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PubMed1982

Lysis of hapten-labeled cells by anti-hapten IgG and complement: effect of cell surface hapten density.

Rabbit anti-methotrexate (MTX) IgG antibody was used to study the effect of MTX density on sheep red cells on the lysis of cells by complement (C). Under conditions in which the density of IgG did not vary, the number of lytic IgG complexes remained approximately the same when the average distance between MTX molecules was between 5 and 7 nm, but decreased rapidly with an average distance less than 10 nm. The results were interpreted to mean that in addition to aggregation for IgG to acquire maximal C in fixing and activating properties, the angle of the Fab arms of the molecule must not be larger than about 50 to 60 degrees. The data also suggested IgG molecules with Fab angles of less than about 30 degrees might also not be able to induce C-mediated cell lysis.

Animals↗
PubMedDate not supplied

LPS greatly enhances the antibody response to hapten-polysaccharide conjugates, but not to hapten-protein conjugates.

In confirmation of earlier findings, we observed that an injection of bacterial lipopolysaccharide (LPS) into mice caused a considerable increase in the serum concentrations of IgM and IgG (total Ig rose three- to four-fold in 7 days), and a corresponding increase in the concentrations of "natural" anti-(3-iodo-4-hydroxy-5-nitrophenyl) acetyl (NIP) and anti-trinitrophenol (TNP) antibodies. Our main purpose was to determine what effect LPS had on antigen-dependent responses. Hapten conjugates of a polysaccharide and of proteins were used as antigens. Hapten-protein conjugates induced a strong anti-hapten antibody response (up to 1 mg/ml of anti-hapten antibodies on day 7). Hapten-polysaccharide conjugates induced only a meagre increase in anti-hapten antibodies from the pre-immunization level (maximal concentration 65 micrograms/ml on day 7). LPS, when injected with the antigen, greatly enhanced the antibody response to the hapten-polysaccharide conjugates (up to 2.6 mg/ml of anti-hapten antibodies on day 7). It had little effect on antibody responses to hapten-protein conjugates. The combination treatment had the same effect on immunoglobulin concentrations as LPS alone.

Animals↗
PubMedDate not supplied

Kinetics of the dissociation of indium-(p-substituted-benzyl)ethylenediaminetetraacetic acid hapten analogues from the monoclonal anti-hapten antibody CHA255.

Half-lives were measured for the dissociation of a series of 20 indium-benzyl-EDTA derivatives from a monoclonal antibody that binds to them. Most haptens gave expected monoexponential dissociation curves with half-lives ranging from approximately 8 to approximately 100 min at 22 +/- 1 degree C. Precise (+/- approximately 2.5%) determinations were made using centrifugal ultrafiltration to separate free from bound hapten. A strong pH dependence of the dissociation half-life was found for the two haptens studied. Activation enthalpies were identical (23 +/- 1 kcal/mol) for the dissociation of four haptens, suggesting that, in contrast to individual rate constants, this parameter is insensitive to hapten modification. The dissociation half-lives provided evidence for the location of a positive charge in the binding site, but gave no clear indication of the role of hydrophobic interactions or of steric requirements in hapten binding. While variations in ionic strength had no effect on the dissociation rate, lowering surface tension with dioxane increased the rate somewhat. Three hapten-antibody complexes showed biexponential dissociation rates. It is postulated that this results from distinct conformations of the complex dissociating at different rates. The dissociation rate constant was found to be an extremely sensitive indicator of the hapten-antibody interaction that can be measured very precisely.

Albumins↗
PubMed2003

Pharmacokinetics and dosimetry studies for optimization of anti-carcinoembryonic antigen x anti-hapten bispecific antibody-mediated pretargeting of Iodine-131-labeled hapten in a phase I radioimmunotherapy trial.

