Analysis of the immunoglobulin isotypes present in the sera of vaccinated mice. (A) Immunoglobulin profile of the sera of vaccinated mice. Three groups of mice were immunized with PBS, BTAA alone, or BTAA combined with NLP weekly for 4 weeks. Seven days after the last injection, the sera were assessed for IgG, IgA and IgM content by ELISA. The data correspond to mean ± SD of four observations. *Significantly higher as compared to the value of the BTAA immunized group (P < 0.01). (B) Isolation of IgG and IgM from the sera of vaccinated mice. Pooled sera from mice immunized with PBS, BTAA alone, or BTAA combined with NLP, were fractionated on a Protein A-Sepharose 4B column. Bound IgG was eluted and flow-through containing Igs (mainly IgM) other than IgG was collected. The OD of these two fractions was adjusted and the relative IgG and IgM content estimated by ELISA. *Significantly higher compared to the value of the BTAA immunized group (P < 0.01). (C) Three groups (n = 4 for each group) of mice (A: Swiss; B: Balb/c) were immunized (i.v.) with PBS, BTAA alone, or BTAA combined with NLP weekly for 4 weeks in total. Seven days after the last injection, sera were assessed for IgG1 and IgG2a anti-BTAA antibodies by ELISA after coating the plate with BTAA. Each bar represents the group mean of IgG2a/IgG1 response.