Monoclonal Antibody Directed Against Neospora Caninum Tachyzoite Carbohydrate Epitope Reacts Specifically With Apical Complex–associated Sialylated Beta Tubulin

Abstract

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

Importance of serological cross-reactivity amongToxoplasma gondii, Hammondiaspp.,Neosporaspp.,Sarcocystisspp. andBesnoitia besnoiti

“…MAbs raised against T. gondii reacted with N. caninum tachyzoites by IFAT, but only at low titres (10–40) (Latif and Jakubek, 2008). A number of reports demonstrated the usefulness of MAbs against T. gondii , which do not cross-react with N. caninum (Baszler et al 2001; Uchida et al 2004; Srinivasan et al 2006; Cunha-Junior et al 2010). …”

A Novel Giardia lamblia Nitroreductase, GlNR1, Interacts with Nitazoxanide and Other Thiazolides

“…Mass spectrometry identification of proteins was performed as previously described (40). Liquid chromatography-tandem mass spectrometry data were acquired using an LTQ ion-trap mass spectrometer (Thermo-Electron, Hemel Hempstead, United Kingdom) coupled online to a Thermo-Finnigan surveyor high-pressure liquid chromatography system equipped with a BioBasic C 18 reversed-phase column (100 ϫ 0.18 mm) and submitted to MASCOT to search predicted open reading frames from GiardiaDB (http://gmod.mbl.edu/perl/site /giardia?pageϭintro) and MSDB.…”

Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum

“…Production of mAbs for diagnostic purposes has been widely reported (Cole et al, 1994;Bjorkman and Hemphill, 1998;Schares et al, 1999;Baszler et al, 2001;Srinivasan et al, 2006;Aguado-Martinez et al, 2010;Sohn et al, 2011). Because mAbs are directed against a specific epitope of a parasite antigen, the probability of cross-reaction with closely related parasites is low.…”

“…The mAb technology has been widely used in diagnostic tests to detect N. caninum infection (Cole et al, 1994;Baszler et al, 1996Baszler et al, , 2001Bjorkman and Hemphill, 1998;Schares et al, 1999;Srinivasan et al, 2006;Aguado-Martinez et al, 2010;Sohn et al, 2011). However, the usefulness and reliability of mAbs in IHC using naturallyinfected tissue were not evaluated yet.…”