Antigen-antibody binding kinetics for biosensor applications. A dual-fractal analysis

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 fit obtained by a dual-fractal analysis is compared with that obtained from a single-fractal analysis. In some cases, the dual-fractal analysis provides an improved fit when compared with a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot (San Rafael, CA). These examples are presented. It is of interest to note that the state of disorder (or the fractal dimension) and the binding rate coefficient both increase (or decrease, a single example is presented for this case) as the reaction progresses on the biosensor surface. For example, for the binding of monoclonal antibody MAb 49 in solution to surface-immobilized antigen, a 90.4% increase in the fractal dimension (Df1 to Df2) from 1.327 to 2.527 leads to an increase in the binding rate coefficient (k1 to k2) by a factor of 9.4 from 11.74 to 110.3. The different examples analyzed and presented together provide a means by which the antigen-antibody reactions may be better controlled by noting the magnitude of the changes in the fractal dimension and in the binding rate coefficient as the reaction progresses on the biosensor surface.     

Analyte-receptor binding kinetics for biosensor applications. An analysis of the influence of the fractal dimension on the binding rate coefficient

The diffusion-limited binding kinetics of antigen (analyte), in solution with antibody (receptor) immobilized on a biosensor surface, is analyzed within a fractal framework. Most of the data presented is adequately described by a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot. A single example of a dual-fractal analysis is also presented. It is of interest to note that the binding-rate coefficient (k) and the fractal dimension (Df) both exhibit changes in the same and in the reverse direction for the antigen-antibody systems analyzed. Binding-rate coefficient expressions, as a function of the Df developed for the antigen-antibody binding systems, indicate the high sensitivity of the k on the Df when both a single- and a dual-fractal analysis are used. For example, for a single-fractal analysis, and for the binding of antibody Mab 0.5 beta in solution to gp120 peptide immobilized on a BIAcore biosensor, the order of dependence on the Df was 4.0926. For a dual-fractal analysis, and for the binding of 25-100 ng/mL TRITC-LPS (lipopolysaccharide) in solution with polymyxin B immobilized on a fiberoptic biosensor, the order of dependence of the binding-rate coefficients, k1 and k2, on the fractal dimensions, Df1 and Df2, were 7.6335 and -11.55, respectively. The fractional order of dependence of the k(s) on the Df(s) further reinforces the fractal nature of the system. The k(s) expressions developed as a function of the Df(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 further emphasize, in a quantitative sense, the importance of the nature of the surface in biosensor performance.     

Surface engineering: optimization of antigen presentation in self-assembled monolayers

The formation of self-assembled monolayers (SAMs) on gold surfaces containing an antigenic peptide (NANP)6 and HS(CH2)11OH, and the specific binding of a monoclonal antibody to these layers were investigated by surface plasmon resonance (SPR). Peptides were synthesized by solid-state phase synthesis and were linked either to cysteine or to an alkyl-thiol to allow covalent attachment to gold. The content of the peptide in the SAMs was systematically varied, and the binding properties of the monoclonal antibody were compared with those measured by microcalorimetry in solution. At a critical peptide concentration in the SAM an optimal antibody binding and complete surface coverage was attained. At lower peptide concentrations, the amount of adsorbed antibody decreased; at higher peptide concentrations, the binding constant decreased. These effects can be explained if the accessibility of the antigenic epitopes depends on the peptide density. Addition of free antigen induced the desorption of bound antibodies and allowed accurate measurements of the dissociation rate constant. Binding constants obtained from steady-state measurements and from measurements of the kinetic rate constants were compared.     

Single- and dual-fractal analysis of hybridization binding kinetics: biosensor applications

The diffusion-limited hybridization kinetics of analyte in solution to a receptor immobilized on a biosensor or immunosensor surface is analyzed within a fractal framework. The data may be analyzed by a single- or a dual-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot. It is of interest to note that the binding rate coefficient and the fractal dimension both exhibit changes in the same direction for both the single-fractal and the dual-fractal analysis examples presented. For example, for a single-fractal analysis and for the hybridization of 10 nM 16CFl (oligonucleotide) to 16B immobilized via sulfosuccinimidyl-6-(biotinamido)hexanoate and streptavidin using chemical and thermal regeneration (Abel, A. P.; Weller, M. G.; Duveneck, G. L.; Ehrat, M. Widmer, H. M. Anal. Chem. 1996, 68, 2905-2912), an increase in the fractal dimension, Df from 1.211 (chemical regeneration) to 1.394 (thermal regeneration), leads to an increase in the binding rate coefficient, k, from 86.53 (chemical regeneration) to 100.0 (thermal regeneration). An increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. When a dual-fractal analysis was utilized, an increase in the fractal dimension value from Df1 to Df2 leads to an increase in the binding rate coefficient value from k1 to k2. The fractional order of dependence of the binding rate coefficient, k1, on (a) the analyte (rRNA) concentration in solution and (b) on the fractal dimension, Df1, for the hybridization kinetics to detect Listeria species (Fliss, R.; St-Laurent, M.; Emond, E.; Simard, R. E.; Lemieux, R.; Ettriki, A.; Pandian, S. Appl. Microbiol. Biotechnol. 1995, 43, 717-724.) further reinforces the fractal nature of the system. The binding rate coefficient(s) expressions developed as a function of the analyte concentration in solution and the fractal dimension are of particular value since they provide a means to better control of biosensor or immunosensor performance.     

