Human IL-7 MAb （Clone 7417） 500ug
Product classification
Monoclonal
Anti-human IL-7 Antibody
ORDERING INFORMATION
Preparation
This antibody was produced from a hybridoma resulting from the fusion of a mouse myeloma
with B cells obtained from a mouse immunized with purified, E. coli-derived, recombinant human
interleukin 7 （rhIL-7）. The IgG fraction of ascites fluid was purified by Protein A affinity
chromatography. IL-7 is a cytokine secreted by stromal cells from various tissues. It binds to the
high affinity IL-7 receptor complex and has a wide variety of effects on B cells, T cells and
monocyte/macrophages.
Catalog Number: MAB207
Clone: 7417
Lot Number: HE05
Size: 500 μg
Formulation
Lyophilized from a 0.2 μm filtered solution in phosphate-buffered saline （PBS） with 5% trehalose.
Formulation: 0.2 μm filtered solution in PBS
with 5% trehalose
Endotoxin Level
< 0.1 EU per 1 μg of the antibody as determined by the LAL method.
Storage: -20o
C
Reconstitution
Reconstitution: sterile PBS
Reconstitute with sterile PBS. If 1 mL of PBS is used, the antibody concentration will be
500 μg/mL.
Specificity: human IL-7
Storage
Immunogen: E. coli-derived rhIL-7 Lyophilized samples are stable for twelve months from date of receipt when stored at -20° C
to -70° C. Upon reconstitution, the antibody can be stored at 2° - 8° C for 1 month without
detectable loss of activity. Reconstituted antibody can also be aliquotted and stored frozen
Ig class: mouse IgG1
Avoid repeated freeze-thaw cycles.
Recommended Applications:
ELISA capture
Specificity Neutralization of bioactivity
Western blot
This antibody was selected for its ability to neutralize the bioactivity of rhIL-7 and for use as a
capture antibody in sandwich ELISAs. When used in combination with the biotinylated polyclonal
detection antibody （Catalog # BAF207） in sandwich ELISAs, no significant cross-reactivity or
interference was observed with other cytokines tested.
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Immunohistochemistry
Figure 1
Applications
Neutralization of Human IL-7 Bioactivity - The exact concentration of antibody required to
neutralize rhIL-7 activity is dependent on the cytokine concentration, cell type, growth conditions
and the type of activity studied. To provide a guideline, R&D Systems has determined the
neutralization dose for this antibody under a specific set of conditions. The Neutralization
Dose50 （ND50） for this antibody is defined as that concentration of antibody required to yield
one-half maximal inhibition of the cytokine activity on a responsive cell line, when that cytokine is
present at a concentration just high enough to elicit a maximum response.
Figure 1: Human IL-7 stimulates
3
H-thymidine incorporation by 5-day PHA-activated
human peripheral blood mononuclear cells in a dose dependent manner. The
ED50 for this effect is typically 0.2 - 0.5 ng/mL.
Figure 2: Approximay 0.4 - 0.8 μg/mL of the antibody will neutralize 50% of the
bioactivity due to 2.5 ng/mL of rhIL-7.
Figure 2 Western Blot - This antibody can be used at 1 - 2 μg/mL with the appropriate secondary
reagents to detect human IL-7. Using a colorimetric detection system, the detection limit for
rhIL-7 is approximay 25 ng/lane under non-reducing conditions. Use of this antibody under
reducing conditions in not recommended. Chemiluminescent detection with WesternGlo
Chemiluminescent Detection Substrate （R&D Systems, Catalog # AR004） will increase
sensitivity by 5 to 50 fold.
ELISA Capture - This antibody can be used as a capture antibody in a human IL-7 ELISA in
combination with biotinylated, human IL-7 affinity purified polyclonal detection antibody
（Catalog # BAF207）. A general protocol is provided on the next page. Using plates coated with
100 μL/well of the capture antibody at 2 μg/mL, in combination with 100 μL/well of the detection
antibody, an ELISA for sample volumes of 100 μL can be obtained. To arrive at the optimal dose
range for this ELISA, set up a two-fold dilution series of the protein standard starting with 0.5 ng/mL.
