Histamine release due to bivalent penicilloyl haptens: control by the basophil plasma membrane.

We describe the characteristics of in vitro histamine release from human basophils passively sensitized with serum from a penicillin-allergic individual. The histamine release is induced by a synthetic bivalent hapten, bis benzylpenicilloyl 1,6 diaminohexane (BPO)2. We present data on the effect of a monovalent hapten, benzylpenicilloyl formyl-L-lysine (BPO)1, on the histamine release. We also examine how histamine release depends on the concentration of serum used for passive sensitization, the source of cells used for passive sensitization, and the time allowed for histamine release. We interpret these experiments in terms of a theory of equilibrium binding of bivalent haptens to cell surface antibody that is presented in the previous paper. The results are consistent with the idea that the amount of histamine release is controlled by the number of cross-linked IgE molecules on the cell surface. In particular, the histamine dose-response curve rises because cross-links rise, has a maximum because the cross-links are a maximum, and falls because the cross-links fall.     

Theory of equilibrium binding of a bivalent ligand to cell surface antibody: The effect of antibody heterogeneity on cross-linking

We investigate the equilibrium binding of symmetric bivalent ligands to a heterogeneous population of symmetric bivalent cell surface receptors. The receptors are heterogeneous in their binding affinities (equilibrium binding constants) for the ligand. For any distribution of receptor binding affinities we show how to calculate the total concentration of receptors that are cross-linked by the ligand, i.e., the concentration of cell surface aggregates composed of two or more receptors, as well as the concentration of any given aggregate. We show that certain qualitative properties of cross-linking which hold for homogeneous antibody populations fail to hold in the heterogeneous case. We use our results to interpret certain in vitro experiments in which synthetic bivalent haptens are used to trigger histamine release from basophils which have on their surface antibody specific for the hapten.This work was performed under the auspices of the Department of Energy and supported by Grant AI 16465 from the National Institute of Allergy and Infectious Diseases.     

Equilibrium and kinetic parameters for the interaction of a monoclonal antibody with liposomes bearing fluorescent haptens

We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.     

Dissociation kinetics of bivalent ligand-immunoglobulin E aggregates in solution

We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented.     

Test of a theory relating to the cross-linking of IgE antibody on the surface of human basophils

Recent mathematical models of bivalent hapten-induced histamine release from basophils predict that under appropriate conditions histamine release is maximum when cross-link formation is maximum, at a hapten concentration equal to 1/(2Ka), where Ka is the average affinity constant of the hapten for a single IgE binding site. To test this prediction we sensitized human basophils with a monoclonal anti-dinitrophenol IgE and generated histamine release dose-response curves with a bivalent hapten, alpha, epsilon-DNP-lysine. The monoclonal IgE has a published affinity constant of 7.1 X 10(7) M-1 for epsilon-DNP-lysine as determined by equilibrium dialysis. From the position of the maximum of the histamine dose-response curves, both in the presence and in the absence of monovalent DNP hapten, we determine that the sensitizing IgE has an intrinsic affinity constant of 6.9 +/- 0.5 X 10(7) M-1 for epsilon-DNP-lysine and 1.2 +/- 0.6 X 10(6) M-1 for alpha-DNP-lysine. The agreement between the two estimates of the epsilon-DNP-lysine affinity constant, one from histamine release experiments involving surface bound IgE and one from binding experiments involving IgE free in solution, 1) is consistent with a central prediction of the theory of cross-linking and 2) indicates that the hapten-binding properties of the IgE are unaffected by its being bound to Fc epsilon receptors on the basophil surface.     

Studies of antigen binding on human basophils. I. Antigen binding and functional consequences

We developed an assay to study simultaneously antigen binding and its functional consequences on human basophils. Using a 125iodine-labeled penicillin-human serum albumin conjugate, we were able to detect as few as 100 molecules of antigen bound to purified basophils. We found that histamine release could be initiated with fewer than 100 molecules of antigen and that the optimum for histamine release occurred at a concentration at which 10 to 15% of the available antibody-binding sites were occupied. Analysis of the binding kinetics in the presence of monovalent hapten allowed a calculation of the affinity constant for the antibody:hapten association; the value of 2 to 3 X 10(6)/Msec confirmed an earlier independent calculation. Binding data also suggested a 25% fraction of the bound antigen was binding in monogamous bivalent form. It is anticipated that studies of this kind will delineate the nature of the antibody-antigen reaction on cell surfaces.     

