Mouse IL-4 ELISA Assay Kit

$405.00

The Eagle Biosciences Mouse Interleukin 4 (IL-4) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of mouse Interleukin 4 (IL-4) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Mouse Interleukin 4 (IL-4) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Mouse IL-4 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl

Additional Information

Assay Principle


The Eagle Biosciences Mouse Interleukin 4 (IL-4) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Interleukin 4 (IL-4) has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Interleukin 4 (IL-4) present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for Interleukin 4 (IL-4) is added to the wells and binds to the combination of capture antibody-IL-4 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of Interleukin 4 (IL-4) present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven Interleukin 4 (IL-4) standard dilutions and Interleukin 4 (IL-4) sample concentration determined.

  1. Prepare all reagents and working standards as directed in the previous sections.
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
  4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. 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.
  5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
  6. Repeat the aspiration/wash.
  7. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
  10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)

Assay Background


Interleukin 4 (IL-4) is a pleiotropic cytokine produced primarily by activated T lymphocytes, mast cells and basophils (1-3).  The cDNA sequence of mouse IL-4 predicts a 140 amino acid (aa) residue precursor protein containing a 20 aa residue signal peptide that is cleaved to form the mature protein (4). At the amino acid sequence level, mature mouse IL-4 is approximately 50% identical to human IL-4 but there is no species cross-reactivity for biological activity for the two proteins (1, 2). Mouse IL-4 also shares approximately 30% amino acid sequence identity to mouse IL-13 and the two cytokines exhibit overlapping biological activities (5, 6). The gene for IL-4 has been mapped to mouse chromosome 11, in close proximity to the genes for IL-3, IL-5, IL-13 and GM-CSF (1, 2).

IL-4 has multiple immune response-modulating activities on a variety of cell types. It is an important regulator of isotype switching, inducing IgE production in B lymphocytes. It is an important modulator of the differentiation of precursor T helper cells to the Th2 subset that mediates humoral immunity and modulates antibody production. In addition, IL-4 has also been shown to have anti-tumor activity both in vivo and in vitro (1-3).

The biological effects of IL-4 are mediated by specific cell surface receptor complexes. Although IL-4 R does not bind IL-13 directly, it has been shown to complex with the low-affinity IL-13 R to form the functional high-affinity receptor complex for IL-13 (7, 8). In addition to the membrane-bound form of IL-4 R, a naturally occurring soluble form of IL-4 R has been identified in human and mouse biological fluids and in mouse cell culture supernates (9-11). Soluble IL-4 R has been to shown to bind IL-4 with high affinity in solution.

Manual

Product Manual


Publications

References


  • Howard, M. and N. Harada (1994) in Guidebook to Cytokines and Their Receptors, Nicola N.A. ed., Oxford University Press, New York, p. 44.
  • Banchereau, J. and M.E. Rybak (1994) in The Cytokine Handbook, 2nd ed., A. Thomson ed., Academic Press, New York, p. 99.
  • Yokota, T. et al. (1990) in Peptide Growth Factors and Their Receptors I, Sporn, M.B. And A.B. Roberts eds., Springer-Verlag, New York, p. 577.
  • Lee, F. et al. (1986) Proc. Natl. Acad. Sci. USA 83:2061.
  • McKenzie, A.N.J. and G. Zurawski (1994) in Guidebook to Cytokines and Their Receptors, Nicola N.A. ed., Oxford Univ. Press, New York, p. 92.
  • Zurawski, G. and J.E. de Vries (1994) Immunol. Today 15:19.
  • Lin, J-X. et al. (1995) Immunity 2:331
  • Zurawski, S.M. et al. (1995) J. Biol. Chem. 270:13869.
  • Mosley, B. et al. (1989) Cell 59:335.
  • Fernandez-Botran, R. and E.S. Vitetta (1990) Proc. Natl. Acad. Sci. USA 87:535.