Molecular heterogeneity of secretory IgA anti-DNP antibodies

Evidence is presented that demonstrates that the microscale sucrose isoelectric focusing technique can be used to assess the molecular heterogeneity of secretory IgA anti-DNP antibodies. The data indicate that substitution of the DNP group on the bacterial carrier, pneumococcus, can limit the structural complexity of secretory IgA anti-DNP antibodies. The data support the concept that factors governing secretory IgA antibody heterogeneity are analogous to those influencing the heterogeneity of serum IgG antibodies.     

Monoclonal IgE-dependent eosinophil cytotoxicity to haptenated schistosomula of Schistosoma japonicum: enhancement of the cytotoxicity and expression of Fc receptors for IgE by Nippostrongylus brasiliensis infection

To obtain direct evidence for the involvement of IgE antibodies in eosinophil-mediated killing of schistosomula of S. japonicum, dinitrophenylated (DNP) schistosomula pretreated with mouse monoclonal IgE antibodies were co-cultured with purified rat peritoneal eosinophils. It was found that the eosinophil-mediated adherence and damage to haptenated schistosomula were dependent on a monoclonal anti-DNP IgE antibody, but not on monoclonal anti-ovalbumin IgE antibody. Moreover, eosinophils from N. brasiliensis-infected rats demonstrated an enhanced ability in the IgE-dependent damage to DNP-schistosomula as compared with the cells from normal rats. The enhancement was associated with an increase in the proportion of eosinophils expressing Fc receptors for IgE.     

Tolerance defects in New Zealand Black and New Zealand Black X New Zealand White F1 mice

The susceptibility of autoimmune NZB and (NZB X NZW)F1 mice to the induction of tolerance by monomeric BSA was compared with several normal mouse strains. Unresponsiveness in T and B lymphocyte compartments was probed by challenging with DNP8BSA and measuring anti-DNP and anti-BSA antibodies separately. Tolerance induced by monomeric BSA was carrier specific, and there was no evidence of epitope-specific suppression. Normal NZW, NFS, and B10.D2 mice were easily rendered tolerant with monomeric BSA and did not produce anti-DNP or anti-BSA antibodies after challenge with DNP8BSA. By contrast, the lack of anti-DNP antibody response in similarly treated NZB mice was dependent on the dose of monomeric BSA, indicating that the helper T cells were partially resistant to tolerance induction. NZB mice treated with a high dose of monomeric BSA produced anti-BSA, but not anti-DNP, antibodies after immunization. Thus, the anti-carrier B cells in NZB mice may have been primed by monomeric BSA. The presence of the xid gene on the NZB background rendered the mice susceptible to induction of tolerance, suggesting that the tolerance defect in NZB mice involves the B cell compartment. This abnormal antibody response was a dominant trait: (NZB X NFS)F1 and (NZB X B10.D2)F1 mice had the same characteristics as NZB mice. These F1 hybrids do not develop autoimmune disease, indicating that resistance to experimental tolerance induction expressed at a B cell level may not be sufficient for disease development. In contrast to NZB and other NZB F1 hybrids, (NZB X NZW)F1 hybrids treated with monomeric BSA and challenged with DNP8BSA responded to both DNP and BSA. The contribution of a B cell defect to the tolerance abnormality of (NZB X NZW)F1 mice was examined by analyzing the effect of the xid gene on the progeny of (NZB.xid X NZW)F1 mice. Unlike the effect of the xid gene on NZB mice, both phenotypically normal heterozygous female and phenotypically xid hemizygous male mice produced anti-DNP and anti-BSA antibodies after tolerance induction and immunization, demonstrating that a major helper T cell abnormality was present in (NZB X NZW)F1 mice. The (NZW X B10.D2)F1 hybrid was rendered tolerant by this procedure, indicating that the helper T cell defect (NZB X NZW)F1 mice may have resulted from gene complementation with the NZB mice contributing partial resistance of T helper cells to tolerance induction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effectiveness of different rat IgG subclasses as IgE-blocking antibodies in the rat basophil leukaemia cell model

