1.   The antigen on interest is coated on an Immulon 2 HB- High Binding plates or strips or other plates designed for ELISAs at a concentration of 1.0 ug/ml in 0.05 M in carbonate-bicarbonate buffer at pH 9.6.  For certain peptide antigens, the manufacturers instructions for peptide immobilization are followed for the use of Reacti-Bind Maleic Anhydride Activated Polystyrene Plates. The coating concentration and buffer may have to be optimized.  Incubate two hours room temperature (RT) or refrigerate overnight.

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

3.  The plates are blocked 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.  Block one hour RT.

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

5.  Serial dilutions of polyclonal sera are prepared in dilution tubes using a 1% BSA/PBS diluant and transferred to the assay plate.  For the first assay, serial dilutions will be done over a broad range such as a single eight five-fold dilution series starting at 1:50.  Follow up ELISAs use a more narrow range of dilutions and include replicates to make more accurate determinations of titers.  Incubate one hour RT.

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

7.  The detection antibody, horseradish peroxidase conjugated anti-IgG is used diluted in 1 % BSA/PBS and incubated for one hour RT.

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

9.  KPL has a ready to use TMB reagent that is convenient and works well according to the manufacturer’s instructions.  Absorbance is determined by dual wavelength absorbance at 450 nm and 620 nm and is captured with Deltasoft JV for export into Microsoft Excel.

If you have any questions regarding ELISAs, please contact Capralogics

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