PURPOSE: Pharmacokinetics and dosimetry of hMN-14 x m734 bispecific monoclonal antibody (BsMAb) and (131)I-labeled di-diethylenetriaminepentaacetic acid-indium ((131)I-hapten) were studied to optimize pretargeted radioimmunotherapy. EXPERIMENTAL DESIGN: Thirty-five patients with carcinoembryonic antigen-expressing tumors were included. In a first group of 12 patients, (131)I-trace-labeled BsMAb doses were escalated from 10 to 100 mg/m(2), and 3.7 GBq of (131)I-hapten were administered 7 days later. In a second group, 12 patients received 75 mg/m(2) BsMAb and 2.6-4.2 GBq of (131)I-hapten 5 days later. The BsMAb dose was then reduced to 40 mg/m(2), and 10 patients received 1.9-5.5 GBq of (131)I-hapten. Blood samples were collected. Biodistribution was monitored by quantitative scintigraphy. RESULTS: Directly labeled BsMAb pharmacokinetics was described by two exponentials: half-lives were 8.1 h (2.0-18.1 h) and 48.2 h (22.8-79.4 h); blood clearance was 123 ml/h (64-195 ml/h). With a 7-day interval, 10 or 30 mg/m(2) BsMAb resulted in fast elimination and very low tumor uptake of hapten, whereas 50 or 100 mg/m(2) resulted in favorable tumor accretion. With 75 mg/m(2) BsMAb and a 5-day interval, hapten clearance was 152 ml/h (81-298 ml/h). Calculated radiation dose to tumor was 3.9 Gy/GBq (0.4-22.4 Gy/GBq) for the hapten, compared with 2.0 Gy/GBq (0.3-3.8 Gy/GBq) for the BsMAb, but hematological toxicity prevented dose escalation. Reduction of the BsMAb dose to 40 mg/m(2) accelerated hapten clearance to 492 ml/h (113-2544 ml/h) and reduced hematological toxicity without compromising tumor uptake [5.2 Gy/GBq (0.5-12.6 Gy/GBq)]. CONCLUSIONS: Optimized BsMAb doses and time interval will allow for the administration of higher, tumoricidal, activity doses.

Adult↗
PubMed1976

Mechanisms by which hapten conjugates of pneumococcal polysaccharide interfere with the challenge of anti-hapten memory cells.

Incubation of trinitrophenylated hemocyanin (TNP-KLH)-primed spleen cells with microgram amounts of 2,4-dinitrophenyl (DNP) or 2,4,6-trinitrophenyl (TNP) conjugates of pneumococcal polysaccharide type 3 (SIII) for as little as 5 min at 4 degrees C results in a specific "block" of the 19 S and 7 S adoptive memory response to TNP-KLH. This hapten-SIII-induced block of anti-hapten memory B cell responsiveness seems to be an example of specific receptor blockade. The block is specific and can be prevented by simultaneous incubation of the primed cells with hapten-protein conjugates which presumably compete with the hapten-polysaccharide for attachment to the B cell surface via anti-hapten Ig receptors. Removal via capping of these Ig receptors by exposure of TNP-KLH-primed memory cells to rabbit anti-mouse Fab serum for 45 min at 37 degrees C renders these cells refractory to the blocking effect of hapten-SIII. Once the hapten-SIII has attached to the memory cells, these blocked cells can be "rescued" (i.e. returned to a state of responsiveness) by incubating these cells with either mouse anti-SIII at 37 degrees C or rabbit anti-DNP serum at 4 degrees C. Since a papain digest of the IgG fraction of rabbit anti-DNP did not rescue the cells while the intact IgG did, a capping off of the TNP-SIII was proposed as the mechanims for this return to responsiveness of the hitherto blocked cells. A rescue was not seen by treatment of recipient mice with such B cell mitogens as dextran sulfate, endotoxin or purified protein derivative of tuberculin.

Animals↗
PubMed1994

ELISA screening of monoclonal antibodies to haptens: influence of the chemical structure of hapten-protein conjugates.

An analysis has been made of the influence of the chemical structure of hapten-protein conjugates on ELISA based screening monoclonal antibodies to haptens. Evidence has been obtained that the size of haptens should be taken into account. For haptens with a relatively large molecular weight, one can use a conjugate with a carrier protein differing from the immunogen. For small haptens, the screening should be conducted with a conjugate differing from the immunogen with respect to both the carrier protein and the chemical linkage between the hapten and the protein. In addition, the hapten-protein coupling should involves different amino acid residues.

Animals↗
PubMed1983

Studies on the immune response in chickens. IV. Generation of hapten-specific memory and absence of increase in carrier-specific helper memory in antibody response to sheep red blood cell antigen and its hapten-conjugate.