Pretargeted radioimmunotherapy using 131I-labelled bivalent hapten-bearing peptides

Summary The advantages of bivalent hapten-bearing peptides for the detection of tumours pretargeted with bispecific antibodies have been demonstrated. This technology is now considered for radioimmunotherapy and bivalent haptens designed to target¹³¹I are needed. We thus synthesised a series of tyrosine-containing peptides bearing the histamine-hemisuccinate hapten. These molecules were tested for their ability to bind simultaneously two anti-hapten antibody molecules. One of these bivalent haptens, AG3.0, with a lysyl-d-tyrosyl-lysine connecting chain, was found to have optimal binding characteristics and was thus selected for further investigations. AG3.0 was shown to efficiently deliver radioactive iodine to human colorectal tumours grafted in nude mice using an anti-carcinoembryonic antigen×anti-histamine-hemisuccinate bispecific antibody. AG3.0 was also targeted to human B lymphoma cells pretargeted with a bispecific antibody specific for membrane IgM. In this system, bivalent ligands such as F(ab′)₂ or IgG are rapidly internalised and covalently linked radioactive iodine is released from target cells as a result of intracellular catabolism. With the pretargeted iodine-labelled bivalent hapten, a fivefold increase in the intracellular activity retention time as compared to¹²⁵I-labelled F(ab′)₂ and IgG was observed. The radiolabelled hapten did not undergo any degradation after internalisation. These results have been confirmed in vivo with an anti-BCL₁ IgM idiotype bispecific antibody and¹³¹I-labelled AG3.0. These reagents injected as a single 300 μCi dose, 7 days after inoculation of 10⁴ BCL₁ lymphoma cells in BALB/c mice, cured 14/16 of the animals and the treatment was well tolerated. Comparatively, the same dose of labelled IgG cured 13/16 of the mice but three mice died of haematologic toxicity. The same dose of labelled F(ab′)₂ or Fab′ was completely inefficient.¹³¹I-labelled bivalent haptens are now used in phase I radioimmunotherapy clinical trials.     

Kinetics of binding reactions of an antibody molecule with haptens on a membrane surface

Direct measurement has been made of the reaction rate of binding of a bivalent antibody and fluorescent haptens, which were covalently bound on a model membrane surface, by a method of stopped-flow fluorometry. The result was interpreted as indicating that the reaction takes place in two steps: (i) binding of a hapten with one of the two antigen-combining sites of an antibody molecule, and (ii) binding of another hapten with the other site of the antibody molecule in question. The rate of the second step was found to depend on the fluidity of the membrane.     

Structural and thermodynamic basis of affinity in anti-dinitrophenyl antibody.

The thermodynamic quantities of the anti-dinitrophenyl antibody-hapten interaction are reported for rabbit, goat, and guinea pig antibodies. Rabbit and goat antibodies had similar exothermic enthalpy changes for their reaction with 2,4-dinitrophenyl-L-lysine (-13.9 and -14.8 kcal/mol, respectively). The enthalpy change with guinea pig antibody was much less exothermic (-8.7 kcal/mol), and this value was the same for two guinea pig antibody preparations that differed in affinity by almost two orders of magnitude. A heterogeneous goat anti-dinitrophenyl antibody preparation was fractionated on a molecular sieve column in the presence of a bivalent ligand, a procedure that has been reported to separate antibodies according to differences in the depth of interaction with the ligand. The relationship of these differences in apparent site depth to changes in interactions with the hapten tail was examined by comparing the affinities of various fractions for two haptens. The results show that the presumed deeper sites have stronger interactions with the hapten tail. These studies suggest that the heterogeneity of anti-dinitrophenyl antibodies with respect to affinity results from differences in entropy driven lysyl side-chain interactions which arise from a heterogeneity in antigen binding site depth.     

