ELISA Measurement of Mouse IL-2   

Abstract

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.