Editing Peptide Conjugation to KLH (section)

== Chemical coupling of synthetic peptides to KLH or BSA ==

The synthetic peptides of figures 1-8 were reduced with lOmM DTT, or with immobilized DTT (Reduetacryl, Calbiochem, San Diego, California, U.S.A., 10 mg Reductacryl per mg peptide) overnight at 4°C, pH8 in lOmM borate or other buffers depending upon peptide solubility, with gentle mixing. KLH was dissolved at a concentration of 10-30 mg/ml in 10 mM phosphate buffer pH 7.2., and BSA was dissolved at a concentration of 20-80 mg/ml for treatment with heterobifunctional linkers MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), SPDP [N-Succinimidyl 3-(2 pyridyldithio)propionate], or Sulfo-SMCC [Sulfo- Succinimidyl 4(N-maleimidomethyl)-cyclohexane-l-carboxylate]. The hetero- bifunctional linkers were added to the protein solutions in a molar excess of linker to BSA of 100, or of linker to KLH of 1000. MBS was dissolved in DMF at lOmg/ml, SPDP was dissolved in absolute ethanol at lOmg/ml and each of these was added dropwise with mixing into the protein solution, whereas the Sulfo- SMCC dry powder was dissolved into the protein solution. Each protein-linker mixture was reacted at room temperature for 15 minutes to 1 hour and then free linker was separated from BSA-linker or KLH-linker by gel chromatography with Sephadex G25 using 50mM phosphate, pH 6 for KLH-MBS, lOmM acetate, 100 mM NaCl, pH 4.6 for BSA-SPDP, or 100 mM phosphate, 1 mM EDTA, pH 6.5 for BSA- SMCC. The reduced peptides were separated from DTT by Sephadex G10 column chromatography when soluble DTT was used, or by centrifugation followed by filtration through sa 0.22 micron filter when Reductacryl was used. Reduced peptides were combined with linker treated KLH or BSA in molar ratios of 100- 1000 peptide/KLH-MBS, 1-50 peptide/BSA-SMCC, and 0.1-10 peptide/BSA-SPDP. Each reduced peptide was added to the linker-treated protein with mixing, overlayed with N2, and then mixed gently at room temperature for one hour, and then overnight at 4°C. The SMCC-BSA conjugates were further treated with a 1 m final concentration of mercaptoethylamine for 4 hours, and then dialyzed overnight against 100 volumes of buffer (20mM phosphate, 1 mM EDTA, 0.05% sodium azide, pH 6.5) at 4°C. All other conjugates were adjusted to a final concentration of sodium azide of 0.05%. Optimal molar ratios of peptide/protein for the conjugates were determined by solubility and antigenicity (as evaluated by EIA). For example, the best ratios were found to be approximately 1:1 (SPDP- BSA), 10:1 (SMCC-BSA), and 100:1 (MBS-KLH).