Impact of hapten presentation on antibody binding at lipid membrane interfaces

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K_D) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K_D(bulk) = 1.7 ± 0.2 nM vs. 2.9 ± 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K_D = 3.6 ± 1.1 nM vs. 2.0 ± 0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K_D value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 ± 1.1 μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K_D = 21 ± 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.     

Determination of an antibody-antigen binding constant by enzyme immunoassay and a theory for analysis of competitive binding of two ligands to heterogeneous receptor

A method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag* and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag* much less than Ab much less than Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the "affinity-average binding constant." A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using beta-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48 +/- 0.24 (S.D.) X 10(8) M-1 for unlabeled BLS.     

Expression of multivalency in the affinity chromatography of antibodies. Appendix: Derivation and evaluation of equations for independent bivalent interacting systems in quantitative affinity chromatography.

The expression of multivalency in the interaction of antibody with immobilized antigen was evaluated by quantitative affinity chromatography. Zones of radioisotopically labeled bivalent immunoglobulin A monomer derived from the myeloma protein TEPC 15 were eluted from columns of phosphorylcholine-Sepharose both in the absence and presence of competing soluble phosphorylcholine. At sufficient immobilized phosphorylcholine concentration, the variation of elution volume of bivalent monomer with soluble ligand was found to deviate from that observed for the univalent binding of the corresponding Fab fragment. In addition, the apparent binding affinity of the bivalent monomer increased with immobilized antigen density. Use of equations relating the variation of elution volume with free ligand concentration for a bivalent binding protein allowed calculation of microscopic single-site binding parameters for the bivalent monomeric antibody to both immobilized and soluble phosphorylcholine. The chromatographic data not only demonstrate the effect of multivalency on apparent binding affinity but also offer a relatively simple means to measure microscopic dissociation constants for proteins participating in bivalent interactions with their ligands.     

Quantification of specific bindings of biomolecules by magnetorelaxometry

The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP), to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX). Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated) serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality.     

Interaction of monoclonal anti-peptide antibodies with lysozyme

The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both. Multivalent interaction with the adsorbed ligand is a key factor in the efficacy of binding. Measurement of binding constants in homogeneous solution, by equilibrium dialysis and energy transfer, demonstrated that lysozyme was bound to an IgG antipeptide antibody with an association constant (4 X 10(2) M-1) 200-fold less than that for the free peptide (8 X 10(4) M-1). It was also inferred for IgM that an association constant of the order of 10(2) M-1 was sufficient to effect selective interaction in a system providing multivalent interaction. The shared conformations between protein and peptide, implied by the specific reactivity of the anti-peptide antibody with the protein, points to structural fluctuations of the surface regions and residues of globular proteins.     

Detection of ligand-induced perturbations affecting the biotinyl group of mammalian acetyl-coenzyme A carboxylase by using biotin-binding antibodies

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.     

Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands

BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.     

Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

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.     

A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.     

Assessment of small ligand-protein interactions by electrophoretic mobility shift assay using DNA-modified ligand as a sensing probe

The interaction between a small ligand and a protein were assessed by electrophoretic mobility shift assay. A sensing probe was created by modifying the model ligand, biotin, with DNA. The complex of DNA-modified ligand and anti-biotin antibody or streptavidin as a target protein was analyzed by agarose gel electrophoresis. The band corresponding to the DNA-modified ligand was shifted in the presence of the target protein, and the intensities of the shifted bands were decreased by adding increasing concentrations of free ligand ranging from 0.1 μM to 100 μM. From this calibration the concentration of ligand in the samples could be determined, allowing for evaluation of the interaction between a small ligand and its target.     

The use of antibody immobilized on supports coated with polyglycidyl methacrylate in saturation analysis

A new procedure for separation of free and bound ligand in saturation analysis (e.g., radioimmunoassay, competitive protein binding analysis) is presented. The antibody was immobilized on different carriers (glass rods, aluminium or polyethylene strips) covered with a thin layer of polyglycidyl methacrylate. The surface of the polymer had been activated by reaction with either ethylene diamine and glutaraldehyde or sulfuric acid and sodium periodate. The antibody was immobilized on this activated polymer by a covalent bond. The advantages of the presented separation methods are rapidity, simplicity, and conservation of the free and bound ligand equilibrium. A comparison with other separation techniques is carried out.     

The binding of monosaccharides to wheat germ agglutinin: fluorescence and NMR investigations

The synthesis of N-acetyl- and N-trifluoroacetyl-glucosaminides was reported. The interaction of these compounds with wheat germ agglutinin, a plant lectin specific for N-acetyl-glucosamine and sialic acid, was investigated by two complementary approaches: 1H and 19F NMR, and fluorescence spectroscopy. This last technique relies on the existence of a competitive equilibrium involving the protein, the ligand and O-(methylumbelliferyl)-N-acetyl-glucosaminide, a fluorescent saccharide. The binding constants and the chemical shifts in the complex were determined and were related to the protein structure.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag

We describe the use of the SBP-tag, a new streptavidin-binding peptide, for both the one-step purification and the detection of recombinant proteins. The SBP-tag sequence is 38 amino acids long and binds to streptavidin with an equilibrium dissociation constant of 2.5 nM. We demonstrate that a single-step purification of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity of the immobilized streptavidin used to purify SBP-tagged proteins is about 0.5 mg per milliliter of matrix, which is high enough to isolate large quantities of proteins for further study. Also, the elution conditions from the streptavidin column are very mild and specific, consisting of the wash buffer plus biotin. This combination of high-affinity, high-yield, mild elution conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/purification procedures, including robotically manipulated protocols with microtiter plates. Additionally, the SBP-tag can be used for detection since a wide variety of streptavidin-conjugated fluorescent and enzymatic systems are commercially available. We also present a new, rapid, method for the measurement of protein-protein, protein-peptide, or protein-small molecule equilibrium dissociation constants that require as little as 1 fmol of labeled protein. We call this method the spin-filter binding inhibition assay.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.     

Evaluation of small ligand–protein interaction by ligation reaction with DNA-modified ligand

A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5′-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.     

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 A) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.