石蜡在多组元粘结剂和高比重合金喂料中的低温热脱脂动力学

为了控制热脱脂过程中的缺陷,研究了石蜡（ＰＷ）在多组元粘结剂体系和高比重合金喂料中的低温热脱脂行为。研究表明,在２５０℃以下,当保温时间相当长时,ＰＷ可以全部脱完。在此温度下,石蜡在聚合物和粉末孔隙中的扩散为石蜡的脱除速度控制,脱脂率与脱脂时间、样品厚度的倒数服从指数关系,即：１－ｍ（ｔ）ｍ（０）＝１＋Ｃ′ｅＫｄｔ。脱脂过程中产生的缺陷主要由此阶段不正确的脱脂引起     

Kinetics model for leaching of ion-adsorption type rare earth ores

This paper aims to establish a mathematical model that can analyze the whole leaching kinetics process of ion-adsorption type rare earth ores. This leaching process is composed of three steps: (1) ammonium ions arrive at the ore particle surface through the diffusion layer; (2) ammonium ions exchange with rare earth ions; and (3) rare earth ions enter into the external solution through the diffusion layer. In the leaching process, it is hypothesized that the ore particle size remains constant. The process of ammonium ions and rare earth ions passing through the diffusion layer was described by the Fick law, and the reversible ion exchange process between ammonium ions and rare earth ions was described by the Kerr model. A leaching kinetics model of rare earth ions by ammonium ions was constructed. Accuracy of this kinetics model was verified with laboratory tests. It is found that the correlation coefficients of all data are greater than 0.9000. The proposed kinetics model is therefore feasible for kinetics analysis throughout the leaching process.     

Surface diffusion of cellulases and their isolated binding domains on cellulose

The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-beta-1-4-glycanase (Cex), an endo-beta-1-4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 x 10(-11) to a maximum of 1.2 x 10(-10) cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimi cellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.     

Hydrogen induced grain boundary fracture in high purity nickel and its alloys—Enhanced hydrogen diffusion along grain boundaries

Embrittlement of nickel by solute hydrogen is usually accompanied by a change in fracture mode from ductile rupture to an intergranular mode. When hydrogen is supplied at the external surface, the kinetics of this embrittlement is shown to be more rapid than can be accounted for by lattice diffusion of hydrogen. The embrittlement kinetics are shown to be consistent with hydrogen diffusion along grain boundaries and the grain boundary diffusivity is derived over the temperature range 274–314 K. The effects of carbon and sulfur grain boundary segregation on the kinetics of grain boundary embrittlement were also investigated. Segregation of carbon decreases the extent of embrittlement while sulfur segregation increases the amount of embrittlement relative to that observed in pure nickel. These effects are interpreted in terms of the effects of segregated solutes on hydrogen grain boundary diffusivity and on the critical hydrogen concentration for intergranular fracture.     

Kinetic study on bastnaesite concentrate mechanochemical decomposition in NaOH solution

Mechanochemical reaction involves simultaneous chemical reaction and particle crushing;the latter increases the effective reaction area and improves the reactivity,thus enhancing its kinetics.The classical shrinking core model was used to model the kinetics of bastnaesite mechanochemical decomposition in NaOH solution,which shows a questionable result.Mechanochemical reaction is a dynamic process,where the particle shape and concentration in reaction interface undergo constant change.Thus,a physically consistent model was applied to describe the kinetics.The variations in OH~-concentration and particle shape were considered in the revision of model.Considering the variation in OH~-concentration in solution with time,the model with varying OH~-concentration agrees better with the experimental data,improving the regression coefficients to between 0.936 and 0.992.By introducing fractal geometry to deal with the irregular system,the model was further optimized,and the regression coefficients increase to between 0.940 and 0.997.All these models considere shrinking particle approaches and controlling mechanisms for the diffusion and chemical reaction.Finally,the fractal model with varying OH~-concentration was selected to describe the mechanochemical decomposition of bastnaesite,which indicates that the process is controlled by chemical reaction.     

Antibody mediated lung targeting of long-circulating emulsions

Monoclonal antibody 34A, which specifically binds to a surface glycoprotein (thrombomodulin) of the pulmonary endothelial cell surface in mice, has been conjugated to the surface of long-circulating emulsions composed of Castor oil, phosphatidylcholine and polyethylene glycol coupled to distearoylphosphatidyl-ethanolamine. These antibody-containing emulsions were found capable of binding to the lung when injected into mice through the tail vein. The level of lung accumulation of these emulsions depends on the amount of antibodies conjugated to the surface of the emulsions. With an input antibody to lipid ratio of 2:1 (w/w), 30% injected emulsions were found in the lung 30 minutes after administration. Such high level accumulation can be blocked by co-administration of free 34A antibody, indicating that the binding is specific and 34A antibody mediated. Kinetic studies showed that emulsion targeting to the lung was very rapid. Five minutes after tail vein injection, the total amount of emulsion found in the lung was the highest among the time points examined, indicating the completion of lung binding. However, about 50% of the initially bound emulsions remained bound for more than 4 hours. These results indicate that the targeted drug delivery using oil-in-water emulsions could be very useful to enhance the therapeutic efficacy of lipophilic drugs.     

