Comparación de algunos métodos para el control de calidad del 123I-Ioflupano / No disponible

Objetivos: Se pretende establecer un método sencillo, preciso y reproducible para determinar la pureza radioquímica del 123I-Ioflupano. Material y Métodos: Se utilizó la radiocromatografía con tres tipos de fases estacionarias (whatmann-17MM, Whatmann-3MM e ITLC-SG) y cinco fases móviles (Éter Dietílico, Metiletilcetona, NaCl al 0,9%, Metanol: Cloroformo (1:9), Metanol:Agua al 85%). Este procedimiento fue realizado con alícuotas de 123I-Ioflupano (DatScan®) y 123INa para estudiar el comportamiento del yoduro libre. Resultados: Se descartó el whatmann-3MM por su mala separación y el ITLC-SG con Metanol: Agua por su baja reproducibilidad. Usando Metiletilcetona como fase móvil en ITLC-SG y Whatmann-17MM no se observa la fracción libre. Únicamente se observó la fracción libre en Whatmann-17MM tanto con Metanol: Cloroformo como con Éter Dietílico y en ITLC-SG con NaCl 0,9% . Conclusiones: La mejor separación del 123I-Ioflupano del 123I- fue observada con: Whatmann-17MM/ Metanol: Cloroformo (1:9) (Rf de la fracción ligada = 0,62±0,03, Pureza Radioquímica = 91,6±2,36) , ITLC-SG/NaCl 0,9% (Rf de la fracción ligada = 0,21±0,05, Pureza Radioquímica = 90,19±3,4) y Whatmann-17MM /Éter Dietílico (Rf de la fracción ligada = 0,62±0,06, Pureza Radioquímica = 92,79±1,65) (AU)

Aim: The aim of this study is to stablish an easy, reproducible, and precise method to determine the radiochemical purity of 123I-Ioflupane. Material and methods: Different systems of radiochromatography were used with three types of stationary phases (Whatmann-17MM, Whatmann-3MM and ITLC-SG) and five mobile phases (Diethylether, NaCl 0.9%, Methanol: Chloroform (1:9), Methanol: Water (85:15) and Methylethylketone). This procedure was made with samples of 123I-Ioflupane and 123I-NaI to study the behaviour of free iodide. Results: Whatmann-3MM was rejected due to an unsuccessful separation and Methanol: Water (85:15) with ITLC-SG was not reproducible. Free fraction wasn`t observed in Methylethylketone as mobile phase with ITLC-SG and Whatmann-17MM. Free fraction was just observed in Whatmann-17MM with Methanol: Cloroform and Diethylether and in ITLC-SG with NaCl 0.9%.Conclusions: Maximum separation between 123I-Ioflupane and 123I- was observed in Whatmann-17MM/Methanol: Cloroform (1:9) (Rf of bound fraction = 0.62±0.03, Radiochemical Purity =91.6±2.36%), ITLC-SG/NaCl 0,9% (Rf of bound fraction = 0.21±0.05, Radiochemical Purity =90.19±3.4%) and Whatmann-17MM/ Diethylether (Rf of bound fraction = 0.62±0.06, Radiochemical Purity =92.79±1.65%) (AU)

Formacion del inmunocomplejo radiactivo en un ria de doble anticuerpo / Radioactive immunocomplex formation in a double antibody ria

Se ha elaborado un modelo para el estudio de la cinética y equilibrio de las reacciones antígeno-anticuerpo implicadas en el radioinmunoanálisis (RIA) de doble anticuerpo correspondiente al Péptido C. Se pretende caracterizar la influencia de las concentraciones iniciales de antígeno marcado (M) y no marcado (Q) sobre la formación del inmunocomplejo (PM) al segundo anticuerpo (J) inmovilizado sobre una bola. Se realizan 30 experiencias para el estudio del efecto de las variables antes mencionadas. Los resultados obtenidos son concordantes con el modelo propuesto(AU)

