Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes.

We describe an amperometric technique for quantification of an enzyme immunoassay in which we use a magnetic working electrode, both to separate bound and free analyte and to monitor the electrochemical response. We used a "two-site" immunometric assay with monoclonal antibodies for human choriogonadotropin (hCG) as a model system in which magnetic particles were used as the solid phase. Separation of bound and free label is readily achieved by localizing the particles at the electrode. Activity of the bound enzyme in the environment of the electrode is determined electrochemically, permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the system is 150 int. units of hCG per litre (1st Int. Ref. Preparation). Correlation between the amperometric measurement of urinary hCG and data for an immunoradiometric assay was r = 0.9. The assay is rapid, requiring a total assay time for each sample of 20 min, which includes 15 min for antibody/antigen binding.

Comparison of antithrombin III assays using biological and chromogenic substrates.

Antithrombin III (At III) levels in plasma samples were determined by incubation of diluted plasma with thrombin, either with or without heparin. Followed by measurement of residual thrombin using clotting and amidolytic methods. All assays were of multidose bioassay design suitable for parallel line analysis. Assays without heparin showed only a small difference between amidolytic and clotting methods, which was not significant at the 95% level. Assays with heparin showed a much larger difference between clotting and amidolytic methods which is shown to be attributable to the heat defibrination step used in the clotting assay. Heat defibrination and ancrod defibrination methods are compared using the amidolytic heparin cofactor assay and it is shown that heat defibrination can cause the loss of nearly 50% of the functional At III in a reconstituted freeze-dried plasma which was used as a standard. No loss occurs when ancrod is used for defibrination. It is an advantage of the amidolytic heparin cofactor assay that defibrination is not required.

Plasmin potency estimates: influence of the substrate used in assay.

A urokinase-activated plasmin (UK-plasmin) preparation was assayed against the International Reference Preparation for Plasmin (IRP-plasmin) using caseinolytic, fibrinolytic, fibrinogenolytic and chromogenic assay methods. The relative potency (using multi-dose bioassays) was estimated by the fibrinolytic method to be about twice that obtained by the caseinolytic, fibrinolytic and chromogenic assay methods. It was found that the UK-plasmin binds to fibrin to a greater extent than does the IRP-plasmin and this is advanced as an explanation for the discrepancy between assay methods. This difference in the binding of the two plasmins to fibrin may mean that it will be difficult to compare the fibrinolytic activities of various plasmin preparations. It is also shown that, during thermal degradation, the IRP-plasmin loses fibrinolytic activity more rapidly than amidolytic activity.

The membrane potential of mouse ascites-tumour cells studied with the fluorescent probe 3,3'-dipropyloxadicarbocyanine. Amplitude of the depolarization caused by amino acids.

1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the "null point" procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519--552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the "null point" value of E within the range--20mV to--100mV. The fluorescence intensity at the "null point" was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the "null point" was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about --80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.

Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites- tumour cells.

1. The tumour cells were incubated at 37 degrees C in Ringer solutions containing glucose, 1 mM-methionine, various concentrations of Na+ and K+ and, in some instances, ouabain or valinomycin to lower the membrane potential generated by the Na+ pump. After about 30 min, when the system had reached a steady state, the ratio [extracellular Na+]/[cellular Na+] varied from about 0.6 to 3.2 with the ionic conditions. The membrane potential, determined by means of the fluorescent probe 3,3'-dipropyloxadicarbocyanine, also varied systemically from about zero to--55mV.2. the ratio [cellular methionine]/[extra-cellular methionine] varied from about 1 to 35 in these circumstances. The Na+ electrochemical gradient, measured in the same units, varied from about 1 to 30. Its magnitude in 46 assays was almost directly proportional to, though slightly smaller than, the methionine gradient. 3. A mathematical model was used to define the relation between these two gradients, which were not in equilibrium, owing to the presence of a leak pathway for the amino acid. On the assumption that the values of [cellular Na+] were correct, the methionine gradient was about 1.8 times larger than this version of the gradient hypothesis predicted.

The effects of ionophores on the fluorescence of the cation 3,3'-dipropyloxadicarbocyanine in the presence of pigeon erythrocytes, erythrocyte 'ghosts' or liposomes.

1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3'-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.

Inhibition of the soluble adenosine triphosphatase from mitochondria by adenylyl imidodiphosphate.

1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.

Use of progress curves to investigate product inhibition in enzyme-catalysed reactions. Application to the soluble mitochondrial adenosine triphosphatase.

1. Several methods of analysing progress curves of enzyme-catalysed reactions are discussed briefly in relation to their usefulness in a situation where a reaction product has a K(i) much lower than the K(m) for the substrate 2. A comparison is made of different methods of estimating initial rates in this situation. 3. The use of a computer curve-fitting routine capable of handling functions of more than one variable for the extraction of kinetic parameters from progress curves is described. 4. This method and that of fitting time as a polynomial in product concentration are applied to progress curves for the soluble mitochondrial adenosine triphosphatase and the results are compared with values obtained by more conventional methods.