Human IL-3 ELISA Development Kit contains the key components required for the quantitative measurement of natural and/or recombinant IL-3 in a sandwich ELISA format. Using the ELISA protocol described below, this kit provides sufficient reagents to assay IL-3 in approximately 1500 ELISA plate wells.
1.) Capture Antibody
2.) Detection Antibody
5.) TMB Liquid Substrate "Ready to Use"
Other Materials and Solutions Required
Additional Required Materials
ELISA microplates (Thermo Fisher Cat. # 456529)
BSA (Sigma Cat. # A-7030)
Dulbecco’s PBS (DPBS) [10x] (Leinco Product No. D388)
Stop Solution: 450 nm Stop Reagent for TMB Microwell (Leinco Product No. T125)
PBS: dilute 10xPBS to 1xPBS, pH 7.2, in sterile water
Wash Buffer: 0.05% Tween-20 in PBS (Leinco Product No. W101)
Reagent Diluent: 1.0% BSA in PBS*
Blocking Buffer 1.0% BSA in PBS (Leinco Product No. B395)
Note: Other acceptable blocking buffers such as Leinco's Ultra-FISH Block (Leinco Product No. B396) or Ultra-Sythetic Block (Leinco Product No. B397) may be used for assay optimization.
Some of the required components may contain acid and/or cause allergic reactions. Breathing in product mist or fumes should be avoided. Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Please refer to the MSDS on our website prior to use.
Mouse Anti-Human IL-3 Capture Antibody: Centrifuge vial prior to opening. Reconstitute in 0.5 mL sterile PBS. Refer to the lot-specific datasheet for amount supplied and dilute in PBS without carrier protein to the working concentration indicated on the C of A. Following reconstitution the capture antibody may be stored at 2 – 8°C for up to 6 months. For long term storage, it is recommended to aliquot into working volumes and store at -70°C in a manual defrost freezer. Avoid repeated freeze and thaw cycles.
Biotin Detection Antibody
Biotinylated Goat Anti-Human IL-3 Detection Antibody: Refer to the lot-specific datasheet for amount supplied. Centrifuge vial prior to opening. Reconstitute with 1.0 mL of Reagent Diluent. Dilute in Reagent Diluent to the working concentration indicated on the C of A. Detection antibodies may be stored at 2 – 8°C for up to 6 months. For long term storage, it is recommended to aliquot into working volumes and store at -70°C in a manual defrost the freezer. Avoid repeated freeze and thaw cycles.
E. coli - expressed Recombinant Human IL-3 Standard: Centrifuge vial prior to opening. Reconstitute each vial with 0.5 mL of Reagent Diluent. Refer to the lot-specific datasheet for amount supplied. The rProtein may be stored at 2 – 8°C for one (1) month or aliquoted and stored at -70°C for up to three months in a manual defrost freezer. Avoid repeated freeze and thaw cycles.
TMB Liquid Substrate "Ready to Use" (TMB Substrate should be at ambient temperature prior to use): 60.0 mL of TMB HRP Microwell Substrate Standard Kinetic One Component "Ready Use" (Leinco Product No. T118) is provided. The high quality of the substrate can be preserved by storing at temperatures between 2 – 8ºC. When properly stored, TMB Microwell Substrate is stable for a minimum of 48 months from the manufactured date.
1. Dilute the capture antibody to the working concentration in PBS without carrier protein and immediately add 100 μL to each ELISA plate well. Seal the plate and incubate overnight at room temperature.
2. Aspirate the wells to remove liquid and wash the plate 4 times using 300-400 μL of wash buffer per well. Note: We recommend using an autowasher, although a squirt bottle or manifold dispenser would suffice.
3. After the last wash, invert plate to remove residual buffer and blot on paper towel.
4. Add 300 μL block buffer to each well and incubate for at least 1 hour at room temperature.
5. Aspirate and wash plate 4 times.
NOTE: Complete removal of liquid at each step is essential for good performance and sensitivity of assay.
Standard/Sample: Add 100 μL of the working dilution with reagent dilution standard or sample to each well (duplicate recommended). Cover plate with an adhesive plate cover and incubate at room temperature for at least 2 hours.
Detection: Aspirate and wash plate 4 times. Add 100 μL of the detection antibody, diluted in Reagent Diluent to each well. Cover with a new adhesive plate cover and incubate at room temperature for 2 hours.
StreptAvidin-HRP Conjugate: Aspirate and wash plate 4 times. Add 100 μL of the working dilution (the dilution factor may require optimization) to each well. Cover and incubate at room temperature for 20-30 minutes. Exposure to direct light should be avoided.
TMB Liquid Substrate: Aspirate and wash plate 4 times. Add 100 μL of TMB HRP Microwell Substrate Standard Kinetic One Component "Ready Use" (Leinco Product No. T118) to each well. Incubate at room temperature for 20-30 minutes and monitor color development. Exposure to direct light should be avoided.
Stop Solution: Add 50-100 μL of Stop Solution (Leinco Product No. T125) to each well. Monitor color development with an ELISA plate reader at 450 nm with wavelength correction set at 540 nm or 570 nm.
IL-3 is a 17 kD pleiotropic cytokine and a member of a family of growth factors that sustains both the proliferation and the development of hematopoietic precursors. It also displays neurotrophic activity and is thought to be associated with neurologic disorders. Signal transmission is assured by high affinity binding to the IL-3 receptor (IL3RA). The receptor contains α and β subunits. IL-3 shares the β subunit with IL-5 and GM-CSF which explains their biological functional similarities. The IL-3/receptor complex induces JAK2/STAT5 cell signalization pathway and it can stimulate both the activation of gene expression and the suppression of apoptosis via transcription factor c‑myc and the Ras pathway, respectively. IL-3 fuels the differentiation of multipotent hematopoietic stem cells into myeloid progenitor cells or lymphoid progenitor cells (when in conjunction with IL-7). Together with other cytokines, including EPO, GM-CSF, and IL-6, IL-3 can stimulate proliferation of all cells in the myeloid lineage. The constitutive expression of IL-3 in some leukemia cell lines (as opposed to selective T-cell expression in response to specific impulses or antigens) is thought to fuel the development of the disease. Post-chemotherapy IL-3 application can promote regeneration of granulocytes and platelets. Furthermore, during high-dose chemotherapy, the combination of IL-3, GM-CSF and stem cell factor increases peripheral blood stem cells. IL-3 is thought to have therapeutic potential in lymphohematopoietic disorders and solid cancers.
Products are for research use only. Not for use in diagnostic or therapeutic procedures.