A model has been produced for the kinetic and equilibrium study of antigen-antibody reactions in the radioimmunoassay (RIA) of C-Peptide double antibody. The aim is (1) to characterise the influence of initial concentrations of labelled (M) and unlabelled (Q) antigen and that of the initial concentration of the antibody in solution (P), and (2) to study the binding of the immunocomplex (PM) to the second antibody (J) immobilised on a bead, and the replacement of M by Q in the PMJ immunocomplex. In order to study the effect of such variables, 44 experiments were conducted. The results are in line with the model proposed(AU)

Formacion del inmunocomplejo radiactivo en un ria de doble anticuerpo / Radioactive immunocomplex formation in a double antibody ria

Se ha elaborado un modelo para el estudio de la cinética y equilibrio de las reacciones antígeno-anticuerpo implicadas en el radioinmunoanálisis (RIA) de doble anticuerpo correspondiente al Péptido C. Se pretende caracterizar la influencia de las concentraciones iniciales de antígeno marcado (M) y no marcado (Q) sobre la formación del inmunocomplejo (PM) al segundo anticuerpo (J) inmovilizado sobre una bola. Se realizan 30 experiencias para el estudio del efecto de las variables antes mencionadas. Los resultados obtenidos son concordantes con el modelo propuesto(AU)

A model has been produced for the kinetic and equilibrium study of antigen-antibody reactions in the radioimmunoassay (RIA) of C-Peptide double antibody. The aim is (1) to characterise the influence of initial concentrations of labelled (M) and unlabelled (Q) antigen and that of the initial concentration of the antibody in solution (P), and (2) to study the binding of the immunocomplex (PM) to the second antibody (J) immobilised on a bead, and the replacement of M by Q in the PMJ immunocomplex. In order to study the effect of such variables, 44 experiments were conducted. The results are in line with the model proposed(AU)

Efecto anzuelo en análisis por radioligando del Anti Ácido Glutámico Descarboxilasa (Anti-GAD65). Influencia de la temperatura e interpretación fisicoquímica / Hook effect in radioligand assay for Anti Glutamic Acid Decarboxylase (Anti-GAD65). Influence of temperature and physicochemical interpretation

Objetivo: El análisis por radioligando es uno de los métodos principales utilizados en ladeterminación analítica del Anti-GAD65. Se ha estudiado la influencia de la temperaturasobre las gráficas de calibración obtenidas por dicha técnica.Material y Métodos: Usamos un kit comercial para Anti-GAD65 y un contador gamma. Losresultados son analizados mediante el programa Statistica.Resultados y Discusión: Las actividades ligadas al anticuerpo aumentan con la temperatura.Se observa una disminución de la actividad para altas concentraciones atribuible al llamado“efecto anzuelo”. Se propone un sencillo modelo fisicoquímico que justificasatisfactoriamente los resultados(AU)

Background: Radioligand assay is one of the principal methods used for analyticaldetermination of the Anti-GAD65 concentration. We studied the influence of temperature onthe calibration curves obtained by such a methodMatherial and Methods: We used a commercially available RIA kit for Anti-GAD65 and agamma counter. Data was analyzed using Statistica software.Results and Discusion: Activities bound to the antibody increase with temperature. Therewas a decrease in activity for high concentrations attributable to the "hook effect". Wepropose a simple physicochemical model that justifies satisfactorily the results(AU)

[Influence of temperature on the calibration curves in IRMA for neuron-specific enolase and its physicochemical interpretation]. / Influencia de la temperatura en las curvas de calibración en IRMA de enolasa neuronal específica y su interpretación fisicoquímica.

BACKGROUND: immunoradiometric assay (IRMA) is one of the principal methods used for the analytical determination of neuron specific enolase (NSE) concentration. We studied the influence of temperature on the calibration curves obtained by this method, and a physicochemical justification based on two theoretical models is proposed. MATERIAL AND METHODS: we used a commercially available RIA kit for NSE and a gamma counter. Data was analysed using Statistical software. RESULTS AND DISCUSSION: activity bound to the antibody increases with temperature, producing results that are consistent with two modifications to the four parameter and Langmuir equations. CONCLUSIONS: the two models used successfully reproduce the results, with the adsorption model being preferable due to its greater simplicity and clearer physical significance.

