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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.