Immunohistochemistry - This antibody was used at a concentration of 25 μg/mL with
appropriate secondary reagents to detect IL-7 in paraffin-embedded human breast cancer tissue
sections. For chromogenic detection of labeling, the use of R&D Systems’ Cell and Tissue
Staining Kits （CTS Series） is recommended.
Optimal dilutions should be determined by each laboratory for each application. ELISA Protocol
Solutions Required
• Wash Buffer - 0.05% Tween 20 in PBS, pH 7.4
• Diluent - 1 % BSA in PBS, pH 7.4
• Substrate Solution - 1:1 mixture of Color Reagent A （H202） and Color Reagent B （Tetramethylbenzidine） （R&D Systems,
Catalog # DY999）
• Stop Solution - 1 M H2SO4
Plate Preparation
1. Transfer 100 μL/well of the capture antibody （diluted to the appropriate concentration in PBS, use immediay） to an ELISA plate. Seal
plate and incubate overnight at room temperature.
2. Aspirate each well and wash with Wash buffer, repeating the process two times for a total of 3 washes. Wash by filling each well with
Wash Buffer （400 μL） using a squirt bottle, multi-channel pipette, manifold dispenser 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 by inverting the plate and
blotting it against clean paper toweling.
3. Block plates by adding 300 μL of PBS containing 1% BSA, 5% sucrose and 0.05% NaN3 to each well. Incubate at room temperature for a
minimum of 1 hour.
4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition. Alternatively, the blocking buffer can be aspirated
after step 3 and the plates can be dried under vacuum. When sealed with dessicant, the plates can be stored at 4 - 8° C for at least
2 months. Assay Procedure
1. Dilutions of unknowns and standards should be carried out in polypropylene tubes. Add 100 μL of sample or standards in an appropriate
diluent, per well. Mix by gently tapping the plate frame for 1 minute. Cover with an adhesive strip and incubate 2 hours at room
temperature.
2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
3. Add 100 μL of the biotinylated detection antibody, diluted in the appropriate diluent, to each well. Cover with a new adhesive strip and
incubate 2 hours at room temperature.
4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
5. Add 100 μL streptavidin HRP （R&D Systems, Catalog # DY998, dilute according to the directions on the vial label） to each well. Cover the
plate and incubate for 20 minutes at room temperature.
6. Repeat the aspiration/wash as in step 2.
7. Add 100 μL of Substrate Solution to each well. Incubate for 20 - 30 minutes at room temperature. Avoid placing the plate in direct light.
8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. If wavelength correction is
available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at
450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be
higher and less accurate.
Calculation of Results
To calculate assay results, average the duplicate readings and subtract the zero standard optical density from the sample optical density.
Create a standard curve using data reduction software capable of generating a four parameter （4P-L） curve fit. Alternatively, plot the optical
density for the standards versus the concentration of the standards and draw the best curve. The data can be linearized by using log-log paper
and regression analysis may be applied to the log transformation. To determine the human IL-7 concentrations for each sample, first find the
absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis
and read the corresponding human IL-7 concentration. If samples have been diluted, the concentration read from the standard curve must be
multiplied by the dilution factor. A standard curve should be generated for each set of samples assayed.
Limitations
It is important that the diluents selected for reconstitution and for dilution of the standard reflect the environment of the samples being
measured. The diluent suggested in the above protocol may be suitable for most cell culture supernate samples. Validate diluents for specific
sample types prior to use.
A basic understanding of immunoassay development is required for the successful use of these reagents in immunoassays. The protocol
provided is for demonstration purposes only. The type of enzyme and substrate and the concentrations of capture/detection antibodies used
can be varied to create an immunoassay with a different sensitivity and dynamic range.
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bFGF acidic, rhG-CSF, rhGM-CSF, rhIL-1α, rhIL-1β, rhIL-2, rhIL-3, rhIL-4, rhIL-6, rmIL-7, rhIL-8, pPDGF, rhTNF-α, rhTNF-β, hTGF-β1, pTGF-β1