CCL21 ELISA Kit

Catalog No. ABIN1979905

Product Details CCL21 ELISA Kit

Target: CCL21 (Chemokine (C-C Motif) Ligand 21 (CCL21))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 30-8,000 pg/mL

Minimum Detection Limit: 30 pg/mL

Application: ELISA

Purpose: The Human 6Ckine ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human 6Ckine in serum, plasma, cell culture supernatants and urine.

Sample Type: Cell Culture Supernatant, Plasma, Serum

Analytical Method: Quantitative

Cross-Reactivity (Details): Cross Reactivity: This ELISA kit shows no cross-reactivity with the following cytokines tested: human Angiogenin, BDNF, BLC, CNTF, ENA- 78, FGF-4, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, IL-309, IP-10, FGF-4, FGF-6, FGF-7, GDNF, GM-CSF, IFN-gamma, IGFBP-2, IGF-BP-3, IGF-BP-4, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIF, MIG, MIP-1alpha, MIP-1 beta, MIP-1delta, PARC, PDGF, RANTES, SCF,SDF-1alpha, TARC, TGF-beta, TIMP-1, TIMP-2, TNF- alpha, TNF-beta, TPO, VEGF.

Sensitivity: < 30 pg/mL

Components: 1. 6Ckine Microplate (Item A): 96 wells (12 strips x 8 wells) coated with anti-human 6Ckine.
2. Wash Buffer Concentrate (20x) (Item B): 25 mL of 20x concentrated solution
3. Standards (Item C): 2 vials of recombinant human 6Ckine.
4. Assay Diluent C (Item L): 30 mL diluent buffer, 0.09 % sodium azide as preservative. For Standard/Sample (serum/plasma) diluent.
5. Assay Diluent B (Item E): 15 mL of 5x concentrated buffer. For Standard/Sample (cell culture medium/urine) diluent.
6. Detection Antibody (Item F): 2 vial of biotinylated anti-human 6Ckine (each vial is enough to assay half microplate).
7. HRP-Streptavidin Concentrate (Item G): 8 µL 20,000x concentrated HRP-conjugated streptavidin.
8. TMB One-Step Substrate Reagent (Item H): 12 mL of 3,3™,5,5™-tetramethylbenzidine (TMB) in buffer solution.
9. Stop Solution (Item I): 8 mL of 2 M sulfuric acid.

Material not included: 1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 µL to 1 ml volumes.
3. Adjustable 1-25 ml pipettes for reagent preparation.
4. 100 ml and 1 liter graduated cylinders.
5. Absorbent paper.
6. Distilled or deionized water.
7. Log-log graph paper or computer and software for ELISA data analysis.
8. Tubes to prepare standard or sample dilutions.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs an antibody specific for human 6Ckine coated on a 96-well plate. Standards and samples are pipetted into the wells and 6Ckine present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human 6Ckine antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of 6Ckine bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent C (Item L) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of cell culture supernates/urine. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent C (for serum/plasma samples) or 1x Assay Diluent B (for cell culture supernates/urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 80 µL 6Ckine standard (50 ng/mL) from the vial of Item C, into a tube with 420 µL Assay Diluent C or 1x Assay Diluent B to prepare a 8,000 pg/mL standard solution. Pipette 300 µL Assay Diluent C or 1x Assay Diluent B into each tube. Use the 8,000 pg/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent C or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 80 µL standard + 420 µL 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 8,000 3,200 1,280 512.0 204.8 81.92 32.76 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 400-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 30 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 400-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: 4 °C/-20 °C/-80 °C

Storage Comment: May be stored for up to 6 months at 2 - 8 °C from the date of shipment. Standard (recombinant protein) should be stored at -20 °C or -80 °C (recommended at -80 °C) after reconstitution. Opened Microplate Wells or reagents may be stored for up to 1 month at 2 - 8 °C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge. Note: the kit can be used within one year if the whole kit is stored at -20 °C.

Expiry Date: 12 months

References for CCL21 ELISA Kit (ABIN1979905)

Gao, Camous, Lu, Lim, Larbi, Ng: "Novel inflammatory markers associated with cognitive performance: Singapore Longitudinal Ageing Studies." in: Neurobiology of aging, Vol. 39, pp. 140-6, (2016) (PubMed).

Target Details for CCL21

Target: CCL21 (Chemokine (C-C Motif) Ligand 21 (CCL21))

Alternative Name: CCL21 / 6ckine (CCL21 Products)

Synonyms: 6Ckine ELISA Kit, CKb9 ELISA Kit, ECL ELISA Kit, SCYA21 ELISA Kit, SLC ELISA Kit, TCA4 ELISA Kit, ccl21 ELISA Kit, Ccl21b ELISA Kit, C-C motif chemokine ligand 21 ELISA Kit, C-C motif chemokine 21 ELISA Kit, CCL21 ELISA Kit, ccl21 ELISA Kit, Ccl21 ELISA Kit

Pathways: Regulation of Actin Filament Polymerization