The role of carrier recognition in the hapten-specific antibody response of chickens to hapten (TNP) conjugate of sheep red blood cells (SRBC) was studied. The anti-TNP antibody response to the challenge with either TNP-SRBC or DNP conjugate of Salmonella enteritidis (DNP-Sal) of chickens previously primed with TNP-SRBC was definitely higher than that of unprimed chickens. Injection of TNP-chicken red blood cells, which did not provoke any significant primary anti-TNP response, also induced the memory for secondary anti-TNP response to the challenge with TNP-SRBC or DNP-Sal. The TNP-specific memory generated by stimulation with TNP-SRBC was adoptively transferable by spleen cells into cyclophosphamide (CY)-treated X-irradiated syngeneic recipients. On the other hand, the SRBC-specific memory for augmentation of anti-TNP response to the challenge with TNP-SRBC was never detected in chickens previously primed with either any of a wide range of doses (10(4)-10(8)) of SRBC, solubilized SRBC or SRBC fixed with formaldehyde, nor in CY-treated X-irradiated chickens receiving SRBC-primed spleen cells. The anti-TNP response of chickens to the challenge with TNP-SRBC was rather suppressed by the SRBC preimmunization. The passively administered anti-SRBC chicken serum also suppressed the response to TNP-SRBC. The carrier-specific suppressor activity generated by SRBC stimulation was not adoptively transferable by spleen cells. It is suggested from these results that the mechanism of amplification of the anti-hapten antibody response of chickens to hapten-SRBC conjugate is primarily mediated by the hapten-recognition, and carrier-recognition is more important for suppression rather than for amplification of the anti-hapten response, although the inductive mechanism for anti-hapten response itself should somehow involve the carrier recognition.

Animals↗
PubMed1992

Dimerization kinetics of the IgE-class antibodies by divalent haptens. I. The Fab-hapten interactions.

The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of monoclonal DNP-specific IgE antibodies with divalent haptens having different spacer length (gamma = 14-130 A). Binding was monitored by quenching of intrinsic tryptophan emission of the Fab. Addition of divalent haptens with short spacers (gamma = 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilibrium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to the fluorescence titrations yielded dissociation rate constants of 7.8 x 10(-3) s-1 and 6 x 10(-3) s-1 for the Fab dimers formed by the flexible divalent haptens N alpha, N epsilon-di(dinitrophenyl)-L-lysine (gamma = 16 A) and bis(N beta-2,4-dinitrophenyl-alanyl)-meso-diamino-succinate (gamma = 21 A). Making the simplifying assumption that a single step binding equilibrium prevails, the corresponding dimer formation rate constants were calculated to be 1.9 x 10(5) M-1 s-1 and 1.1 x 10(4) M-1 s-1, respectively. In contrast, all haptens with spacers longer than 40 A (i.e., bis(N alpha-2,4-dinitrophenyl-tri-D-alanyl)-1,7-diamino-heptane, and di(N epsilon-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)n-L-l ysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (greater than 7 x 10(6) M-1 s-1). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants. Thus, ligands having better access to the binding site would react faster.

Animals↗
PubMed1978

Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.

Binding Sites, Antibody↗
PubMed1996

Hapten-carrier interactions and their role in the production of monoclonal antibodies against hydrophobic haptens.

The coupling of haptens to carrier proteins is required for the generation of antihapten antibody response. To date, different polypeptides (such as gelatin, albumin, and keyhole limpet hemocyanin) have been used to increase the immune response against haptens. Different methods of conjugation have also been used to increase the specificity of the antibody response. Our results, obtained through an analysis of the antibody response at both the polyclonal and the monoclonal levels, show that carrier proteins specifically modulate the antibody titer, the specificity of the response, the fusion efficiency, and the number of specific clones. Moreover, the affinity constants of both serum and randomly selected hybridomas directed against different haptens have been found to be exclusively modulated by carrier proteins. In addition, the results show that the carrier protein should be selected considering its possible physicochemical interaction with haptens. Data obtained suggested that any carrier is suitable for hydrophilic haptens, while the choice of the carrier for hydrophobic haptens is critical in obtaining a specific immune response. The use of specific carriers allowed the production of highly specific antibodies: even IgMs, obtained by using these carriers, were able to specifically react with hydrophobic haptens; the case of some IgM monoclonal antibodies specifically reacting with protoporphyrin IX is reported.

Amino Acid Sequence↗
PubMed1982

Immunogenicity of liposomal model membranes sensitized with spin-labeled haptens and topographical distribution of haptens on the membranes.

