Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Antibody-antigen interactions measured by surface plasmon resonance: global fitting of numerical integration algorithms

The interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sensograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BIAcore measurements. The difference in results obtained with solid-phase BIAcore and competition BIAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BIAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BIAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.     

Detection of low-molecular-weight heparin oligosaccharides (Fragmin) using surface plasmon resonance

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.     

Preservation of immobilized bacterial cell-matrix by drying for direct use in microbial sensors

A simple and effective method for the drying of immobilized bacterial cells to be used directly in a microbial biosensor for measurement of activity is reported. As a case example, plasmid-bearing cells of Alcaligenes eutrophus JMP 134, DSM 4058 were immobilized on various carriers and liquid-dried. The dried cell-matrix was used directly after rehydration/reactivation as the biological component of a biosensor for determining the concentration of xenobiotic compounds in the environment. Good viability results were obtained after long-term storage and cells exhibited no loss of plasmids responsible for the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The activity of the cells for 2,4-D was proved using a respiration electrode. No time-consuming, repeated cell cultivation and harvesting was required, as the cells preserved from a single batch served as a continuous source for activity measurements. Many other microbial cultures can be preserved by this method and the cells preserved in the form of immobilized dried cell-matrix can be used directly to perform enzymatic tests, complex biochemical conversions and for production in the reactors. The dried cell-matrix can serve as a stable interchangeable component for a multipurpose biosensor.     

Detection of Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.     

Evaluation of progesterone-ovalbumin conjugates with different length linkers in enzyme-linked immunosorbant assay and surface plasmon resonance-based immunoassay

A series of progesterone-4-ovalbumin (OVA) conjugates with different length linkers (4-, 11-, and 18-atoms long) were synthesized by successive aminocaproic acid homologation of 3-(pregn-4-ene-3,20-dione-4-yl)thiopropanoic acid (1) before conjugation to ovalbumin. The performance studies of these progesterone-4-ovalbumin conjugates showed that the effects of the length of linker on the antibody binding are dependent upon different immunoassay formats. In a rapid flow biosensor surface, on a BIAcore Surface Plasmon Resonance (SPR) instrument, antibody-binding capacities and response rate were dramatically increased for progesterone-4-ovalbumin conjugates when the length of the linker was incremented from 4 atoms to 11 or 18 atoms. Thus, highly sensitive SPR-based immunoassays for progesterone over a range of 0.1-50 ng ml(-1) were developed using biosensor surfaces immobilized with progesterone-ovalbumin conjugates having extended linkers. The SPR-based assays were fully competitive with conventional enzyme-linked immunosorbant assay (ELISA) but much more rapid and simple. However, there were little changes in antibody-binding performance using a conventional ELISA for the same conjugates. The progesterone-4-ovalbumin conjugate (1-OVA) had better antibody binding than its progesterone-7alpha-ovalbumin analog (2-OVA) in the SPR-based assay, but with a conventional ELISA there was no significant difference between these two isomeric conjugates.     

Growth and nutrient uptake by immobilised tissue culture cells of celery (Apium graveolens)

Celery cell suspension immobilized in Ca-alginate and maintained in a medium containing 0.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,5-dichlorophenoxyacetic acid (3,5-D) as the auxin sources were compared with freely suspended cells in the same media. In a 2,4-D-containing medium the immobilized cells showed a reduced dry weight and a more uniform respiration rate, whereas the uptake of sucrose, phosphate and ammonia nitrogen was similar in both immobilized and freely suspended cells. In a 3,5-D medium, no increase in dry weight occurred in the immobilized cells, but a small increase in respiration rate showed that the cells were still viable. Uptake of sucrose, phosphate and ammonia nitrogen was reduced in the immobilized cells. The role of slow growing partially differentiated immobilised cells in the synthesis of plant secondary products is discussed.     

Transport effects on surface–volume biological reactions

Many cellular reactions involve a reactant in solution binding to or dissociating from a reactant confined to a surface. This is true as well for a BIAcore, an optical biosensor that is widely used to study the interaction of biomolecules. In the flow cell of this instrument, one of the reactants is immobilized on a flat sensor surface while the other reactant flows past the surface. Both diffusion and convection play important roles in bringing the reactants into contact. Usually BIAcore binding data are analyzed using well known expressions that are valid only in the reaction-limited case when the Damk?hler number Da is small. Asymptotic and singular perturbation techniques are used to analyze dissociation of the bound state when Da is small and O(1). Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. In addition, effective rate constants are derived that illustrate the effects of transport on the measured rate constants. All these expressions provide a direct way to estimate the rate constants from BIAcore binding data.     

