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ELISA Measurement of Mouse IL-2   

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Abstract

Interleukin-2 (IL-2) is a cytokine secreted by T cells that is essential for immune system activation. This protocol is routinely used for quantification of IL-2 concentration in the supernant of cultured lymphocytes under various stimulations and co-culturing conditions. Upon slightly modification and optimization, this protocol can also be adapted to quantitatively measure other secreted proteins and bio-molecules.

Materials and Reagents

  1. BD OptEIA ELISA set including IL-2 standard, capture antibody, detection antibody/enzyme reagent (BD Biosciences, catalog number: 555148 )
  2. Assay diluent (BD Biosciences, catalog number: 555213 ) or medium made of half RPM1640/10% FCS/PS and half RPM1640
  3. Substrate solution (BD Biosciences, catalog number: 555214 )
  4. NaCl
  5. NaHCO3
  6. Na2CO3
  7. Na2HPO4
  8. KH2PO4
  9. KCl
  10. Tween-20
  11. H2SO4
  12. Coating buffer (see Recipes)
  13. Wash buffer (PBST, pH 7.0) (see Recipes)
  14. Stop solution (see Recipes)

Equipment

  1. ELISA plates (100 plates/case) (BD Biosciences, catalog number: 353279 ) and plate sealers (100 plates/case) (PGC Scientifics, catalog number: 045-826 )
  2. 96-well plates for dilution (SARSTEDT AG, catalog number: 82.1583 )
  3. Multichannel pipette and pipette tips (eBay, catalog number: RT-L200F )
  4. Reagent reservoir (50 ml, 5/bag) (Corning, Costar®, catalog number: 4870 )
  5. ELISA micro plate reader

Procedure

  1. Prepare coating buffer. Dilute capture antibody in coating buffer (1:250 for lot #0000052895) and add 50 μl to each well. Seal plates and incubate overnight at 4 °C.
  2. Wash 3 times.
  3. Block: 200 μl Assay diuent to each well. Room temperature (RT) 1 h.
  4. Wash 3 times.
  5. Add 50 μl standard or sample to each well. RT 2 h.
    Standard: make 800 pg/ml standard in assay diluent from original 135 ng/ml stock and aliquote 0.4 ml each (enough for one assay) and store at -80 °C.
    135 ng/ml x 23.7 μl = 800 p/ml x (3.98+0.0237) ml
    Pg/ml               0         50         100      200     400      800
    800 pg/ml (μl)  0        9.38       18.8     37.5     75       150
    Diluent (μl)      150     140.6     131.2   112.5    75       0
    50 μl each
  6. Wash 5 times.
  7. 15 min before use, dilute detection antibody and avidin-conjugated HRP in Assay diluent (1:330 for lot #0000052895, 8 μl each/6 ml for one 96-well plate). RT 1 h.
  8. Wash 7 times.
  9. Prepare substrate solution (1:1 of A and B, 6 ml for one 96-well plate). Add 50 μl to each well. RT 30 min in dark.
  10. Add 25 μl stop solution. Read at 450 nm and 570 nm within 30 min.

Recipes

  1. Coating buffer (0.1 M carbonate buffer) (pH 9.5)
    4.20 g NaHCO3
    1.28 g Na2CO3 / 0.5 L
    Use within 7 days and store at 4 °C.
  2. Wash buffer (PBST, pH 7.0)
    16 g NaCl
    2.32 g Na2HPO4
    0.4 g KH2PO4
    0.4 g KCl, 1 ml
    Tween-20 / 2 L per two 96-well plates
    Use within 3 days and store at 4 °C.
  3. Stop solution
    2 N H2SO4 (1 M)

Acknowledgments

This work was supported by National Institute on Drug Abuse (NIDA; DA00266, DA10309) and the National Institute of Mental Health (NIMH; MH068830).

References

  1. Huang, G. N., Huso, D. L., Bouyain, S., Tu, J., McCorkell, K. A., May, M. J., Zhu, Y., Lutz, M., Collins, S., Dehoff, M., Kang, S., Whartenby, K., Powell, J., Leahy, D. and Worley, P. F. (2008). NFAT binding and regulation of T cell activation by the cytoplasmic scaffolding Homer proteins. Science 319(5862): 476-481.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Huang, G. N. (2012). ELISA Measurement of Mouse IL-2. Bio-101: e169. DOI: 10.21769/BioProtoc.169.
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