The degranulation of mast cells in an allergic response is initiated by the aggregation of high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. Recently it has been shown that such degranulation can be inhibited by cross-linking Fc epsilon RI and low-affinity IgG receptors (Fc gamma RII) which are also expressed by mast cells. The ability of various monoclonal antibodies to block the degranulation of rat basophil leukaemia (RBL) cells sensitized with IgE antidinitrophenyl (DNP) antibodies has been investigated. Sensitized cells were challenged with immune complexes formed using varying concentrations of antigen, and of both high- and low-valency antigen. It is reported here that rat IgG1 antibodies, which are associated in the rat with a Th1-type response, act as highly effective blocking antibodies over a wide concentration range. Rat IgG2a antibodies, which are associated with a Th2-type response, were able only to inhibit degranulation when immune complexes were formed with very low concentrations of high-valency antigen (DNP32-HSA). Under these conditions, some inhibitory activity was seen with high-affinity murine IgA anti-DNP but not with low-affinity rat IgG2b anti-DNP antibody-containing immune complexes. In addition to this inhibitory activity, IgG2a antibodies were shown to be capable of inducing degranulation of cells via unoccupied Fc epsilon RI. These results demonstrate that blocking activity may arise via both inhibitory receptors and by masking of antigen.     

Studies on the regulation of igm immune response. VII. changes in the affinity of antibodies and cell receptors after immunization with the T-cell independent antigen DNP-Ficoll.

A/J-mice immunized by a single injection with DNP21-Ficoll respond on the humoral level exclusively with IgM antibodies. The intrinsic association constants (K0) of IgM anti-DNP to monovalent hapten E-DNP-L-lysine are within the range of 105-106 M-1 and do not change significantly during the immune response. On the other hand, the functional association constants (KF) of pentameric IgM to multivalent DNP-T4 bacteriophage increase from 1010 M-1 at 3rd day up to 1012 M-1 at 8th day. Subsequently, a decrease of KF to 1011 M-1 can be observed. This rise and fall of the affinity of IgM antibodies of multivalent DNP-conjugate can be detected at the cellular level also by inhibition of plaque formation. The concentration of DNP15-BSA needed for 50% inhibition of plaque formation (I50) decreases from second day to 8 th day by 4 orders, which represents a strong increase of functional affinity. In contrast, the I50 of E-DNP-L-lysine slightly decreases only until day 4 and does not change until day 21. the inhibition of rosette formation by mono- and multivalent ligands was used to study the affinity of lymphocyte receptors. In the course of immunization antigen-binding cells carrying receptors with increasingly higher affinity for multivalent DNP-conjugates occur. These results are discussed with regard to the importance of functional affinity of lymphocyte receptors for the antigen-driven selection of high affinity anti-DNP-cell clones producing IgM antibodies.     

Contact hypersensitivity reactions to dinitrofluorobenzene mediated by monoclonal IgE anti-DNP antibodies

