Antigen Recognition and the Immune Response

Abstract

Based on the original observation that L-tyrosine-azobenzene-p-arsonate (RAT) I induced specific delayed hypersensitivity in guinea pigs (1), we have shown that this small molecule could serve as a carrier for a macromolecular hapten (2). Conjugates of RAT and poly-3'-n-glutamic acid (PGA) raised humoral antibody specific for the PGA determinant and cellular immunity directed against the RAT determinant in guinea pigs. In addition, prior immunization with RAT potentiated the anti-hapten response (2). These observations prompted the conclusion that RAT, although having a molecular weight of only 409, is a true immunogenThe R A T -P G A conjugates had, on the average, 9-12 R A T groups per molecule of PGA. In the present communication, we report the preparation and use of better defined, small bifunctional antigen molecules composed either of one R A T carrier moiety and one dinitrophenyl (DNP) haptenic group or of two R A T moieties, separated b y spacers of varying size. These molecules permitted an exploration of the spatial relationships between carrier and hapten required for a humoral antibody response, as well as the question of "self-help." The latter refers to the ability of a carrier determinant to cooperate in the humoral response to a second identical determinant. In addition, the structural features of R A T which are essential for immunogenicity have been investigated.

Antigen Recognition and the Immune Response

“…DISCUSSION" RAT is probably the smallest molecule which has been shown to be immunogenic and, as such, offers an excellent model for study of the immune response at molecular and cellular levels. It has been shown that RAT can serve as a carrier for haptens (2,3), and bifunctional antigen molecules have been fabricated to explore various molecular parameters of the immune response such as minimum essential spacing of determinants and the capacity to induce "selfhelp": the cooperation of an immunogenic determinant in the humoral response to a second identical determinant (3). In addition, the antihapten antibodies produced in response to small bifunctional antigens appear to be substantially less heterogeneous by isoelectric focusing than antibodies provoked by conventional hapten-protein conjugates (Roelants, G. E., and J. W. Goodman, unpublished observations), suggesting that RAT may have general utility as a carrier for the preparation of relatively homogeneous anti-hapten antibody of virtually any specificity.…”

“…Modifications at the arsonate position yielded immunogens with distinctive specificities (3). Preliminary findings with compounds modified at the tyrosine position indicated that one, but not both, charged groups were essential for immunogenicity (3).…”

“…Indeed, the activity of RAT parallels that of conventional macromolecular immunogens. In addition to the induction of delayed cutaneous sensitivity, it triggered the release of migration-inhibitory factor and a proliferative response by cells from sensitized animals, and it prepared guinea pigs for secondary anti-hapten responses (2,3).…”

Antibodies to Hepatitis B Surface Antigen (HBsAg) Elicited by Immunization with a Synthetic Peptide Covalently Linked to Liposomes

“…Treatment of stearylamine-containing liposomes with glutaraldehyde provides reactive groups suitable for linkage of synthetic peptides and at the same time increases the rigidity of the lipid membranes. Such liposomes, especially when containing carrier function-enhancing RAT sites (Alkan et al, 1971(Alkan et al, , 1972, appear to be a promising tool for preparing fully synthetic immunogens for eliciting antiviraI antibodies.…”

Antigen Recognition and the Immune Response

“…Included among these are the minimum separation between haptenic and carrier determinants required for an anti-hapten antibody response and whether "self-help," the capacity of an immunogenic determinant to cooperate in the humoral response to a second identical determinant, is possible (3). Asymmetric bifunctional molecules consisting of one RAT determinant and one dinitrophenyl (DNP) determinant separated by spacers of various size consistently induced anti-DNP antibody and cellular immunity specific for the RAT group (3). On the other hand, symmetrical bifunctional molecules composed of two RAT determinants separated by the same spacers were unable to provoke either primary or secondary anti-RAT humoral responses (3), although it is well known that the arsonate group is a potent hapten.…”