Wongwairot Sirima, Kueakhai Pornanan, Changklungmoa Narin, Jaikua Wipaphorn, Sansri Veerawat, Meemon Krai, Songkoomkrong Sineenart, Riengrojpitak Suda, Sobhon Prasert
Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd, Ratchathewi, Bangkok, 10400, Thailand.
Parasitol Res. 2015 Jan;114(1):133-40. doi: 10.1007/s00436-014-4170-8. Epub 2014 Oct 17.
Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.
组织蛋白酶Ls（CatLs）是片形吸虫分泌的主要半胱氨酸蛋白酶，对寄生虫的消化和组织侵袭至关重要。巨片形吸虫组织蛋白酶L1H（FgCatL1H）是在迁移和侵袭早期表达的同种型。在本研究中，利用用重组前FgCatL1H（rproFgCatL1H）免疫的BALB/c小鼠的脾细胞，通过杂交瘤技术制备了抗重组巨片形吸虫组织蛋白酶L1H（rFgCatL1H）的单克隆抗体（MoAb）。该MoAb是具有κ轻链同种型的免疫球蛋白（Ig）G1。该MoAb与rproFgCatL1H、尾蚴和新脱囊幼虫（NEJ）全身提取物中分子量（MW）为38至48 kDa的天然FgCatL1H特异性反应，并通过免疫印迹和间接ELISA与2周龄和4周龄幼虫、成虫以及成虫排泄-分泌（ES）组分的WB中MW为25至28 kDa的rFgCatL1和天然FgCatLs交叉反应。它与其他寄生虫的WB组分中的抗原无交叉反应，这些寄生虫包括扩展巨槽绦虫、鹿同盘吸虫、瘤胃腹袋吸虫、胰阔盘吸虫、唇乳突丝虫和柯氏菲策吸虫。通过免疫定位，抗rFgCatL1H的MoAb与尾蚴和NEJ肠道中的天然蛋白反应，也与2周龄和4周龄幼虫以及成年巨片形吸虫中的CatL1交叉反应。因此，FgCatL1H及其MoAb可用于反刍动物和人类早期和晚期片形吸虫病的免疫诊断。