Several studies have suggested a possible role for IgE antibodies in the pathogenesis of cutaneous hypersensitivity reactions that reach maximum intensity 24 to 48 hr after antigen challenge. The recent availability of murine monoclonal IgE anti-hapten antibodies has made possible the direct examination of the range of cutaneous inflammatory reactions that can be mediated by such antibodies. We have examined the effects of passively sensitizing BALB/c mice with monoclonal IgE anti-dinitrophenyl (DNP) antibody 48 hr before antigen challenge. Inflammatory responses were assessed by measuring ear swelling in mice challenged on the ears with the reactive hapten 2,4-dinitrofluorobenzene (DNFB). Compared with unsensitized controls, the ears of mice passively sensitized with IgE anti-DNP displayed a biphasic pattern of ear swelling after DNFB challenge. An early, transient response (present within 15 to 30 min of challenge and returning to control levels within 4 to 9 hr) was followed by a second, more persistent increase in ear swelling that peaked 24 to 48 hr after challenge. This biphasic pattern of ear swelling seen in IgE-sensitized mice was temporally indistinguishable from that observed in mice conventionally sensitized for allergic contact dermatitis reactions by epicutaneous application of DNFB 5 days before DNFB ear challenge. Antigen specificity of the IgE-mediated contact hypersensitivity reactions was demonstrated by the failure of mice passively sensitized with IgE anti-DNP to display early or delayed ear swelling greater than unsensitized controls when challenged with either of two noncross-reacting haptens, fluorescein isothiocyanate or oxazolone. Mice passively sensitized with a monoclonal IgA anti-DNP antibody (MOPC 315) 48 hr before DNFB challenge failed to display early or delayed ear swelling greater than unsensitized controls. Heat inactivation of the IgE anti-DNP ascitic fluid at 56 degrees C for 30 min completely abolished its capacity to passively sensitize mice for contact hypersensitivity reactions after DNFB challenge. These results document the existence of an antigen-specific, IgE-mediated, delayed-in-time cutaneous hypersensitivity response that can be elicited by epicutaneous challenge (contract) with a reactive hapten.     

In vivo activity of affinity-purified helper factor from antigen-specific helper clone

Supernatant from culture of a virally transformed OVA-specific helper T clone (C-41) was examined for the presence of soluble helper factor. Inoculation of helper clone supernatant into DNP-KLH-primed mice enhanced the IgG anti-DNP response when given with DNP-OVA. The C-41 supernatant did not trigger the DNP-primed B cells in mice when injected with hapten (DNP) coupled to an unrelated carrier (BSA). The carrier-dependent helper activity of C-41 supernatant in vivo demonstrates the presence of an antigen-specific T helper factor in the media of the cultured helper clone. Extensive immunization of F1(C57BL X BALB/c) mice with the helper clone resulted in the production of anti C-41 antibodies. Monoclonal antibodies prepared from the immunized mice were screened for specificity of binding to other transformed T lines and clones, some specific to OVA. Monoclonal antibodies that stained the C-41 cells exclusively were considered clone-specific. Supernatants of the helper clone were passed over columns of anti-clone-specific antibodies. The eluates from three antibodies were active as antigen-specific helper factor, i.e., they elevated the IgG anti-DNP response in vivo in a linked recognition fashion in the presence of DNP-OVA. The affinity-purified factor was inactive when injected with DNP-BSA or DNP-BSA + OVA. Thus, we describe the antigen-specific immune function of a clone-produced helper factor in normal mice.     

Stimulation of the in vivo dinitrophenyl antibody response to the DNP conjugate of L-glutamic acid60-L-alanine30-L-Tyrosine10 (GAT) polymer by a synthetic adjuvant, muramyl dipeptide (MDP): target cells for adjuvant activity and isotypic pattern of MDP-stimulated response

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.     

Preservation of polyclonal antibody production by hybridization

The fusion of unimmunized (Balb/c × SJL)_F1, mouse spleen cells in which a polyclonal response had been induced by bacterial lipopolysaccharide with a myeloma cell line resulted in hybrid cell populations. The hybrid populations obtained elaborated antibody activity to human hemoglobin A, Keyhole Limpet hemocyanin, the dinitrophenyl (DNP) hapten, and human erythrocytes, Thus, hybridization allowed preservation of the normally transitory polyclonal response induced in mouse B cells by lipopolysaccharide. Furthermore, monospecific production of anti-DNP antibody was successfully factored out of the polyspecific production of antibodies by cloning. The expansion and subsequent injection of one of these clones into a (Balb/c × SJL)_F1 mouse resulted in the formation of an antibody-producing tumor that was successfully passed to other (Balb/c × SJL)_F1 recipients. Collection of the serum from tumor-bearing mice provided useful quantities of an anti-DNP antiserum without resort to any program of immunization whatsoever.     

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.     

