Kinetic measurements of molecular interactions by spectrofluorometry

The kinetics of antibody–antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti-fluorescein antibodies are featured in the review as model proteins to explore fluorescence-based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti-fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k₁) and dissociation (k₂). The near diffusion-limited k₁ rates and the prolonged k₂ rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti-metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformational substates.     

Immunological characterization of xenogenic anti-metatype antibodies

High affinity murine anti-fluorescein monoclonal antibody (Mab) 4-4-20 (K alpha = 1.3 x 10(10) M-1; IgG2a; kappa), affinity labeled with fluorescein isothiocyanate (I), served as the immunogen to elicit a xenogenic (rabbit) polyclonal "anti-metatype" reagent in a rabbit specific for the liganded state. The reagent did not bind Mab 4-4-20 in its nonliganded (idiotypic) conformation and demonstrated no reactivity with fluorescein ligand. Interaction of the anti-metatype reagent with liganded 4-4-20 caused a significant decrease in the dissociation rate of fluorescyl ligand bound to Mab 4-4-20 and a single-chain antibody derivative of 4-4-20. Additionally, most members of the 4-4-20 idiotype family displayed the same kinetic effect in the presence of anti-metatype. Members most closely related to Mab 4-4-20 (based on concentration in Mab required to inhibit the 4-4-20 idiotype/4-4-20 anti-idiotype interaction 50%) showed relatively greater decreases in the dissociation rate, establishing a quantitative correlation between idiotype and metatype. The immunologically distinct metatypic state is discussed in terms of conformational changes in or near the active site upon binding ligand, possibly involving two amino acid residues which "close off" the mouth of the antibody-active site upon binding fluorescein.     

Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.     

Surface plasmon resonance (SPR) as a tool for antibody conjugate analysis

Surface plasmon resonance (SPR) analysis was used to assess the immunoreactivity of anti-biotin (4) and anti-fluorescein (5) monoclonal antibody after conjugation with the N-hydroxysuccinimide ester of acridinium-9-carboxamide 1. Only minor changes in the apparent equilibrium dissociation constants of the antibody conjugates for their ligands resulted from the conjugation process. However, comparison of the initial binding rate of the conjugates with their ligands with those of the unmodified antibodies over a range of concentrations showed that the antibody conjugates were partially inactivated. The anti-fluorescein conjugates retained at least 90% of their immunoreactivity over the range of modification tested, while anti-biotin conjugates showed a progressive loss of reactivity with increased substitution by the label.     

Comparison of variable region primary structures within an anti-fluorescein idiotype family

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.     

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.     

Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities

Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′)₂ antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′)₂ were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′)₂ were used to map the progressive depth of the bound F(ab′)₂ layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′)₂ was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′)₂ front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′)₂ samples.     

Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody.

Anti-metatype antibodies have been described as antibodies which recognize ligand-induced conformational changes in the antibody variable region. Additionally, anti-metatype antibodies, produced by multiple immunizations with liganded high affinity monoclonal anti-fluorescein antibody 4-4-20, enhanced the lifetime of monoclonal antibody 4-4-20-fluorescein complex. To better understand the mechanism of the delayed dissociation rate, deuterium oxide was used to probe the liganded active site. The rate and extent of deuterium oxide-mediated fluorescence enhancement of bound ligand served to monitor the conformational dynamics of the active site in the presence and absence of anti-metatype antibodies. Results showed that anti-metatype antibodies reduced the rate and extent of deuterium oxide-mediated fluorescence enhancement of 4-4-20, a single-chain derivative of 4-4-20 (consisting of the variable domains and a polylinker), and idiotypically related monoclonal anti-fluorescein antibodies suggesting that anti-metatype stabilized the liganded active site. Size exclusion liquid chromatography was utilized to isolate the liganded antibody-anti-metatype complex. Liganded single chain antibody 4-4-20 was mixed with 10-fold molar excess anti-metatype Fab fragments, and a major complex eluted with an apparent M(r) 249,000. The apparent molecular weight of this complex inferred that one liganded single chain antibody was bound by five antimetatype Fab fragments. Spectral analysis confirmed these results and the characteristic delayed rate of ligand dissociation was also observed for the isolated complex. The results suggest that anti-metatype antibodies stabilize the liganded conformation by forming a large, stable, macromolecular complex.     

Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.     

