Polyclonal antisera against recombinant E. histolytica Nbp35 or Cfd1 was raised in adult rabbit by three repeated subcutaneous injection. Pre-immune sera was collected before immunization and first dose of 300 µg Nbp35 or Cfd1 proteins emulsified in complete Freund’s adjuvant was injected 8–10 places subcutaneously; followed by three booster doses of 250 µg of proteins emulsified in Freund’s incomplete adjuvant. Anti-Nbp35 & Cfd1 titres were checked by ELISA after three weeks of final immunization. Finally, serum was collected from rabbits and stored at −30°C in small aliquots. Working antibodies were stored at 4°C. Prior animal ethical committee approval was taken and recommendations were strictly followed.
Cell lysate of E. histolytica (∼1×107 cells/ml) was prepared using lysis buffer (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1.0 mM EDTA, 1.0 mM DTT, 10% glycerol) as described previously [24] (link). The protein fractions were resolved by 10% SDS-PAGE and electro-blotted on to nitrocellulose membrane. The membrane was probed with polyclonal anti-Nbp35 or Cfd1 sera (1∶2000) raised in rabbit as mentioned above. ALP-conjugated goat anti-rabbit IgG (1∶2000) was used as secondary antibody and blot developed with BCIP/NBT (Santa Cruz), as per manufacturer’s instructions [48] .
Cell lysate of E. histolytica (∼1×107 cells/ml) was prepared using lysis buffer (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1.0 mM EDTA, 1.0 mM DTT, 10% glycerol) as described previously [24] (link). The protein fractions were resolved by 10% SDS-PAGE and electro-blotted on to nitrocellulose membrane. The membrane was probed with polyclonal anti-Nbp35 or Cfd1 sera (1∶2000) raised in rabbit as mentioned above. ALP-conjugated goat anti-rabbit IgG (1∶2000) was used as secondary antibody and blot developed with BCIP/NBT (Santa Cruz), as per manufacturer’s instructions [48] .