Biomedical subjects

E W Voss

Publications and source records attributed to E W Voss.

At least 109 records · Page 6Linked to original sources

PubMed1985

Identification of recurrent idiotypes within the unrestricted anti-fluorescein immune response.

The BALB/cV immune response to fluorescein (F1) was analyzed for expression of serologically defined idiotypes (Id) present on two anti-F1 hybridoma proteins, 3-13 and 3-17. After a I degree, II degree, or III degree immunization with F1-KLH, Id 3-13 and 3-17 were expressed in 15 of 28 and 22 of 28 mice, respectively. Solid-phase Id inhibition assays, performed by using pooled or individual anti-F1 antibody, indicated Id 3-13 and 3-17 represented less than 1.0% of the I degree, II degree, or III degree serum anti-F1 repertoire. Hybridoma proteins 3-13 and 3-17 had similar but distinct active sites as indicated by similar patterns of Id inhibition, fine specificity of binding, and affinity for F1. Analyses of 11 anti-F1-AECM-Ficoll hybridoma proteins (predominantly mu, lambda) indicated 4 of 11 clones were weakly cross-reactive with 3-13 and 3-17. Anti-F1-KLH II degree antibody from mice of various Igh allotypes was also screened and indicated these Id were expressed in each strain but at lower levels than in BALB/cV mice and were not strictly allotype associated. Id 3-13 and 3-17 represent the first demonstration of repertoire-specific recurrent Id expressed during a T-dependent immune response lacking a dominant Id.

Animals↗

PubMed1985

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids.

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.

Animals↗

PubMed1985

Characterization of monoclonal anti-fluorescein antibodies from autoimmune New Zealand mice.

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 secondary response to fluorescein (FL) presented on T-dependent carrier, demonstrated a high binding affinity for FL (KA = 2.9 X 10(10) M-1) and cryoprecipitation, which could be abrogated upon FL binding. Based on these unusual properties and their possible association with defective immune regulation in lupus-prone mice, further studies were carried out to evaluate the basis of 18-2-3 cryoprecipitation, expression of characteristics related to the 18-2-3 clonotype, and structure/function aspects of additional homogeneous IgM and IgG antibodies of similar origin and specificity. Solubility experiments in which the effect of ionic strength on macroscopic aggregation was measured indicated that 18-2-3 intrinsically possessed both cryoglobulin and euglobulin properties in the absence of auxiliary gamma-globulin components. Rates of hapten fluorescence quenching by 18-2-3 were limited by factors other than diffusion and were dependent on solution temperature and ionic strength. Thirty-seven additional IgM and IgG monoclonal antibodies were shown to possess normal low-temperature solubility and hapten fluorescence-quenching properties, suggesting that 18-2-3 was derived from a relatively rare B cell progenitor. Collective results from FL binding and spectrotype analyses indicated that the majority of proteins were diverse with respect to variable region structure and binding mechanisms but unusually restricted in binding affinities (KA less than 5 X 10(6) M-1). Relative subclass frequencies for 30 monoclonal IgG proteins (IgG1 greater than IgG2b greater than IgG2a greater than IgG3) were consistent with polyclonal IgG subclass expression in normal mice in response to T-dependent immunogen.

Animals↗

PubMed1984

Prolonged weightlessness and humoral immunity.

Preflight, inflight, and postflight serum samples obtained from crewmen aboard STS-9 were analyzed for immunoglobulin content. Control studies for circadian rhythm were conducted to further validate the analyses. Quantitation of immunoglobulins G, M, A, D, and E indicated relatively minor fluctuations in the concentration of each class of immunoglobulin during the experiment. Thus, microgravity effects on immunoglobulin levels during a 10-day flight were considered insignificant.

Antibody Formation↗

PubMed1984

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA.

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.

Antibodies↗

PubMed1984

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein.

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.

Animals↗

PubMed1984

The molecular mechanism of cryoimmunoglobulin precipitation--II. Thermodynamic basis for self-association as determined by fluorescence polarization.

Protein MAT is a homogeneous human IgM (lambda) cryoprecipitating cold agglutinin wherein the Fabmu and Fcmu5 fragment interaction is facilitated at low temps. Thermal dependence of the association of 2,5 DNS labeled Fab MAT with unlabeled Fcmu5 MAT was examined by fluorescence depolarization. The association data, treated by the method of van't Hoff, showed a nonlinear increase in binding with decreased temp, suggestive of certain dynamic changes in the system. Temperature effects on the rotational diffusion of five different 1,5 DNS labeled Fabmu fragments (including two derived from cryoglobulins) were also examined by fluorescence polarization. The linear nature of the Perrin plots derived from the data failed to reveal temp-induced hydrodynamic changes in any of the Fabmu fragments studied. Involvement of carbohydrates in the low-temp self-association of protein MAT was established by the finding that glycopeptides isolated from another IgM molecule (BAZ) could inhibit (as judged by depolarization of fluorescence) the interaction of 2,5 DNS labeled Fabmu MAT and unlabeled Fcmu5 BAZ fragments. These findings indicate that, although cryoprecipitation of protein MAT seemingly involves an antigen-antibody-like reaction between a site on the Fab region and carbohydrate moieties on the Fcmu5 region, no direct evidence for a low-temp-induced conformational change in the Fab region was obtained.

