Location of destroyed antigenic sites of Gly m Bd 60 K after three processing technologies

Gly m Bd 60 K, which is the α subunit of β-conglycinin, is a major soybean (Glycine max) allergen. We used high hydrostatic pressure (HHP), thermal techniques, and glycation to treat β-conglycinin, which can effectively reduce the antigenicity of β-conglycinin. β-conglycinin was used to immunize New Zealand rabbits, and the antiserum had a titer > 1: 1 × 10⁵ and an IC₅₀ of 2.254 μg/mL. β-conglycinin was subjected to HHP, thermal techniques, and glycation and mixed with rabbit antiserum against β-conglycinin to obtain the site-specific antiserum. The overlapping gene fragments of Gly m Bd 60 K were amplified by polymerase chain reaction (PCR), then cloned into a T7 phage vector and packaged in vitro, the recombinant T7 phages were constructed. Indirect ELISA (iELISA) was used to locate the destroyed antigenic sites and, after three rounds of segment expression and identification, the C2-1 and C2-2 fragments were identified as destroyed antigenic sites of Gly m Bd 60 K. Allergenicity analysis showed that the C2-1 and C2-2 fragments reacted with allergic patients' serum, which indicated that the destroyed sites were allergic sites.