Proteomic Analysis of Eluted Antigens from Crude Somatic Antigens of Trichinella Spiralis Muscle Larva by Igg-ELISA, Two-Dimensional Gel Electrophoresis, and Mass Spectrometry

Abstract Background: Trichinellosis is caused by Trichinella spiralis muscle larvae (ML), which in swine is the main source of transmission. Trichinellosis is detected by enzyme-linked immunosorbent assay (ELISA) using excretory–secretory antigens (ESAg). Preparation of ESAg is difficult, and it produces a low content. Furthermore, the sensitivity and specificity of the test depends on the quality of the antigen that is produced in each batch. Crude somatic antigens (CSAg) of T. spiralis ML at molecular weights (MWs) of 101, 79, and 43 kDa were previously positive for swine trichinellosis sera, with no cross-reaction observed for normal sera, except that 79 and 43 kDa each reacted with one of the coccidiosis and with mixed infections containing trichuriasis and coccidiosis. Therefore, the current study aimed to obtain eluted antigens of T. spiralis ML CSAg, which were analyzed by IgG-ELISA for detecting swine trichinellosis and identification by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC MS-MS).Methods: In this study, T. spiralis larvae CSAg at 101, 79, and 43 kDa (TsCSAg-101, TsCSAg-79, and TsCSAg-43, respectively) were eluted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivities and specificities. In addition, three specific antigens were identified by 2-DE and LC MS-MS.Results: Sensitivity of IgG-ELISA using three eluted antigens was persistent at 100%. Specificities were 90.63%, 95.54%, and 97.77% following TsCSAg-101, TsCSAg-79, and TsCSAg-43, respectively. The LC MS-MS results showed that 18/20 spots of the antigens were identified for 11 different proteins. One protein has several isoforms; for example, a serine proteinase and the phosphoenolpyruvate carboxykinase protein.Conclusions: TsCSAg-43 showed the highest specificity compared to the other two eluted antigens, which indicated that these specific proteins (a 45k antigen–trichina [fragment], a DNA topoisomerase 2-alpha, an antigen targeted by protective antibodies, and a conserved hypothetical protein [gi339234223]) should be developed and produced in large volumes in further study.