Monoclonal antibody against non-histone chromosomal protein high mobility group 1 Co-migrates with high mobility group 1 into the nucleus

Non-histone chromosomal protein high mobility group 1 (HMG-1) rapidly migrates into the nucleus when injected into the cytoplasm of bovine fibroblasts and HeLa cells by red cell-mediated microinjection (Rechsteiner, M., and Kuehl, L. (1979) Cell 16, 901-908). We isolated hybridomas secreting monoclonal antibodies against HMG-1. One of these monoclonal antibodies, FR-1, inhibited in vitro binding of 125I-HMG-1 to chromatin isolated from FL cells. When 125I-HMG-1 was co-introduced with antibody FR-1 by red cell-mediated microinjection, antibody FR-1 did not prevent the accumulation of 125I-HMG-1 in the nucleus. When 125I-antibody FR-1 or fluorescein isothiocyanate antibody FR-1 was introduced into the cytoplasm of FL cells, most of the antibody did not accumulate in the nucleus. But when 125I- or fluorescein isothiocyanate antibody FR-1 was co-introduced with HMG-1 into the cytoplasm of FL cells, it did migrate into the nucleus.     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.

Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interestingly, the observed degrees of quenching were nearly independent of the lipid membrane model studied, but directly correlated with the chemical structure of the lipids. In all cases, the antibody recognized and quenched most efficiently a lipid based on dioctadecylamine where fluorescein is attached to the headgroup via a long, flexible hydrophilic spacer. Dipalmitoyl phosphatidylethanolamine containing a fluorescein headgroup demonstrated only partial binding/quenching. Egg phosphatidylethanolamine with a fluorescein headgroup showed no susceptibility to antibody recognition, binding, or quenching. Formation of two-dimensional protein domains upon antibody binding to the fluorescein-lipids in monolayers is also presented. Chemical and physical requirements for these antibody-hapten complexes at membrane surfaces have been discussed in terms of molecular dynamics simulations based on recent crystallographic models for this antibody-hapten complex (Herron et al., 1989. Proteins Struct. Funct. Genet. 5:271-280).     

Generation and application of a fluorescein-specific single chain antibody

A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.     

基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体*

在马的免疫学研究领域中,由于目前市场上缺乏商业化的马IgG单克隆抗体,使得对马的B细胞研究受到很大阻碍,IgG是B细胞受体(BCR)的重要构成成分,与B细胞分化成熟相关。为了获得马IgG特异性单克隆抗体,利用单个B细胞扩增技术进行抗体筛选。首先,将马IgG蛋白(EqIgG1-C)密码子优化后合成到真核表达载体pcDNA3.4上,纯化出抗原蛋白。随后,使用蛋白质免疫小鼠,分离脾细胞后利用流式细胞术分离特异性单个B细胞,扩增出抗体重链和轻链的可变区基因,用overlapping PCR方法扩增出线性化的完整抗体,并进行鉴定。结果从80个B细胞中获得了27株特异性重组单克隆抗体,并挑选出3株线性结合活性最强的抗体基因构建到表达载体上,共转染Expi293F^TM细胞后表达纯化,经过ELISA和Western blot验证,显示获得的抗体可以和EqIgG1-C蛋白有良好的结合作用。使用该方法可以省时高效的获得特异性抗体,为马的免疫学研究提供了重要研究工具,为鼠单克隆抗体筛选提供了技术拓展。     

Discrete bathochromic shifts exhibited by fluorescein ligand bound to rabbit polyclonal anti-fluorescein Fab fragments

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by 14 to 15 nm and/or energies of 1.5 kcal mol^–1 relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.     

Location and dynamics of a membrane-bound fluorescent hapten. A spectroscopic study

In the preceding paper (Petrossian, A. and Owicki, J.C. (1984), Biochim. Biophys. Acta 776, 217–227), we describe the binding of a monoclonal anti-fluorescein antibody to a membrane bound fluorescein-lipid hapten. Those results suggest that some of the hapten fluorescein moiety is extended away from the membrane surface and is available for antibody binding, while some of the hapten is sequestered and not immediately available for antibody binding. In this paper, we carry out a spectroscopic study of the membrane-bound hapten and show that there is more than one physically distinct fluorophore environment, with the sequestered hapten associated with the phospholipid headgroup region. The amount of membrane-associated fluorophore depends upon the membrane lipid composition: most of the fluorophore is associated when the lipid is unsaturated or branched-chain phosphatidylcholines (PC), whereas the hapten is largely extended for PC/cholesterol mixtures. The effect of cholesterol on the availability of membrane-bound hapten to antibody binding is not unique to this system. The conversion between sequestered and extended hapten is slow (minutes).     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

Monoclonal antibodies to red cell cytoskeletal proteins

Monoclonal antibodies to human red cell cytoskeletal proteins were produced following immunization of mice with Triton shells produced from intact red cells. Two lines producing antibodies binding to spectrin and actin, respectively, were subcloned and further characterized. Clones producing the anti-spectrin antibody were stable. The antibody was monoclonal and specific for spectrin band 2. The anti-actin clones were unstable.     

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.