Kinetics of factor VIII-von Willebrand factor association

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.     

Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions

Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.     

The cortisol response to psychological stress in temporomandibular dysfunction

Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.     

A membrane-based displacement flow immunoassay

The use of a membrane-based continuous flow displacement immunoassay for detection of nanomolar quantities of explosives is demonstrated, and the kinetics of this system are characterized through experimentation. Antibodies of 2,4,6-trinitrotoluene (TNT) are immobilized onto a porous membrane with surface reactive sites designed to facilitate the covalent binding of the antibody. After saturating the immobilized antibody binding sites with labeled antigen, target analyte is introduced in flow, and the displacement reactions are monitored using a fluorometer. The displaced labeled antigen detected is proportional to the concentration of the analyte introduced to the antibody-labeled antigen complex. Multiple assays were performed at flow rates of 2.0, 1.0, 0.50, and 0.25 mL/min using membranes saturated with varying TNT antibody concentrations. The signal intensity (i.e. the concentration of displaced labeled antigen) was independent of antibody concentration at 1.0 mL/min, but proportional to antibody concentration at 0.25 mL/min. Our data suggests that the lower flow rate created a longer interaction time between the injected analyte and the antibody-labeled antigen complex, resulting in greater displacement of the labeled antigen and higher signal intensities than seen at higher flow rates.     

微尺度氧化铁粉的低温还原机理

用热重分析法研究低温条件下(450、500、550和600℃),氢气还原微尺度氧化铁的还原动力学行为。结果表明:随氧化铁粉粒径减小和反应温度升高,初始反应速率加快,后期反应速率减慢。这是因为反应后期生成大量铁须,铁须之间形成搭桥,导致还原后的粉末严重烧结并致密化,阻碍气体的扩散,致使反应速率减慢。且随着粉体粒径减小,粉体表面吸附能增大,粉体致密程度提高,反应后期的粘结现象更加严重,反应速率相应减慢。采用Hancock-Sharp方法分析微尺度氧化铁粉恒温还原的动力学过程,发现前期阶段Fe2O3→Fe3O4,在500℃以下,相界面化学反应的阻力所占的比例较大,表明此阶段的反应控速环节为界面化学反应,温度超过500℃时,则由界面化学反应机理和相转变机理共同控制,点阵结构由Fe2O3的斜方六面体结构转变为Fe3O4的立方结构;后期阶段Fe3O4→Fe,由于粉体发生粘结,还原反应的控速环节转变为扩散控速。     

Effect of stimulation on the stabilization of platelet-fibrinogen interactions

The interaction between fibrinogen and stimulated platelets is a multiphasic process that culminates in the stabilization of ligand binding and reduced accessibility of bound fibrinogen to exogenous antibody. The present study was designed to further explore platelet-fibrinogen interactions by examining the effect of agonist on bound fibrinogen expression and interaction with stimulated platelets as a function of time after ligand binding. Two agents were identified, Zn2+ and phorbol myristate acetate (PMA), which support progressive decreases in bound fibrinogen expression on platelets, but fail to support the stabilization of fibrinogen binding. Sixty min after binding to platelets, approximately 80% of bound fibrinogen remained reversibly associated with Zn(2+)- or PMA-treated platelets and failed to associate with the Triton X-100 insoluble cytoskeleton. In contrast, polyclonal anti-fibrinogen antibody binding decreased by more than 66%. Over the same time course, fibrinogen binding to control platelets, stimulated with thrombin or ADP, was not only accompanied by a 70% decrease in antifibrinogen antibody binding, but also an inability of EDTA or excess exogenous fibrinogen to dissociate more than half of platelet-associated fibrinogen, as well as the progressive association of bound fibrinogen with the platelet cytoskeleton. Costimulation of platelets with ZnCl2 and thrombin or ZnCl2 and ADP enhanced overall fibrinogen binding but not the EDTA-resistant component, and prevented the recovery of irreversibly bound fibrinogen with the Triton X-100 insoluble cytoskeleton. Costimulation of PMA- or Zn(2+)-treated platelets with low doses of A23187, however, restored the stabilization of platelet-fibrinogen interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding kinetics of monoclonal antibody using antigen-beta-galactosidase hybrid protein: application to measurement of peptide antigenicity

A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the beta-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (KD), and the association (Kass) and dissociation (Kdiss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (KA) of the peptide can be estimated from the KD value of the antigen-beta-galactosidase-antibody interaction, and from the IC50 value of the peptide.     

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.     

Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows: antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.