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PubMed2006

The behavior of mixtures of paralytic shellfish toxins in competitive binding assays.

Organisms that contain paralytic shellfish toxins (PSTs) may contain many members of this toxin family. PSTs block voltage-gated sodium channels (Na channel) and elicit neurotoxicity. Animals, including humans, may encounter PST mixtures via consumption of tainted seafood, contaminated water, or the microalgae that produce the toxins. PST binding by the Na channel as well as other proteins such as antibodies and saxiphilin have been used to develop biomolecular assays for PSTs. An equation that predicts the combined effects of binary and ternary PST mixtures has been experimentally validated for two unrelated STX-binding proteins, the rat brain Na channel and a saxiphilin from the xanthid crab Liomera tristis. It was found that the most potent toxin or toxins in any mixture profoundly affect the cumulative potency of the mixture, overwhelming weaker toxins with the transition from strong to weak toxicity and changing in a curvilinear manner. Less active PSTs must be several orders of magnitude more concentrated than stronger toxins for the mixture to reflect their potency. This behavior is important in understanding how toxin mixtures may act at the Na channel receptor via which PSTs exert their neurotoxicity and that the presence of weaker toxins does not dilute the effect of stronger toxins in a linear fashion. This strong dominance of a mixture by the most potent toxins also has implications for measurement of toxic test samples and for standards that may contain low levels of highly potent bioactive impurities. This equation has been extended to mixtures of PSTs containing more than three toxins and may be applicable to other natural contaminants and any competitive binding assays used to detect their presence and measure their concentration.

Animals↗
PubMed1996

Cocaine concentration-effect relationship in the presence and absence of lidocaine: evidence of competitive binding between cocaine and lidocaine.

UNLABELLED: To better understand the interaction between cocaine and lidocaine, we studied the cocaine's concentration-effect relationship for action potential duration (APD) and the rate of rise of phase 0 of the action potential (Vmax) of canine papillary muscle in the presence and absence of lidocaine. We measured APD and Vmax during programmed stimulation and superfusion with normal Tyrode's, 30 microM cocaine and 30 microM cocaine + 30 microM lidocaine. Using two microelectrodes, we simultaneously recorded action potentials from two sites during programmed stimulation and measured the conduction velocity and effective refractory period during exposure to normal Tyrode's, cocaine and cocaine + lidocaine. Cocaine with or without lidocaine delayed the plateau of the APD restitution curve. At 1000 msec cycle length, the addition of 30 microM lidocaine to the superfusate containing 30 microM cocaine shortened the time constant for reactivation of Vmax from 514 +/- 63 to 234 +/- 28 msec (P < .01). Lidocaine also improved the conduction velocity decreased by cocaine, but did not significantly change the effective refractory period. The configuration of cocaine concentration-effect curve for APD was biphasic. For cocaine concentrations < 100 microM, APD progressively shortened prolonged with increasing concentrations. As cocaine concentrations increased > 100 microM, APD progressively shortened. The addition of lidocaine to the superfusate with cocaine > 100 microM tended to attenuate the progressive APD shortening due to cocaine. Lidocaine shifted the curve correlating cocaine concentration and reduction of Vmax rightward, but preserved Emax at cocaine concentration > 225 microM. These findings suggest competitive antagonism between cocaine and lidocaine at a single sodium channel receptor. CONCLUSION: lidocaine displaces cocaine from the sodium channel receptor through competitive binding. Lidocaine may prove to be beneficial in reversing cocaine-induced slowing of ventricular conduction.

Action Potentials↗
PubMed1979

Serodiagnosis of viral hepatitis A by a modified competitive binding radioimmunoassay for immunoglobulin M anti-hepatitis A virus.

A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.

Acute Disease↗
PubMedDate not supplied

A spin-column procedure for estrogen receptor equilibrium and competition binding analysis.

Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.

Alphavirus Infections↗
PubMed2003

Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate.

The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress.

Binding, Competitive↗
PubMedDate not supplied

Cooperative and competitive binding in synergistic mixtures of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A.

Synergism between cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 degrees C show that binding levels of cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 degrees C, however, most of the cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 degrees C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE < 1 was generally observed for the reactions at 5 degrees C indicating anti-synergistic behavior. Degrees of synergistic binding (DSB) were also calculated, where anti-synergistic mixtures had DSB < 1 and synergistic mixtures had DSB>1. We conclude that the lower extents of binding at 5 degrees C are due to competition for binding sites by the cellulase components in the mixtures and the enhanced binding extents at 50 degrees C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.

Actinomycetales↗
PubMed1997

Inhibition of peroxisome proliferator signaling pathways by thyroid hormone receptor. Competitive binding to the response element.

