CX3CL1 ELISA Kit

Catalog No. ABIN1380025

Product Details CX3CL1 ELISA Kit

Target: CX3CL1 (Chemokine (C-X3-C Motif) Ligand 1 (CX3CL1))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 625-20000 pg/mL

Minimum Detection Limit: 625 pg/mL

Application: ELISA

Purpose: The OmniKine? Human Fractalkine ELISA Kit contains the components necessary for quantitative determination of natural or recombinant Human Fractalkine concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a "Sandwich" Enzyme- Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a "sandwich" format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on Human Fractalkine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and "sandwiching" of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3', 5, 5'-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Brand: OmniKine™

Sample Type: Cell Lysate, Serum, Plasma

Analytical Method: Quantitative

Specificity: The Human Fractalkine ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human Fractalkine proteins.

Cross-Reactivity (Details): The Human Fractalkine ELISA is capable of recognizing both recombinant and naturally produced Human Fractalkine proteins. The antigens listed below were tested at 500 ng/mL and did not exhibit significant cross- reactivity or interference. Murine: Fractalkine (Chemokine domain aa 22-105), Fractalkine (Chemokine domain and mucin stalk), Fractalkine (Chemokine domain aa 25-105)

Characteristics: The Human Fractalkine ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human Fractalkine proteins within the range of 625-20000 pg/mL.

Components:

• Microstrips Coated w / Capture Antibody: 12 x 8-Well Microstrips
• Protein Standard: Lyophilized (100 ng), Red container
• Biotinylated Detection Antibody: Lyophilized, Yellow container
• 400x Streptavidin-HRP: 30 μL, Blue container
• Wash Buffer (10x): 50 mL, Clear containter
• Assay Diluent: 50 mL, Clear container
• Ready-to-Use Substrate: 12 mL, Brown container
• Stop Solution: 12 mL, Clear container
• Adhesive Plate Sealers: 4 Sheets
• Technical Manual 1 Manual

Material not included: The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
Microplate reader able to measure absorbance at 450 nm (with correction wavelength set to 540 nm or 570 nm)
Micropipettes with capability of measuring volumes ranging from 1 μl to 1 mL
Deionized or sterile water
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent paper or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 mL
Bench
top centrifuge (optional)
Bench
top vortex (optional)
Orbital shaker (optional)

Application Details

Plate: Pre-coated

Protocol: This particular immunoassay utilizes the quantitative technique of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a sandwich format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on the Human Fractalkine cytokine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and sandwiching of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3’, 5, 5’-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Sample Preparation:

If samples are to be used within 24 hours, aliquot and store at 4 °C. If samples are to be used over a long period of time, aliquot and store between -20 °C and -80 °C, depending on the duration of storage.
Note: Samples containing a visible precipitate or pellet must be clarified prior to use in the assay.
Caution: Avoid repeated freeze/thaw cycles to prevent loss of biological activity of proteins in experimental samples.

• Cell Lysate and Supernatants:
  Remove large cell components via centrifugation and perform the assay. Cell lysates and supernatants require a dilution using Assay Diluent. A serial dilution may be performed to determine a suitable dilution factor for the sample. For future use of the sample, follow the sample storage guidelines stated above.
• Serum:
  Allow samples to clot in a serum separator tube (SST) for 30 minutes. After sufficient clotting, centrifuge at 1000 x g for 15 minutes and remove serum from SST in preparation for the assay. Serum samples require at least a 1:50 dilution using Assay Diluent. For future use of the sample, follow the storage guidelines above.
• Plasma:
  Use heparin, citrate or EDTA as an anticoagulant to gather plasma from original biological sample. After collection of the plasma, centrifuge for 15 minutes at 1000 x g. This step must be performed within 30 minutes of plasma collection. Plasma samples require at least a 1:50 dilution using Assay Diluent. Afterwards, perform the assay or for future use of the sample, follow the storage guidelines stated above.

Assay Procedure:

Note: If possible, all incubation steps should be performed on an orbital shaker to equilibrate solutions when added to the microplate wells. Also, all provided solutions should be at ambient temperature prior to use.
Note: Avoid adding solutions into wells at an angle, always keep pipette tip perpendicular to plate bottom.

