Antibodies production

Rabbit anti-CS21, anti-LngB, and anti-LngC sera were produced by immunization of New Zealand white rabbits with either purified CS21 obtained from the ETEC strain E9034AΔlngR::km (Saldaña et al., unpublished data) or C-terminal His-Tag LngB and LngC purified proteins. Rabbits were immunized every 2 weeks, and emulsions in complete Freund's adjuvant (1 dose with 1 μg of antigen in 500 μl of PBS + 500 μl of adjuvant) or incomplete Freund's adjuvant (3 doses with 0.5 μg of antigen in 500 μl of PBS + 500 μl of adjuvant) were subcutaneously administered. The antisera obtained were adsorbed 8 times against E9034AΔlngA, E9034AΔlngB, and E9034AΔlngC strains respectively, to remove nonspecific antibodies and increase the specificity. The antisera were used in Western blot (WB), immunofluorescence (IF), and immunogold assays, as described below.