Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to Sepharose beads or nitrocellulose paper strips.

Two novel immunization methods designed for immunization with small quantities of antigen, immobilized on a solid matrix and without the use of adjuvant, are presented. The major test antigen used in order to evaluate these methods was bovine serum albumin (BSA). It was deposited in the spleen of mice and rabbits, either attached to Sepharose beads (Pharmacia Sepharose 4B) or to nitrocellulose (NC) paper strips (Millipore). BSA was attached to NC by direct application or by electroblotting after SDS-polyacrylamide gel electrophoresis. The antibody response in mouse and rabbit serum, after intrasplenic immunizations with various quantities of antigen, was analyzed in an ELISA standard procedure. In mice, an antibody response in serum was detected after three intrasplenic immunizations with a total quantity of 73.6 ng BSA bound to Sepharose beads and after two immunizations with a total quantity of 800 ng BSA attached to NC. Determination of the antigen-binding to NC and the clearance rate of antigen attached to NC deposited in the spleen of mice was performed with 125I-labeled BSA. In rabbits, an antibody response in serum was detected after a single intrasplenic immunization with 2.6 micrograms BSA attached to NC. When testing human insulin and sheep prolactin, attached to NC, as antigens in intrasplenic immunization of rabbits, an antibody response was found after a total quantity of 3.2 micrograms insulin and 10.5 micrograms prolactin, respectively.