A method for the preparation of protein-protein conjugates of predetermined composition.

A novel procedure for the synthesis of well-defined protein-protein conjugates is described using ovalbumin (OA) and IgG as test proteins. This procedure involves the highly selective and rapid reaction of alkyl halide and sulfhydryl groups, which have been grafted, respectively, onto the proteins to be conjugated. Accordingly, iodoacetylated IgG, (ICH2CO)nIgG, was prepared by the reaction of the epsilon-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA), the degree of iodoacetylation (n) being proportional to the concentration of NHIA. OA was reacted with S-acetylmercaptosuccinic anhydride (SAMSA) under conditions yielding, on the average, a monosubstituted derivative. Following removal of the protective S-acetyl group, the resulting -SH derivative of OA was reacted with (ICH2CO)nIgG. The OAx-IgG conjugates so produced were characterized by gel filtration, specific radioactivity (using tritiated OA) and immunodiffusion. It was found that the average number of OA molecules coupled per IgG molecule could be controlled by varying the degree of iodoacetylation of IgG.

The role of antigen-presenting cells in the IgE antibody response. I. The induction of high titer IgE antibody responses in IgE high-responder and low-responder mice by the administration of antigen-pulsed macrophages in the absence of adjuvants.

The administration of 2 i.p. injections of 2 x 10(7) ovalbumin- (OA) pulsed, syngeneic, adherent, peritoneal exudate cells (OA-pulsed macrophages [M phi]) to (C57BL/6 x DBA/2)F1 (B6D2F1) mice promoted a persistent and high-titer anti-OA IgE response in these mice. Similarly, 2 i.p. injections of OA-pulsed DBA/2 parental M phi also induced the generation of an anti-OA IgE response in B6D2F1 mice; an identical procedure using OA-pulsed C57BL/6 parental M phi, on the other hand, elicited in most cases a very weak to negligible anti-OA IgE response. The administration of 3 i.p. injections of OA-pulsed syngeneic M phi to SJL mice also induced these "IgE nonresponder" mice to develop an anti-OA IgE response that was both boosterable and of moderate to high titer. The significance of these results with regard to the induction and regulation of the IgE antibody response is discussed.

Regulation of the murine IgE antibody response. III. Analysis of the IgE response to insulin species variants.

IgE responses to insulin species variants i.e. to bovine, ovine and porcine insulins (respectively BI, OI and PI), were investigated. H-2d mice developed an anti-BI IgE response when this antigen was administered i.p. with A1(OH)3 gel, and H-2b and H-2a,d,k mice generated anti-BI and anti-OI IgE responses, respectively, when these antigens were injected i.p. with Freund's complete adjuvant (FCA). H-2d mice, on the other hand, developed only weak to negligible anti-PI IgE responses, when this antigen was administered in A1(OH)3 or in FCA. Antibody cross-reactivity indicated that at least two populations of IgE antibodies were produced in response to BI and/or OI; one population specific for the A chain loop and a second population specific for a determinant common to several insulins. Finally, i.v. injections of conjugates of insulin with spleen lymphocytes markedly suppressed the capacity of mice to develop an anti-insulin IgE response.

Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation.

Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.

The suppression of insulin-specific immune responses by the administration of insulin-derivatized syngeneic cells and soluble lysates from these cells.

The results of this study demonstrated that the i.v. administration of insulin, in the form of a conjugate with either syngeneic spleen cells (SC) or peritoneal exudate cells (PEC), markedly reduced the capacity of recipient mice to develop insulin-specific immune responses, as manifested by diminished in vivo IgE antibody production and by depressed in vitro, lymph node cell proliferation responses, respectively. Furthermore, it was shown that i.v. injection of insulin-PEC conjugates induced the activation of suppressor cells that had the capacity to downregulate insulin-specific IgG plaque-forming cell (PFC) responses. Finally, it was also determined that freezing and thawing of the insulin-PEC conjugates resulted in the release of a soluble tolerogenic molecule and/or membrane preparation that could also markedly depress insulin-specific IgG antibody production.

Immunochemical properties of some monoclonal IgE antibodies to 4-hydroxy-3-nitrophenylacetyl (NP).

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (gamma, kappa) tumour cells with spleen cells from (BALB/c X C57B1/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-epsilon-3.57, NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31 all possessed lambda 1 (or possibly lambda 3) light chains; and that NP-epsilon-3.57 possessed lambda 2 light chains; NP-epsilon-95.31 also expressed the P3-X20 derived, MOPC-21 kappa light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-epsilon-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells.