Determining the parameters for receptor-ligand interaction by serial dilution method for the case when the ligand and receptor are in a pre-existing mixture

New methods of determining the binding parameters for ligand-receptor interaction are considered. The considered approaches are based on the earlier suggested method of serial dilution and application of so-called coordinates of dilution. It was shown that the suggested methods allow to evaluate affinity constant and ligand concentration even for the case, when the receptor and corresponding ligand of unknown concentration are in a mixture and their separation from each other is impossible. In this connection the suggested methods are especially useful for studying the ligand-receptor interaction if the receptor is very liable and its purification from the ligand would cause drastic changes of its binding properties.     

Electrochemical modulation of antigen-antibody binding

The label-free amperometric detection of a rabbit IgG antigen by an anti-rabbit IgG antibody is achieved by observing the electrochemistry at a glassy carbon electrode modified with antibody entrapped in an electrodeposited polypyrrole membrane. In a flow injection apparatus the electrode is pulsed between -0.2 and +0.4 V versus Ag/AgCl. The pulsing of the electrode switches the polypyrrole membrane between the oxidised and reduced states. When antigen is injected into the flow stream a change in current is observed at the electrode despite the antigen or antibody being redox inactive at the potentials employed. It is proposed that this current is due to a change in the flux of ions into and out of the polypyrrole matrix during a pulse when the poly-anionic antigen is present. The immunoreaction was reversible because the 200 ms pulse at each potential was too short to allow secondary bonding forces (hydrogen bonding and hydrophobic forces) which are responsible for the strength of the antibody-antigen complex to be established. The consequence of the reversibility of the antigen-antibody binding is a low apparent affinity constant but an easily regenerated recognition interface.     

Modeling micropatterned antigen-antibody binding kinetics in a microfluidic chip

The reaction kinetics of antigen-antibody binding in the electrokinetically controlled microfluidic heterogeneous immunoassays has been investigated by numerical simulations. A two-dimensional computational model was employed to include the mass transport (convection and diffusion) and binding reaction between the antigen in the bulk flow and the immobilized antibody at the channel surface. The influence of the bulk velocity, the concentrations of the antibody and antigen, and the geometry of the microchips was studied for a variation of conditions and the guidance for designing of microfluidic immunoassay was provided. The model also shows that electrokinetically driven immunoassays have better reaction kinetics than pressure-driven ones, resulting from the plug-like velocity profile. Finally, a multi-patch immunoassay chip was analyzed and the reaction kinetics was optimized by rearranging the reaction patches at the channel surfaces.     

Determination of binding parameters in the presence of coupled reactions

Components of a binding reaction may undergo nonbinding reactions: receptors may be degraded, internalized, or exchanged with cryptic sites; ligand may be degraded or compartmented. In such cases the parameters that characterize the system are not obtained from the usual equilibrium analyses. We have simulated the reactions of such systems and generated association curves, "Scatchard" plots, and "Scatchard-like" plots that permit the calculation of binding affinity and receptor number not normally calculable under nonequilibrium binding conditions. In particular, we show that certain coupled reactions produce local maxima and sigmoid shapes in association curves and that the maxima can be used to obtain affinities and receptor numbers.     

Determination of dependence of binding parameters on receptor occupancy

Measurements of the binding of ligand to receptors that are macromolecules, either free or components of biomembranes, often show deviation from what is expected of a simple reaction described by an association and a dissociation rate constant. A more versatile model and more discriminating experiments are required for a satisfactory explanation. This paper is based on a general model of the binding reaction in which the rate constants and equilibrium constant are dependent upon occupancy of receptors. The analysis of the model leads to three kinds of experiments: (1) equilibrium measurements which permit quantitative determination of a dissociation equilibrium parameter as a function of receptor occupancy; (2) measurements prior to equilibrium which yield the same information; and (3) measurements prior to equilibrium which reveal quantitatively the dependence of both association and dissociation rate parameters separately, on occupancy.     

A single- and a dual-fractal analysis of antigen-antibody binding kinetics for different biosensor applications.

The diffusion-limited binding kinetics of antigen (or antibody) in solution to antibody (or antigen) immobilized on a biosensor surface is analyzed within a fractal framework. The data is adequately described by a single- or a dual-fractal analysis. Initially, the data was modelled by a single-fractal analysis. If an inadequate fit was obtained then a dual-fractal analysis was utilized. The regression analysis provided by Sigmaplot, 1993 (Scientific Graphing Software: User's Manual. Jandel Scientific, San Rafael, CA) was utilized to determine if a single-fractal analysis is sufficient, or a dual-fractal analysis is required. In general, it is of interest to note that the binding rate coefficient and the fractal dimension exhibit changes in the same direction (except for a single example) for the antigen-antibody systems analyzed. Binding rate coefficient expressions as a function of the fractal dimension developed for the antigen-antibody binding systems indicate a high sensitivity of the binding rate coefficient on the fractal dimension when both a single -as well as a dual-fractal analysis is used. For example, for a single-fractal analysis and for the binding of human endothelin-1 (ET-1) antibody in solution to ET-1(15-21) x BSA (bovine serum albumin) immobilised on a surface plasmon resonance surface, the order of dependence of the binding rate coefficient, k on the fractal dimension, Df is 7.0945. Similarly, for a dual-fractal analysis and for the binding of parasite L. donovani diluted pooled sera in solution to fluorescein isothiocyanate-labeled anti-human immunoglobulin IgG immobilized on an optical fibre, the order of dependence of k1 and k2 on Df1 and Df2 were 6.8018 and -4.393, respectively. Binding rate coefficient expressions are also developed as a function of the analyte (antigen or antibody) concentration in solution. The binding rate coefficient expressions developed as a function of the fractal dimension(s) are of particular value since they provide a means to better control biosensor performance by linking it to the heterogeneity on the surface, and emphasize in a quantitative sense the importance of the nature of the surface in biosensor performance.     

