No abstract

Effect of high-affinity mouse monoclonal antidigoxin antibodies D22 on digoxin-induced cardiac disturbances is studied in guinea pigs injected with 0.5 mg/kg digoxin. Five minutes after digoxin injection, bradycardia, isolated extrasystoles, conduction disturbances, and changes in the ST segment and QRS complex appear; digoxin concentration attains 0.2-0.4 gg/ml. Antidigoxin monoclonal antibodies injected 10 min after digoxin slightly reduce heart rate, prevent isolated extrasystoles, improve atrioventricular conduction, and normalize the amplitude of the ST segment and the shape of the QRS complex. Serum digoxin concentration decreases to 0.015-0.03 gg/ml. These data suggest that monoclonal antidigoxin antibodies can be effectively used in acute digoxin intoxication.

Digoxin is a cardiac glycoside exhibiting a pronounced cardiotonic action, which is used for the therapy of acute and chronic cardiac insufficiency. However, this drug is characterized by a very narrow therapeutic interval of concentrations (I -2 ng/ml) in the blood. Even a slight increase in the drug content above this level leads to toxicity manifestations. At the same time, the metabolism of digoxin exhibits strongly individual character: a several-fold difference in the concentration of digoxin may be observed in the blood of different patients upon receiving the same dose of the drug. This leads to the necessity of thoroughly controlling the digoxin content in the blood and selecting individual drug doses for each patient. The digoxin concentration is usually measured by immunoassay techniques [1].In recent years, there is a pronounced trend to replace polyclonal antibodies by monoclonal antibodies, which is explained by the obvious advantages of the latter: high specificity, affinity, amd homogeneity. Earlier [2] we have developed a mice hybridoma line D-18 capable of producing high-affinity specific antidigoxin antibodies with Kafr = 5 x 109 M-1. In previous work [3] devoted to phenobarbital determination, we demonstrated a number of advantages offered by the methods of immunoassay using labeled antibodies as compared to the conventional ELISA technique.The purpose of this work was to develop an indirect ELISA method of digoxin determination based on the competition between digoxin from a sample and the drug sorbed in 96-well plates (in the form of a bovine serum albumin conjugate) for binding to peroxidase-labeled Fab-fragrnents of monoclonal antibodies. EXPERIMENTAL PARTBovine serum albumin (BSA), horseradish peroxidase, digoxin, Tween-20, sodium periodate, sodium borohydfide, papain, EDTA, cysteine, and iodoacetic acid were obtained from Siam'ha (USA); tetramethylbenzidine (H-Roche, Switzerland); 96-well sample plates (Nunc, Denmark); DEAE- Preparation of BSA conjugated digoxin. To 14 mg of digoxin, dissolved in a mixture of 150 ~tl DMSO and 550 ~tl ethanol, was added dropwise with stirring 700 ~tl of an 0.1 M NaIO4 solution and then (in 1 h) 20 ~tl of 1 M ethylene glycol. The mixture was allowed to stand for 5 rain and then added to a solution of 10 mg BSA in 100 ~tl of 0.05 M earbonate-bicarbonate buffer (pH 9.6). The reaction mixture was held at room temperature with stirring for 1.5 h. Then a solution of 3 mg NaBI-I4 in 150 ~tl water was added and the mixture was allowed to stand for 20 min. Finally, the mixture was dialyzed for a week against cold flowing water.Sorption of dig-BSA conjugate on polystyrene plates. Aliquots of the doubly diluted conjugate with a concentration of 2~tg/ml in 0.05M carbonate-bicarbonate buffer (pH 9.6) were incubated with a solid sorbent phase for 2 h at 20~ and then for 18 h at 4~ Then the plates were washed with water, incubated for 1 h in an 0.1% BSA solution, washed, and dried.Purification of antidigoxin monoclonal mice antibodies. The hybridoma line D-18 producing...

The binding of 2G3 mouse antitheophylline monoclonal antibodies (affinity constant K.n=2x 101~ liter/tool) to theophylline is studied using isothermal titration microcalorimetry. Thermodynamic parameters of the binding are: enthalpy change AH=-0.23 kcal/mol; Gibbs free energy change AG =-16.42 kcal/mol; entropy change AS=0.054 kcal/(mol x K). Regression analysis shows a two-site kinetic binding model. A great contribution of entropy component to the free energy change of 2G3 antibody binding to theophylline is indicative of an entropy-dependent process. The entropy-dependent nature of the binding presumably determines the binding kinetics.