Development of an Indirect ELISA and a Microsphere Detection Test Strip for the Detection of Spirometra mansoni

There is currently no highly sensitive and specific serological detection method for Spirometra mansoni infection, nor is there a rapid test strip available for onsite diagnosis. Therefore, in this study, an indirect enzyme-linked immunosorbent essay (ELISA) method suitable for detecting S. mansoni was developed, and a colour microsphere test strip based on pyruvate kinase (PK) recombinant protein (rSmPK) was developed. First, the excretory-secretory (ES) protein (ESP) of the plerocercoid was collected as the coating antigen to establish an ELISA method (SmES-ELISA). Moreover, PK was screened as the target antigen, and an ELISA based on rSmPK (rSmPK-ELISA) was performed. Finally, rSmPK was labelled with NHS-biotin, and NHS-biotin-rSmPK was linked to coloured microspheres containing streptavidin (SA), successfully establishing a colour microsphere detection test strip. The optimal reaction conditions for SmES-ELISA and rSmPK-ELISA were screened by systematically optimising conditions such as the coating concentration, serum dilution, blocking conditions and dilution of the enzyme-labelled secondary antibody. Sensitivity testing revealed that SmES-ELISA and rSmPK-ELISA still had good sensitivity at dilutions of 25,600 and 800 times, respectively. Specific detection was conducted using eight parasites that may be coinfected with S. mansoni, and the results showed that SmES-ELISA did not exhibit cross-reactivity, whereas rSmPK-ELISA only showed weak cross-reactivity with Clonorchis sinensis. The colour microsphere detection test strip based on rSmPK had good sensitivity but weak cross-reactivity with Hymenolepis nana, C. sinensis and Schistosoma japonicum. However, in actual testing, there have been no reports of hosts simultaneously infected with these four parasites or even co-infected with any two parasites. Therefore, the detection test strip constructed in this study still has high application value. SmES-ELISA and rSmPK-ELISA compensate for the shortcomings of serological diagnostic methods for S. mansoni infection. Moreover, the colour microsphere detection test strip based on rSmPK provides the possibility for large-scale epidemiological investigations.