Manne Stenberg

Kinetics of antigen-antibody reactions at solid-liquid interfaces

The kinetics of antigen-antibody reactions is reviewed with special attention paid to the specific properties at solid-liquid interfaces. Theories of possible diffusion limitation in forward reaction rates are compared to experiments. It is found that the intrinsic forward reaction rate in the bimolecular antigen-antibody reaction is normally not limited by diffusion either in solution or at the solid-liquid interface. However, reactions at the solid-liquid interface can be diffusion limited due to depletion of reactants close to the surface. This effect depends on geometry, intrinsic reaction rate and surface concentration of receptor molecules. Normally cell surface reactions are not diffusion limited whereas reactions at artificial surfaces often are limited by diffusion. When not limited by diffusion it is also found that the intrinsic forward and reverse reaction rates are lower for surface reactions compared to reactions in solution. Antigen-antibody reactions at solid-liquid interfaces can often be considered as practically irreversible and limited by mass transport or steric interactions.

External diffusion in solid-phase immunoassays

Calculations are presented describing the influence of external diffusion in the kinetics of solid-phase immunoassays. The analysis is concerned with systems where one reactant is immobilized at the surface of a sphere of arbitrary radius. The solution for a plane surface is found as a limiting case. The factors determining whether the reaction is diffusion or reaction controlled are found to be sphere radius, surface concentration of binding sites, forward reaction rate and diffusion constant of reacting species. Means of determining whether the reaction is diffusion or reaction controlled from observable quantities are described. When applied to heterogeneous antibody-antigen binding it is found that normally the binding to cell-size spheres is not limited by external diffusion. However, when applied to solid-phase assays with high surface concentrations of binding sites immobilized at plane surfaces or macroscopic spheres the binding is found to be diffusion limited. The importance of a mass transfer analysis in this case is also discussed.

Kinetics of antibody-binding to surface-immobilized antigen: Influence of mass transport on the enzyme-linked immunosorbent assay (ELISA)

Abstract The kinetics of antibody-binding to surface-immobilized antigen was studied by ellipsometry, using bovine serum albumin as antigen. The results show that the initial binding of antibody was linear to the square root of time rather than time, indicating an initial diffusion-rate limitation of the reaction. The antibody-binding rate was retarded at a surface concentration of 0.2 μg/cm2 and became reaction-rate limited above this surface concentration. The reverse reaction was shown to be too slow to be measured after 43 h dissociation. A Scatchard plot of the data showed time dependence which gave a further indication that the antibody-binding was not an equilibrium reaction. The results obtained from ellipsometry experiments were applied to experiments with the enzyme linked immunosorbent assay (ELISA) which by the sensitive detection system measures the initial antibody-binding to immobilized antigen. It was shown that the dose-response curve of the ELISA could be explained assuming a diffusion-rate limited, irreversible antibody-binding to the immobilized antigen.

A receptor-ligand reaction studied by a novel analytical tool—The Isoscope ellipsometer☆

Abstract A receptor-ligand binding reaction, cholera toxin-ganglioside GM 1 , was studied by diffusion of the ligand over a receptor-coated surface. The surface reaction was analyzed by the use of a novel analytical tool, the Isoscope ellipsometer. The thickness profile of the receptor-ligand binding reaction can be directly visualized and photographed with this instrument. Experimental thickness profiles were compared to different theoretically calculated thickness profiles assuming different kinetics of the receptor-ligand reaction. Experimental data were best fitted to theoretical calculations assuming an irreversible and diffusion rate-limited surface reaction. The use of the Isoscope ellipsometer for further studies on receptor-ligand reactions is discussed.