Influencia de la temperatura en las curvas de calibración en IRMA de enolasa neuronal específica y su interpretación fisicoquímica / Influence of temperature on the calibration curves in IRMA for neuron specific enolasa and its physicochemical interpretation

Objetivo: el análisis inmunorradiométrico (IRMA) es uno de los métodos principales utilizados en la determinación analítica de la enolasa neuronal específica (NSE). Se ha estudiado la influencia de la temperatura en las gráficas de calibración obtenidas por dicha técnica y se propone una justificación fisicoquímica basada en dos modelos teóricos. Material y métodos: usamos un kit comercial de IRMA para NSE y un contador gamma. Los resultados son analizados mediante el programa Statistica. Resultados y discusión: las actividades ligadas al anticuerpo aumentan con la temperatura, lo que da resultados que se ajustan a sendas modificaciones de las ecuaciones de los cuatro parámetros y Langmuir. Conclusiones: los dos modelos utilizados reproducen satisfactoriamente los resultados, pero es preferible el modelo de adsorción debido a su mayor simplicidad y significado físico más claro(AU)

Background: immunoradiometric assay (IRMA) is one of the principal methods used for the analytical determination of neuron specific enolase (NSE) concentration. We studied the influence of temperature on the calibration curves obtained by this method, and a physicochemical justification based on two theoretical models is proposed. Material and methods: we used a commercially available RIA kit for NSE and a gamma counter. Data was analysed using Statistical software. Results and discussion: activity bound to the antibody increases with temperature, producing results that are consistent with two modifications to the four parameter and Langmuir equations. Conclusions: the two models used successfully reproduce the results, with the adsorption model being preferable due to its greater simplicity and clearer physical significance(AU)

Influencia de la temperatura sobre las curvas de calibración en IRMA de IGF I. Interpretación mediante dos modelos no excluyentes / Influence of the temperature on calibration curves of IGF I in IRMA. Interpretation by two models non excluding

Se estudia la influencia de la temperatura sobre las gráficas de calibraciónutilizadas en la determinación analítica de IGF I mediante IRMA. Los resultadosse analizan mediante la ecuación de los cuatro parámetros y una modificación dela isoterma B.E.T. Tales ecuaciones corresponden, respectivamente, a los modelosde interacción ligando (antígeno)-receptor (anticuerpo) y adsorción del antígenosobre la pared del tubo recubierta de anticuerpo. Los ajustes a ambas ecuacionesson satisfactorios con algunas ventajas para el modelo de absorción que lo hacenpreferible en cuanto a la interpretación de los resultados. Se determinan tambiénlos parámetros termodinámicos para la disociación del inmunocomplejo

The influence of temperature on the calibration graphs used in the analyticaldetermination of IGF I by IRMA has been studied. The results were analyzed usingthe equation of the four parameters and a modification of the isotherm BET. Suchequations correspond, respectively, to the models of interaction ligand (antigen)-receiver (antibody) and adsorption antigen on the wall of the tube coated antibody.The adjustments to both equations are satisfactory with some advantages for theadsorption model that make it preferable in the interpretation of results. It alsodetermine thermodynamic parameters for the immunocomplex dissociation

Prediction of calibration curves in ria of digoxin; Influence of some variables.

BACKGROUND: Competitive protein binding radioimmunoassay (CPB-RIA) is a principal method for quantifying serum digoxin concentration. The accuracy of this method is critically dependent on factors that influence the substitution reaction between unlabelled (Q) antigen (digoxin) with (125)I-labelled antigen (M) bound to anti-digoxin antibody (P). We studied the influence of initial concentration of M, ionic strength, and viscosity on the substitution reaction between M and Q. In addition, we propose a kinetic model for this reaction. METHODS: We used a commercially available CPB-RIA for digoxin, a gamma counter, and a viscosimeter to study the effect of initial concentration of M, ionic strength, viscosity, and temperature on the substitution reaction between M and Q. Data were analyzed using Statistica software. RESULTS: The apparent rate constant for the reaction between M and Q in the formation of PM is dependent on the initial concentration of M, and the ionic strength, viscosity, and temperature of the reaction medium, and independent of the concentration of Q. CONCLUSION: A kinetic model for the displacement of the (125)I-digoxin by the digoxin in its union to a specific antibody is proposed. Such model adjusts satisfactorily to the results and allows the prediction of the calibration curves of RIA (activity bound to the antibody vs. concentration of digoxin) showing the influence of the concentration of both species, the time of incubation, the viscosity and the ionic strength of the medium, on the sensitivity of the method of RIA on which the analytical determination of the digoxin is based.

