“…Alongside the Western blotting results provide evidence of targeted reactivity with the native parvalbumins by IgG (Figure ). The results are consistent with prior studies of antigenicity demonstrated for purified turbot and flounder parvalbumin by its homologous anti-parvalbumin antibodies. , 1 SDS-PAGE profile and Western blot of purified parvalbumin. SDS-page is shown in blue color (a, c, e) and Western blot is shown in gray (b, d, f).…”
Section: Resultssupporting
confidence: 90%
“…The results are consistent with prior studies of antigenicity demonstrated for purified turbot and flounder parvalbumin by its homologous antiparvalbumin antibodies. 14,15 Seven types of different species of different orders of commonly ingested fish were considered in our study. The protein profile of crude extracts of these fish was separated using SDS-PAGE (Figure 2).…”
Section: Resultsmentioning
confidence: 99%
“…With regards to isoforms immunodetectability, two isoforms of parvalbumin in turbot 14 had 62 and 67% sequence identity with cod parvalbumin. Among the two isoforms of tilapia parvalbumin, both shared higher sequence identity (80%) with all three parvalbumins: cod, turbot, and flounder.…”
Section: Anti-parvalbumin Antibody Reactivity To Muscle Extractsmentioning
The findings of this study emphasize the complexity of detecting fish parvalbumin using immunochemical methods. Different antibodies, processing methods, fish species, and measurement techniques all play a role in influencing the detectability of fish species' parvalbumin. Among the generated IgG, the anti-turbot antibody was particularly suitable for immunoassay approaches to detect parvalbumin in treated extracts of various fish species, including extracts that the cod antibody and flounder antibody could not detect. The anti-turbot antibody exhibited broad reactivity across different treatments and fish species. Heat treatments that were homogeneous and effective (e.g., frying) and those with higher heat penetration capacity (e.g., microwaving) resulted an improved immunological response to parvalbumin monomers in fish extracts, as demonstrated by Western blot analysis of binding intensity. Further, our study observed higher molecular weight structures, possibly indicating the formation of oligomer bands on the Western blot, for fish species treated with cooking methods such as oven-baking, steaming, frying, and heated raw extracts. Fish species treated with certain cooking methods (e.g., frying, oven-baking, microwaving) might experience heat-induced structural changes that lead to alterations in parvalbumin's binding properties. Fish species such as mackerel and basa, treated with frying, oven-baking, or microwaving, are more affected by altered binding properties.
“…Alongside the Western blotting results provide evidence of targeted reactivity with the native parvalbumins by IgG (Figure ). The results are consistent with prior studies of antigenicity demonstrated for purified turbot and flounder parvalbumin by its homologous anti-parvalbumin antibodies. , 1 SDS-PAGE profile and Western blot of purified parvalbumin. SDS-page is shown in blue color (a, c, e) and Western blot is shown in gray (b, d, f).…”
Section: Resultssupporting
confidence: 90%
“…The results are consistent with prior studies of antigenicity demonstrated for purified turbot and flounder parvalbumin by its homologous antiparvalbumin antibodies. 14,15 Seven types of different species of different orders of commonly ingested fish were considered in our study. The protein profile of crude extracts of these fish was separated using SDS-PAGE (Figure 2).…”
Section: Resultsmentioning
confidence: 99%
“…With regards to isoforms immunodetectability, two isoforms of parvalbumin in turbot 14 had 62 and 67% sequence identity with cod parvalbumin. Among the two isoforms of tilapia parvalbumin, both shared higher sequence identity (80%) with all three parvalbumins: cod, turbot, and flounder.…”
Section: Anti-parvalbumin Antibody Reactivity To Muscle Extractsmentioning
The findings of this study emphasize the complexity of detecting fish parvalbumin using immunochemical methods. Different antibodies, processing methods, fish species, and measurement techniques all play a role in influencing the detectability of fish species' parvalbumin. Among the generated IgG, the anti-turbot antibody was particularly suitable for immunoassay approaches to detect parvalbumin in treated extracts of various fish species, including extracts that the cod antibody and flounder antibody could not detect. The anti-turbot antibody exhibited broad reactivity across different treatments and fish species. Heat treatments that were homogeneous and effective (e.g., frying) and those with higher heat penetration capacity (e.g., microwaving) resulted an improved immunological response to parvalbumin monomers in fish extracts, as demonstrated by Western blot analysis of binding intensity. Further, our study observed higher molecular weight structures, possibly indicating the formation of oligomer bands on the Western blot, for fish species treated with cooking methods such as oven-baking, steaming, frying, and heated raw extracts. Fish species treated with certain cooking methods (e.g., frying, oven-baking, microwaving) might experience heat-induced structural changes that lead to alterations in parvalbumin's binding properties. Fish species such as mackerel and basa, treated with frying, oven-baking, or microwaving, are more affected by altered binding properties.
“…P. trituberculatus muscles were homogenized with cold acetone and aired, then extracted using PBST (10 mM PBS, 0.05% Tween 20, pH 7.4) in 1:20 (m/v) at 4 °C for 12 h. The solution was analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE western blot, which was performed with sera from 5 crab-sensitized patients and 2 healthy individuals as described . Detailed methods of SDS-PAGE were described in the Supporting Information.…”
Section: Methodsmentioning
confidence: 99%
“…The PCR amplification products were sequenced by BGI Genomics. The rSCP was procured based on the method reported . Briefly, the target sequence (5′-6 × His tag-enterokinase site-SCP CDS-3′) was ligated into Nde I and Xho I of pET-30a through Tsingke Biotechnology Co., Ltd. (Beijing, China).…”
Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgEbinding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4−70 °C and pH 3−10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.
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