Characterization of a self-assembled monolayer of thiol on a gold surface and the fabrication of a biosensor chip based on surface plasmon resonance for detecting anti-GAD antibody

A biosensor chip utilizing surface plasmon resonance (SPR) was fabricated for detecting anti-glutamic acid decarboxylase (GAD) antibody, which is an indicator of the presence of type I diabetes mellitus. The sensor surfaces were constructed from various thiol mixtures of different molar ratios of 3-mercaptopropionic acid (3-MPA) to 11-mercaptoundecanoic acid (11-MUA). To determine the surface characteristics of the different alkanethiol monolayers, several quantitative and kinetic measurements were carried out. The extent of immobilization of streptavidin (SA) and biotin-GAD (the anti-GAD receptor) and the immune response of anti-GAD antibody against GAD were measured using the SPR biosensor. The terminal functional group of a thiol has different effects on the adsorption and covalent binding of protein depending on the steric hindrance. The protein chip described herein permits simple, rapid detection of anti-GAD antibody.     

Bioconjugation of CdTe quantum dot for the detection of 2,4-dichlorophenoxyacetic acid by competitive fluoroimmunoassay based biosensor

Quantum dots (QD) are semiconductor fluorescent nanoparticles, which can be made use of for environmental monitoring with high sensitivity. In view of the alarming levels of pesticides and herbicides being used in agriculture practices, there is a need for their rapid, sensitive and specific detection in food and environmental samples, as pesticides and herbicides are harmful to living beings even at trace levels. Present study was carried out to develop a reliable and rapid method for analysis and detection of 2,4-D (herbicide) using cadmium telluride quantum dot nanoparticle (CdTe QD). Fluoroimmunoassay based on the fluorescent property of quantum dot was used along with immunoassay to detect 2,4-D. CdTe capped with mercaptopropionic acid, was conjugated using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS) to alkaline phosphatase (ALP) which was in turn conjugated to 2,4-D molecule. Anti 2,4-D-IgG antibodies were immobilized in an immunoreactor column using Sepharose CL-4B as an inert matrix. The detection of 2,4-D was carried out by fluoroimmunoassay-based biosensor using competitive binding between conjugated 2,4-D-ALP-CdTe and free 2,4-D with immobilized anti 2,4-D antibodies in an immunoreactor column. It was possible to detect 2,4-D upto 250pgmL(-1). Present study also emphasizes on the resonance energy transfer between ALP and CdTe QD as a result of bioconjugation, which can be used for future biosensor development based on quantum dot-biomolecular interactions.     

Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease

Porphyromonas gingivalis , a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin–avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of l -Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae–Arg interaction (a novel host–parasite relationship).     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Clinical application of a novel sliver nanoparticles biosensor based on localized surface plasmon resonance for detecting the microalbuminuria

In order to explore the clinical application of the nanobiosensor based on localized surface plasmon resonance (LSPR), we used our LSPR biosensor to detect the microalbuminuria in this work. The sliver nanoparticles were fabricated by using nanosphere lithography. The anti-human albumin antibody was immobilized on the sensor surface by amine coupling method. The different concentrations of commercial albumin and albumin in urine samples from three mild preeclampsia patients were determined according to the peak of LSPR extinction spectra. Under optimum conditions, our results showed that the biosensor displayed a detection limit of 1 ng/ml and wide dynamic range of 1 ng/ml to 1 μg/ml. Furthermore, the microalbuminuria of three patients was determined by our biosensor within a short assay time, without sample purification. This biosensor proposed herein is easy to prepare and could be used for low-cost, rapid, label-free, and sensitive screening of the microalbuminuria. This approach provides a promising platform for developing clinical diagnostic applications.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Bacterial, Fungal, and Actinomycete Populations in Soils Receiving Repeated Applications of 2,4-Dichlorophenoxyacetic Acid and Trifluralin

Soil samples were collected from an untreated plot and plots receiving repeated applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and alpha,alpha,alpha-trifluoro-2, 6-dinitro-N,N-dipropyl-p-toluidine (trifluralin); they were then plated on media specific for bacteria, fungi, and actinomycetes. The actinomycete colony count in the trifluralin-treated plot was greater than the control, but the same as the control in the 2,4-D-treated plot. The bacterial count was lower in both treated plots. Fungal colonies in the trifluralin-treated plots were greater than the control, but not different from the control in the 2,4-D-treated plot.     

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.     

Direct ring conjugation of catecholamines and their immunological interactions

Catecholamine derivatives were synthesized with potential applications as coating antigens in biosensors or in the raising of specific antibodies. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine, and epinephrine that attach carboxylic acid functionalities directly to the aromatic ring via an easily incremented linker chain were synthesized by an electrochemical method. These derivatives were purified by convenient ion-exchange chromatography, exact positions of conjugation determined by NMR, and a dopamine derivative immobilized in situ in a BIAcore surface plasmon resonance (SPR) biosensor and its antibody binding studied in comparison with immobilization via the catecholamine primary amine. Binding of an antibody raised to an amine-conjugated protein conjugate showed clear distinction between conjugations at different positions on the catecholamine, illustrating the importance of rational conjugate design in immunosensing of the catecholamines.     

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.