Description
Human CD5 antigen-like (CD5L) ELISA Kit
The Human CD5 antigen-like (CD5L) ELISA Kit is specifically designed for the precise measurement of CD5L levels in human samples such as serum, plasma, and cell culture supernatants. With a high level of sensitivity and specificity, this ELISA Kit guarantees accurate and consistent results, making it an invaluable tool for various research purposes.CD5L is an important protein that plays a role in immune response regulation and cell survival. It is known to be involved in autoimmune diseases, inflammation, and cancer progression, making it a key biomarker for studying these conditions and exploring potential therapeutic interventions.
Overall, the Human CD5 antigen-like (CD5L) ELISA Kit offers researchers a reliable and efficient method for detecting CD5L levels in human samples, providing valuable insights into its physiological functions and its potential implications in various diseases.
| Product Name: | Human CD5 antigen-like (CD5L) ELISA Kit |
| SKU: | HUEB2360 |
| Size: | 96T |
| Target: | Human CD5 antigen-like (CD5L) |
| Synonyms: | Apoptosis inhibitor expressed by macrophages, CT-2, IgM-associated peptide, SP-alpha, hAIM, UNQ203/PRO229, API6 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 78-5000pg/mL |
| Sensitivity: | 33 pg/mL |
| Intra CV: | 4.3% | ||||||||||||||||||||
| Inter CV: | 8.4% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Secreted protein that acts as a key regulator of lipid synthesis: mainly expressed by macrophages in lymphoid and inflammed tissues and regulates mechanisms in inflammatory responses, such as infection or atherosclerosis. Able to inhibit lipid droplet size in adipocytes. Following incorporation into mature adipocytes via CD36-mediated endocytosis, associates with cytosolic FASN, inhibiting fatty acid synthase activity and leading to lipolysis, the degradation of triacylglycerols into glycerol and free fatty acids (FFA). CD5L-induced lipolysis occurs with progression of obesity: participates in obesity-associated inflammation following recruitment of inflammatory macrophages into adipose tissues, a cause of insulin resistance and obesity-related metabolic disease. Regulation of intracellular lipids mediated by CD5L has a direct effect on transcription regulation mediated by nuclear receptors ROR-gamma (RORC). Acts as a key regulator of metabolic switch in T-helper Th17 cells. Regulates the expression of pro-inflammatory genes in Th17 cells by altering the lipid content and limiting synthesis of cholesterol ligand of RORC, the master transcription factor of Th17-cell differentiation. CD5L is mainly present in non-pathogenic Th17 cells, where it decreases the content of polyunsaturated fatty acyls (PUFA), affecting two metabolic proteins MSMO1 and CYP51A1, which synthesize ligands of RORC, limiting RORC activity and expression of pro-inflammatory genes. Participates in obesity-associated autoimmunity via its association with IgM, interfering with the binding of IgM to Fcalpha/mu receptor and enhancing the development of long-lived plasma cells that produce high-affinity IgG autoantibodies (By similarity). Also acts as an inhibitor of apoptosis in macrophages: promotes macrophage survival from the apoptotic effects of oxidized lipids in case of atherosclerosis (PubMed:24295828). Involved in early response to microbial infection against various pathogens by acting as a pattern recognition receptor and by promoting autophagy (PubMed:16030018, PubMed:24223991, PubMed:24583716, PubMed:25713983). |
| Uniprot: | O43866 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human CD5 antigen-like |
| Sub Unit: | Interacts with FASN; the interaction is direct (By similarity). Interacts with IgM; protecting CD5L from renal excretion and leading to increased CD5L levels in circulating blood (PubMed:8034987, PubMed:24804991). |
| Research Area: | Cancer |
| Subcellular Location: | Secreted Cytoplasm Secreted by macrophages and circulates in the blood (PubMed:24223991, PubMed:24804991). Transported in the cytoplasm via CD36-mediated endocytosis (By similarity). |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | CD5L: May play a role in the regulation of the immune system. Seems to play a role as an inhibitor of apoptosis. |
| UniProt Protein Details: | Protein type:Receptor, misc.; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q23.1 Cellular Component: extracellular space; membrane; extracellular region Molecular Function:scavenger receptor activity Biological Process: receptor-mediated endocytosis; apoptosis; cellular defense response |
| UniProt Code: | O43866 |
| NCBI GenInfo Identifier: | 20177834 |
| NCBI Gene ID: | 922 |
| NCBI Accession: | O43866.1 |
| UniProt Secondary Accession: | O43866,Q6UX63, A8K7M5, |
| UniProt Related Accession: | O43866 |
| Molecular Weight: | 347 |
| NCBI Full Name: | CD5 antigen-like |
| NCBI Synonym Full Names: | CD5 molecule-like |
| NCBI Official Symbol: | CD5L |
| NCBI Official Synonym Symbols: | AIM; API6; PRO229; Spalpha; SP-ALPHA |
| NCBI Protein Information: | CD5 antigen-like; CT-2; apoptosis inhibitor 6; igM-associated peptide; CD5 antigen-like (scavenger receptor cysteine rich family) |
| UniProt Protein Name: | CD5 antigen-like |
| UniProt Synonym Protein Names: | CT-2; IgM-associated peptide; SP-alpha |
| Protein Family: | CD5 antigen |
| UniProt Gene Name: | CD5L |
| UniProt Entry Name: | CD5L_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. |
| ELISA |
| Human CD5L ELISA Kit |
