Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten鈥揚rotein Conjugates with Sulfhydryl Linkages

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• 作者：Eric C. Peterson ; Michael D. Hambuchen ; Rachel L. Tawney ; Melinda G. Gunnell ; James L. Cowell ; Jackson O. Lay ; Jr. ; Bruce E. Blough ; F. Ivy Carroll ; S. Michael Owens
• 刊名：Bioconjugate Chemistry
• 出版年：2014
• 出版时间：December 17, 2014
• 年：2014
• 卷：25
• 期：12
• 页码：2112-2115
• 全文大小：243K
• ISSN：1520-4812

文摘

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses ¹⁴C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten鈥揃SA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r² = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten鈥損rotein conjugations, and choosing the most effective epitope densities for conjugate vaccines.