Chemistry: electrical and wave energy – Apparatus – Electrolytic
Reexamination Certificate
2000-06-07
2003-06-03
Warden, Jill (Department: 1755)
Chemistry: electrical and wave energy
Apparatus
Electrolytic
C204S616000, C204S606000
Reexamination Certificate
active
06572746
ABSTRACT:
BACKGROUND OF THE INVENTION
Zone electrophoresis techniques on agarose gel can separate protein constituents contained in samples, in particular biological samples such as serum, blood, urine, cereprospinal fluid, etc. In a medium in the form of a gel and containing a buffer solution, proteins deposited on the gel surface ionise and migrate at different rates depending on their respective charge under the effect of an electric field. The thinness of the bands obtained after electrophoresis, corresponding to the separated constituents of the sample and hence the resolving power of the procedure, depends mainly on the thinness of the loading of the sample.
Different types of sample applicators can be used to improve the thinness of the loading. Such applicators, often termed sample combs, can take a number of different forms, as illustrated by the following devices:
“Groove” combs where the teeth carry a groove the edges of which are parallel to the plane of application, which means that a drop of biological sample can be taken by capillary action. The comb is then applied to the electrophoresis gel under the effect of its own weight. The teeth are then in contact with the gel surface and the sample is transferred from the tooth to the gel. Such combs can be formed from a metallic or plastics material.
“Strip” combs, whose principle is identical to that of groove combs, but the teeth of which comprise two parallel strips disposed at their extremity in a plane perpendicular to the plane of application. The sample is taken by capillary action into the space delimited by the two strips and loaded on the gel in the same manner as when using a groove comb. As before, such combs can be formed from a metallic or plastics material.
Membrane combs whose teeth are constituted by a microporous membrane. Such combs have been described in European patents EP-A-0 493 996 and in U.S. Pat. Nos. 5,464,515 and 5,405,516. The teeth are impregnated with the sample until the microporous membrane is saturated. The teeth of the comb are then brought into contact with the gel surface in the same manner as in the above two cases. In this case, the sample is loaded by diffusion of the proteins from the microporous membrane towards the gel.
Such different applicator types can all produce thin loading at a greater or lesser efficiency.
The sample is usually diluted when carrying out electrophoresis, in particular for immunofixation. The most routinely used dilutions are from ⅓ to {fraction (1/10)}. In general, the diluting solution is physiological water to keep the proteins in solution. After applying the diluted sample to the gel using an applicator as described above for an average period of 30 seconds to 120 seconds, preferably 60 seconds to 120 seconds depending on the applicator and the dilution of the sample, the location where the tooth comes into contact with the gel is observed to exhibit a compression mark which does not disappear during migration. After the staining step, this compression mark results in an absence of staining or a reduction in the intensity of staining described above.
Considering their weight (from a few grams to tens of grams, depending on the embodiment), application of such combs to the gel surface compresses the gel at the location where the teeth are in contact with the gel, resulting in a deformation in the form of a compression mark on the surface. If care is not taken, this deformation subsists during migration and becomes visible after staining the gel, which is carried out to reveal the electrophoretic profile. That deformation results in a zone, which is less, stained or not stained which can be considerably deleterious to interpretation of the electrophoregram.
SUMMARY OF THE INVENTION
The aim of the invention is to provide a means for eliminating this loading mark whatever the type of applicator used.
The inventors have observed that the loading mark produced on the electrophoresis support, in particular on the gel, can be eliminated or at the very least neutralised so that it does not affect the interpretation of the results obtained following electrophoresis, by hydrating the electrophoresis support at the sample loading zone compressed by application of the sample applicator. This hydration must be carried out under conditions enabling the constituents, and in particular the proteins contained in the sample being tested, to penetrate into this electrophoresis support zone to undergo electrophoretic migration.
Thus the electrophoresis support zone compressed by the applicator used to load the sample has to be re-hydrated sufficiently rapidly and effectively for the constituents of the sample to be able to penetrate into the electrophoresis support and migrate under the applied electric field.
When electrophoresis is carried out over a certain period of time, hydration must occur in a period corresponding to 10% to 20% of that electrophoresis period (this latter corresponding to the period during which a voltage is applied to the electrophoresis support), timed from application of the voltage to the electrophoresis support. Advantageously, re-hydration can occur more rapidly, i.e., within a period less than 10% of the electrophoresis period, timed from application of the voltage.
The inventors have identified certain compounds which, when introduced into the sample diluent and thus loaded onto the electrophoresis support at the same time as the sample, are capable of causing the clear mark left by compression of the electrophoresis support to disappear. These compounds remove or at least attenuate this distinct mark from the first moments of electrophoretic migration, while this mark persists throughout migration in the absence of said compounds.
The inventors have observed that these compounds cause the electrophoresis support to re-swell at the location marked by loading the sample, resulting from rapid re-hydration of the compressed zone.
The invention thus provides a composition for use in a process for separating the constituents of a sample by electrophoresis on an electrophoresis support, comprising one or more ionic compounds which, on application of an electric field to an electrophoresis support having negative surface charges, causes hydration of the loading zone of the sample to be separated, when said zone exhibits a compression mark resulting from loading the sample. This compression results from the pressure exerted by the applicator on the support.
The presence of negative surface charges on the electrophoresis support is the sign of non-zero electroendosmosis (or electroosmotic flow).
Compression of the electrophoresis support resulting from loading the sample using an applicator can be observed with the naked eye and corresponds to a compression mark on the support. Such a mark can, for example, be described as being V shaped, generally with a depth of less than 100&mgr; and with a width of about 1 mm between the two top portions of the V for an electrophoresis support constituted by a 0.8% agarose gel, when the pressure of the applicator is more than 0.1 g/mm
2
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Depending on the case, the ionic compounds used to prepare the composition of the invention can be displaced or remain essentially immobile during electrophoretic migration.
The absence of displacement in the electric field is attributable either to the nature of the compound or to its concentration in the composition, or to a combination of these factors.
For a given electrophoresis support, in particular one where the electroendosmosis index (−rm index, relative mobility index) is known, the concentration of ionic compounds present in the composition is determined as a function of the nature of these compounds, in particular their capacity or otherwise to migrate in the electrophoresis support after application of the electric field. This concentration must be selected so as to enable hydration of the sample loading zone in the electrophoresis support from the first moments of electrophoretic migration.
As indicated above, the desired hydration or re-hydration effect o
Nouadje Georges
Vincent Laurent
Brown Jennine
Lerner David Littenberg Krumholz & Mentlik LLP
Sebia
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