Size and structure of antigen-antibody complexes: thermodynamic parameters

The role of antigen-antibody (Ag-Ab) complexes in the immune response depends, in part, on the size of the complexes. Previously, we combined electron microscopy with classical and quasi-elastic light scattering to characterize the molecular weight distribution and the conformation of Ag-Ab complexes made from bovine serum albumin (BSA) and pairs of anti-BSA monoclonal antibodies at a single concentration and Ag:Ab molar ratio. In this report, the molecular weight distribution of Ag-Ab complexes was determined by classical light scattering at a single Ag:Ab ratio and over a range of concentrations, and binding of BSA to pairs of MAb was determined by radioimmunoassay at several Ag:Ab molar ratios. A thermodynamic model was developed for the equilibrium size distribution of Ag-Ab complexes formed between a pair of MAb, each with unique affinity and specificity, and an Ag containing a single epitope for each of the pair of MAb. The combined experimental data were used in conjunction with the model to determine the values of cyclization and polymerization constants. Successful determination of the parameters required data from both classical light scattering and electron microscopy. Cyclization constants were lower than those reported in other studies of Ag-Ab complexes; this may be attributable to our use of a protein Ag, as compared to a divalent hapten. In two out of three cases, cyclization constants increased with increasing number of Ab in the complex, in contrast to previous assumptions. The validity of the thermodynamic model was further shown by its ability, in combination with conformational and hydrodynamic model, to predict the hydrodynamic radius of the complexes over a wide range of experimental conditions.     

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.     

Ligand-receptor interactions: a new method for determining the binding parameters

A new experimental procedure and new plot coordinates that allow determination of the binding parameters of ligand-acceptor interaction have been proposed. Instead of titration of a constant concentration of receptors with changing concentrations of ligand, as requested by the well-known methods of Klotz and Scatchard, a series of sequential dilutions of the reacting ligand-receptor mixture is suggested. This allows the application of a new coordinate system that transforms the binding isotherms into straight lines. The case of one acceptor with two classes of receptors with different binding constants is also considered briefly, where the correspondent graphs are nonlinear. It is suggested that in some cases this approach can be a simple and convenient substitute of the broadly used methods of Klotz and Scatchard.     

Determination of rate constants of antigen-antibody reaction. A theory

A new method of determination of rate constants for antigen-antibody interactions is proposed. This method is based on a solid phase immunoenzymatic analysis of the dynamics of elution of immobilized antigen-bound antibodies in the presence of a free antigen. The kinetics of this process is described by a system of differential equations, whose solution results in expression defining the dynamics of antibody interaction with immobilized and free antigens. Simple formulas were derived for the calculation of the rate and equilibrium constants for the antibody-antigen reaction on the basis of experimental kinetic curves. The use of theoretical kinetic curves for antibody elution showed that these formulas reflect with a high degree of accuracy the kinetic properties of the reaction under study.     

Determination of a dimethylaniline mixture in the intermediate products for the production of semisynthetic penicillins]

A gas-chromatographic method for determination fo the content of dimethylaniline (DMA) in 6-aminopenicillanic acid, a semi-product for production of semi-synthetic antibiotics, was elaborated. The chloroform extracts of DMA from the alkaline solutions of the preparation were analysed in a gas chromatograph with a flame-ionization detector on a stainless steel column 2 m long and the inner diameter of 2 mm filled with 10 per cent OV-17 on chromosorb W-HMDS at a temperature of 112 degrees C. The minimum detectable amount of DMA was 5.10(-9) g. The assay error was +/- 5.35 per cent. The method may be used for the assay of other semi-synthetic antibiotics.     

Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.     

Determination of the parameters for an athetotic arm model

A model is developed to represent elbow motions of a cerebral palsied arm with athetotic movements. The parameters of the model are defined and determined. The resulting computer model can then be used to either generate athetotic motions or to regenerate prerecorded experimental data.     

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.     

Determination of quantitative parameters of the binding of F-protein (phosphofructokinase) with myosin and localization of binding sites on the myosin molecule

To determine the localization of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM K-phosphate pH 6.5 was studied, using sedimentation, electron microscopic and optical diffraction methods. Sedimentation experiments showed that F-protein binds to myosin and myosin rod rather than to light meromyosin or S-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of F-protein binding sites on myosin calculated per chain weight of 80 000 is 5 +/- 1. The sedimentation results were confirmed by electron microscopic data. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added before or after filamentogenesis. A comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones revealed a marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case.