antigen ME491 ELISA Kit; Melanoma associated antigen MLA1 ELISA Kit; Melanoma-associated antigen ME491 ELISA Kit; MGC72893 ELISA Kit; MLA 1 ELISA Kit; MLA1 ELISA Kit; NGA ELISA Kit; Ocular melanoma associated antigen ELISA Kit; Ocular melanoma-associated antigen ELISA Kit; OMA81H ELISA Kit; PTLGP40 ELISA Kit; Tetraspanin 30 ELISA Kit; Tetraspanin-30 ELISA Kit; Tspan 30 ELISA Kit; Tspan-30 ELISA Kit; TSPAN30 ELISA Kit
Abbreviation CD63
Uniprot No. P08962
Species Homo sapiens (Human)
Sample Types serum, plasma, tissue homogenates
Detection Range 0.625 ng/mL-40 ng/mL
Sensitivity 0.156 ng/mL
Assay Time 1-5h
Sample Volume 50-100ul
Detection Wavelength 450 nm
Research Area Immunology
Assay Principle quantitative
Measurement Sandwich
Precision
Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision (Precision between assays): CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
Linearity
To assess the linearity of the assay, samples were spiked with high concentrations of human CD63 in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.
 SampleSerum(n=4)
1:1Average %96
Range %92-104
1:2Average %87
Range %81-94
1:4Average %98
Range %92-106
1:8Average %96
Range %92-103
Recovery
The recovery of human CD63 spiked to levels throughout the range of the assay in various matrices was evaluated. Samples were diluted prior to assay as directed in the Sample Preparation section.
Sample TypeAverage % RecoveryRange
Serum (n=5) 10498-107
EDTA plasma (n=4)9389-103
Typical Data
These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed.
ng/mlOD1OD2AverageCorrected
402.639 2.538 2.589 2.428
201.947 1.846 1.897 1.736
101.301 1.312 1.307 1.146
50.776 0.765 0.771 0.610
2.50.473 0.462 0.468 0.307
1.250.336 0.347 0.342 0.181
0.6250.246 0.240 0.243 0.082
00.162 0.160 0.161  
Materials provided
  • A micro ELISA plate --- The 96-well plate has been pre-coated with an anti-human CD63 antibody. This dismountable microplate can be divided into 12 x 8 strip plates.
  • Two vials lyophilized standard ---Dilute a bottle of the standard at dilution series, read the OD values, and then draw a standard curve.
  • One vial Biotin-labeled CD63 antibody (100 x concentrate) (120 μl/bottle) ---Act as the detection antibody.
  • One vial HRP-avidin (100 x concentrate) (120 μl/bottle) ---Bind to the detection antibody and react with the TMB substrate to make the solution chromogenic.
  • One vial Biotin-antibody Diluent (15 ml/bottle) ---Dilute the Biotin-antibody.
  • One vial HRP-avidin Diluent (15 ml/bottle) ---Dilute the HRP-avidin solution.
  • One vial Sample Diluent (50 ml/bottle)---Dilute the sample to an appropriate concentration.
  • One vial Wash Buffer (25 x concentrate) (20 ml/bottle) ---Wash away unbound or free substances.
  • One vial TMB Substrate (10 ml/bottle) ---Act as the chromogenic agent. TMB interacts with HRP, eliciting the solution turns blue.
  • One vial Stop Solution (10 ml/bottle) ---Stop the color reaction. The solution color immediately turns from blue to yellow.
  • Four Adhesive Strips (For 96 wells) --- Cover the microplate when incubation.
  • An instruction manual
Materials not provided
  • A microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
  • An incubator can provide stable incubation conditions up to 37°C±5°C.
  • Centrifuge
  • Vortex
  • Squirt bottle, manifold dispenser, or automated microplate washer
  • Absorbent paper for blotting the microtiter plate
  • 50-300ul multi-channel micropipette
  • Pipette tips
  • Single-channel micropipette with different ranges
  • 100ml and 500ml graduated cylinders
  • Deionized or distilled water
  • Timer
  • Test tubes for dilution
Troubleshooting
and FAQs
ELISA kit FAQs
Storage Store at 2-8°C. Please refer to protocol.
Lead Time 3-5 working days

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 Q&A
Q:

Please let me ask one question.
Can this kit detect only human CD63? Or is there some cross-reactivity with CD63 of other species(mouse, rat)?

A:
Thanks for your inquiry.
#CSB-E14107h,Human cluster of differentiation 63,CD63 ELISA Kit
This kit is developed based on matrix of human and it is directed to human samples.Human CD63 is a different protein from rat/mouse CD63.The sequence of human CD63 and matrix are different from that of rat/mouse CD63.
And we did not do analysis of cross-reactivity on human DC63 and rat/mouse CD63.If you are going to test rat/mouse sample,the kit of respective species is advised.
If further questions,pls do ont hesitate to cotnact us.
Q:

I want to lyse my sample and I will use RIPA buffer for lysis.
Does RIPA buffer has any effect to ELISA result?

