XXXX : purification of saline-extractable proteins by isoelectrofocusing.

@entity19 @entity1735 @entity8857 @entity2975 @entity12196 @entity291 @entity214
A procedure was developed for separating antigens associated with a saline extract of @entity8857 serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the @entity1735 antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and @entity214 - @entity2975 gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of @entity19 with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent @entity8857 was seen in @entity19 injected with antigen Groups 2 and 4. Six @entity291 were immunized with saline extract. These @entity291 had greater resistance to experimental pneumonic @entity12196 than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated @entity291 by an enzyme-linked immunosorbant assay.