Mechanism of adjuvant activity of poly A:U on antibody responses to hapten-carrier conjugates

The adjuvant action of poly A:U has been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. Administration of poly A:U at the time of primary immunization with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) shortens the induction period for, and heightens the magnitude of peak anti-DNP antibody and specific memory cell production. In order to define the cellular locus of poly A:U action, the effect of this adjuvant on adoptive secondary anti-DNP antibody responses was studied. Spleen cells from DNP-KLH-primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine gamma globulin (BGG), can be stimulated to do so when an appropriate dose of poly A:U is administered with DNP-BGG. The capacity for poly A:U to exert this effect requires the presence of T lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro prior to adoptive transfer abrogates the response to DNP-BGG plus poly A:U. Moreover, evidence is presented that demonstrates that poly A:U exerts its adjuvant action on the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of poly A:U to augment adoptive secondary anti-DNP responses to the DNP derivative of a nonimmunogenic copolymer of d-glutamic acid and d-lysine (d-GL) for which there are few or no specific, functional T cells.     

Genetic control of the immune response of mice to highly dinitrophenylated human gamma-globulin (DNP56HGG)

The immune response to highly dinitrophenylated human gamma-globulin (DNP56HGG) was tested in inbred strains of mice. Significant differences in the anti-DNP response among inbred strains were found, including the magnitude of serum antibody and the location of plaque-forming cells (spleen or lymph nodes). The strain differences persisted when the dose and adjuvant were changed. The genetic control of the anti-DNP response to DNP56HGG was investigated. The analysis of the response of congenic and F1 hybrid mice to DNP56HGG suggests that at least two genes are involved in the control of the anti-DNP response. The two genes are demonstrated by complementation in the F1 generation, and show no correlation with H-2 haplotype or IgG2a allotype. A third gene may be implicated by differences in response observed between male and female mice.     

Genetic control of the immune response to staphylococcal nuclease

Summary Antibody responses of inbred strains of mice to staphylococcal nuclease were studied by isoelectric focusing in polyacrylamide gels followed by in situ labeling of focused antibodies with radioactive antigen. All A/J mice examined produced antinuclease antibodies of limited heterogeneity, and although there was individual variation in the focusing patterns observed, a characteristic spectrotype produced by all of the animals could be discerned. In order to determine the possible relationship between this characteristic spectrotype and the cross-reactive idiotypes of A/J antinuclease antibodies previously described (7), focused antibodies were also examined with a radioactively labeled pig anti-(A/J antinuclease) anti-idiotypic antibody preparation. Using this reagent, similar spectrotypes to those observed for antigen binding were seen in all of the individual A/J sera, suggesting that cross-reactive idiotype expression is a reflection of the characteristic spectrotypes observed. The same labeled anti-idiotypic reagent revealed characteristic but different spectrotypes when used to develop focused antinuclease antibodies from individual mice of other strains, suggesting that the use of similar variable region structures may be a common feature of the antinuclease response in mice of different allotypes. These studies thus provide a structural basis for the genetics of idiotype expression defined previously by serologic analysis.     

Induction of suppressor T cells and tolerant B cells in vitro by DNP-IgG

Intravenous injection at proper time of irradiated reticulum cell sarcoma cells into SJL mice immunized with dinitrophenylated (DNP) keyhole limpet hemocyanin inhibits the production of anti-DNP IgG₁ and IgG₂ antibodies.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Behavior of the idiotypic network in conventional immune responses. II. Affinity and heterogeneity of idiotypic and anti-idiotypic antibodies following immunization with T-independent and T-dependent antigens.

The relative affinity and heterogeneity of affinity of idiotypic and anti-idiotypic antibodies in mice immunized with the T-independent antigen DNP-Ficoll and the T-dependent antigen DNP-HGG were measured by a plaque inhibition assay. Idiotypic plaque-forming cells (PFC) were detected by a conventional assay utilizing DNP-coated SRBC. Anti-idiotypic PFC were detected with SRBC coated with affinity-purified anti-DNP antibody of rabbit origin. It was found that both idiotypic and anti-idiotypic antibodies elicited by immunization with the T-independent antigen had lower affinity and were less heterogeneous than the corresponding antibodies originating in mice immunized with the T-dependent antigen. In addition, the affinity and heterogeneity values of the idiotypic antibodies were correlated with the affinity and heterogeneity values of the anti-idiotypic antibodies from the same mice. This finding indicates that idiotypic and anti-idiotypic antibodies mutually regulate each other, thus pointing to internal immunoregulatory effects of the idiotypic network with respect to these parameters.     