Agglutinins↗

PubMed1984

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.

Animals↗

PubMed1983

Specificity of anti-DNA antibodies in SLE-I. Definition and gross specificity of antibody populations in human SLE plasma.

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations of anti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonucleotides (average length tetranucleotides). For one of the plasma tested (PS), pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.

Antibodies, Antinuclear↗

PubMed1983

Specificity of anti-DNA antibodies in SLE--II. Relative contribution of backbone, secondary structure and nucleotide sequence to DNA binding.

Fine specificity of a population of anti-DNA antibodies which bound both ssDNA and dsDNA with apparently equal affinity was studied in two SLE plasma. Sensitivity of DNA binding to increasing sodium chloride concentration indicated that electrostatic interactions occurred between antibody and phosphate moieties of DNA. Secondary nucleic acid structure was important to DNA binding as double-stranded synthetic deoxynucleotide polymers were more effective inhibitors than their substituent single-stranded polymers. Nucleotide bases were also found to play a role in recognition of DNA by these cross-reactive antibodies, as ssDNA binding was sensitive to increasing temperature which caused unstacking of the nucleotide bases. Differing patterns of reactivity with synthetic deoxynucleotide polymers with similar secondary structures but different nucleotide compositions further indicated the importance of nucleotide bases to dsDNA binding by cross-reactive anti-DNA antibodies in SLE plasma.

Antibodies, Antinuclear↗

PubMed1983

Determination of dissociation constants and ligand specificity of detergent solubilized surface membrane immunoglobulin A from MOPC-315.

Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.

Animals↗

PubMed1983

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: localization and characterization of idiotypic determinants.

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (Ka values ranging from 5 X 10(6) to 10(10) M-1) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (gamma, kappa) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15-20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained gamma 1 heavy (H) chains and 4-6-10 contained both gamma 1 and gamma 2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which defined binding mechanism diversity. The location of determinants recognized by anti-idiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotype by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (approximately 25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the VH- and VL-chain interaction must exist for the expression of these idiotypes.

Animals↗

PubMed1983

Expression of defined idiotypes throughout the BALB/c anti-fluorescyl antibody response: affinity and idiotype analyses of heterogeneous antibodies.

Heterogeneous BALB/c anti-fluorescyl antibodies were shown to display increases (greater than 50-fold) in binding affinity from the primary through the tertiary responses. The structural basis of such affinity maturation and the diversity exhibited by anti-fluorescyl antibodies was examined by idiotypic analysis using a panel of anti-idiotype reagents specific for seven different monoclonal antifluorescyl antibodies. Because these clones exhibited binding affinities characteristic of a secondary or hyperimmune response, it was possible to examine the mechanism of affinity maturation by determining the prevalence of the seven idiotypes (Id-4-4-20, Id-20-19-1, Id-20-20-3, Id-6-10-6, Id-20-4-4, Id-4-6-10 and Id-6-19-1) in specifically purified heterogeneous preparations with low (i.e. primary response) or high (i.e. secondary and tertiary responses) binding affinities. Four of the idiotypes were not detected in heterogeneous preparations and thus each represented less than 0.1% of the total anti-fluorescein repertoire. Although results indicated that each of three other clones expressed unique or private idiotypic determinants not present in the heterogeneous population, these idiotypes (Id-4-4-20, Id-6-10-6, Id-6-19-1) were detected and ranged from approximately 0.2 to 2.0% of the repertoire. However, results indicated that each clone expressed unique or private idiotypic determinants not present in the heterogeneous population. Determinants expressed by such high-affinity monoclonal antibodies were expressed equally in all heterogeneous preparations examined. Because those determinants which were expressed were found in either low- or high-affinity heterogeneous antibodies, it is likely that the higher affinities exhibited by monoclonal antibodies derived from a secondary response are associated with unique idiotypic determinants which were not detected in polyclonal preparations. Hence, the process of affinity maturation may find as its structural correlate a mechanism such as somatic mutation which generates individual or unique idiotypes.

Animals↗

PubMed1983

Analysis of heterogeneous dissociation kinetics in polyclonal populations of rabbit anti-fluorescyl-IgG antibodies.