Biexponential model for the kinetics of the substitution reaction in the radioimmunoassay of triiodothyronine.

The influence of initial concentrations, ionic strength, viscosity and temperature on the substitution reaction of 125I-triiodothyronine (T3) (M) by unlabelled T3 (Q) in the immunocomplex PM (P = anti-T3 antibody) has been studied, and a kinetic model is proposed. The apparent rate constant is dependent on the initial concentration of labelled and unlabelled T3, viscosity and temperature, and independent of ionic strength. The reaction is endothermic and is not controlled by diffusion. The results obtained are in line with the proposed model. We propose some modifications for optimizing the technique.

Cinética de la estabilidad del radiofármaco 99mTc-DTPA. Influencia de la temperatura / Stability kinetics of the radiopharmaceutical 99mTc-DTPA. Influence of the temperature

La pureza radioquímica del radiofármaco 99mTc-DTPA en función del tiempo seestudia por radiocromatografía en las primeras horas transcurridas tras su preparación.Los resultados se ajustan a ecuaciones exponenciales indicativas de que ladegradación del radiofármaco sigue una cinética de orden uno. Las constantes obtenidasa diferentes temperaturas siguen la ecuación de Arrhenius, cuyos parámetrosse calculan. Se proponen sendos métodos, analítico y gráfico, para el cálculodel tiempo de validez (t95) en función de la temperatura

The radiochemical purity of the radiopharmaceutical 99mTc-DTPA dependingon the time is studied by radiocromatography in the first hours passed after itspreparation. Results adjust to exponential equations that indicate order one forthe degradation of radiopharmaceutical. The constants obtained for differenttemperatures follow the equation of Arrhenius, whose parameters are calculated.Analytical and graphical methods are provided for the calculation of the validitytime (t95) depending on temperature

Modelo general y cuatro ecuaciones para el ajuste de curvas de calibración en RIA. Comparación de resultados / General model and four equations for the fit of calibration curves in RIA. Comparison of results

Se presenta un modelo teórico para la justificación de las ecuaciones de los cuatro parámetros y logarítmico-logística, utilizadas empíricamente en el ajuste de las curvas de calibración en RIA. De dicho modelo se deducen otras dos ecuaciones. Se analiza el ajuste de 192 curvas de RIA para las cuatro ecuaciones y se interpretan los resultados en términos de las posibles características de enlace en la unión antígeno-anticuerpo

A theoretical model for the justification of the equations of the four parameters and logarithmic-logistic, used empirically in the fit of the calibration curves in RIA, is presented. From this model other two equations are deduced. The fit of 192 curves of RIA is analyzed for the four equations and the results are interpreted in terms of the possible characteristics of binding in the union antigen-antibody

Kinetics of androstendione-radioactive immunocomplex substitution reaction.

A kinetic model is put forward for the study of the antigen-antibody reactions involved in the coated tube radioimmunoassay (RIA) of androstendione. Twenty experiments were conducted to determine the influence of initial concentrations, ionic strength, viscosity, and temperature on the substitution reaction of 125I-androstendione (M) by unlabelled androstendione (Q) in the immunocomplex PM (P = anti-androstendione antibody). The results obtained are in line with the proposed model. The concentration of radioactive immunocomplex is directly proportional to the initial concentration of labelled androstendione and independent of the concentration of unlabelled androstendione, ionic strength, and viscosity. The reaction is not diffusion-controlled.

Comparison between mono- and bi-exponential models for reaction kinetics in the immunoradiometric assay of neuron-specific enolase.