The relation between the in vitro immunogenicity of phosphatidylcholine liposomes containing 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) as a hapten and the topographical distribution of the haptens on lipid membranes was studied. In distearoylphosphatidylcholine liposomes, the immunogenicity increased with increase of cholesterol content in the liposomal membranes. The electron spin resonance spectra of spin-labeled DNP-Cap-PE in distearoylphosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled DNP-Cap-PE on the membranes. In the absence of cholesterol, a considerable amount of haptens was clustered on the distearoylphosphatidylcholine membranes, but with increase of cholesterol, random distribution of the haptens on the membranes increased. The cholesterol-dependent change in the topographical distribution of the haptens on the membranes paralleled the change of immunogenicity, i.e., the immunogenicity was low when haptens were clustered on the liposomal membranes. Haptens arranged at a proper distance on the membranes may be required for optimum immune response.

Animals↗
PubMed1983

Interaction of hapten-sensitized liposomes with cells bearing hapten-specific receptors.

The binding of liposomes sensitized with 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) to MOPC-315 cells, which secrete and bear on their surfaces anti-DNP immunoglobulins, was studied. The binding was affected by cholesterol content, phospholipid composition and hapten density of liposomes: The binding of distearoylphosphatidylcholine liposomes sensitized with 5 mol% hapten to the cells increased with increasing cholesterol content in liposomes. The amount of liposomes composed of phospholipid with a higher transition temperature (such as distearoylphosphatidylcholine), which bound to MOPC-315 cells, was much higher than that of liposomes composed of phospholipid with a lower transition temperature (such as egg yolk phosphatidylcholine). The amount of distearoylphosphatidylcholine liposomes containing equimolar cholesterol, which bound to the cells at 0 degrees C, increased with increasing amount of the hapten in liposomes up to 2.5 mol%. The binding became maximum at 2.5 mol% and decreased with higher hapten density in liposomes. The immunogenicity of hapten-sensitized liposomes is known to be affected by the liposomal composition such as cholesterol content, phospholipid composition and hapten density. This model study suggests that the binding of liposomes to cells is important for expressing the immunogenicity of hapten-sensitized liposomes.

Animals↗
PubMed1979

Attempts to modulate the immune response to a hapten-carrier complex with various hapten-containing compounds.

The immune response to a hapten-carrier conjugate appears to be a complex phenomenon where reactions of the T-cell population are not restricted to the carrier and where the reactions of the B-cell population are not limited to the hapten determinant of the antigen molecule. To get a better understanding of the different cell interactions during the immune response to a hapten-carrier complex, the effects of immunogenic or tolerogenic injections of various hapten-containing compounds on the responses induced by immunization with the same hapten coupled to protein carriers were studied. The results indicate that T cells involved in delayed hypersensitivity and T cells involved in contact dermatitis could belong to distinct subclasses and confirm that hapten and carrier moieties of the antigen molecule could compete, probably at the macrophage level, for both delayed hypersensitivity to the carrier and antibody synthesis to the hapten.

Animals↗
PubMed1976

The induction of hapten-specific T cell tolerance by using hapten-modified lymphoid cells. I. Characteristics of tolerance induction.

BALB/c mice were made tolerant to the T cell-dependent phenomenon of contact sensitivity to DNFB by i.v. injection of syngeneic lymphoid cells which had been previously modified with DNFB in vitro. The highly efficient unresponsiveness, as measured by ear challenge and in vitro antigen-induced cell proliferation, was shown to follow dose-response kinetics both in vivo and in vitro and to be exquisitely specific for the DNP moiety. The kinetics of tolerance induction were shown to be very rapid and had been previously shown to be long lasting. Unresponsiveness was more efficient when the hapten-modified cells were introduced by the i.v. route and tolerance could be increased by repeated injection of tolerogen. The tolerance could be transferred to normal syngeneic recipients by spleen and/or lymph node cells from tolerant donors. A wide variety of hapten-modified lymphoid cells, including mixed cell populations and enriched populations of T cells, B cells, and macrophages, were capable of inducing tolerance. The unresponsiveness was dependent merely on the association of DNP to the lymphoid membrane proteins and not upon the viability of the hapten-modified cells. These experiments support the hypothesis that in hapten-specific T cell sensitivity, as exemplified by contact sensitivity to DNFB, specific T cell tolerance in actively induced by hapten on self-membrane. In other hapten-specific antibody-forming systems, tolerance appears to be most readily induced by hapten on soluble self protein or on a nonimmunogeneic carrier.

Animals↗