Peroxisome proliferators (e.g. clofibric acid) and thyroid hormone play an important role in the metabolism of lipids. These effectors display their action through their own nuclear receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid hormone receptor (TR). PPAR and TR are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related nuclear receptor superfamily. The present study focused on the convergence of the effectors on the peroxisome proliferator response element (PPRE). Transcriptional activation induced by PPAR through a PPRE was significantly suppressed by cotransfection of TR in transient transfection assays. The inhibition, however, was not affected by adding 3,5,3'-triiodo-L-thyronine (T3). Furthermore, the inhibition was not observed in cells cotransfected with retinoic acid receptor or vitamin D3 receptor. The inhibitory action by TR was lost by introducing a mutation in the DNA binding domain of TR, indicating that competition for DNA binding is involved in the molecular basis of this functional interaction. Gel shift assays revealed that TRs, expressed in insect cells, specifically bound to the 32P-labeled PPRE as heterodimers with the retinoid X receptor (RXR). Both PPAR and TR bind to PPRE, although only PPAR mediates transcriptional activation via PPRE. TR.RXR heterodimers are potential competitors with PPAR.RXR for binding to PPREs. It is concluded that PPAR-mediated gene expression is negatively controlled by TR at the level of PPAR binding to PPRE. We report here the novel action of thyroid hormone receptor in controlling gene expression through PPREs.

Animals↗
PubMed1984

Specificity of human IgM M-proteins that bind to myelin-associated glycoprotein: peptide mapping, deglycosylation, and competitive binding studies.

Ten patients with neuropathy and IgM M-proteins that bind to the myelin-associated glycoprotein (MAG) were studied to determine whether the M-proteins bind to common regions of MAG and whether the reactive determinants contain carbohydrate residues. The M-protein of one patient was biotinylated, and binding to human MAG was quantitated by enzyme-linked immunosorbent assay (ELISA) by using avidin-biotin-peroxidase complexes. Serum from the same patient and nine others, but not from controls, competed with the biotinylated M-protein for binding to human MAG. Bovine MAG was digested with staph protease or cleaved with cyanogen bromide, and the resultant fragments were separated by electrophoresis and were transferred onto nitrocellulose sheets. Serum from all patients immunostained the peptide fragments identically. Bovine MAG was deglycosylated by trifluoromethanesulfonic acid, and binding of the M-proteins to MAG and to deglycosylated MAG was tested by immunoblotting. None of the patient's M-proteins bound to deglycosylated MAG. Deglycosylated MAG was visualized by using a mouse monoclonal antibody, GEN-S3, directed at the polypeptide core of MAG. The effectiveness of deglycosylation was ascertained by electrophoresis and by binding of biotinylated concanavalin A. These data and the observed identical species specificity of the M-proteins suggest that the respective anti-MAG M-proteins all bind to the same region in MAG and that the reactive determinants may contain carbohydrate moieties.

Animals↗
PubMed1997

Mechanism of quenching of phototransduction. Binding competition between arrestin and transducin for phosphorhodopsin.

Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh*), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh*. While the beta-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of >/=2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of approximately 0.9 mol/mol. Moreover, while beta-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh* (50% inhibition at approximately 1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by arrestin on P-Rh*-stimulated transducin activation (50% inhibition at approximately 1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.

Animals↗
PubMed1991

Determination of cyclosporine by a competitive binding assay with cyclophilin.

A competitive protein-binding assay for cyclosporine based on use of the intracellular cyclosporine-binding protein cyclophilin (CYP) was used to measure cyclosporin A (CsA) and its bioactive metabolites in whole blood. CYP from cytoplasmic extracts or erythrocyte lysates was applied in the binding assay with use of [3H]CsA as tracer and charcoal adsorption for separating bound from free tracer. Binding affinities of various CsA analogs and metabolites correlated well with their reported in vitro immunosuppressive activities. The assay detected as little CsA as 50 micrograms/L (1 g = 0.832 mmol of CsA), analytical recovery was greater than 80%, and CVs were less than 8% for intra-assay and less than 11% for interassay precision in the range of 150-1000 micrograms/L. We used this assay to measure CsA concentrations in blood and compared the results with those measured by HPLC or by CsA-specific (monoclonal) and CsA-nonspecific (polyclonal) radioimmunoassays. Binding assay results were, in nearly all cases, less than those measured by the nonspecific RIA and frequently greater than 20% above the values determined by the CsA-specific assays. Individual patients had pronounced differences in the relative proportions of CsA, CYP-binding (bioactive) metabolites, and cross-reacting CsA metabolites. Because the presence of bioactive metabolites may considerably contribute to the immunosuppressive activity of CsA, we consider the binding assay clinically useful for measuring CsA in biological fluids.

