Resumen
[0001] The present invention relates to a method and a kit for evaluating the surface activity of a test preparation comprising: incubating a labelled test preparation with a non-surface active extinguishing agent which blocks the detectable signal of the labelled preparation; detecting the signal produced by the labelled preparation adsorbed into interfacial air-liquid interface; and comparing the signal obtained with the signal produced by a standard surfactant. It also relates to a method for determining the influence of experimental parameters, and the influence of inhibitor/activators on the surface activity; to a method for screening a substance library, and to a method for controlling the quality of a surfactant.
Reivindicaciones
1. A method for evaluating the surface activity of a test preparation comprising the following steps: a) incubating the test preparation with a detectable substance to obtain a labelled preparation which provides a detectable signal, b) preparing an aqueous solution comprising a non-surface active extinguishing agent which is able to block the detectable signal of the substance in step a), c) combining the labelled preparation of step a) with the solution of step b), and mixing the obtained solution for a period of time sufficient to allow the labelled preparation to adsorb into the air-liquid interface and form a surface film, d) detecting the signal produced by the labelled preparation adsorbed into interfacial air-liquid interface, and e) comparing the signal obtained in step d) with the signal produced by a standard surfactant. 2. The method according to claim 1, wherein the test preparation is a pulmonary surfactant. 3. The method according to claim 2, wherein the aqueous solution is a buffered solution adjusted to meet physiological conditions. 4. The method according to any of the claims 1-3, wherein the detectable substance is a molecule being or containing a detectable moiety, and this molecule interacts stably with the test preparation. 5. The method according to any of claims 1-4, wherein the detectable substance comprises a fluorophore, and the extinguishing agent is a fluorescence extinguisher. 6. The method according to claim 5, wherein the detectable substance comprising a fluorophore is 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine, and the fluorescence extinguisher is tetrasodium (6Z)-4-acetamido-5-oxo-6-[[7-sulfonato-4-(4-sulfonatophenyl)diazenylnaphthalen-1-yl]hydrazinylidene]-naphthalene-1,7-disulfonate. 7. Kit for evaluating the surface activity of a test preparation by the method as defined in any of the claims 1-6 comprising: a) a detectable substance with molecular properties making it suitable for stable incorporation into test preparations, b) a solution comprising a non-surface active extinguishing agent which is able to block the detectable signal of the substance in step a), and c) a standard surfactant. 8. A method for evaluating the surface activity of a test preparation comprising carrying out the method according to any of claims 1-6 and at least a method for measuring the surface tension. 9. A method for determining the influence of an experimental parameter on the surface activity of a surfactant preparation, comprising evaluating the surface activity according to the method as defined in any of the claims 1-6 in response to variations of the experimental parameter. 10. The method according to claim 9, wherein when the experimental parameter is ionic strength, then the method further comprises the step of adding a ionic salt at different concentrations to the aqueous solution of step b); when the experimental parameter is pH, then the method further comprises the step of adding different buffers to the aqueous solution of step b); and when the experimental parameter is presence of Ca, then the method further comprises the step of adding a calcium salt at different concentrations to the aqueous solution of step b). 11. A method for determining the influence of a surfactant inhibitor on the surface activity of a surfactant preparation, the method comprising evaluating the surface activity according to the method as defined in any of the claims 1-6, further comprising the step of adding the inhibitor either to the labelled preparation of step a), or alternatively, to the aqueous solution of step b), and in step e) comparing the signal obtained in step d) with the signal produced either by the standard surfactant, or by the surfactant preparation in the absence of inhibitor. 12. The method according to claim 9, wherein the inhibitor is bovine serum albumin (BSA). 13. A method for determining the influence of a surfactant activator on the surface activity of a surfactant preparation, the method comprising evaluating the surface activity according to the method as defined in any of the claims 1-6, further comprising the step of adding the activator either to the labelled preparation of step a), or alternatively, to the aqueous solution of step b), either in the presence or absence of an inhibitor, and in step e) comparing the signal obtained in step d) with the signal produced either by the standard surfactant, or by the surfactant preparation in the absence of activator. 14. A method for screening a compound library to determine the ability of the compounds to inhibit or activate the surface activity of a surfactant preparation, the method comprising evaluating the surface activity according to the method as defined in any of the claims 1-6, further comprising the step of adding each compound to be screened either to the labelled preparation of step a), or alternatively, to the aqueous solution of step b). 15. A method for controlling the quality of a surfactant preparation, the method comprising evaluating the surface activity according to the method as defined in any of the claims 1-6.
