Palmieri G, Cassani G, Fassina G
Tecnogen SCpA, Piana di Monte Verna (CE), Italy.
J Chromatogr B Biomed Appl. 1995 Feb 3;664(1):127-35. doi: 10.1016/0378-4347(94)00353-7.
Two different procedures were developed for the non-covalent immobilization of peptide antigens on calcium alginate beads. The antigenic peptide is first synthesized in a multimeric form starting from a polydentate lysine core, and then immobilized on alginate beads (average volume 0.05 ml) by entrapment or simply by non-covalent adsorption. Coupling yields, as monitored by RP-HPLC analysis of the immobilization time course and/or by amino acid analysis of derivatized beads, were close to 1-2 mg of peptide per ml of gel. Immobilization yields were not dependent on the peptide net charge, hydrophobicity or length, but mainly on the extent of peptide multimerization. After immobilization on alginate gel, peptide antigenic properties were fully retained, as clearly demonstrated by the batchwise micropreparative purification of anti-peptide antibodies in good yields and with a high degree of purity, directly from crude sera in a single adsorption-elution step. Derivatized beads were sufficiently stable towards repeated washing-equilibration procedures, allowing very limited peptide leakage from the matrix. Peptide beads were also successfully used for the development of solid-phase immunoassays in test-tubes to characterize the corresponding antibodies, with the immobilization yield and signal-to-noise ratio being greatly enhanced in comparison with other types of conventional supports.
开发了两种不同的方法用于将肽抗原非共价固定在海藻酸钙珠上。抗原肽首先从多齿赖氨酸核心开始以多聚体形式合成，然后通过包埋或简单的非共价吸附固定在海藻酸钙珠（平均体积0.05 ml）上。通过对固定化时间进程的RP-HPLC分析和/或对衍生化珠子的氨基酸分析监测，偶联产率接近每毫升凝胶1-2毫克肽。固定化产率不依赖于肽的净电荷、疏水性或长度，而主要取决于肽的多聚化程度。固定在海藻酸凝胶上后，肽的抗原特性得以完全保留，这在单步吸附-洗脱步骤中直接从粗血清中以高收率和高纯度分批微量制备纯化抗肽抗体时得到了明确证明。衍生化珠子对重复的洗涤-平衡程序具有足够的稳定性，使得从基质中泄漏的肽非常有限。肽珠还成功用于开发试管中的固相免疫测定以表征相应抗体，与其他类型的传统支持物相比，固定化产率和信噪比大大提高。
