Peptide ELISA

jack_horner at my-deja.com jack_horner at my-deja.com
Sun Feb 6 20:15:59 EST 2000


>From the method I use:
1. Antigen (peptide/protein) was diluted to 5 mg/ml in
carbonate-bicarbonate buffer (see MATERIALS below). For example, 5 ml of
peptide from a 10 mg/ml peptide stock was added to 10 mls of
carbonate-bicarbonate buffer (enough for one plate). 100 ml per well was
added to flat bottomed polyvinyl chloride microplates (Flow Laboratories
Inc.) and incubated overnight at 4oC or 90 minutes at 37oC.

2. Antigen was flicked off the plate and the wells were blocked with 200
ml of 5% skim milk in PBS-Tween 20 (see MATERIALS below) overnight at
4oC or 90 mins at 37oC.

Carbonate-bicarbonate coating buffer
15 mM Na2CO3  (1.6 g)
35 mM NaHCO3   (2.9 g)
1 litre H2O
The pH was adjusted to 9.5

And now a question: wouldn't a method that used drying of Ag to the
plate result in free antigen (that would competitively bind antibodies)
being released when the test serum was added? Or do you wash it off
using the blocking agent.
martinC at qimr.edu.au


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