A:
Thanks for your inquiry.
Usually RIPA would contains components SDS、Triton X-100、NP-40,ect.Pls kindly be noted that it must not contain SDS which would destroy the hydrogen bonds of protein macromolecules and causes denaturation of proteins .And Triton X-100、NP-40 should be controlled in the concentration range (usually not over 1% which was not validated ever).
For RIPA buffer,we did not ever use this kind of treatment method.You can only try by yourself and do pre-testing.On our side,we use the freeze-thaw method on PBS and we suggest you do the same.
Regarding EGTA and EDTA,they can both be used as chelat to eliminate the interfere of metal ions.They can affect the results from BCA method,but the affect to elisa method is not clear so far.
For 1 mM Na3,we think it might be typed wrong,which should be NaN3.Rigt?NaN3 can not be contained in the sample.Or it will be easily to get false negative result.
For the other components in your RIPA Buffer,we are not clear about their affects to the results.We suggest you use the suggested method.If you have prepared well your sample,you can go on try and do pre-testing.If the sample is not prepared well yet,pls use the suggested method.
For further questions,pls contact us.
Q:

I have a one question. Is there any difference in composition between biotin ab. diluent and HRP-avidin diluent?

A:
Thanks for your inquiry.
biotin ab. diluent and HRP-avidin diluent are 2 different components
Biotin-antibody Diluent is for dilutions of Biotin-antibody
HRP-avidin Diluent is for dilutions of HRP-avidin.Pls let me know if you have any further questions. Thank you.
Q:

If I use biotin-antibody diluent for HRP-avidin, then Is there any important problem?!

A:
May I know why you would use biotin-antibody diluent for HRP-avidin?Usually we do not suggest that.

