Editing 293F Protein Production (section)

==== ELISA<br>  ====

<br>1. 96-well ELISA microtiter plates with flat bottom.<br>2. Blocking buffer: 0.5% casein hydrolysate (Applichem) and 0.05% Tween 20 (Sigma-Aldrich) in PBS (pH 7.1).<br>3. Capture antibody: Goat anti-human kappa light chain (AbD Serotec).<br>4. Coating solution: For each 96-well plate, 11 L of capture antibody is mixed with 11 mL of PBS (pH 8.0).<br>5. Washing buffer: PBS (pH 8.0) with 2% Tween 20.<br>6. Detection antibody: Alkaline phosphatase-conjugated goat anti-human gamma heavy chain (Biosource).<br>7. Standard: Human IgG, whole molecule (ChromPure, Jackson ImmunoResearch). The standard is diluted in blocking buffer to 40 ng/mL and then serially<br>diluted 1:2 with blocking buffer.<br>8. Substrate buffer: Add 97 mL diethanolamine (Sigma-Aldrich) to 700 mL H2O and adjust pH to 9.8 with 2 M HCl. Add 0.5 mL 1 M MgCl2 and 2 g NaN3. Adjust volume to 1 L.<br>9. Substrate: 4-nitrophenyl phosphate disodium salt (NPP) (Applichem) is dissolved in substrate buffer to 1.5 mg/mL.<br>10. Stop solution: 3 M NaOH.<br> 

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