Descriptive statistics included the calculation of the means and S.D. cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in ABT-199 (Venetoclax) P15 (Assemblage E) trophozoites. Conclusions We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other. Background em Giardia lamblia /em is a flagellated unicellular microorganism that causes Giardiasis, a generally self-limited clinical illness [1]. Typically, the infection is characterized by diarrhea, abdominal cramps, bloating, Plat weight loss, and malabsorption, although asymptomatic infection also frequently occurs [2]. em G. lamblia /em infection is transmitted by the faecal-oral route and results from the ingestion of cysts through the consumption of contaminated food or water or from person-to-person transmission. em Giardia /em is ABT-199 (Venetoclax) distributed globally and has been detected in nearly all classes of vertebrates, including domestic animals, wildlife and in marine vertebrates [3,4]. Since the 80’s, differences have been observed between different isolates of em Giardia /em , both in isoenzyme studies and in surface-antigen, as well as in the DNA banding pattern after endonuclease restriction analysis, giving rise to the hypothesis that these differences might explain the various clinical manifestations, host responses and treatment efficacy of human Giardiasis [5-7]. Nowadays, advances in molecular epidemiology have enabled specialized genetic groups (i.e., assemblages) to be identified that are relatively species-specific. Among the eight defined genotypes of em Giardia /em , only assemblages A and B are known to infect humans, and these two have shown differences related to axenic em in vitro /em culture conditions [8-10], metabolism, biochemistry, DNA content, and clinical features, among others [4,11-13]. All these biological differences may be explained by genetic as well as genomic differences, such as the presence of isolate-specific proteins, unique patterns of allelic sequence divergence, differences in genome synteny and in the promoter region of encystation-specific genes and differences in VSP repertoires [14]. It has, therefore, been suggested that assemblages A and B could be considered to be two different em Giardia /em species. During the vegetative stage of the parasite, the trophozoite attaches to the intestinal microvilli to colonize and to resist peristalsis. The ventral disc allows the parasite to orient, ventral side down, to biological or inert substrates, and is a concave cytoskeletal structure surrounded by a plasma membrane, composed of ABT-199 (Venetoclax) 3 distinct features (microtubules that are coiled around a bare area; microribbons that protrude into the cytoplasm; and cross-bridges that connect adjacent microtubules) [15]. Three gene families of giardins generally localize to the ventral disc including: (i) annexins (i.e. -giardins) that are localized at the outer edges of microribbons [16-21]; (ii) striated fiber-assemblins such as -giardin, which are closely associated with microtubules and -giardin (a component of microribbons) [22,23]; and (iii) -giardin, which is also a microribbon protein [24]. Alpha-giardins form a large class of proteins encoded by 21 different genes (named -1 to -19). All of these 21 alpha-giardin genes in WB were found to be conserved in GS along with the genome synteny, although the structural protein alpha-2 giardin was postulated to be an assemblage A-specific protein of human infective em G. lamblia /em [25]. However, in a recent study, Franzn et al. encountered a -2 giardin-like gene in the assemblage B GS strain, with a 92% aa identity in a syntenic position [14]. Differences occurring in the structural proteins may explain the differences observed in key infection processes such as adhesion and motility between both assemblages. To date, the intracellular localization of giardins in em G. lamblia /em has been performed using rabbit polyclonal antisera or by the use of epitope tagged -giardins [19,26]. However, both these methods have limitations when attempting to study assemblages A and B because polyclonal antibodies have shown cross-reaction with other proteins, while transfection experiments are difficult to carry out on GS assemblages [27]. Therefore, we developed monoclonal antibodies (mAbs) against the.