The binding of phosphorylcholine-carrying antigens to the anti-phosphorylcholine monoclonal antibody TEPC 15. A fluorescence spectroscopic study

The intrinsic fluorescence of the anti-phosphorylcholine monoclonal antibody TEPC 15 has been used to study its interaction with the hapten phosphorylcholine and some phosphorylcholine-carrying lipids. Spectral conditions were selected so as to obtain fluorescence emission attributable mainly to tryptophan residues. Upon addition of the hapten, the fluorescence intensity increases, and the emission maximum is shifted towards lower wavelengths, in a hyperbolic and saturable process. These effects seem to be specific for phosphorylcholine, since they are not produced by the analogue phosphorylethanolamine. The quenching results suggest that a conformational change occurs in the protein upon interaction with the hapten. Upon addition of a variety of phosphorylcholine-carrying lipids, it is shown that the antibody interacts with the hapten when the lipid exists in the form of micelles, but not when it is present in the lamellar phase.     

Improving the antigen affinity of an antibody Fv-fragment by protein design.

The affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone. On their basis the hapten was placed into a model of the Fv-fragment built according to the principles of canonical antibody structures. From the model, unfavourable contacts between hapten and an aspartyl side-chain in complementarity-determining region 3 of the heavy chain were predicted. Substitution of the aspartyl residue by alanine resulted in a threefold increase in affinity of the antibody Fv-fragment for two hapten derivatives when compared with the wild-type. Nuclear magnetic resonance analysis of the improved Fv-fragment revealed an interaction between the alpha-carbon proton of alanyl residue with the ligand, which was not seen for the aspartyl residue. This interaction is not entirely in accordance with the model, which predicts an interaction between the side-chain of this residue and the hapten. However, it shows that by combined use of nuclear magnetic resonance analysis and molecular modelling, a residue that is critical for antigen binding was identified, whose mutation allowed the design of an improved antibody combining site.     

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.     

Structural basis for antibody catalysis of a cationic cyclization reaction

Antibody 4C6 efficiently catalyzes a cationic cyclization reaction. Crystal structures of the antibody 4C6 Fab in complex with benzoic acid and in complex with its eliciting hapten were determined to 1.30A and 2.45A resolution, respectively. These crystal structures, together with computational analysis, have elucidated a possible mechanism for the monocyclization reaction. The hapten complex revealed a combining site pocket with high shape complementarity to the hapten. This active site cleft is dominated by aromatic residues that shield the highly reactive carbocation intermediates from solvent and stabilize the carbocation intermediates through cation-pi interactions. Modeling of an acyclic olefinic sulfonate ester substrate and the transition state (TS) structures shows that the chair-like transition state is favored, and trapping by water directly produces trans-2-(dimethylphenylsilyl)-cyclohexanol, whereas the less favored boat-like transition state leads to cyclohexene. The only significant change observed upon hapten binding is a side-chain rotation of Trp(L89), which reorients to form the base of the combining site. Intriguingly, a benzoic acid molecule was sequestered in the combining site of the unliganded antibody. The 4C6 active site was compared to that observed in a previously reported tandem cyclization antibody 19A4 hapten complex. These cationic cyclization antibodies exhibit convergent structural features with terpenoid cyclases that appear to be important for catalysis.     

Antibody buffering in the brain

The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.     

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.     

Cooperation between Carrier-reactive and Hapten-sensitive Cells <Emphasis Type="Italic">in vitro</Emphasis>

THE mechanism, known as the carrier effect, whereby immunity to one or more determinant groups enhances the response to other determinants on the same multivalent antigen, was first recognized in delayed hypersensitivity to haptens, in which, for an appreciable response, the hapten must be coupled to the same protein carrier for priming and challenge^{1, 2}. Carrier specificity has also been demonstrated in the secondary antibody responses to hapten protein conjugates³. Two alternative hypotheses have been advanced to explain this specificity. The “local environment” hypothesis supposes that the hapten-sensitive cell recognizes both the hapten and the carrier determinants. However, the antihapten antibodies produced do not distinguish details of the carrier molecule and so do not reflect the specificity of the cellular receptor. Furthermore, inert spacer molecules inserted between hapten and carrier do not interfere with carrier specificity in the antibody response³. Reflecting current views on the cooperation between thymus-derived (T) and bone marrow derived (B) lymphocytes in the antibody response to various antigens⁴, the second hypothesis invokes two or more cells, one with receptors directed towards the hapten (hapten-sensitive cell), the others specific for the carrier molecule proper (carrier-reactive cells). Supporting this is the observation that pre-immunization to a particular protein carrier alone could potentiate the primary or secondary antihapten response to a hapten conjugated to that protein⁵. In an adoptive transfer system, moreover, the efficiency of antihapten antibody production by cells primed to a particular hapten-protein conjugate and stimulated with the hapten conjugated on a heterologous protein, is significantly enhanced by the introduction of cells primed to the heterologous carrier alone. Anti-carrier serum antibody does not cause such enhancement⁶. The carrier-reactive cells must therefore cooperate in increasing the efficiency of the hapten-sensitive cells in some way other than by providing humoral anti-carrier antibody. Recent work strongly suggests that carrier reactive cells are thymus-derived^{6, 7}.     

