Mutations of an epitope hot-spot residue alter rate limiting steps of antigen-antibody protein-protein associations.

The antibodies, HyHEL-10 and HyHEL-26 (H10 and H26, respectively), share over 90% sequence homology and recognize with high affinity the same epitope on hen egg white lysozyme (HEL) but differ in degree of cross-reactivity with mutant lysozymes. The binding kinetics, as measured by BIAcore surface plasmon resonance, of monovalent Fab from both Abs (Fab10 and Fab26) to HEL and mutant lysozymes are best described by a two-step association model consistent with an encounter followed by docking that may include conformational changes. In their complexes with HEL, both Abs make the transition to the docked phase rapidly. For H10, the encounter step is rate limiting, whereas docking is also partially rate limiting for H26. The forward rate constants of H10 are higher than those of H26. The docking equilibrium as well as the overall equilibrium constant are also higher for H10 than for H26. Most of the free energy change of association (Delta G degrees) occurs during the encounter phase (Delta G1) of both Abs. H10 derives a greater amount and proportion of free energy change from the docking phase (Delta G2) than does H26. In the H10--HEL(R21Q) complex, a significant slowing of docking results in lowered affinity, a loss of most of Delta G2, and apparently faster dissociation. Slower encounter and docking cause lowered affinity and a loss of free energy change primarily in the encounter step (Delta G1) of H26 with mutant HEL(R21Q). Overall, in the process of complex formation with lysozyme, the mutations HEL(R21X) affect primarily the docking phase of H10 association and both phases of H26. Our results are consistent with the interpretation that the free energy barriers to conformational rearrangement are highest in H26, especially with mutant antigen.

Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,).

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.

Experimental design for analysis of complex kinetics using surface plasmon resonance.

Using BIAcore surface plasmon resonance technology, we found that the real-time association kinetics of Fabs specific for hen egg-white lysozyme did not conform to a 1:1 Langmuir association model. Heterogeneity of the components is not the source of the complex kinetics. Informed by independent structural data suggesting conformational flexibility differences among these antibodies, we chose global mathematical analysis based on a two-phase model, consistent with the encounter-docking view of protein-protein associations. Experimental association times (T(a)) from 2 to 250 min revealed that initial dissociation rates decreased with increasing T(a), confirming a multiphasic association. The relationship between observed dissociation rate and T(a) is characteristic of each antibody-antigen complex. We define a new parameter, T(50), the time at which the encounter and final complexes are of equimolar concentration. The observed T(50) is a function of analyte concentration and the encounter and docking rate constants. Simulations showed that when the ligand is saturated at high analyte concentrations, T(50) reaches a minimum value, T(50)(MIN), which can be used to compare antigen-antibody complexes. For high-affinity complexes with rapid rearrangement to a stable complex, T(50)(MIN) approaches T(1/2) of the rearrangement forward rate constant. We conclude that experiments with a range of T(a) are essential to assess the nature of the kinetics, regardless of whether a two-state or 1:1 model is applicable. We suggest this strategy because each T(a) potentially reveals a different distribution of molecular states; for two-step analysis, a range of T(a) that brackets T(50) is optimal.

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).

Antibody recognition imaging by force microscopy.

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody-antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.

Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5.

Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.

Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties.

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.

Immunotolerance against a foreign antigen transgenically expressed in the lens.

PURPOSE: To extend our knowledge concerning immunotolerance against autologous lens crystallins, transgenic (Tg) mice that express a foreign antigen in their lens were generated, and the immune response against the antigen in these mice was analyzed. METHODS: Conventional techniques were used to generate lines of Tg mice that express soluble (S-) or membrane-bound (M-) hen egg lysozyme (HEL) under the control of the alphaA-crystallin promoter. The presence of HEL in various organs was determined by the particle concentration fluorescence immunoassay (PCFIA), and reverse transcription-polymerase chain reaction technique was used to detect mRNA transcripts of the molecule. To examine the development of immunity (or tolerance), Tg mice and their wild-type controls were immunized with HEL (25 microg) in Freund's complete adjuvant and 14 days later were tested for immune response against the antigen. Cellular immunity was measured by the lymphocyte proliferation assay and cytokine production, and humoral immunity was determined by enzyme-linked immunosorbent assay. RESULTS: Eyes of the high copy number M-HEL Tg mice were dystrophic, with disrupted lens, whereas no morphologic changes were detected in the eyes of the other Tg mouse lines. All Tg mice exhibited tolerance to HEL by their cellular and humoral immune compartments. The state of immunotolerance to HEL was retained in the Tg mice for as long as 10 months after removal of the main depot of this protein, by enucleation. Measurable amounts of HEL were found in the eyes of all Tg mice, but the protein could not be detected in the serum or in other organs by the sensitive PCFIA (with a threshold of 1 ng/ml). Yet, HEL mRNA was found in the thymus of the Tg mice, suggesting that minute amounts of the protein are expressed in this organ. CONCLUSIONS: The unresponsiveness to HEL in the Tg mice seems to be due to a "central" mechanism of tolerance, mediated by a minuscule amount of HEL in the thymus. Conversely, the much larger amounts of HEL in the peripheral depot, the eyes, play a minor role if any in the tolerogenic process. It is further proposed that a similar mechanism of central tolerance is responsible for the immunotolerance against autologous lens crystallins.

