The eukaryotic expression of SAP-2 shows higher sensitivity than the prokaryotic expressed SAP-2 protein in the detection of antibodies against bovine fasciolosis.

Bovine fasciolosis, caused by Fasciola gigantica and Fasciola hepatica, represents a major economic burden to the livestock industry. Developing a reliable diagnostic antigen is crucial for advancing diagnostic kits for bovine fasciolosis, which could effectively mitigate these economic losses. FgSAP-2 has demonstrated​​​ considerable potential as a diagnostic antigen when expressed in prokaryotic systems. Computational analyses suggest that FgSAP-2 undergoes glycosylation, promoting the investigation of whether eukaryotic expression systems--capable of performing post-translational modifications--might enhance its antigenic properties and improve its suitability for diagnostic kit development. To explore this possibility, FgSAP-2 was expressed in Pichia pastoris (reFgSAP-2), purified and used to establish an indirect ELISA. The sensitivity, specificity, and stability of the ELISA were subsequently evaluated. Field serum samples from Guangxi were tested using the reFgSAP-2-based ELISA and compared to results from ELISAs employing prokaryotically expressed FgSAP-2 (rpFgSAP-2) and Excretory-Secretory Products (FgESP). The reFgSAP-2 ELISA exhibited positive detection at a serum dilution of 1:1600, with a coefficient of variation (CV) below 10 % in both intra-batch and inter-batch repeatability tests. Furthermore, no cross-reactivity was observed with sera positive for Schistosoma japonicum and Toxoplasma gondii. The positive detection rates of the reFgSAP-2-ELISA were comparable to those of the FgESP-ELISA, both surpassing the detection performance of the rpFgSAP-2-ELISA. CONCLUSION: An indirect ELISA detection method based on eukaryotically expressed FgSAP-2 was successfully developed, demonstrating high sensitivity, specificity, and repeatability. This approach shows promise for further development in the preparation of diagnostic kits.