Four to six week-old female BALB/c mice were immunized via the footpad with 10 µg Stx1 or 3 µg Stx2 toxoid adsorbed to 250 µg aluminum hydroxide. The immunization protocols consisted of three booster injections of the toxoid (10 µg) in 0.01 M phosphate buffered saline, pH 7.4 (PBS) at four-week intervals for Stx1 toxoid, and two booster injections (15 µg) with a 15-day interval for Stx2 toxoid. The experiments were conducted in agreement with the Ethical Principles in Animal Research, adopted by the Brazilian College of Animal Experimentation, and they were approved by the Ethical Committee for Animal Research of Butantan Institute (469/08).
The mouse with the highest antibody titer was boosted with 10 µg Stx1 or 15 µg Stx2 toxoid three days prior to cell fusion. Serum samples were obtained just before the first immunization by the retro-orbital sinus method to be used as the negative control in specific antibody evaluation. Serum samples were also obtained ten days after the last antigen injection and subsequently analyzed by ELISA.
The popliteal lymphnode cells were fused with SP2/O-Ag14 mouse myeloma cells (2:1) using polyethylene glycol 1500 [37 ], with modifications. Hybrids were selected in RPMI 1640 medium plus 3% HAT containing 10% FBS at 37 °C and 5% CO2. The supernatant fluids were screened for species-specific antibodies by indirect ELISA.
For ELISA, hybridoma supernatant (100 µL) was added to wells of a 96-well plate previously coated with 0.1 µg-purified toxins to screen cultures for antibody production. Antibody-secreting cells were expanded and cloned twice at limiting dilution. Hybridomas secreting MAbs were selected using STEC and other non-producing Stx isolates by capture ELISA.
The mouse with the highest antibody titer was boosted with 10 µg Stx1 or 15 µg Stx2 toxoid three days prior to cell fusion. Serum samples were obtained just before the first immunization by the retro-orbital sinus method to be used as the negative control in specific antibody evaluation. Serum samples were also obtained ten days after the last antigen injection and subsequently analyzed by ELISA.
The popliteal lymphnode cells were fused with SP2/O-Ag14 mouse myeloma cells (2:1) using polyethylene glycol 1500 [37 ], with modifications. Hybrids were selected in RPMI 1640 medium plus 3% HAT containing 10% FBS at 37 °C and 5% CO2. The supernatant fluids were screened for species-specific antibodies by indirect ELISA.
For ELISA, hybridoma supernatant (100 µL) was added to wells of a 96-well plate previously coated with 0.1 µg-purified toxins to screen cultures for antibody production. Antibody-secreting cells were expanded and cloned twice at limiting dilution. Hybridomas secreting MAbs were selected using STEC and other non-producing Stx isolates by capture ELISA.
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