Reconstitution of Provided Materials:

1. Reconstitute the Biotin-Conjugated Detection Antibody in 67 µL of ddH₂O for a concentration of 180 µg/ml.
2. Reconstitute the Protein Standard in 100 µL of ddH₂O for a concentration of 340 ng/ml.
3. Dilute the 50 mL of 10x Wash Buffer in 450 mL of ddH2O for 500 mL of 1x Wash Buffer.

Addition of Known Standard and Unknown Sample to Immunoassay:

The OmniKine™ Human CD163 ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human CD163 proteins

Calculation of Results:

Generation of Standard Curve and Interpretation of Data
1. Average the duplicate or triplicate readings for each standard, control and sample and subtract the average zero standard optical density.
2. Generate a standard curve by using Microsoft Excel or other computer software capable of establishing a 4- Parameter Logistic (4-PL) curve fit. If using Excel or an alternative graphing tool, plot the average optical density values in absorbance units (y-axis) against the known standard concentrations in pg/ml (x-axis). Note: Only use the values in which a noticeable gradient can be established. Afterwards, generate a best fit curve or trend-line through the plotted points via regression analysis.

Restrictions: For Research Use only

Handling

Precaution of Use: Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Handling Advice: This ELISA kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind.
Materials included in this kit should NOT be used past the expiration date on the kit label.
Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.
Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided.
The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.

Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Storage: 4 °C

Storage Comment: Note: If used frequently, reagents may be stored at 4 °C.

• Unopened Kits: Store at 4 °C for 6 months.
• Microstrips Coated w/ Capture Antibody, 400x Streptavidin-HRP Wash Buffer (10x), Assay Diluent Ready-to-Use Substrate, Stop Solution: 6 Months at 4 °C
• Protein Standard, Biotinylated Detection Antibody: Lyophilized: 6 Months (if Reconstituted: 1 Month) at 4 °C

Target Details for CX3CL1

Target: CX3CL1 (Chemokine (C-X3-C Motif) Ligand 1 (CX3CL1))

Alternative Name: Fractalkine (CX3CL1 Products)

Synonyms: ABCD3 ELISA Kit, CX3CL1 ELISA Kit, DKFZp459K117 ELISA Kit, Cx3c ELISA Kit, Scyd1 ELISA Kit, ABCD-3 ELISA Kit, C3Xkine ELISA Kit, CXC3 ELISA Kit, CXC3C ELISA Kit, NTN ELISA Kit, NTT ELISA Kit, SCYD1 ELISA Kit, fractalkine ELISA Kit, neurotactin ELISA Kit, AB030188 ELISA Kit, AI848747 ELISA Kit, CX3C ELISA Kit, Cxc3 ELISA Kit, D8Bwg0439e ELISA Kit, ATP binding cassette subfamily D member 3 ELISA Kit, C-X3-C motif chemokine ligand 1 ELISA Kit, chemokine (C-X3-C motif) ligand 1 ELISA Kit, ABCD3 ELISA Kit, CX3CL1 ELISA Kit, Cx3cl1 ELISA Kit, Abcd3 ELISA Kit

Background: Fractalkine, also known as C-X3-C motif chemokine 1 (CX3CL1), is a 397 amino acid single-pass type I membrane protein. The processed fractalkine is secreted. Fractalkine belongs to the intercrine delta family. The soluble form is chemotactic for T-cells and monocytes, but not for neutrophils. The membrane-bound form promotes adhesion of those leukocytes to endothelial cells. Fractalkine may play a role in regulating leukocyte adhesion and migration processes at the endothelium. It binds to CX3CR1 and can be found in the small intestine, colon, testis, prostate, heart, brain, lung, skeletal muscle, kidney and pancreas. Fractalkine is induced by TNF and IL1/interleukin-1 in pulmonary endothelial cells and umbilical vein endothelial cells. A soluble short 95 kDa form may be released by proteolytic cleavage from the long membrane-anchored form. Fractalkine is O-glycosylated with core 1 or possibly core 8 glycans after post- translational modification. Source: Entrez Gene, Swiss-Prot

Pathways: Synaptic Membrane