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ELISA for titering the serum

  1. Coat 100uL target protein [target protein: coating buffer containing 10uL Antigen] in parallel and incubate at 4°C overnight, .  
  2. Wash the wells 2 times with the washing buffer [PBST: PBS containing 0.05% Tween-20].
  3. Block the wells with 350 μL blocking buffer [PBSM: PBS containing 4% milk] for 2 h at room temperature.
  4. Shake out the blocking buffer and wash the plate 5 times with the washing buffer, slapping the plate face-down onto a clean section of paper towel each time.
  5. Add 100 μL of diluted serum and incubate at room temperature for 1 hour. Wash 5 times with the washing buffer.
  6. Dilute Sheep anti-Rabbit IgG [HRP conjugate] in PBS. Add 100 μL of diluted antibdy per well and incubate at room temperature for 1 hour.
  7. Prepare the HRP substrate solution as follows: a stock solution of OPD can be prepared in advance by dissolving 22 mg OPD (Sigma) in 100 mL of 50 mM sodium citrate, pH 4.0. Filter sterilize and store at 4°C. Immediately prior to the detection step, add 36 uL 30% H2O2 to 21 mL of OPD stock solution.
  8. Add 100 uL of substrate solution per well and incubate at room temperature for 30 minutes.
  9. Read plates using a microplate reader set at 490nm.

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