Mesoscale Modeling of Nucleosome-Binding Antibody PL2-6: Mono- versus Bivalent Chromatin Complexes

Interactions of chromatin with bivalent immunoglobin nucleosome-binding antibodies and their monovalent (papain-derived) antigen-binding fragment analogs are useful probes for examining chromatin conformational states. To help interpret antibody-chromatin interactions and explore how antibodies might compete for interactions with chromatin components, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model. We analyze interactions and fiber structures for the antibody-chromatin complexes in open and condensed chromatin, with and without H1 linker histone (LH). Despite minimal and transient interactions at physiological salt, we capture significant differences in antibody-chromatin complex configurations in open fibers, with more intense interactions between the bivalent antibody and chromatin compared to monovalent antigen-binding fragments. For these open chromatin fiber morphologies, antibody binding to histone tails is increased and compaction is greater for bivalent compared to monovalent and antibody-free systems. Differences between monovalent and bivalent binding result from antibody competition with internal chromatin fiber components (nucleosome core and linker DNA) for histone tail (H3, H4, H2A, H2B) interactions. This antibody competition for tail contacts reduces tail-core and tail-linker interactions and increases tail-antibody interactions. Such internal structural changes in open fibers resemble mechanisms of LH condensation, driven by charge screening and entropy changes. For condensed fibers at physiological salt, the three systems are much more similar overall, but some subtle tail interaction differences can be noted. Adding LH results in less-dramatic changes for all systems, except that the bivalent complex at physiological salt shows cooperative effects between LH and the antibodies in condensing chromatin fibers. Such dynamic interactions that depend on the internal structure and complex-stabilizing interactions within the chromatin fiber have implications for gene regulation and other chromatin complexes such as with LH, remodeling proteins, and small molecular chaperones that bind and modulate chromatin structure.     

The binding of haptens by the polypeptide chains of rabbit antibody molecules

1. The binding of haptens by the polypeptide chains derived from two rabbit immunoglobulin G antibodies was examined by gel chromatography and equilibrium dialysis. 2. The gamma chains were examined in a dilute sodium acetate buffer, pH5.4, in which they exist as a monodisperse solution of dimers; aggregation of the protein promoted by some haptens had to be avoided. These chains exhibited variable extents of binding, reflecting the specificities of the parent antibody molecules, usually with only small increments above the binding by gamma chains from normal immunoglobulin G. 3. The light chains existed as an interconverting mixture of monomers and dimers in all buffers of near neutral pH that were examined. They bound small amounts of hapten, again broadly reflecting the specificities of the parent antibody molecules. 4. For both the gamma and light chains the dimeric state appeared necessary for appreciable binding of hapten. Apparently in each case the partners in the dimer interact in a manner analogous to the gamma chain-light chain interaction in the parent antibody molecule, to give a site analogous to the antibody site. This implies that the binding of antigens by isolated chains has a large fortuitous element, providing no reliable indication of their contributions to the original antibody sites.     

Effects of charged groups in haptens on adsorption equilibrium of hapten antibody

Antisera against charged (p-azobenzoate and p-azoben zenesulfonate) and uncharged (dinitrophenyl) haptenic groups were produced in rabbits, and the equilibrium characteristics of hapten-antibody were measured by use of immunoadsorbents. The antibody to the uncharged hapten formed a stable binding with the hapten to the changes in ionic strength and pH. On the other hand, the antibodies to the charged haptens showed affinities sensitive to the changes in pH and ionic strength. Therefore, the effect of the pK(a) of ionizable haptens on the pH dependence of the hapten-antibody binding was studied by comparing the interactions between a series of para-substituted benzoic acids and the anti-p-azobenzoate antibody. The pH dependence of the interactions was strongly affected by the pK(a) of ionizable groups in haptens. Furthermore, the equilibrium characteristics of anti-p-aminobenzoyl dipeptides were compared. The characteristics of interactions were affected by the features of amino acid residues.     

Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces

In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG) antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i) Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii) The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion), which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii) One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts.     

Interaction of antibodies with liposomes bearing fluorescent haptens

Kinetic and equilibrium aspects of the recognition of antigenic model membranes by antibodies have been studied. Monoclonal anti-fluorescein IgG and its monovalent Fab fragment were allowed to interact with a fluorescein-lipid hapten that was incorporated into phospholipid vesicles. The binding was assayed in the nanomolar hapten concentration range by monitoring the quenching of hapten fluorescence by antibody. The rate and strength of the binding depended on the lipid composition of the vesicles; cholesterol enhanced both. The biphasic binding kinetics observed at high antibody concentrations for some compositions, plus additional spectroscopic evidence, led us to hypothesize that the hapten existed in a composition-dependent equilibrium between at least two conformations: (1) extended away from the membrane surface, available for binding, and (2) sequestered at or in the surface, unavailable for binding. The rate and strength of IgG binding were always greater than those of Fab, indicating bivalent binding by the IgG. This binding was intra-vesicular, since no agglutination of the vesicles was detected.     