A mutational analysis of binding interactions in an antigen-antibody protein-protein complex.

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine. Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively. The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

Association of the anti-hen egg lysozyme antibody HyHEL-5 with avian species variant and mutant lysozymes.

The energetics of association of the murine anti-hen egg lysozyme antibody HyHEL-5 with bobwhite quail lysozyme, California quail lysozyme, and the Arg45-->Lys mutant of hen egg lysozyme was characterized by isothermal titration calorimetry. The association of each lysozyme with HyHEL-5 is enthalpically driven in the temperature range 10 degrees C to 37 degrees C. The calorimetric results indicate that the salt-links between Arg45 and Arg68 of hen egg lysozyme and GluH50 on the HyHEL-5 paratope are energetically important in HyHEL-5/HEL association. In contrast to previous studies, the results suggest that the three characteristic 'quail' mutations affect the energetics of antibody/antigen association, even though they are buried and not in direct contact with the antibody.

Plasmacytoma-refractory BALB/cAnPt mice have naive T cell and highly specific B cell responses to antigen.

Recently, a reduction in the incidence of pristane-induced plasmacytomas in BALB/cAnPt (BALB/c) mice that were kept in viral specific pathogen-free (SPF) conditions has been reported. Environmentally, these SPF-BALB/c mice differed from conventionally-housed (CON) mice only in viral exposure and diet (i.e. sterilization of mouse chow), since microbial colonization of the intestinal tract was seen to be equivalent. This report assessed the ability of SPF- and CON-BALB/c mice to respond to immunologic challenge with soluble antigen, i.e. hen egg white lyzosyme (HEL), as a means of evaluating differences in T and B cell function and, indirectly, evaluating the possible effects these differences might have on plasmacytoma development. When cultured in vitro for 5 days with HEL, HEL-primed lymph node cells (LNC) from SPF-BALB/c mice proliferated to a significantly lesser extent than HEL-primed CON-BALB/c LNC. Moreover, HEL-induced production of IFN-gamma and IL-5 was significantly lower in SPF LNC. Serum IgG1 levels were 10-fold lower in SPF-BALB/c mice with, or without prior immunization with HEL and were not reconstituted by repeated injections of HEL in adjuvant. Serum IgM levels of SPF- and CON-BALB/c mice were equivalent. This reduction in immune responses could not be attributed to a lack of colonization of secondary lymphoid organs, since flow cytometric analysis of LNC revealed no difference in the number of recoverable cells and the proportion of lymphocyte subsets (CD4+, CD8+ and CD45+ cells) obtained from SPF- and CON-BALB/c mice. However, only CON LNC were induced to increase surface expression of CD44 after antigenic or mitogenic stimulation in vitro. Antibody responsiveness to HEL, as evidenced by serum anti-HEL binding or splenic hybridoma studies, demonstrated higher levels of IgG1 antibodies in CON BALB/c mice than in SPF mice. However, a greater proportion of the SPF IgG1 antibodies present were specifically directed against HEL, so that specific activity was greater in SPF-BALB/c mice. Therefore, while SPF BALB/c mice have a more restricted response to HEL than CON-BALB/c mice, those antibodies that are produced are more specifically directed against HEL with very little apparent bystander/polyclonal activation of multireactive cells. Resistance to plasmacytomas in SPF-BALB/c mice, therefore, may stem from a reduced number of circulating memory T and B cells, which are capable of reacting and/or crossreacting with a chronic inflammatory stimulus.

Specificity of monoclonal antibodies elicited by mucosal infection of BALB/c mice with virulent Shigella flexneri 2a.

Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment.

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an antihen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes.

Molecular recognition of lysozyme by monoclonal antibodies.