Neutralization of bacterial viruses by antibodies of young animals

The development of the avidity of 19S and 7S neutralizing antibodies was studied after the immunization of young rabbits with ΦX 174 bacteriophage. Both 19S and 7S antibodies formed during the entire course of the immunization process were able to neutralize bacteriophage ΦX 174 without the participation of thermolabile serum components: neither heat inactivation at 56°C nor addition of piglet complement into the neutralizing system influenced the titer in the neutralization test. In both immunoglobulin classes (IgM and IgG) the first antibodies formed possessed a low avidity that increased in the course of the immunization process so that in the secondary response antibodies of both 19S and 7S type were formed possessing a high binding activity towards the specific antigen. Following the immunization with a high dose of the phage antigen (10¹⁰ PFU of ΦX 174 phage) 19S antibodies were formed at a relatively fast rate, the avidity of which did not further increase during immunization. When a low antigen dose was used for immunization the avidity of antibodies significantly increased in the course of immunization.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.     

Suppression of reaginic antibody formation. III. Relationship between immunogenecity and tolerogenicity of hapten-carrier conjugates.

From the study of the effect of epitope density on the immunogenicity of haptenated ovalbumin (DNP-OA) it was concluded that the lightly haptenated conjugate, DNP0-5-OA, induced, on the one hand, only low titers of anti-DNP hemagglutinating antibody and no reaginic antibodies to the hapten and, on the other, high reaginic and high hemagglutinating antibody responses to the carrier. The conjugate with a slightly higher degree of haptenation, i.e., DNP2.3-OA, induced both reaginic and hemagglutinating antibodies to both the hapten and the carrier. By contrast, the heavily haptenated conjugate, DNP20-OA, elicited reaginic and hemagglutinating antibodies only against the hapten but not against the carrier. Specific suppression of anti-hapten reaginic antibody formation had been achieved by treatment of mice with a tolerogen consisting of the hapten (DNP) conjugated covalently to isologous gamma globulins (MgammaG). The epitope density of the DNPx-MgammaG conjugates was shown to play a dominant role in determining whether or not the conjugate was tolerogenic. Thus, lightly haptenated conjugates (DNP0.5-MgammaG, DNP1.3-MgammaG or DNP1.9-MgammaG) were not tolerogenic, moderately haptenated conjugates (DNP4.2-MgammaG, DNP8-MgammaG, and DNP 14-MgammaG) were tolerogenic, and heavily haptenated conjugates (DNP32-MgammaG and DNP53-MgammaG) were immunogenic, being capable of priming the recipients for the DNP hapten. Further evidence for the nonimmunogenicity of DNP 8-MgammaG conjugate was inferred from its rate of clearance in tolerized and normal mice. Thus, the half-life of 125I-labeled DNP8-MgammaG in circulation was not significantly different for normal and tolerized mice; it was 3.7 and 3.5 days, respectively, which is within the range of data reported for clearance of normal MgammaG. These results suggest that DNP8-MgammaG was catabolized at a rate similar to that of nonconjugated, isologous MgammaG. Moreover, there was no significant difference in the localization of DNP8-MgammaG in identical difference in the localization of DNP8-MgammaG in identical organs (spleen, thymus, kidney, and liver) of normal and tolerized mice. All the multivalent DNPx-MgammaG conjugates were shown to be able to elicit passive cutaneous anaphylaxis (PCA) reaction on i.v. challenge of rats which had been pre-sensitized i.d. with anti-DNP reaginic antibodies.