Production of potent salmonella H antisera by immunization with flagellae, isolated by immunosorption

For serotyping Salmonella H sera should exhibit a high H titer and preferably a low O titer which does not interfere with the H slide agglutination at the working dilution of the serum. By conventional immunization with motile bacteria OH sera are always produced. We isolated flagellae from highly motile Salmonella cultures, which were detached from the bacterial cells by a waring blendor, with an immunosorbent, i.e. cyanobromide-activated Sepharose to which the respective anti H antibody was coupled. The desorption of the flagella from the immunosorbent was done by lowering the pH to 2.3 with glycine-HC1 buffer. 4.2 mg of flagellar protein were recovered from 20 agar swarm plates. 10 mug of the antigen incorporated into complete Freund adjuvant was found to immunize a rabbit successfully. The H titers varied from 10,000-160,000 and the majority of the O titers did not exceed 160. Only one immunosorbent was necessary to isolate a group of related H antigens (i.e., lv, lw, lz13, lz28). After regeneration the immunosorbent can be reused many times.