Viability of the trophozoites after treatment was evaluated, leaving the cultures for ten days and analyzing the adherent living cells. Descriptive statistics included the calculation of the means and S.D. of the control and experimental groups. Average counts were compared between Ab treatments for statistical differences using the independent GSK621 molecular weight samples Student’s t-test from the SPSS Statistic program. Results and
discussion Polyclonal antibodies against WB trophozoites are also reactive against GS trophozoites Antibodies against variable specific-surface proteins (VSPs) as well as metabolic enzymes were found in patients infected with Giardia in both an endemic region (León, Nicaragua) and in a non-endemic area during a waterborne outbreak (Sälen, Sweden). There was also strong immunoreaction to antigens associated with the cytoskeleton, including giardins learn more [31, 32]. Therefore, to produce mAbs against giardins, we purified a fraction enriched in cytoskeletal proteins from a lysate of G.
lamblia trophozoites of the WB strain. After subcellular fractionation, each fraction was analyzed, using mAbs against VSP9B10 (non-cytoskeletal proteins) and tubulin (cytoskeletal protein), by dot-blotting (Figure 1A). The VSP9B10 mAb recognized a VSP that is expressed in WB trophozites, Selleckchem ZIETDFMK labeling the surface of the trophozoites, including the flagella . The P1a to P1c fractions were collected, and used as the antigen for mouse immunization. Figure 1 Polyclonal antibody production. (A) Dot-blotting of the subcellular fractionation of WB trophozoites
shows that surface proteins localized mainly in fractions P3 (samples e-g) and weakly in fraction P1 (samples c-e), while cytoskeleton proteins were found in P1 (samples a-c). P1, P2, and P3 corresponded to the fractions of pellet centrifuged at 1,000 × g, 20,000 × g, and 105,000 × g, respectively. (B) Antibody reactivity. Western blotting of a total WB, GS and Portland-1 Giardia lysate incubated with the pre-immune (PI) or the immune polyclonal (pAb) serum. Lane 1: standards of the indicated molecular weights. (C) Reactivity of polyclonal antibodies Ureohydrolase determined by indirect immunofluorescence in WB, GS and Portland-1 trophozoites. PI: control with pre-immune serum. Scale bar: 10 μm. The screening of the polyclonal serum was performed by Western blot and immunofluorescence, in G. lamblia WB and Portland-1(assemblage A) and GS (assemblage B) trophozoites. Western blotting showed several bands in WB and Portland-1, but fewer in GS trophozoites (Figure 1B), with the main band of about 30 kDa found in all samples possibly representing the common immunoreactive protein that has been repeatedly identified in natural Giardia infections [18, 34–36].