Human CD40 / TNFRSF5 ELISA Kit
- SKU:
- HUFI00269
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P25942
- Sensitivity:
- 11.719pg/ml
- Range:
- 19.531-1250pg/ml
- ELISA Type:
- Sandwich
- Synonyms:
- CD40, TNFRSF5, B cell surface antigen CD40, Bp50B cell-associated molecule, CD40 antigen, CD40 type II isoform, CD40L receptor, CDw40, MGC9013, p50, tumor necrosis factor receptor superfamily member 5,
- Reactivity:
- Human
Description
system_update_altDatasheet - तकनीकी मैनुअलsystem_update_altMSDS - तकनीकी मैनुअल
| Product Name: | Human CD40 / TNFRSF5 ELISA Kit |
| Product Code: | HUFI00269 |
| Size: | 96 Assays |
| Alias: | CD40, TNFRSF5, B cell surface antigen CD40, Bp50B cell-associated molecule, CD40 antigen, CD40 type II isoform, CD40L receptor, CDw40, MGC9013, p50, tumor necrosis factor receptor superfamily member 5 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human TNFRSF5/CD40 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 11.719pg/ml |
| Range: | 19.5-1250pg/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human TNFRSF5/CD40 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNFRSF5/CD40 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TNFRSF5/CD40 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | 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 | P25942 |
| UniProt Protein Function: | CD40: a member of the TNF-receptor superfamily. This receptor for CD40L mediates a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. Defects in CD40 are the cause of hyper-IgM immunodeficiency type 3 (HIGM3). HIGM3 is an autosomal recessive disorder which includes an inability of B cells to undergo isotype switching, one of the final differentiation steps in the humoral immune system, an inability to mount an antibody-specific immune response, and a lack of germinal center formation. Two alternatively spliced isoforms have been reported. Isoform I is a type I membrane protein; isoform II is secreted. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, cytokine Chromosomal Location of Human Ortholog: 20q12-q13.2 Cellular Component: cell surface; integral to plasma membrane; plasma membrane Molecular Function:enzyme binding; protein binding; receptor activity; signal transducer activity; tumor necrosis factor receptor activity; ubiquitin protein ligase binding Biological Process: activation of NF-kappaB transcription factor; cellular calcium ion homeostasis; immune response; multicellular organismal development; positive regulation of GTPase activity; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of MAP kinase activity; positive regulation of protein amino acid phosphorylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tyrosine phosphorylation of Stat1 protein; protein complex assembly; regulation of cell proliferation; regulation of immune response; response to lipopolysaccharide; tumor necrosis factor-mediated signaling pathway Disease: Immunodeficiency With Hyper-igm, Type 3 |
| NCBI Summary: | This gene is a member of the TNF-receptor superfamily. The encoded protein is a receptor on antigen-presenting cells of the immune system and is essential for mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. AT-hook transcription factor AKNA is reported to coordinately regulate the expression of this receptor and its ligand, which may be important for homotypic cell interactions. Adaptor protein TNFR2 interacts with this receptor and serves as a mediator of the signal transduction. The interaction of this receptor and its ligand is found to be necessary for amyloid-beta-induced microglial activation, and thus is thought to be an early event in Alzheimer disease pathogenesis. Mutations affecting this gene are the cause of autosomal recessive hyper-IgM immunodeficiency type 3 (HIGM3). Multiple alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported. [provided by RefSeq, Nov 2014] |
| UniProt Code: | P25942 |
| NCBI GenInfo Identifier: | 116000 |
| NCBI Gene ID: | 958 |
| NCBI Accession: | P25942.1 |
| UniProt Secondary Accession: | P25942,Q53GN5, Q5JY15, Q5U007, Q7M4Q8, Q86YK5, Q9BYU0 E1P5S9, |
| UniProt Related Accession: | P25942 |
| Molecular Weight: | 22,259 Da |
| NCBI Full Name: | Tumor necrosis factor receptor superfamily member 5 |
| NCBI Synonym Full Names: | CD40 molecule |
| NCBI Official Symbol: | CD40 |
| NCBI Official Synonym Symbols: | p50; Bp50; CDW40; TNFRSF5 |
| NCBI Protein Information: | tumor necrosis factor receptor superfamily member 5 |
| UniProt Protein Name: | Tumor necrosis factor receptor superfamily member 5 |
| UniProt Synonym Protein Names: | B-cell surface antigen CD40; Bp50; CD40L receptor; CDw40; CD_antigen: CD40 |
| Protein Family: | CD40 ligand |
| UniProt Gene Name: | CD40 |
| UniProt Entry Name: | TNR5_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
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.
| Step | 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. |
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. |
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