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10⁸ clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

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.     

Selection of phage-displayed peptides that bind to a particular ligand-bound antibody

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.     

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.     

Carrier-specific unresponsiveness in reaginic antibody formation. II. Suppression of hapten and carrier responsiveness in presensitized rats.

By inducing carrier-specific tolerance to sheep γ-globulin (SGG) in rats challenged with TNP-SGG in alum, it has been possible to study the effect of helper T-cell Unresponsiveness on IgE anti-TNP antibody formation. Rats primed to either the carrier (SGG) or the hapten (TNP as TNP-KLH) were treated with a single high dose (10 mg) of soluble SGG resulting in a suppression of both IgE anti-TNP and anti-SGG antibody which was maintained following a normally immunogenic secondary challenge with TNP-SGG in alum. This suppression was relatively long lasting, with no detectable IgE responsiveness to hapten or carrier observed for up to 8 weeks after tolerance induction. Suppressed animals were able to respond to the hapten when challenged with TNP-KLH, indicating that the induced effect did not directly involve the IgE antibody producing cells, but rather the carrier-specific helper cells. These results parallel our previous findings for IgM and IgG responses in a similar system. Such relatively long lasting and easily induced suppression in IgE antibody formation to specific protein antigens in primed animals may eventually provide a clinically useful means of allergic desensitization to large protein allergens.     

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.     

Photoreversible antigen-antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.     

上皮生长因子受体和癌胚抗原分子内血型Y配基表达的免疫生化分析

以抗CEA、EGF-γ和血型前体Y配基抗原的单抗进行分析,结果表明:结肠癌组织中表达CEA和血型前体Y配基较强,而EGF-γ表达水平极低;进一步鉴定分析,识别血型前体Y配基抗原决定簇的单抗C_(14)与肿瘤具有较好的反应性。并发现这种决定簇存在于各种生物大分子中,包括以糖蛋白和糖脂形式存在,并表达于EGF-γ与CEA分子中。这一结果提示血型前体抗原过量表达于肿瘤细胞中,不但是血型组织相容抗原,可能也参与到一些特殊生物分子中,应深入研究和鉴定其功能。     

A rapid approach to 11 alpha-hemisuccinylprogesterone synthesis

The synthetic hapten 11 alpha-hemisuccinylprogesterone (11 alpha-hemisuccinyl-pregn-4-ene-3,20-dione), when linked to the appropriate macromolecular carrier, has been used successfully as a solid-phase antigen for progesterone detection in immunoassay. In this study the synthesis of 11 alpha-hemisuccinylprogesterone from 11 alpha-hydroxyprogesterone has been improved by using 4-dimethylaminopyridine (DMAP in refluxing dioxane, a highly nucleophilic polar solvent.     

Theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody: application to histamine release from basophils.

We present a theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody in the presence or absence of monovalent hapten. Bivalent haptens can link together antibodies to form linear chains or rings on cell surfaces. We show how to calculate the amount of any complex of bound bivalent hapten, monovalene fraction of antibody involved in complexes made up of two or more antibodies, i.e., the fraction of antibody that is cross-linked (Xpoly). We treat the case when the antibody on the cell surface, which is specific for the hapten, is homogeneous. For this case we prove a number of general properties about Xpoly: 1) Xpoly approaches zero at both high and low bivalent hapten concentration. 2) Xpoly becomes a maximum when the bivalent hapten concentration equals Amax, where Amax = 1/H + B/2. H is twice the equilibrium constant for the binding of a single hapten site to a single antibody site and B is the monovalent hapten concentration. 3) a plot of Xpoly vs the log of the bivalent hapten concentration is symmetric about the maximum value of Xpoly. We use these and other properties of Xpoly in this paper to clarify the relationship between cross-link formation and histamine release.     

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.     

Magnetic resonance studies of the binding site interactions between phosphorylcholine and specific mouse myeloma immunoglobulin

The interaction of phosphorycholine-binding mouse myeloma protein M603 and the isotopically substituted hapten phosphoryl[methyl-13C] choline has been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13C and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known x-ray structure of the M603 Fab'-phosporylcholine complex (Segal,D.M., Padlan, E.A., Cohen G.H., Rudikoff S., Potter,M., and Davies, D.R. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4298).