HEL was one of the first proteins to be mapped antigenically using mAb, and panels of mAb have been used as a measure of antigenicity in order to study regulation of the immune response and the apparent 'antigenic structure' of HEL. These studies have confirmed the multideterminant hypothesis derived from pAb. However, although the entire surface of HEL is potentially antigenic, the mature immune response appears to be dominated by three functionally nonoverlapping antigenic regions, defined operationally by antibody complementation assays. Recent structural studies have confirmed the existence of three distinct epitope clusters. Functional epitopes, defined by immunoassays, are generally only a subset of the structural epitope, the 14-17 amino acid residues which contact antibody in the X-ray structure of the complex. An even smaller subset of 'critical residues', the 'energetic' epitope, may predominate the interaction energetically. Antibody complex formation with HEL is enthalpically driven, and is accompanied by an unfavorable entropy change. Mutations of either antibody or antigen which lower affinity appear to do so primarily by increasing dissociation rates, and also appear to be accompanied by entropy/enthalpy compensation. The current availability of six structurally defined antibody-lysozyme complexes presents excellent opportunities for comparative studies in order to understand the structural bases of affinity, specificity, and thermodynamic properties, as well as the interrelationships of functional, structural, and energetic epitopes.

Structure of an antibody-lysozyme complex unexpected effect of conservative mutation.

The structure of the complex between the Fab HyHEL-5 and chicken lysozyme revealed a large interface region containing 23 lysozyme and 28 Fab residues. Arg68 of the lysozyme is centrally placed in this interface and theoretical studies together with binding assays of this Fab to different avian lysozymes have previously shown that this arginine residue is an important contributor to the binding. The Arg68-->Lys mutant binds 10(3) times less well to the HyHEL-5 Fab. We have examined the refined crystal structure of the complex of this mutant lysozyme with the Fab. No global changes occur, but there is an introduction of a new water molecule into the interface that mediates the hydrogen bonding interactions between the lysine and residues on the Fab. These data are compared with the effects of similar changes on the inhibition of serine proteases such as trypsin where the energetic effects of this substitution are small.

Parvovirus particles as platforms for protein presentation.

Empty capsids of the human pathogenic parvovirus B19 can be produced in a baculovirus system. B19 capsids are composed mainly of major capsid protein (VP2) and a small amount of minor capsid protein (VP1); VP1 is identical to VP2 but contains an additional 227-aa N-terminal region ("unique" region). A portion of that region of VP1 is external to the capsid, and VP1 is not required for capsid formation. We substituted the unique region with a sequence encoding the 147 aa of hen egg white lysozyme (HEL) and constructed recombinant baculoviruses with variable amounts of retained VP1 sequence joined to the VP2 backbone. After cotransfection with VP2 baculovirus and expression in insect cells, capsids were purified by density sedimentation. Purified recombinant capsids contained HEL. External presentation of HEL was demonstrated by immunoprecipitation, ELISA, and immune electron microscopy using anti-lysozyme monoclonal antibodies or specific rabbit antisera. Empty particles showed enzymatic activity in a micrococcal cell wall digestion assay. Rabbits inoculated with capsids made antibodies to HEL. Intact heterologous protein can be incorporated in B19 particles and presented on the capsid surface, properties that may be useful in vaccine development, cell targeting, and gene therapy.

High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis.

The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21HEL, Asp-101HEL, and Gly-102HEL) and at a partially buried residue (Asn-19HEL) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101GHEL mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the delta delta Gdissoc is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with delta delta Gdissoc at position 21. Although Gly-102HEL and Asn-19HEL are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody.

Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme.

We tested 49 BALB/c antilysozyme mAb from seven intervals during the immune response to lysozyme for patterns of specificity and avidity. We found that the antibody epitopes in composite covered at least 80% of the lysozyme surface, and their patterns of overlap suggest a continuum of potential antibody epitopes. Previously observed regional specificities, which emerged at different times in the immune response, were more discretely defined in late response antibodies, when the majority of mAb could be assigned to one of three functionally nonoverlapping complementation groups. The area covered by each antigenic region may be greater than an individual epitope, and may include multiple epitopes that overlap structurally and functionally to varying degrees. Connectivity between antigenic regions was seen in interactions among early and late stage antibodies, and among secondary stage mAb, but not among tertiary stage mAb from hyperimmunized mice. Patterns of overlap of early and late response antibodies suggest that the organization of antibody specificities change during the progression from primary to secondary to tertiary response. Over the same period in the response, the average relative avidity of IgG1 kappa mAb did not increase, suggesting that "affinity maturation" of serum antibodies reflects an increase in the number and diversity of antibodies, rather than an overall increase in the avidity of individual antibodies.