Target Background

Function
(From Uniprot)
Functions as cell surface receptor for TIMP1 and plays a role in the activation of cellular signaling cascades. Plays a role in the activation of ITGB1 and integrin signaling, leading to the activation of AKT, FAK/PTK2 and MAP kinases. Promotes cell survival, reorganization of the actin cytoskeleton, cell adhesion, spreading and migration, via its role in the activation of AKT and FAK/PTK2. Plays a role in VEGFA signaling via its role in regulating the internalization of KDR/VEGFR2. Plays a role in intracellular vesicular transport processes, and is required for normal trafficking of the PMEL luminal domain that is essential for the development and maturation of melanocytes. Plays a role in the adhesion of leukocytes onto endothelial cells via its role in the regulation of SELP trafficking. May play a role in mast cell degranulation in response to Ms4a2/FceRI stimulation, but not in mast cell degranulation in response to other stimuli.
Gene References into Functions
  1. Signaling focal contacts via CD63 were identified to facilitate cell adhesion, migration and mediate extracellular matrix-physical cues to modulate hematopoietic stem cells and progenitor cells function. PMID: 28566689
  2. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. PMID: 29208635
  3. our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking. PMID: 27578500
  4. CD63 and exosome expression is altered in scleroderma dermal fibroblasts PMID: 27443953
  5. CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling. PMID: 27974566
  6. TIMP1 signaling via CD63 leads to activation of hepatic stellate cells, which create an environment in the liver that increases its susceptibility to pancreatic tumor cells. PMID: 27506299
  7. Concentrations of Cd63 were elevated in gingival crevicular fluid of patients with pre-eclampsia. PMID: 26988336
  8. CD163(+) TAMs in oral premalignant lesions coexpress CD163 and STAT1, suggesting that the TAMs in oral premalignant lesions possess an M1 phenotype in a Th1-dominated micromilieu. PMID: 26242181
  9. Our findings reveal that elevated levels of TIMP-1 impact on neutrophil homeostasis via signaling through CD63. PMID: 26001794
  10. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway PMID: 26213230
  11. It was shown that treatment of macrophages with anti-CD63 inhibits CCR5-mediated virus infection in a cell type-specific manner, but that no inhibition of CXCR4-tropic viruses occurs. PMID: 25658293
  12. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface. PMID: 25033048
  13. Diesel exhaust exposure during exercise induces platelet activation as illustrated by a dose-response increase in the release of CD62P and CD63. PMID: 25297946
  14. Data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity. PMID: 25354204
  15. CD63 tetraspanin is a negative driver of epithelial-to-mesenchymal transition in human melanoma cells. PMID: 24940653
  16. these results indicated that high glycosylation of CD63 by RPN2 is implicated in clinical outcomes in breast cancer patients PMID: 24884960
  17. Collectively, these findings indicate that CD63 may support Env-mediated fusion as well as a late (post-integration) step in the HIV-1 replication cycle. PMID: 24507450
  18. Data suggest that TIMP1 (tissue inhibitor of metalloproteinase-1) acts as chemoattractant for neural stem cells (NSC); TIMP1 enhances NSC adhesion, migration, and cytoskeletal reorganization; these effects are dependent on CD63/CD29 (integrin beta1). PMID: 24635319
  19. Timp1 is assembled in a supramolecular complex containing CD63 and beta1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway. PMID: 23522389
  20. Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation PMID: 18727065
  21. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin. PMID: 21803846
  22. shRNA knockdown in B lymphoblastoid cell line results to increased CD4(+) T-cell recognition PMID: 21660937
  23. decreased levels of CD63 were associated with distant and lymph node metastasis status and does play a direct role in human gastric carcinogenesis. PMID: 21521534
  24. C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-kappaB overstimulation. PMID: 21527913
  25. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step. PMID: 21315401
  26. ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin beta1 PMID: 21149578
  27. Surface expression of the novel CD63 variant is a distinguishing feature of mast cells, which are are stable, multiple-use cells capable of surviving and delivering several consecutive hits PMID: 20337613
  28. this work provides the first evidence of a TIMP-4/CD63 association in astrocytoma tumor cells PMID: 20693981
  29. CD63 expression results from only the anaphylactic degranulation form of histamine release. PMID: 20633031
  30. Serum sCD163 is a homogenous protein covering more than 94% of the CD163 ectodomain including the haptoglobin-hemoglobin -binding region. PMID: 19581020
  31. Results show that AP-3 is absolutely required for the delivery of CD63 to lysosomes via the trans-Golgi network. PMID: 11907283
  32. downregulation of CD63 antigen is associated with breast tumor progression PMID: 12579280
  33. possible role in HIV-1 infections specific for macrophages PMID: 12610138
  34. Post-translational modification of CD63 may be involved in the functional and morphological changes of MHC class II compartments that occur during dendritic cell maturation PMID: 12755696
  35. relationships between the expression levels of CD61, CD63, and PAC-1 on the platelet surface and the incidences of acute rejection and tubular necrosis as well as the recovery of graft function after renal transplantation PMID: 12826159
  36. Upon platelet interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells; cholesterol-rich raft aggregation was accompanied by concentration of c-Src and CD63 in these cell domains PMID: 12871315
  37. The study on CD63 included its chemistry eg. if it had and O-linked carbohydrate that was digested with O-glycanase. PMID: 12974720
  38. CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell. PMID: 14660791
  39. results suggest that CD9, CD63, CD81, and CD82 could play a role in modulating the interactions between immature DCs and their environment, slowing their migratory ability. However, only CD63 would intervene in the internalization of complex antigens PMID: 15130945
  40. Constitutive expression of CD63 may indicate that this factor does not play a direct role in thyroid carcinogenesis PMID: 15375577
  41. CD63 represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion. PMID: 15528334
  42. The linkage of CD63 with PI 4-kinase may result in the recruitment of this signaling enzyme to specific membrane locations in the platelet where it influences phosphoinositide-dependent signaling and platelet spreading. PMID: 15711748
  43. This study identifies a trafficking pathway from CD63-positive multivesicular bodies to the bacterial inclusion, a novel interaction that provides essential lipids necessary for maintenance of a productive intracellular infection. PMID: 16410552
  44. CD63 is recruited to already-budded Weibel-Palade bodies by an AP-3-dependent route PMID: 16683915
  45. CD63-syntenin-1 complex is abundant on the plasma membrane PMID: 16908530
  46. CD63 is a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex. PMID: 16917503
  47. Chronic urticaria serum-induced CD63 expression assay performed on atopic whole blood by means of tricolor flow cytometry could be the most useful tool for identification of a subset of patients with autoimmune chronic urticaria. PMID: 16918509
  48. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy. PMID: 17275019
  49. positive correlation between CD63 and erythrocyte sedimentation rate in rheumatoid arthritis PMID: 17279322
  50. Results suggest that CD63 can be a biomarker for predicting the prognosis in early stages of lung adenocarcinoma. PMID: 17350713

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Subcellular Location Cell membrane, Multi-pass membrane protein, Lysosome membrane, Multi-pass membrane protein, Late endosome membrane, Multi-pass membrane protein, Endosome, multivesicular body, Melanosome, Secreted, exosome, Cell surface
Protein Families Tetraspanin (TM4SF) family
Tissue Specificity Detected in platelets (at protein level). Dysplastic nevi, radial growth phase primary melanomas, hematopoietic cells, tissue macrophages.
Database Links

HGNC: 1692

OMIM: 155740

KEGG: hsa:967

STRING: 9606.ENSP00000257857

UniGene: Hs.445570

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