Targets for the diagnosis of <i>Acanthamoeba</i> eye infections include four cyst wall proteins and the mannose-binding domain of the trophozoite mannose-binding protein

<i>Acanthamoebae</i>, which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not quickly diagnosed and effectively treated. The walls of <i>Acanthamoeba</i> cysts contain cellulose and have two layers connected by conical ostioles. Cysts are identified by <i>in vivo</i> confocal microscopy of the eye or calcofluor-white- or Giemsa-labeling of corneal scrapings, both of which demand great expertise. Trophozoites, which use a mannose-binding protein to adhere to keratinocytes, are identified in eye cultures that delay diagnosis and treatment. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here, confocal microscopy of rabbit antibodies (rAbs) to recombinant Luke, Leo, Jonah, and laccase supported localizations of GFP-tagged proteins in walls of transfected <i>Acanthamoebae</i>. rAbs efficiently detected calcofluor white-labeled cysts of 10 of the 11 <i>Acanthamoeba</i> isolates tested, including six T4 genotypes that cause most cases of keratitis. Further, laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. Structural and experimental methods identified the mannose-binding domain (ManBD) of the <i>Acanthamoeba</i> mannose-binding protein, while rAbs to the ManBD efficiently detected DAPI-labeled trophozoites from all 11 <i>Acanthamoeba</i> isolates tested. We conclude that antibodies to four cyst wall proteins and the ManBD efficiently identify <i>Acanthamoeba</i> cysts and trophozoites, respectively.IMPORTANCEFree-living amoeba in the soil or water cause <i>Acanthamoeba</i> keratitis, which is diagnosed by identification of unlabeled cysts by <i>in vivo</i> confocal microscopy of the eye or calcofluor-white (CFW) labeled cysts by fluorescence microscopy of corneal scrapings. Alternatively, <i>Acanthamoeba</i> infections are diagnosed by the identification of trophozoites in eye cultures. Here, we showed that rabbit antibodies (rAbs) to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently identify CFW-labeled cysts of 10 of the 11 <i>Acanthamoeba</i> isolates tested. Further, laccase released into the medium by encysting <i>Acanthamoebae</i> was detected by an enzyme-linked immunoassay. We also showed that rAbs to the mannose-binding domain (ManBD) of the <i>Acanthamoeba</i> mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identify DAPI-labeled trophozoites of all 11 <i>Acanthamoeba</i> isolates tested. In summary, four wall proteins and the ManBD appear to be excellent targets for the diagnosis of <i>Acanthamoeba</i> cysts and trophozoites, respectively.