We recommend that investigators do optimization studies to develop the best ELISA for their application.  This ELISA format works with human serum and plasma samples containing native IL-21.  Please note that some lots of commercially available lots of rhIL-21 do not work for an ELISA standard curve due possibly to folding differences from disulfide bonds.

1. For the capture antibody use product CI0140, goat anti-human IL-21 N-terminus antibody, for coating microtiter plates such as Immulon 2 HB- High Binding plates or strips designed for ELISAs at a concentration of 0.5 to 1.0 ug per well overnight. However, further dilutions up to 0.1 ug per well may be the optimal for the particular ELISA. A 0.05 M sodium carbonate–bicarbonate buffer at pH 9.6 works well.

2. Wash plate three times with phosphate buffered saline (PBS) containing 0.05 % Tween 20.

3. The plates are blocked for two hours at room temperature with a high grade of BSA (bovine serum albumin) that is recommended for ELISA use. A 3 % BSA/PBS concentration works well.  Blocking with 4 % non-fat milk in PBS is also effective.

4. Wash plate three times with PBS containing 0.05 % Tween 20.

5. For human samples, initial serial dilutions should be done ranging from 0.2 ug to 0.1 ng per well.

6. Wash plate three times with PBS containing 0.05 % Tween 20.

7. The detection antibody, product CI0139, rabbit anti-human IL-21 C-terminus antibody, is used initially at a 1:1000 dilution in 1 % BSA/PBS incubated for one hour at room temperature.

8. Wash plate three times with PBS containing 0.05 % Tween 20.

9. Use a goat anti rabbit HRP according to the manufacturer's instructions with 1 % BSA/PBS as the diluant.

10. Wash plate three times with PBS containing 0.05 % Tween 20.

11. Use substrate of choice such as ABTS or TMB and follow manufacturer’s instructions. KPL has a ready to use TMB reagent that is convenient and works well. A stop solution of 1 N sulfuric acid can be used.

If you have any questions regarding this protocol, please contact Capralogics

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