Finally, five MAbs recognized a truncated ORF2 encompassing the C-terminal 394C660 aa region however the target epitope was not detected in any of its three sub-fragments examined (Table 1, Figure 4)

Finally, five MAbs recognized a truncated ORF2 encompassing the C-terminal 394C660 aa region however the target epitope was not detected in any of its three sub-fragments examined (Table 1, Figure 4). DNA was cloned into the vector pCR?-Blunt by the Zero Blunt PCR Cloning Kit (Life Technologies, Carlsbad, CA, USA) and sequenced. 2.2. Production of Recombinant Antigens A series of recombinant proteins encompassing the entire ORF2 antigen were expressed: the entire ORF2 (1C660 aa), its C-terminal portion (394C660 aa), and five 100C120 aa long ORF2 peptides (named peptide 1 to 5) were expressed in and baculovirus were conducted by sub-cloning the coding region of interest into pQE30 (Qiagen, Hilden, Germany) and pFastBac (Life Technologies, Carlsbad, CA, USA) vectors, respectively. The production of the proteins was described previously [21]. The proteins expressed in were purified by His-tag at the N-terminal position using a Ni-NTA resin according to the manufacturers instructions (Qiagen Hilden, Germany). All the proteins expressed were checked by SDS-PAGE and indirect ELISA. The entire (1C660 aa) and the truncated ORF2 (112C608 aa) were analyzed by western blotting using serum from an experimentally infected pig. The western blotting is described below. 2.3. Production and Characterization of Monoclonal Antibodies Two BALB/c mice were immunised with 2C3 doses of 80 g per dose of the purified ORF2 1C660 aa protein. RKI-1313 The fusion of mouse splenocytes and NS0 myeloma cells was conducted according to standardised procedures within the laboratory. Hybridomas supernatants were screened by indirect ELISA against the homologous antigen ORF2 (1C660 aa). The isotype of the MAbs was determined using a commercial ELISA kit (BD-Pharmingen, Franklin Lakes, NJ, USA) following the manufacturers instructions. MAbs were characterized by western blotting and mapped by indirect ELISA assays against all the recombinant constructs expressed by of bovine albumin and 0.05% of Tween 20. MAb binding was detected by incubation with HRP-labelled rabbit anti-mouse IgG and the chemiluminescent substrate Amersham ECL plus (GE healthcare, Chicago, IL, USA). Swine sera were diluted 1/20 in the phosphate buffer described above. Antibody binding was detected by incubation with an HRP-conjugated anti-swine IgG monoclonal antibody using a chemiluminescent substrate as described above. 2.6. Indirect ELISA An Indirect ELISA was conducted to map the MAbs and study the immunological profile of five ORF2 peptides and the ORF3 antigen with swine field serum samples. Optimal coating dilutions RKI-1313 of all the antigens used for MAbs mapping were previously determined using the serum from immunized mice, ORF2 peptides and ORF3 antigen were also pre-titrated using known HEV positive and negative pig sera. The purified antigens expressed in were adsorbed in a carbonate-bicarbonate buffer onto ELISA microplates (Nunc Maxisorp, Thermo Scientific, Waltham, MA, USA) by overnight incubation at 4 C. After washing, MAbs (hybridomas cultures Rabbit Polyclonal to DUSP16 supernatants 1/2 diluted) or serum samples (1/100 dilution) were added and their reaction was monitored after washes by HRP-conjugated anti-mouse or anti-swine IgG at the appropriate dilution. The diluting buffer was PBS with 0.05% Tween 20 and 1% yeast extract; 50 L/well of reagent and 1 h of incubation at 37 C were the conditions adopted for both steps. Then, the substrate OPD (o-Phenylenediamine 0.5 mg/mL in phosphate-citrate buffer pH 5.6 supplemented with 0.02% H2O2) was added for 10 min at room temperature. The reaction was stopped with 50 L of 1M H2SO4 and absorbance values were read at 492 nm. When serum samples were tested, sera were delivered in duplicate wells, one coated RKI-1313 with the antigen and one conditioned with carbonate buffer alone as a negative antigen control, and a 1% of negative extract was added to the serum dilution buffer to block non-specific reactions when expressed proteins were used. For each tested and control serum, the RKI-1313 net Optical Density (OD) value was calculated by subtracting the OD value of the well without antigens from the OD of the well containing the antigen. The variation between plates and experiments was normalized by calculation of the percentage of positivity to a positive control serum present in each plate according to the formula: (net OD sample/net OD positive control) 100. 2.7. MAb Competitive Binding ELISA The assay was conducted in ORF2 (1C660 aa) coated microplates (Nunc Maxisorp, Thermo Scientific, Waltham, MA, USA). Fifty microliters of hybridoma culture supernatants were incubated sequentially diluted from 1/2, followed by the addition of 25 L of HRP-conjugated MAb. After washing, the colorimetric reaction was developed as previously described. Results were expressed as the percentage inhibition of the binding of HRP-conjugated MAb. The amount.