Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails

In order to target specifically double-Ag-positive cells in vivo, we synthesized chemically two mAb conjugates with specificities for both an allelic murine B cell-surface Ag and for a synthetic hapten. One conjugate was designed for its specificities for I-Ek and for N-epsilon-(2,4-DNP)-amino-caproate, and the other one for its reactivity to Lyb-8.2 and to indium-diethylenetriamine pentaacetate. A radiolabeled tracer, containing both the N-epsilon-(2,4-DNP)-amino-caproate and the indium-diethylenetriamine pentaacetate haptens, was obtained by reacting diethylenetriamine pentaacetic acid dianhydride with mono-[N-epsilon-(2,4-DNP)-amino-caproyl]-tyrosyl-lysine and labeling with indium-111. Mice from various strains (CBA/N: I-Ek+, Lyb-8.2+; AKR/N: I-Ek+, Lyb-8.2-; BALB/c: I-Ek-, Lyb-8.2+; and DBA/2: I-Ek-, Lyb-8.2-) were given simultaneous i.v. injections of microgram amounts of less than anti-[N-epsilon-(2,4-DNP)-amino-caproate], anti-I-Ek greater than and of [anti-(indium-diethylene-triaminepentaacetate), anti-Lyb-8.2] antibody conjugates and picomole amounts of the tracer. As expected, specific uptake of the tracer by the spleen was observed in strains where spleen cells expressed at least one Ag (CBA/N, AKR/N, and BALB/c). Furthermore, spleen cells from the double-Ag-positive mouse strain (CBA/N), when compared with spleen cells from single-positive mouse strains, exhibited a significantly higher uptake of the bivalent hapten. This specificity for double-Ag-positive cells, it is suggested, occurs through the formation of stable complexes between both cell-surface Ag, both conjugates, and the asymmetric bivalent hapten. The use of such asymmetric bivalent haptens, together with matched (anti-hapten, anti-cell) antibody conjugates, is proposed as a general method for increasing the in vivo specificity of immunoimaging and radioimmunotherapy.     

An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.     

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.     

Determination of antibody-hapten association kinetics: a simplified experimental approach.

A simplified method has been developed for the determination of antibody-hapten association kinetics that permits the study of high affinity interactions with second order forward rate constants of the order of 10-7 to 10-8 M-1 sec-1. Use of tritiated haptens of high specific activity and antibodies of high affinity allows reactions to be run at initial hapten and antibody concentrations of the order of 10-9 to 10-10M, well below the level at which mixing becomes the rate-limiting step. Separation of antibody-bound from free hapten by the use of dextran-coated charcoal can be carried out with sufficient rapidity (2 sec) that the systems under investigation are not appreciably disturbed. With this technique, the association of 3-H-ouabain with rabbit ouabain-specific antibody was found to occur with a rate constant of 0.8 times 10-7 M-1 sec-1, similar to association rates of dye haptens with antibodies of substantially lower affinity. The ratio of this association rate constant to the independently determined dissociation rate constant was 5.4 times 10-9 M-1, in satisfactory agreement with a ko value of 3.5 times 10-9 M-1 determined by Sips analysis of data obtained under equilibrium conditions. This approach should be applicable to the direct kinetic assessment of numerous high affinity antibody-hapten systems of current interest.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Affinity electrophoresis and its applications to studies of immune response

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.     

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.     

The antibody-enzyme analogy. Characterization of antibodies to phosphopyridoxyltyrosine derivatives.

Stable analogs of the crucial Schiff base intermediate of enzymatic and nonenzymatic pyridoxal phosphate catalysis have been used as haptens for induction of specific antibodies. N-(5-phosphopyridoxyl)-3'-amino-L-tyrosine and its conformationally distinct cyclized derivative resemble the Schiff base formed upon mixing tyrosine with pyridoxal phosphate. These compounds were covalently coupled to a protein carrier via the 3'-amino group so as to confer a prescribed orientation, with the coenzyme region farthest removed from the carrier. A third antigen, with the phosphopyridoxyl group alone as the hapten, was prepared by linkage of pyridoxal phosphate directly to free amino groups on the carrier protein. Antibodies elicited for each determinant were purified by means of appropriate affinity columns. Antibody heterogeneity was observed in that different species could be separated from a given serum by sequential elution from the affinity columns with 1 M sodium phosphate buffers of pH 7.6, 5.2, 2.6 and 1.5. In assays of quantitative precipitation, inhibition of precipitation, equilibrium dialysis, and fluorescence quenching, antibodies to the phosphopyridoxyltyrosine haptens showed specificity for the phosphorylated form of the coenzyme and binding activity for both the coenzyme and tyrosine portions of the hapten. Antibodies to the phosphopyridoxyl groups alone did not display a similar reactivity toward the tyrosine portion of the complex haptens. The cyclic and noncyclic conformations of the hapten were serologically distinct, as antibody to each reacted preferentially with the homologous form.     

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.     

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.     

Isolation and affinity maturation of hapten-specific antibodies

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.     

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.     

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.     

Design of acridinium-9-carboxamides and anti-acridinium antibodies for chemiluminescent signal enhancement

A novel system of signal enhancement is presented in which every labeled antibody is capable of generating a signal. Three chemiluminescent acridinium-9-carboxamide haptens (1, 2, and 3) which incorporated differences in charge and location of the linker were designed and synthesized. Anti-acridinium polyclonal antibodies for each hapten were screened using surface plasmon resonance instrumentation to determine specificity for each hapten. Anti-acridinium 2 antibodies were found to be non-cross-reactive to acridinium 1. This property was exploited to design secondary antibody conjugates which would bind to primary antibodies labeled with 2 yet could still be labeled with the structurally similar acridinium 1. Consequently, both layers contributed to the overall chemiluminescent signal. This format is an advance over other signal amplification formats which employ non-signal-generating, labeled antibodies to construct multilayered systems.     

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.     

Interaction of antibodies specific towards the 2,4-dinitrophenyl group and of their fragments with hapten

The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.     

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.     

Synthesis and application of organomercury haptens for enzyme-linked immunoassay of inorganic and organic mercury

Two organomercury haptens were synthesized via the classical oxymercuration reaction. An intramolecular oxymercuration reaction was the strategy employed to prepare a structurally simple, but chemically robust, organomercury hapten that was conjugated to chicken immunoglobulin G (IgG). The resulting immunogen afforded mouse anti-mercury antibodies that were evaluated in an enzyme-linked immunosorbent assay (ELISA). Antibodies demonstrating high titers were obtained, and various immunoassay parameters were investigated. The sensitivity and selectivity of the resulting antibodies were evaluated by exploring different cross-coupling chemistries and solid-phase synthetic variations. A second hapten was prepared with the intermolecular oxymercuration reaction, and the resulting compound, once coupled to carrier protein, afforded a solid-phase conjugate that revealed the versatility of the mouse anti-mercury antibody. The anti-mercury antibody developed in this study was capable of detecting both mercury(II) salts and organomercury compounds.     

Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens

The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.