3.2.1. Adsorption of Peptide to Microtiter Plates
1. When using free peptide as an antigenic target, use at a final concentration of approx 50 pmol/mL in adsorption buffer (this corresponds to a dilution of 1 in 2000 of a 100 |g/mL stock solution of a 10-mer peptide). If using a peptide-carrier conjugate as the target, dilute the conjugate in adsorption buffer to a peptide concentration of about 10 |g/mL. The precise amount of peptide conjugate may need eventually to be titrated to give optimal signals.
2. Add 100 |L of diluted peptide solution to each well.
3. Leave at room temperature in a moist environment overnight (a seal-able box containing a small amount of water is suitable).
4. Shake out any unadsorbed peptide, and wash the plate three times in TBS by immersion of the plate in TBS. Immerse the plate at an angle to avoid trapping air bubbles. Shake the plate dry.
5. Add 150 |L of TM buffer per well and leave at room temperature for at least 30 min. If required, store the plates at this stage at -20°C.
1. Empty the wells by shaking the plate dry and add 100 |L of antibody diluted in TMT per well. A starting dilution of 1 in 50 is suitable for most antisera. Serially dilute antibody in doubling dilutions down one row of the microtitre plate (i.e., eight dilutions in all).
2. Leave for 30-60 min at room temperature.
4. Add 100 |L per well of appropriate second-layer reagent diluted in TMT. For most commercially available HRP-anti-Rabbit Ig conjugates a dilution of 1 in 200 should suffice, although titration may help to optimize signals.
5. Leave for 30-60 min at room temperature.
6. Wash three times in TBS as before.
7. Add 100 ^L of substrate solution per well.
8. Incubate at room temperature. Peroxidase reactions take about 5-30 min to develop. Judge the reaction time by eye (see Notes 7-9). Reactions may be stopped by adding 100 ^L of stop solution to each well. The SDS in the stop solution also solubilizes any precipitated products formed in the HRP reaction.
9. Read the optical density (OD) on an ELISA plate reader. Green ABTS reaction product should be read at OD406.
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