, 2002). The assays were performed in triplicate and the venom encapsulation efficiency (EE%) was calculated using Eq. (1) ( Gan et al., 2005): equation(1) EE%=(totalamountprotein−freeamountproteininsupernatant)(totalamountprotein)×100

Experimental mice were immunized for 6 weeks with 100 μL of subcutaneous injections administered bilaterally in the lumbar region with T. serrulatus venom in different concentrations (5.0 or 10.0%), encapsulated in chitosan nanoparticles or associated with the aluminum hydroxide. The experimental mice were bled by cardiac puncture. Blood samples in the absence of an anticoagulant, were incubated at 37 °C for 30 min and then at 4 °C for Thiazovivin molecular weight 2 h for clot retraction. Then the samples were centrifuged at 15,000 × g for 5 min at 4 °C and the supernatant (serum) collected and stored at −20 °C. Antigen-specific

serum antibody responses were measured 1 week following the booster vaccination by ELISA. The ELISA assay was performed following the protocol of eBioscience. The plate was sensitized with 100 μL/well of capture antibody in coating buffer, the plate was sealed and incubated overnight at 4 °C. After this step, the wells were washed twice with wash buffer solution with 400 μL. The wells were blocked with 250 μL of blocking buffer and incubated at room temperature for 2 h. After washing the plate again two-fold serial dilutions of the standards were performed with assay click here buffer to make the standard curve. For each well 100 μL of assay buffer were added to the blank wells and 90 μL added to the sample wells, after this 10 μL/well of prediluted samples were added in assay buffer to the appropriate wells and 50 μL/well of diluted detection-antibody

was added to all wells. The plate was sealed and incubated at room temperature for 3 h. After washing, substrate was added and the plate incubated at room temperature for 15 min. The reaction was stopped and the plate read at 450 nm. The molecular protein profile of T. serrulatus venom was determined by polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS-PAGE) either and is shown in Fig. 1. This data showed several protein fractions, divided into low and medium molecular weight comprising of around 30.0–60.0 kDa. The particles were formed spontaneously by intra- and intermolecular bonds between the phosphate groups of TPP and the amine of chitosan (Gan and Wang, 2007). The formation of an opalescent final dispersion was observed, characteristic of the presence of nanoparticles (Gan et al., 2005), which was confirmed by particle size analysis. The nature of interactions between the chitosan and TPP was established with FT-IR spectroscopy, since any kind of physicochemical interaction between chitosan and TPP will automatically lead to frequency shifts or splitting in absorption bands. FT-IR spectra of chitosan nanoparticles and chitosan matrix are shown in Fig. 2.