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Biomedical subjects

H B Halsall

Publications and source records attributed to H B Halsall.

At least 37 records · Page 2Linked to original sources
PubMed1991

Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent.

The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.

2,6-Dichloroindophenol↗
PubMed1991

Electrochemical determination of azidothymidine in human whole blood.

An electrochemical method based on differential pulse voltammetry is presented for the determination of AZT in whole blood of fasted subjects. A protein-free supernatant of whole blood is prepared using HClO4 precipitation followed by neutralization with phosphate buffer. The AZT is reduced at a hanging mercury drop electrode. The linear dynamic range of standards in buffer is from the detection limit of 4.1 nM to 206.5 microM (1.1 to 55,200 ng/ml). However, in spiked blood samples the linear dynamic range is from 0.029 to 0.29 microM (7.75 to 77.5 ng/ml). The whole blood assay yields a recovery of 92.30 +/- 5.92% compared to the standard solution assay. After a 30-min preparation time, each sample can be analyzed in 10 min by a manual procedure.

Chemical Precipitation↗
PubMed1991

Electrochemical enzyme immunoassay for phenytoin by flow-injection analysis incorporating a redox coupling agent.

Using phenytoin as a model analyte, we demonstrate an electrochemical enzyme immunoassay based on flow-injection analysis and incorporating 2,6-dichloroindophenol (DCIP) as a redox coupling agent. DCIP reacts with NADH to form NAD+ and DCIPH2, the reduced form of the coupling agent. The production of DCIPH2 is monitored at +250 mV vs Ag/AgCl. This low applied potential improves selectivity in the biological matrix, differentiating against components that are oxidizable at the more-positive potentials required for direct electrochemical detection of NADH. The kinetics-based assay also eliminates other common interferences, mainly from ascorbic acid and glutathione. This system does not require precolumns or analytical columns for isolation of the NADH response. Good agreement with a routine clinical laboratory procedure for phenytoin is obtained for clinical samples (r = 0.95), illustrating the feasibility of such an approach.

2,6-Dichloroindophenol↗
PubMed1990

Electrochemical immunoassay: an ultrasensitive method.

Hydrodynamic electrochemical techniques such as liquid chromatography and flow injection analysis with electrochemical detection are very effective for the rapid determination of the enzyme-generated product in enzyme immunoassays. The authors have used this detection method in various assay formats using both alkaline phosphatase and glucose-6-phosphate dehydrogenase as labels. Assays for digoxin will be used illustratively. Recently, the authors have used 70 mL microcapillary hematocrit tubes as the immunoassay reaction vessel and alkaline phosphatase as the labeling enzyme. The assay, complete in 30 min, had a detection limit of 5,6 x 10 -20 moles of IgG in serum. The linear range was four orders of magnitude. This low detection limit is due to a combination of the favorable geometry of the reaction vessel and the suppression of nonspecific adsorption by the addition of ion-pairing blocking agents. Even lower detectable amounts should be achievable with smaller reaction vessels. The capability for detecting such small amounts of analyte is potentially useful for the analysis of extremely small samples such as single cells and blood samples from premature infants.

Alkaline Phosphatase↗
PubMed1990

Competitive heterogeneous enzyme immunoassay for theophylline by flow-injection analysis with electrochemical detection of p-aminophenol.

A competitive enzyme-linked immunoabsorbent assay based on the flow-injection amperometric detection of p-aminophenol has been investigated with use of the materials and general procedure of a commercial kit for the determination of theophylline in human serum. The antibody is immobilized on glass beads, and the enzyme label is alkaline phosphatase (EC 3.1.3.1). The high currents generated during the electrochemical detection allowed a rapid (35 min) and simple determination of theophylline throughout its therapeutic range (10-20 mg/L) and also in the subtherapeutic range (detection limit of about 80 micrograms/L).

Aminophenols↗
PubMed1989

Solid-phase electrochemical enzyme immunoassay with attomole detection limit by flow injection analysis.

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 x 10(-8) to 1.0 x 10(-5) M, a detection limit of 2.4 x 10(-8) M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml-1 by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.

Aminophenols↗
PubMed1988

Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level.