Structural and dynamical aspects of membrane immunochemistry using model membranes.

Three different phospholipid haptens have been synthesized, in which the haptenic group is the paramagnetic nitroxide (spin-label) group. These lipid haptens differ from one another in the length and composition of the molecular chain linking the 2,2,6,6-tetramethylpiperidinyl-N-oxy moiety to the phosphodiester group of the lipid. These lipid haptens have been incorporated at low molar concentrations (0.01 to 0.5 mol %) in liposomes containing various proportions of cholesterol and dipalmitoylphosphatidylcholine (DPPC). A study has been made of specific antinitroxide IgG (and Fab) binding to these liposomes, and the fixation of complement. From these studies we conclude: (a) For lipid haptens whose possible extension above the bilayer plane is limited (e.g., approximately 10-20 A), antibody binding and complement fixation depend strongly on the hapten structure and host lipid composition, because of steric limitations on the accessibility of lipid haptens to the binding sites in the protein. (b) Complement fixation by specific IgG antibodies directed against the nitroxide group as part of a lipid hapten depends strongly on the lateral mobility of the lipid hapten when its molar concentration in the plane of the membrane is of the order of 0.1 mol % or less. It is likely that this conclusion applies to many lipid haptens, and possibly other membrane components. (c) The inclusion of cholesterol in lipid membranes has at least two distinct effects on complement fixation involving lipid haptens. Through a steric effect on bilayer structure (probably involving lateral molecular ordering) cholesterol in phosphatidylcholine bilayers can enhance hapten exposure to antibody binding sites, enhance antibody binding, and thereby enhance complement fixation. It is likely that cholesterol also affects complement fixation at low hapten concentrations through a modification of membrane fluidity.     

Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold

A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds approximately 5 kDa. Additionally, for ligands with much higher molecular masses (>10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.     

Oligomerization and ring closure of immunoglobulin E class antibodies by divalent haptens

Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.     

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.     

Thermodynamics of antibody-antigen reactions. 1. The binding of simple haptens to two classes of antibodies fractionated according to affinity

The thermodynamic parameters which characterize the binding of dinitrophenylglycine and dinitrophenylmethoxypoly(ethylene glycol) to selected affinity classes of equine IgG and IgG(T) antibodies were determined by fluorescence quenching and flow calorimetry. The binding enthalpies and entropies were in all cases large and negative, falling in the ranges -14 to -17 kcal/mol and -18 to -25 eu, respectively. The differences in the enthalpies and entropies of binding for different affinity classes and for different haptens are discussed with reference to differences in the structures of the haptens studied and as indications of differences in binding site structure. In addition, the apparent existence of fluorescent side chains which can transfer energy to either hapten binding site in IgG(T) antibodies but not in IgG antibodies is interpreted as indicative of a smaller average interbinding site distance in IgG(T) than in IgG antibodies.     

A monoclonal antibody specific for the Thomsen-Friedenreich cryptic T antigen

A monoclonal antibody (49H.24) is described that was made after immunization of BALB/c mice with human neuraminidase-treated erythrocytes (NE-RBC). 49H.24 is an IgM and reacts with NE-RBC but not untreated, normal human RBC. As little as 100 pg of antibody protein produced detectable direct agglutination of, or binding to, NE-RBC. The fine specificity of 49H.24 was determined by using a series of synthetic sugar haptens as inhibitors of agglutination or binding of 49H.24 to NE-RBC. Only synthetic T hapten (beta Gal(1 leads to 3)alpha GalNAc) produced complete inhibition of agglutination or binding, and no inhibition was produced by several other closely related haptens. Synthetic T hapten-coated silica beads (Synsorb) were used to affinity purify 49H.24. Affinity-purified antibody was radiolabeled with 125I and used in a sensitive competition assay to detect natural T antigen associated with cell membranes or in soluble form. The potential use of this or similar monoclonal antibodies as probes for an important human tumor marker is discussed.