Torres Oscar B, Alving Carl R, Matyas Gary R
Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.
US Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, 20817, USA.
Methods Mol Biol. 2016;1403:695-710. doi: 10.1007/978-1-4939-3387-7_39.
The ability to prepare hapten-carrier conjugates reproducibly with consistent lot-to-lot hapten densities and protein yields is a critical component of hapten vaccine development. This entails the development of appropriate coupling chemistries that do not cause protein precipitation and the development of methods to quantify hapten density. Recently, extensive efforts have been devoted to design vaccines against drugs of abuse. We describe, herein, a method for conjugation of a morphine-like hapten (MorHap) to tetanus toxoid (TT), which involves conjugation of MorHap to the surface lysines of TT through the N-hydroxysuccinimide portion of a heterobifunctional linker and the subsequent attachment of the thiol on MorHap to the maleimide portion of the cross-linker. Methods are described for the analytical quantification of the hapten density of the conjugates using modified Ellman's test, trinitrobenzenesulfonic acid (TNBS) assay, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
能够以批次间一致的半抗原密度和蛋白质产量可重复地制备半抗原-载体缀合物，是半抗原疫苗开发的关键组成部分。这需要开发不会导致蛋白质沉淀的合适偶联化学方法，以及开发定量半抗原密度的方法。最近，人们为设计抗滥用药物疫苗付出了巨大努力。在此，我们描述了一种将吗啡样半抗原（MorHap）与破伤风类毒素（TT）偶联的方法，该方法包括通过异双功能连接子的N-羟基琥珀酰亚胺部分将MorHap与TT的表面赖氨酸偶联，随后将MorHap上的硫醇连接到交联剂的马来酰亚胺部分。描述了使用改良的埃尔曼测试、三硝基苯磺酸（TNBS）测定法和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析定量缀合物半抗原密度的方法。