Electrochemical enzyme immunoassay methodology has been developed to take advantage of the selectivity of antibody reactions, the amplification feature of an enzyme-based assay, and the ease with which small amounts of the enzyme-generated product can be detected electrochemically. A heterogeneous sandwich enzyme immunoassay was used in this work as the model assay. In this type of assay, the antigen is sandwiched between the enzyme conjugate and a primary antibody that is adsorbed to the solid phase. Alkaline phosphatase is a suitable enzyme for electrochemical assays since it catalyzes the conversion of electroinactive phenyl phosphate to electroactive phenol. The product, phenol, is then quantitated by liquid chromatography with electrochemical detection in a thin-layer flow cell with a carbon paste electrode at 0.895 V vs Ag/AgCl. The current produced by the oxidation of phenol is directly proportional to the analyte (antigen) concentration. The problem associated with these types of solid-phase immunoassays is that the adsorption of the primary antibody is desired while the adsorption of other assay proteins is not. The detection limits are generally defined by the ability to control this nonspecific adsorption. The detection limit of a previous electrochemical assay for rabbit IgG was 100 pg/ml and was limited by a large background current observed in the absence of antigen. In the present study, each step of the assay was examined in order to determine the sources of this background current, and it was found that the major contribution was from the nonspecific adsorption of the enzyme conjugate. Using combinations of Tween 20 and bovine serum albumin as blocking agents, the level of nonspecific adsorption was reduced by 96%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Sequestration electrochemistry: the interaction of chlorpromazine and human orosomucoid.

A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 X 10(6) M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

Binding Sites↗
PubMed1986

Alpha-2-plasmin inhibitor comprises a single domain.

The reaction between plasmin and alpha 2PI (alpha-2-plasmin inhibitor), which constitutes a major regulatory step in fibrinolysis, involves both interactions between the kringle structures of plasmin and a complementary site on the inhibitor, and also between the inhibitor reactive center and the enzyme active site. An attempt was made to distinguish calorimetrically the two functional domains of alpha 2PI. Only one transition was seen, both in the DSC and UV experiments, contrary to the expected two. This transition was unaffected by K4 of plasminogen but was abolished in the presence of anhydrotrypsin. This suggests the major domain structure in alpha 2PI is associated with the reactive center.

Binding Sites↗
PubMed1986

The influence of N-acetylneuraminic acid on the properties of human orosomucoid.

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.

Cesium↗
PubMed1986

Collagen fibril formation in the presence of dexamethasone disodium phosphate.

Dexamethasone disodium phosphate was found to inhibit in vitro fibrillogenesis in a buffered collagen solution that otherwise formed in vivo like fibrils. A nonlinear relationship was observed between the steroid salt concentration and the kinetic parameter half transition time. Full fibril inhibition occurred at dexamethasone phosphate concentrations above 15 mM. At lower concentrations, sample buffers that also contained inorganic phosphate were not different from the control in activation energy of 224.3 +/- 29.3 kJ/mol (53.6 +/- 7.0 kcal/mol). The idea is advanced that the soluble steroid salt associates directly to the collagen and prevents the formation of lateral, hydrophobic interactions between adjacent collagen aggregates.

Animals↗
PubMed1986

Interaction between human serum albumin and alpha 1-acid glycoprotein in the binding of lidocaine to purified protein fractions and sera.

The authors have determined the binding parameters for [14C] lidocaine to several preparations of purified human serum alpha 1-acid glycoprotein (AAG) and human serum albumin (HSA). The authors find that the average Kd for the three different purified AAG preparations was 15.2 +/- 0.5 microM, with an average of 0.567 +/- 0.009 binding sites (N) per AAG molecule, when the concentration of AAG was determined by a standard colorimetric protein assay. The number of binding sites per AAG increased to 0.99 +/- 0.06 when the concentration of AAG was determined immunologically. On the other hand, delipidized AAG had a Kd of 28.0 microM, with an N of 1.72 based on the immunological assay. Various preparations of HSA had Kd/N values ranging from 4.3 to 17.3 mM. The number of binding sites per HSA molecule could not be determined because the maximum concentration of lidocaine that can be used is 10 mM, which is only approximately equal to the Kd. The Kd to HSA was so low and the capacity so high that, at therapeutic concentrations, the binding was in the linear range. Solutions containing both purified AAG and HSA had Kd values of 30.2 +/- 1.8 microM, with 2.01 +/- 0.04 binding sites per AAG molecule. With HSA and delipidized AAG, the Kd was 60 microM, with 3.16 sites per delipidized AAG molecule. This increase in the binding parameters appeared to be due to a direct interaction between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