HK1058396B - Mask for depositing and spreading reactant on an analysis support - Google Patents

Description

The invention relates to a mask for depositing and spreading one or more reagents on an analytical medium, including an electrophoresis medium, e.g. an agarose gel.

The invention is applicable, for example, in the field of detection and characterization of constituents present in a biological sample, in particular a biological fluid such as serum, urine, cerebrospinal fluid. In particular, this detection may be achieved by separating the said constituents from the biological sample, for example by electrophoresis. Detection may then be achieved in particular by known immunofixation techniques which require the contact and incubation of reagents with the separate constituents of the sample, to allow an immunological recognition reaction of the separate constituents of the biological sample with the reagents, in specified areas of a sample medium.

The invention is useful for routine analysis, particularly in clinical trials.

The invention also relates to a mask for the deposition and dispersion of one or more reagents on an analytical medium, combined in a device with positioning devices, which allow the mask to be positioned in relation to the analytical medium, in the vicinity of that medium, when the mask is used to deposit and disperse the reagents.

These means of positioning may also be combined with means of guidance, or may include means of guidance, for the movement of the mask when placed near the test medium, so as to allow the dispersion of reagents over defined areas of the medium, including so-called incubation zones of the reagents with the constituents of the sample.

The device of the invention may allow the deposit and dispersal of reagents in manual mode or may be designed to allow the deposit and dispersion of these reagents by automated means.

The step of loading the reagents at the mask level can also be carried out manually or automatically, depending on the embodiments of the invention.

The invention also concerns a process for depositing and spreading reagents on an analytical medium.

Such a process is, according to a particular embodiment of the invention, used for the deposition and dispersion of reagents, intended to achieve immunofixation, for the detection and, if necessary, quantification of specific constituents contained in a biological sample, these constituents being previously separated by electrophoresis on a medium such as an agarose gel.

The invention also relates to a process of immunofixation, which implements the aforementioned mask.

The invention also concerns a kit including a mask according to the invention.

A kit according to the invention is advantageously suited for the implementation of an immunofixation process by means of the mask of the invention.

The invention also relates to the means of positioning and guiding the mask.

It should be recalled that the immunofixation technique, which allows in particular the analysis of biological samples for the type-approval of paraproteins contained in them, is a commonly used analysis, particularly in laboratories for clinical analysis.

This technique, which combines electrophoresis with the formation of precipitates on the electrophoresis gel, has been known for a long time. This technique was described by Alper CA and Johnson AM Vox. Sang. 17:445 (1969), Cawley LP et al. Clin. Chem. 22: 1262 (1976), Ritchie RF and Smith R. Clin. Chem. 22:497,1735,1982 (1976). It allows the identification of abnormalities in various biological samples, especially in biological fluids, for example in serum, urine or cerebrospinal fluid.

This technique mainly consists of the following steps: 1) separation of protein constituents from the serum or test liquid by electrophoresis on a medium such as a gel, e.g. on agarose gel; 2) immunological reaction with specific antibodies to the separated proteins; 3) detection of the immune complexes formed.

The conditions for carrying out these steps were described in the previous art.

European patent application EP 1 335 201 A1 describes, for example, a system in which a gel and a plate form physical contact, the latter comprising projections which cut through the gel, or at least create a depression on the surface of the gel, in order to form cavities with the base formed by the gel, the sides by the projections, and the top by the lower part of the plate.

The devices used also include the possibility of making a control track on the same electrophoresis medium, in particular the same gel, by fixing all the separate proteins present in the sample with a protein fixer including, for example, a multi-purpose antiserum.

The new semi-automatic techniques of application of biological samples to be analysed, temperature-controlled migration and deposit of reagents (including, for example, antiserums and fixators) allow for miniaturisation of immunofixation profiles while maintaining satisfactory sensitivities and resolutions.

Thus, on an electrophoresis gel of 8 x 10 cm, one to nine immunofixations were carried out in a few years (for example, by means of the immunofixation kit marketed under the trade name Hydragel 9IF by SEBIA).

However, in order to perform, for example, 9 immunofixations on the same electrophore gel in 3 rows of 3 samples by means of the mask described in EP 0 526 271 B1, it is necessary, for each sample, to perform the sampling of 6 active substances in total (Anti-Gigitis, Anti-Gigitis, Anti-Igitis, Anti-Igitis and Anti-Igitis, Antis-Igitis and Antis-Igitis).

These hand pipetages can be time-consuming and tedious, despite the possible use of pipettes for repeated distribution.

The present invention is intended in particular to improve the conditions for depositing and spreading reagents on a test medium by means of masks, by proposing a mask which in particular reduces the number of pipetages of these reagents and reduces the amount of reagents used. The means proposed in the present invention can be used in any analytical technique requiring the controlled deposit of reagents on an analytical medium. In this respect, immunofixation techniques are mentioned following electrophoretic separation, techniques for spreading a particular substrate to develop enzymatic exposure, for example for the analysis of lactodehydrogenase (HLD) or creatine kinase (CK).

