Abstract Recently, increasing interest has been focused on the origin and transmission of silk. However, ancient silks have usually undergone severely deterioration and investigations of the sequential and spatial structural changes in the degradation process have rarely been reported. In this paper, a novel immunoassay was proposed for investigating the structural changes of silk fibroin (SF) at sequential level during an alkali aging process. Three polyclonal and one monoclonal antibodies, i.e., HAPJ0449, HAPH1223, HAPJ0409, and HAMK0111, were accurately prepared through animal immunization. Next, a sensitive indirect enzyme-linked immunosorbent assay (ELISA) based on the antibodies was performed to characterize the aged silks. The ELISA results indicated that the macromolecular structure of silk fibroin degraded gradually with an increase in the alkali aging time interval. The antibodies produced by immunizing rabbits with hapten-keyhole limpet hemocyanin (KLH) conjugate exhibit superior sensitivity to those obtained by immunization with silk fibroin. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and attenuated total reflectance—Fourier transform-infrared spectroscopy (ATR-FTIR) results showed the molecular weight and crystallinity index of silk samples decreased during the aging process. Thus, combined with conventional analytical tools, the immunological method has the potential to provide an accurate and reliable quantitative index for aging assessment of severely degraded silks, which may serve as a reference for studying the origin and transmission of these materials.

中文翻译：

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人工老化过程中通过免疫测定法测定的丝素蛋白的结构和性质变化

摘要 近年来，人们对丝绸的起源和传播越来越感兴趣。然而，古代丝绸通常经历了严重的退化，并且很少有关于退化过程中顺序和空间结构变化的研究报道。在本文中，提出了一种新的免疫测定法，用于研究碱老化过程中丝素蛋白 (SF) 在连续水平上的结构变化。通过动物免疫，准确制备了HAPJ0449、HAPH1223、HAPJ0409和HAMK0111三种多克隆抗体和一种单克隆抗体。接下来，进行基于抗体的灵敏的间接酶联免疫吸附测定 (ELISA) 来表征老化的丝绸。ELISA结果表明，随着碱老化时间间隔的增加，丝素蛋白的大分子结构逐渐降解。用半抗原-钥孔血蓝蛋白 (KLH) 偶联物​​对兔子进行免疫接种所产生的抗体比用丝素蛋白进行免疫所获得的抗体具有更高的敏感性。此外，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和衰减全反射-傅里叶变换红外光谱（ATR-FTIR）结果表明，丝绸样品的分子量和结晶度指数在老化过程中下降。因此，结合常规分析工具，免疫学方法有可能为严重降解的蚕丝老化评估提供准确可靠的定量指标，