Description
Human CD86/B7-2 ELISA Kit
The Human CD86 (B7-2) ELISA Kit is a powerful tool for detecting levels of CD86 (B7-2) in human samples such as serum, plasma, and cell culture supernatants. With high sensitivity and specificity, this kit delivers accurate and reproducible results, making it perfect for a variety of research applications.CD86 (B7-2) is a critical co-stimulatory molecule involved in immune response regulation.
It plays a key role in T cell activation and differentiation, making it a valuable marker for studying immune system function and dysfunction. By measuring CD86 (B7-2) levels, researchers can gain insights into various autoimmune diseases, inflammatory disorders, and transplant rejection, paving the way for new therapeutic approaches.
| Product Name: | Human CD86/B7-2 ELISA Kit |
| Product Code: | HUFI00476 |
| Size: | 96 Assays |
| Alias: | B7-2, CD86, Activation B7-2 antigen, B70, B7-2 antigen, B-lymphocyte activation antigen B7-2, BU63, CD28 antigen ligand 2, CD28LG2, CD86 antigen, CD86 molecule, CTLA-4 counter-receptor B7.2, FUN-1, LAB72, T-lymphocyte activation antigen CD86 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human CD86 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human CD86 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CD86 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CD86 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 | P42081 |
| UniProt Protein Function: | CD86: Receptor involved in the costimulatory signal essential for T-lymphocyte proliferation and interleukin-2 production, by binding CD28 or CTLA-4. May play a critical role in the early events of T-cell activation and costimulation of naive T-cells, such as deciding between immunity and anergy that is made by T- cells within 24 hours after activation. Isoform 2 interferes with the formation of CD86 clusters, and thus acts as a negative regulator of T-cell activation. 4 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Immunoglobulin superfamily; Membrane protein, integral Chromosomal Location of Human Ortholog: 3q21 Cellular Component: cell surface; intracellular membrane-bound organelle; plasma membrane; integral to membrane; external side of plasma membrane Molecular Function:protein binding; coreceptor activity; receptor activity; receptor binding Biological Process: positive regulation of lymphotoxin A biosynthetic process; negative regulation of T cell anergy; T cell activation; viral reproduction; nerve growth factor receptor signaling pathway; positive regulation of transcription, DNA-dependent; positive regulation of interleukin-2 biosynthetic process; myeloid dendritic cell differentiation; positive regulation of activated T cell proliferation; positive regulation of interleukin-4 biosynthetic process; positive regulation of T-helper 2 cell differentiation; cell-cell signaling; T cell proliferation during immune response; positive regulation of cell proliferation; response to yeast; defense response to virus; aging; response to drug; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; phosphoinositide-mediated signaling; B cell activation; T cell costimulation; toll-like receptor signaling pathway; innate immune response; immune response |
| NCBI Summary: | This gene encodes a type I membrane protein that is a member of the immunoglobulin superfamily. This protein is expressed by antigen-presenting cells, and it is the ligand for two proteins at the cell surface of T cells, CD28 antigen and cytotoxic T-lymphocyte-associated protein 4. Binding of this protein with CD28 antigen is a costimulatory signal for activation of the T-cell. Binding of this protein with cytotoxic T-lymphocyte-associated protein 4 negatively regulates T-cell activation and diminishes the immune response. Alternative splicing results in several transcript variants encoding different isoforms.[provided by RefSeq, May 2011] |
| UniProt Code: | P42081 |
| NCBI GenInfo Identifier: | 317373339 |
| NCBI Gene ID: | 942 |
| NCBI Accession: | P42081.2 |
| UniProt Secondary Accession: | P42081,Q13655, Q6FHB1, Q6GTS4, Q7M4L5, A0N0P0, B7Z2F3 B7Z702, E7ETN5, E9PC27, |
| UniProt Related Accession: | P42081 |
| Molecular Weight: | 329 |
| NCBI Full Name: | T-lymphocyte activation antigen CD86 |
| NCBI Synonym Full Names: | CD86 molecule |
| NCBI Official Symbol: | CD86 |
| NCBI Official Synonym Symbols: | B70; B7-2; B7.2; LAB72; CD28LG2 |
| NCBI Protein Information: | T-lymphocyte activation antigen CD86; BU63; FUN-1; CTLA-4 counter-receptor B7.2; B-lymphocyte activation antigen B7-2; CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) |
| UniProt Protein Name: | T-lymphocyte activation antigen CD86 |
| UniProt Synonym Protein Names: | Activation B7-2 antigen; B70; BU63; CTLA-4 counter-receptor B7.2; FUN-1; CD_antigen: CD86 |
| UniProt Gene Name: | CD86 |
| UniProt Entry Name: | CD86_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. |
