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Protein A immunocapture assay detecting antibodies to fluke cysteine proteinases for immunodiagnosis of human paragonimiasis and fascioliasis

Enzyme-linked immunosorbent assays (ELISAs) which detect specific antibodies to fluke cysteine proteinases have provided good sensitivity and specificity for the immunodiagnosis of trematode diseases. To detect specific antibodies without the need for purified proteinase antigens, an immunocapture assay using Protein A was applied for the immunodiagnosis of paragonimiasis and fascioliasis. ELISA plate wells were coated with Protein A, incubated with diluted patient sera, then incubated with a preparation containing fluke cysteine proteinases, excretory–secretory (ES) products of adult Paragonimuswestermani or Fasciola sp. The activity of fluke cysteine proteinases bound on the wells was measured by adding fluorogenic peptidyl substrate, Z-Phe-Arg-MCA or Boc-Val-Leu-Lys-MCA. This assay detected specific immunoglobulin G to cysteine proteinases of P. westermani and Fasciola sp. by measuring proteinase activity on the plate wells. Patient sera showed significant high values of proteinase activity when the wells were treated with the respective homologous ES products, whereas the sera had low values after treatment with the heterologous ES products. The sera of patients with other parasitoses and uninfected healthy individuals also showed low values after treatment with the above fluke ES products. Thus, Protein A immunocapture assay, which detected IgG specific for fluke cysteine proteinases, provided a high sensitivity and specificity for immunodiagnosis of paragonimiasis and fascioliasis.