Dissociation of antibodies bound to surface-immobilized antigen

The dissociation of antibodies bound to surface-immobilized antigen was investigated by the ELISA, using a hapten (TNP) as antigen. Antibody binding was found to be stable, and no half-time of dissociation could be defined within 69 h. The role of the bivalence of antibodies and the difference between a homogeneous and a heterogeneous reaction was investigated by comparing the dissociation rate of antigen-antibody complexes formed by monovalent Fab fragments from surface-immobilized antigen and the dissociation rate of TNP-lysine from antibodies in a homogeneous liquid phase. Fab fragments were found to dissociate with a half-time value of about 16 h, whereas the homogeneous binding of TNP-antibody dissociated with a half-time of less than 4 h, indicating that both the bivalence of antibodies and the solid phase contributed to the stability of surface-bound antigen-antibody complexes. Qualitative differences between antibodies produced by different clones in a polyclonal antibody response to TNP was investigated by a spot assay. The results indicated that a minority of the antibodies produced had the capacity of binding practically irreversibly to solid-phase-immobilized antigen. The impact of the results on the interpretation of data from solid-phase assays is discussed together with the biological importance of the findings.

A diffusion limited reaction theory for a microtiter plate assay

Calculations are presented describing the diffusion limited kinetics of a solid-phase immunoassay in which reactants are immobilized at the inner surface of a cylindrical well. The calculations refer to an unstirred situation and simplified expressions are presented which can be used for calibration and optimization of the assay.

Surface-induced aggregation of ferritin: Kinetics of adsorption to a hydrophobic surface

The adsorption of ferritin from a water solution to a hydrophobic methylised quartz surface was studied by transmission electron microscopy, allowing direct examination of the iron core of the molecule without further preparation. The initial adsorption was seen to result in small clusters of molecules, the number of sites/cm(2) being concentration dependent. The adsorption process continued via cluster growth. The rate of adsorption increased and the process became mass transport limited. The clusters formed initially had low fractal dimensions (D approximately 1.0) and a coordination number, cn of 2.6-2.8, which increased with time. These clusters were abruptly restructured at a coordination number of 3.5, and the apparent rate of adsorption decreased during the reorganisation of the adsorbed layer. Finally, an equilibrium level was reached which was stable for at least 24 h. The distribution of ferritin molecules at equilibrium was in clusters with a fractal dimension of D = 1.14 +/- 0.16 and D= 1.33 +/- 0.08, respectively, for ferritin concentrations in the bulk of 10 and 100 microg/ml. Rinsing of adsorbed ferritin layers with buffered salt solution resulted in a rapid transient condensation of the clusters, but the net dissociation of protein was slow with the rate of dissociation being proportional to the logarithm of time. The condensed clusters were slowly restructured to linear polymers of ferritin molecules with a coordination number of 1.9 after 24 h of rinsing. The dissociation of protein molecules continued slowly for more than 3 days of rinsing. The results of the present study indicate that the rate of protein adsorption and desorption is strongly related to the supramolecular structure of the adsorbed protein film. Dense clusters of protein are not stable and this phenomenon may explain the formation of a dynamic equilibrium in spite of the fact that protein adsorption to a solid phase may appear to be practically irreversible.

Determination by ellipsometry of the affinity of monoclonal antibodies

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

Calibration by ellipsometry of the enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) was analyzed with regard to possible diffusion limitations of the binding reaction. The absorbance values of the assay were found to follow the time and concentration relations that would occur when diffusion of antibody to the surface is the rate limiting step. This relationship was used in order to calibrate the absorbance values of the ELISA with antibody concentration by ellipsometry, which itself allows direct measurement of the amount of antibody bound to the solid phase.

Visual detection of organic monomolecular films by interference colors

Thin (<10-nm) adsorbed organic films are visible to the unaided eye, if the substrate is first covered with a dielectric film giving a strong interference color, a so-called sensitive color. It is found that a single low-index dielectric film on an absorbing substrate gives optimal sensitivity. A detection limit in the subnanometer range is predicted and confirmed by experiments. Two practical designs using silicon and glass substrates are discussed. These slides can be produced by industrial methods and have proved to give good visualization of monomolecular protein films, e.g., antigen–antibody layers. They have a detection limit of 0.7 nm or 100 ng of protein/cm2 surface.