Administration, Oral↗
PubMed2002

NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer.

500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.

Antibiotics, Antineoplastic↗
PubMed1974

Competitive binding of bilirubin and drugs to human serum albumin studied by enzymatic oxidation.

The mechanism of drug-induced displacement of bilirubin from the blood into tissues was studied. A model of simple, competitive binding of bilirubin and drug to one site on serum albumin was established. Variations of the free bilirubin concentration after addition of drugs were studied in vitro by measuring velocities of oxidation with hydrogen peroxide and horseradish peroxidase. In all cases, the results were in agreement with the model. The competitive effects of 20 drugs were measured and expressed quantitatively as binding constants to the bilirubin site on human serum albumin. Several drugs caused changes of the bilirubin-albumin light absorption spectrum, indicating simultaneous binding of both ligands, without an effect on the free bilirubin concentration. Noncompetitive site-to-site effects on bilirubin binding could not be demonstrated. An equation is proposed for calculation of the maximal displacing effect of a drug from knowledge of its plasma concentration, the above-determined binding constant, and the degree of protein binding of the drug. Comparison of these results with previous observations of bilirubin displacement in newborn humans and in experimental animals indicates a general agreement with a simple competitive mechanism of binding of bilirubin and drug to one site on the albumin molecule. Binding of drugs to other, noncompetitive sites is common.

Anti-Bacterial Agents↗
PubMed1993

Thrombospondin is a tight-binding competitive inhibitor of neutrophil elastase.

Thrombospondin, a glycoprotein of three identical disulfide-bonded subunits, is a constituent of platelet alpha-granules and a variety of normal and transformed cells and binds to cell surfaces and becomes incorporated into extracellular matrix. It has been implicated in processes such as wound healing and tumor growth and metastasis. In addition, thrombospondin was shown recently to be an inhibitor of the fibrinolytic enzyme, plasmin. In the cause of studying the effects of thrombospondin on other serine proteinases, we found that thrombospondin binds neutrophil elastase in an active-site-dependent manner and competitively inhibits the activity of the enzyme. In a competitive binding assay, neutrophil elastase bound to thrombospondin with a dissociation constant of 17 +/- 7 nM, expressed per mole of thrombospondin trimer, or 52 +/- 20 nM, expressed per mole of thrombospondin subunit. In kinetic studies of the inhibition of the amidolytic activity of neutrophil elastase by thrombospondin, 2.7 +/- 0.3 mol of elastase interacted with 1 mol of thrombospondin trimer with a site-binding constant of 57 +/- 13 nM. Lower limits for the on rate constant of 5 x 10(6) M-1 s-1 and off rate constant of 0.27 s-1 were established. Affinity of binding of neutrophil elastase to thrombospondin was sensitive to ionic strength and calcium ions. Thrombospondin was cleaved by neutrophil elastase, but the site(s) of the limited cleavage are independent of the competitive inhibition of elastase activity by thrombospondin. Neutrophil elastase inactivated with phenylmethylsulfonyl fluoride did not compete with active elastase for binding to thrombospondin, implying that a functional active site is important for the interaction of elastase with thrombospondin. Thrombospondin protected fibronectin from cleavage by neutrophil elastase. In summary, the binding of neutrophil elastase to thrombospondin is tight, reversible, and close enough to the active site of elastase to exclude small synthetic tripeptidyl p-nitroanilide substrates and macromolecular protein substrates. Two potential reactive centers that may be involved in binding elastase have been identified in the calcium-binding type 3 domains of thrombospondin. Neutrophil elastase is the enzyme primarily responsible for degrading and solubilizing connective tissue during inflammatory processes. These findings suggest a previously unsuspected mechanism for regulation of elastase activity at inflammatory sites.

Amino Acid Sequence↗
PubMed1981

Adenylate cyclase activation and competitive binding with renal tissue using synthetic eel calcitonin analog and its fragments.

The structure-function relationship of calcitonin (CT) was investigated using a synthetic eel CT (E-CT) analog and its fragments. Adenylate cyclase activation and competitive binding to rat renal receptors were used as parameters of function. [Asu1,7]E-CT analog, synthesized by replacing the S-S bond of Cys1-Cys7 in the natural hormone with ethylene linkage of 1-amino suberic acid (Asu), and E-CT fragment 11-32 had about 1/5th and 1/50th the potencies of synthetic E-CT, respectively, in both adenylate cyclase activation and competitive inhibition of 125I-labeled [Asu1,7]iodo-E-CT binding on rat renal plasma membranes. The minimal chain length required to activate adenylate cyclase in rat renal plasma membranes was between 12-18 amino acids, and the minimal chain length required to affect the CT receptor binding was between 6-12 amino acids, near the C-terminus. Fragments, including the C-terminus, show a disproportionate and potent inhibition of the binding compared with the potency required for adenylate cyclase activation. Thus, the amino acid sequence near the C-terminus probably plays an important role in the binding of CT to the receptor. (Endocrinology 108: 698, 1981.)

