Editing Peptide Conjugation to KLH (section)

== ELISA ==

Synthetic peptide (2.5 μM) was prepared in carbonate buffer (pH 9.6) and incubated on a microtiter plate overnight at 4°C. Wells were washed three times with PBS containing 0.05% Tween-20. The remaining sites on the wells were blocked by BSA (10 mg/mL) in PBS/Tween-20 at 37°C for 1 h. After subsequent washing, 300 μL of 1:1000 dilutions of antipeptide antiserum was added to wells and incubated for 2 h at 37°C. The wells were washed three times with PBS/Tween-20 to remove the unbound antibody and then incubated with 1:500 dilution of alkaline phosphatase conjugate of goat anti-rabbit IgG in PBS/Tween-20 for 2 h at 37°C. Wells were washed three times with PBS/Tween-20, and 50 μL of enzyme substrate (p-nitrophenyl phosphate) prepared in water was added for 10 min at 37°C. The plate was read at λ = 405 nm with a micotiter plate reader (StatFax-2100, Awareness Technology, Palm City, FL).

&nbsp;<br/>The improved procedure for producing peptide/carrier protein conjugates used in this study consisted of three major modifications of the traditional protocol.

DMF was used as the solvent to solubilize peptides instead of PBS or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7).<br/>The last desalting or dialyzing step to remove uncoupled peptides in the traditional method was eliminated.<br/>Three milliliters of 0.1 M ammonium bicarbonate was added to the carrier protein conjugated peptide solution before lyophilization, which helped the lyophilization process.
[[Category:Antibodies]]