This paper studies the kinetics of the antigen-antibody reactions involved in the analytical determination of neuron-specific enolase (NSE) by means of radiometric immunoassay (IRMA). For the global process, kinetics were found to be dependent on analyte and labelled antibody concentrations, such dependence fitting with the models described in previous papers. Viscosity results clearly indicate its negative influence on the direct reaction rate. Ionic strength shows noticeable but not too relevant effects, which suggests that the variation caused by the glycerol addition is not due to the influence of the dielectric constant of the solutions used. The effect of temperature shows activation parameters similar to the viscous flow energy of water, which suggests diffusion control for the global process. The analysis of the kinetic data of the experiences conducted can be explained by admitting that the antigen-antibody binding takes place through two different binding site types.

Kinetics and equilibrium in insulin radioimmunoassay.

The kinetics of insulin reaction has been studied with its specific antibody immobilized on the inner wall of the reaction tube; the radioimmunoanalytical determination of such a substance is based on the reaction. Independent variables were labelled and unlabelled insulin concentrations, temperature, viscosity, and the medium's ionic strength. Biexponential kinetics was found to be dependent on the concentrations fitted to the models discussed in the paper. The effect of temperature shows activation parameters similar to the viscous flow energy of water, which suggests that the reaction is diffusion-controlled. The results of the viscosity analysis points at the clearly negative influence of viscosity upon the direct reaction rate. Ionic strength has a noticeable, though not relevant, effect which seems to indicate that the variation resulting from the glycerol addition is not due to the influence of the dielectric constant in the solutions used.

Kinetics and equilibrium in the immunoradiometric assay (IRMA) of thyroglobuline.

This paper studies the kinetics of the thyroglobuline reaction with its specific antibody immobilised on the inner wall of the reaction tube, and the subsequent binding of the immunocomplex formed with a second 125I-labelled antibody. These reactions are used in the immunoradiometric determination of thyroglobuline. Independent variables were analyte and labelled antibody, temperature, viscosity, and the medium's ionic strength. For the global process, mono-exponential kinetics were found to be dependent on the concentrations, such dependence fitting with the models discussed in the paper. Viscosity results clearly indicate its negative influence on the direct reaction rate. Ionic strength shows noticeable, but not too relevant, effects, which suggests that the variation caused by the glycerol addition is not due to the influence of the dielectric constant of the solutions used. The effect of temperature shows activation parameters similar to the viscous flow energy of water, which suggests diffusion control for the global process.

Influence of viscosity and ionic strength on the reaction kinetics of aldosterone and androstendione and their specific antibodies.

This paper analyses the influence of viscosity and ionic strength on the kinetics and equilibrium of the reactions of (125)I labelled androstendione and aldosterone with their specific antibodies used in the radioactive immunoassay determination of such hormones. Bi-exponential and irreversible kinetics is found for androstendione, and single-exponential and reversible ones for aldosterone. The results of the viscosity analysis reflect clear negative influence on direct reaction rate. Ionic strength excerpts some influence but not in a significant way, which suggests that the variation resulting from the effect of the glycerol addition is not due to the influence of the dielectric constant of the solutions used. The apparent product of the electrical charges is 0.228 for aldosterone, and 0.230 and -0.230 for androstendione. Results show diffusive control for both cases.

La Difusión en la reacción Antígeno-Anticuerpo / Diffusion in the antigen antibody reaction

En trabajos realizados anteriormente se han observado algunos indicios sobre la importancia de la difusión molecular en la reacción antígeno–anticuerpo. Para el tratamiento de los resultados se utilizaron exclusivamente ajustes polinómicos, los cuales tienen la ventaja de su sencillez y generalidad, pero son poco informativos en cuanto a las características inherentes al proceso. En el presente trabajo se partió de los resultados obtenidos en varias series de experiencias tendentes al estudio de la cinética de distintas reacciones antígeno anticuerpo. En la mayor parte de ellas, se utilizaron antígenos marcados con I125 y en algunos casos, anticuerpos con este mismo radionúclido. En todos los casos se siguió el curso de la reacción midiendo la radiactividad del inmunocomplejo Ag-Ac formado a diferentes tiempos, directamente proporcional a su concentración, porque los datos de partida son siempre tablas: Actividad, en cuentas por minuto (CPM), representada por A, frente al tiempo (t) en minutos. Los resultados obtenidos para un total de dieciséis reacciones se analizan utilizando exlusivamente el método integral mediante los modelos de Stenberg y Karlsson, cada uno de los cuales suministra una ecuación de velocidad integrada que ha sido adaptada para aplicarla a los valores disponibles. Se observa control por difusión en un elevado tanto por ciento de los casos estudiados. (AU)