Adenylyl Cyclases↗
PubMed1995

Properties of the Sp1 zinc finger 3 peptide: coordination chemistry, redox reactions, and metal binding competition with metallothionein.

Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn-(II) in transcription factors containing zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 (+/- 3) x 10(-10), 3 (+/- 1) x 10(-7), and 4 (+/- 1) x 10(-6), respectively) and found to be less than those of an optimized zinc finger peptide (Krizek, B. A., Merkle, D. L., and Berg, J. M. (1993) Inorg. Chem. 32, 937-940) but greater than those of the second finger of transcription factor IIIA (Berg, J. M., and Merkle, D. L. (1989) J. Am. Chem. Soc. 111, 3759-3761). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidation of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the alpha domain of human liver MT-2 (alpha-hMT2) indicates a similar affinity for Zn(II). However, alpha-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.

Amino Acid Sequence↗
PubMed2004

Determination of dissociation constants by competitive binding in partial filling capillary electrophoresis.

The determination of dissociation constands (K(d)) by competitive ligand binding in partial filling capillary electrophoresis is demonstrated. Two different strategies were applied, one of which only uses a single reporter ligand and a more elaborated one which suppresses systemic disturbances by using a racemic mixture as reporter. The dissociation constants obtained by both alternatives were virtually identical and in good agreement with those previously reported.

Alprenolol↗
PubMed1997

Glucocorticoid-dependent transcriptional repression of the osteocalcin gene by competitive binding at the TATA box.

The human osteocalcin gene is transcriptionally repressed by glucocorticoids. A specific binding element for the glucocorticoid receptor (GR) overlapping the TATA box of the human osteocalcin promoter has previously been identified. In the present study, the function of this element has been further characterized by competitive gel mobility-shift assay and transfection experiments. The GR and TATA-binding protein (TBP) bound to the cognate overlapping elements in a mutually exclusive manner. The GR preferentially inhibited the binding of TBP. The isolated DNA-binding domain of the GR is sufficient to compete for TBP binding. The integrity of both half-sites of the glucocorticoid response element (GRE) is required to effectively compete for TBP binding, and competitive binding of the GR is dependent on dimerization. Transient overexpression of TBP overrides the transcriptional repression of the osteocalcin promoter by glucocorticoids. We conclude that the repressive effect of glucocorticoids on this promoter is the result of competitive DNA binding to a basal transcriptional element and that it does not appear to require direct protein-protein interaction between the competitive factors.

Base Sequence↗
PubMed2006

A rapid competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) test strip for microcystin-LR (MCLR) determination.

A competitive binding nonseparation electrochemical enzyme immunoassay (NEEIA) is described for the determination of microcystin-LR (MCLR) using a double-sided microporous gold electrode in cartridge-type cells. A gold film sputtered on one side of porous nylon membrane constitutes a working electrode, while another gold film formed on the opposite side serves as a pseudo reference electrode. After immobilizing MCLR antibody on working electrode by physical adsorption, the double-sided electrode was placed simply in a diffusion U-type or within a dry strip-type cell with a conjugate pad pre-loaded with a glucose oxidase labeled MCLR (GOx-MCLR) on working electrode side. Assays were performed in two steps: an MCLR-containing sample mixed with a known amount of GOx-MCLR conjugate either in buffer solution or in pre-loaded dry pad was incubated for an appropriate period (about 10 min) to induce competitive reaction with an immobilized anti-MCLR antibody on working electrode, and a fixed concentration of glucose solution (substrate) was then added to the backside of the working electrode. Due to the competitive nature of the assay, enzymatically generated product, hydrogen peroxide (H2O2), was detected at the working gold electrode (at +800 mV versus Au) by oxidation, and the magnitude of amperometric current was inversely proportional to the concentration of MCLR in the sample. The response time after substrate addition was about 30s. Mean recovery of MCLR added to tap water was 93.5%, with a coefficient of variation (CV) of 6.6%. The proposed competitive NEEIA system is in general comparable to existing heterogeneous enzyme immunoassays with a similar detection limit (100 pg/mL MCLR), and suitable for developing a disposable type biosensor for on-site monitoring of environment.

Biosensing Techniques↗