Hapten dissociation kinetics were used to characterize the expression of hyperimmune anti-fluorescyl-IgG antibodies within a population of 14 New Zealand White (NZW) rabbits. Anti-fluorescyl-IgG antibody preparations, obtained from individual hyperimmune NZW rabbits, exhibited either three or four discrete dissociative components. The fastest component was typically heterogeneous with respect to dissociation rate, exhibiting an average dissociative lifetime of 8 sec, where dissociative lifetime was the reciprocal dissociation rate constant. Longer-lifetime components exhibited lifetimes of several hundred seconds, and became progressively more homogeneous with increasing lifetime. Comparison of dissociative components, within the NZW rabbit population, revealed the presence of four distinct groupings of dissociative lifetimes, which were approximately Gaussian in their distribution. Average lifetimes of 8, 52.6, 313 and 1417 sec were observed for the four groupings, which corresponded to free-energy (delta G0) values of -9.6, -10.6, -11.6 and -12.42 kcal/mole, respectively (using an association rate value of 5 X 10(6) M-1 sec-1). These findings implied that affinity maturation of the anti-fluorescyl-IgG antibody response involved the generation of discrete high-affinity subpopulations of antibodies rather than a continuous spectrum of high-affinity antibodies.

Animals↗

PubMed1983

An exact procedure for the analysis of heterogeneous kinetics in polyclonal antibody populations.

Antibody populations with heterogeneous binding properties exhibit complex first-order dissociation kinetics. An analytical method has been developed to determine the average dissociation rate constant and the heterogeneity index of a specific antibody population. This procedure was based on Laplace transformation of the gamma distribution function, which yielded an exact, macroscopic rate law for the entire antibody population. Linearization of the macroscopic rate law is achieved by plotting data points versus their numerical derivatives using log-log axes. Linear regression of such plots yields the average dissociation rate constant from the Y-intercept, and heterogeneity index from the slope. This analytic method is transparent to the antibody system and kinetic assay employed, requiring only a programmable calculator to perform the necessary calculations. The usefulness of this analytic method was demonstrated by the evaluation of dissociation kinetics in murine monoclonal and rabbit polyclonal anti-fluorescyl-IgG antibody populations.

Animals↗

PubMed1983

Monoclonal IgM antibody exhibiting high-affinity binding and cryoglobulin properties.

A monoclonal IgM antibody (18-2-3) derived from cell fusion of (NZB X NZW) F1 splenocytes following secondary immunization with fluorescein-conjugated keyhole limpet hemocyanin was shown to exhibit high intrinsic binding affinity and cryoinsolubility. Affinity-purified preparations were determined to be IgM by immunochemical, electrophoretic, and chromatographic analyses. An intrinsic association constant (Ka) of 2.9 X 10(10) M-1 (at 2 degrees C) was measured by first-order dissociation-rate analysis. Antibody solubility at low concentration (approximately equal to 50 micrograms/ml) was shown, by absorption spectroscopy, to be temperature dependent between 4 degrees C and 32 degrees C. Insolubility at low temperature (4 degrees C) was reversible in the presence of homologous fluorescyl hapten, indicative of active site involvement in the mechanism of cryoglobulin-18-2-3 complex formation. Characteristics of clone 18-2-3 are discussed in terms of (i) its potential use as a model for examining the mechanism of cryoprecipitation and (ii) the proposed relationship between affinity maturation and the IgM to IgG class switch.

Animals↗

PubMed1982

A stable, high capacity, F-actin affinity column.

A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds 125I-heavy meromyosin and 125I-tropomyosin. The associations between the F-actin affinity matrix and the iodinated F-actin binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

Actins↗

PubMed1982

Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies.

Binding of fluorescyl ligand by five IgG anti-fluorescyl hybridoma proteins (4-4-20, 6-10-6, 20-4-4, 20-19-=1, 20-20-3) was examined. Relative reduction in fluorescence of bound fluorescein, deuterium oxide (D2O)-induced enhancement of fluorescence, and the effects of pH on binding kinetics were measured for each clone. Individual hybridoma proteins (all of which bind fluorescein with relatively high affinity) exhibited significant differences in the relative contribution of various forces (hydrophobicity, hydrogen bonding, ionic interactions) to binding and hence, affinity. The extent of such variations in binding mechanisms among monoclonal antibodies binding the same hapten is indicative of the extreme functional diversity of active sites. In addition, ligand binding by clone 20-20-3 was examined in greater detail. ABsorption spectra of ligand bound by purified intact antibody, Fab fragments, and reassociated heavy and light chains indicated that protonation of the fluorescyl ligand by a residue within the active site contributed significantly to the binding free energy. Comparative dissociation rates of fluorescein and a structural analog, rhodamine 110, were used to quantitatively substantiate the contribution of this interaction. Association and dissociation rate studies with fluorescein and antibody indicated that: 1) the active site appeared to undergo a conformational change upon ligand binding, and 2) neither intact disulfides nor intersite cooperativity affected the dissociation rate of bound ligand. Observed mechanisms of ligand binding are discussed in terms of proposed mechanisms of antibody affinity maturation and diversity.

Antibodies, Monoclonal↗