Cinética y equilibrio de la competición trazador-analito en radioinmunoanálisis de androstendiona / Kinetics and equilibrium of tracer-analyte competition in radioimmunoasay of androstendiona

Se ha estudiado la cinética de la reacción entre la androstendiona marcada con 125I y su anticuerpo específico utilizado en la determinación radioinmunoanalítica de dicha sustancia, encontrándose resultados ajustables a un modelo michaeliano. La presencia de androstendiona no marcada afecta a la reacción de un modo competitivo. Se encuentran cinéticas biexponenciales que sugieren reacciones con epítopos distintos. La influencia de la temperatura indica un control por difusión. Los resultados de equilibrio se ajustan al modelo de unión de dos ligandos sobre sitios independientes. Se desarrollan modelos teóricos compatibles con los resultados experimentales (AU)

[Relation between the total plasma IgE concentration and the prevalence of the various RAST (radioallergosorbent) classes of allergy]. / Estudio de la relación entre la concentración plasmática total de IgE y la prevalencia de las distintas clases RAST (Radio-Allergo-Sorbent-Test) de alergia.

The aim of this study was to analyze the prevalence of every RAST (Radio-Allergo-Sorbent-Test) class in terms of total IgE serum level, and to establish the existing relationship between the total IgE level and the probability to obtain a definite specific IgE concentration. The analyzed sample was 352 total IgE values obtained by means of RIA in 611 patients of both sexes and ages from 5 to 15 years [corrected], and the RAST results of every one of them. As mathematical model, Logit was used. It was established a mathematical function [p = 1/(1 + K(-1).IgE-beta)], which allows to calculate the probability to obtain a definite RAST class of allergy for a definite total IgE level. The propounded model establishes that, for a plasmatic IgE level lower or equal to 17.0 kU/l (11.5-23.3), the probability to obtain a specific IgE concentration lower than 0.35 kU/l (absence of allergy) is 0.95. For a total IgE level of 23.32 kU/l (16.43-30.91) there is a probability of 0.95 that RAST result to be negative or slightly positive. Finally, for a total IgE level of 62.6 kU/l (49.20-76.55) specific IgE levels of classes 0 or 1 or 2 would be obtained. The mathematical model allows to predict the most probably RAST class of allergy result once known the total IgE level. It makes easier the allergological diagnostic and facilitates, in some cases, to omit the RAST, with the resulting saving of time and money.

[Hepatobiliary clearance of glycocholic acid in Gilbert's disease]. / Aclaramiento hepatobiliar de ácido glico-cólico en la enfermedad de Gilbert.

AIM: To research the behaviour of one biliary acid (glyco-cholic) i.v. injected in patients with Gilbert's disease and in healthy controls, so that contribute to the knowledge of the pathophysiological correlate between bilirubin and biliary acids. PATIENTS AND METHODS: We include 15 patients with Gilbert's disease and 7 healthy voluntary ones. We injected i.v. glycocholic acid and obtained the clearance curve (CG-RIA Abbot method). We evaluated the possible biostatistically significant differences between the obtained values of both groups though the non-parametric method of Mann-Whitney. RESULTS: The clearance curve of both groups had a similar profile; biostatisticaly there are not significant differences between the serum values of glyco-cholic acid in both groups. CONCLUSIONS: The clearance of the glyco-cholic acid in patients with Gilbert's disease had a similar behaviour as in healthy controls, without biostatisticaly significant differences between both groups.