Site-directed immobilization of proteins.

To determine if immobilization chemistry can be used to orient antibody on a support so that the bivalent binding potential can be fully utilized, we developed three activated matrices that couple to different functional groups on the molecule. When AminoLink Gel was used to couple antibody randomly through primary amino groups, the molar ratio of immobilized antibody to recovered antigen averaged 1:1. Iodoacetyl groups on SulfoLink Gel couple through sulfhydryls in the hinge region of the antibody molecule, in theory leaving the antigen binding site available. However, the antibody-to-antigen molar ratio was only slightly improved. Hydrazide groups on CarboLink Gel couple to aldehyde groups generated by oxidation of carbohydrate moieties that are located primarily on the Fc portion of the antibody molecule. The molar ratio of immobilized antibody to purified antigen using CarboLink Gel reached the optimum of 1:2. CarboLink Gel is most effective at orienting antibody for better antigen purification capability.

Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells.

Cationization of BSA generates a molecule that mounts antibody responses of increased magnitude and duration and induces T cell proliferation at concentrations 500 times less than native BSA (nBSA). To explain the alteration in immunogenic properties of this Ag, the uptake of nBSA and cationized BSA (cBSA) by splenic APC has been investigated. T cell proliferation assays were conducted with nBSA and cBSA preparations with varying degrees of substitution. An inverse correlation between the degree of cationization and the amounts of Ag needed for optimal T cell reactivity was observed. To determine whether affinity for APC resulted in an increased uptake of cBSA, splenic APC were incubated with nBSA or cBSA for varying amounts of time. Comparisons were made at each time point between untreated Ag-pulsed APC (Ag uptake) and paraformaldehyde-fixed Ag-pulsed APC (processed Ag). Proliferation of T cells primed with nBSA or cBSA increased in proportion to the amount of time of APC exposure to high concentrations of nBSA, first appearing after a 2-h pulse and peaking at 8 h. Conversely, untreated APC needed only a 30-min cBSA exposure to induce either nBSA- or cBSA-primed T cell proliferation, indicating a rapid uptake of cBSA. Comparisons with proliferation induced by paraformaldehyde-fixed cBSA APC indicate that nBSA T cells recognize a lag phase-processed form of cBSA, whereas a majority of cBSA T cells recognize either a rapidly processed form of cBSA, or a membrane-processed cBSA molecule without a classical lag phase processing event. When monensin was used as an inhibitor of fluid phase pinocytosis in splenic APC, the presentation of nBSA was inhibited by 85%, but the presentation of cBSA was inhibited by only 20%. These results imply that nBSA enters the cell by fluid phase pinocytosis, whereas cBSA enters by a nonspecific adsorptive mechanism. The different modes of cellular entry for the two molecules, nBSA and cBSA, resulting in a rapid uptake of cBSA, may have important ramifications on T cell activation and immunoregulation.

Cationization of protein antigens. III. Abrogation of oral tolerance.

The immunoregulatory effects of oral pretreatment of BDF1 mice with cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been compared. Oral administration of nBSA suppressed the antibody response to both forms of the antigen. In contrast, oral pretreatment with cBSA greatly enhanced the anti-BSA response in animals subsequently challenged i.p. with either the cationized or native form of the molecule. The enhancement observed with cBSA pretreatment was more pronounced than the suppression observed with the same amount and number of feedings of nBSA. As little as a single oral dose of 10 mg of cBSA produced a significant increase in antibody concentration. Cell transfer of spleen cells into irradiated syngeneic recipients demonstrated that both T cells and B cells were involved in the generation of the response, with a greater degree of enhancement provided by cBSA-pretreated T cells. These data extend our previous findings and demonstrate that administration of cBSA by a normally "tolerogenic" route results in enhancement rather than suppression of the immune response.

Cationization of protein antigens. II. Alteration of regulatory properties.

Immunoregulatory effects of cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been investigated. Intravenous administration of nBSA to BDF1 mice substantially suppressed the antibody response to subsequent immunization with either nBSA or cBSA, whereas pretreatment with cBSA by the same route significantly enhanced the responses to both antigens. The functional properties of BSA-specific T and B cells from mice immunized with cBSA or nBSA were examined in reconstitution experiments in which splenic T populations together with B cells were transferred into irradiated syngeneic recipients. Transfer of splenic T cells from mice primed with nBSA caused profound suppression of the response to subsequent immunization with nBSA or cBSA, whereas transfer of either B or T cells from cBSA treated mice produced an enhanced response to both antigens. C57BL/6 mice, which are considered to be low responders to BSA, produced a significant antibody response to BSA when immunized with cBSA. In contrast, immunization with nBSA did not produce measureable amounts of antibody in mice of this strain. Our data clearly demonstrate that cationized BSA exhibits unique immunogenic properties due to alterations in the self-regulation of the immune response.