The invention is primarily concerned with a mask, suitable for depositing and spreading reagents on an analytical medium, the design of which takes into account a use including its movement, for the purpose of carrying out the stage of spreading reagents on specified areas of the analytical medium. The mask of the invention can therefore be presented as a mobile mask in use. The present invention also has the effect of limiting the consumption of reagents, especially antiserums which are expensive products and the fixative in the case of immuno-fixation reactions, and thus of reducing the cost of the limited analyses. It also has the effect of facilitating the loading of reagents into the mask, in particular by performing pipet loading and/or automating the loading of the mask.

In addition, the proposed mask ensures a consistent quality of the result under improved or even simplified handling conditions. In particular, it becomes unnecessary after the incubation phase of the reagents spread by the device of the invention to remove excess reagents that remain between the gel and the mask as is necessary when using a mask as proposed in EP 0 526 271 B1.

With the mask according to the invention, at the end of the distribution and dispersion of the reagents, there is no longer any free reagent between the mask and the test medium, since all the initially introduced reagents have been deposited on the test medium, so that it is not necessary to pump any excess reagents that would be present on the test medium.

The invention therefore concerns a mask, suitable for depositing and spreading reagents on a biological sample analytical medium, comprising: a lower surface and an upper surface at least partially parallel to each other, separated by a distance equal to the thickness of the mask,one or more runways, corresponding to delimited zones, located at the level of the lower surface of the mask and comprising a protruding element protruding from the lower surface of the mask, each protruding element comprising a part which forms a slope relative to a horizontal plane,associated with each runway, an opening for loading and depositing reagents,the opening through the entire thickness of the mask from an upper loading orifice at the top of the mask to an under-deposit orifice, the under-deposit orifice being located in the slope, near the lowest point of the slope of the runway, the mask is such that the trace or traces it contains allow the capillary maintenance of reagents loaded at each opening and deposited on the test medium between the trace and the surface of the test medium against which the mask would be placed.

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The dimensions of the mask of the invention are preferably such that it does not cover the entire surface of the test medium on which the reagents loaded in the mask are to be deposited and spread when the mask is placed near the test medium for use. The width of the mask (which includes the length of the tracks) is in particular less than the length of the electrophoretic migration paths of the test medium, since the length of the mask tracks is less than the length of the electrophoretic migration paths of the test medium; the stacking of the reagents on these tracks results from the displacement of the mask over the test medium as described below in conjunction with the advanced analysis of the said reactive paths on the test medium.

The mask of the invention is therefore intended to be moved over the test medium to allow the reagents to be spread.

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The lower opening is located in the slope, near the lowest point of the slope. By being located near the lowest point - relative to a horizontal plane - of the slope of the runway, the lower opening allows the liquid reagent to be spread at runway level, by a rise of the reagent along the runway.

The upper opening may be at the vertical of the lower opening or it may be positioned in a plane inclined with respect to this vertical, provided that it allows the reagent to be introduced at the level of the lower opening under conditions compatible with the deposit and spread of the reagent on the test medium.

A circular opening, opening at the top of the mask and passing through it from side to side, is advantageously associated with a track opening, which is formed by a circular opening, opening at the top of the mask, which is extended by a trunk opening, for example, ending by a cylindrical opening at the bottom of the mask, through an opening in the slope of the track, near the lowest point of the slope.

The trunk opening also provides an advantageous guide for a filling pipette and ensures that the pipette end is sealed when the reagent is injected between the track and the test medium.

If necessary, the opening shall be modified from the previous one, so that the conical part extends to the upper opening of the opening by a cylindrical part with a circular section.

Depending on a particular mode of the invention, the opening thus described may also be such that the opening at the top of the mask surface is larger than the opening at the bottom, e.g. the diameter of the first opening is larger than the diameter of the second if these openings are circular.

A particular mask according to the previous definitions of the invention, suitable for the application of reagents on a biological sample analytical medium, may be defined as comprising: a lower surface and an upper surface at least partially parallel to each other, separated by a distance equal to the thickness of the mask,one or more runways, each comprising an elongated protruding element, emerging from below the lower surface of the mask, that protruding element comprising a part which forms a slope relative to a horizontal plane,associated with each runway, with an opening through the mask on its entire thickness from an upper orifice on the upper surface of the mask to a lower orifice, the latter being situated in the runway, close to the lowest point of the slope of the runway, the mask is such that the trace or traces it contains allow the capillary maintenance of reagents loaded at the opening and deposited on the test medium between the trace and the surface of the test medium against which the mask would be placed.

The slopes of the mask with an elongated shape can also be called ramps . Their slopes are all inclined in the same direction. These slopes are intended to hold the reagent by capillarity as shown above and to ensure that the reagent is collected at the lowest point of the slope so that, when the mask is moved, it can spread.

The application describes another mask falling within the above definitions, including: a lower surface and an upper surface at least partly parallel to each other, separated by a distance equal to the thickness of the mask,one or more runways each comprising a protruding element emerging from below the lower surface of the mask, consisting of a truncated protrusion of a truncated parallel-epipedal shape, that protruding part comprising a part which forms a slope relative to a horizontal plane,associated with each runway, an opening through the mask on its entire thickness from an opening above the upper surface of the mask to a lower opening, the lower opening being situated in the runway, near the lowest point of the slope of the runway, the mask is such that the trace or traces it contains allow the capillary maintenance of reagents loaded at the opening and deposited on the test medium between the trace and the surface of the test medium against which the mask would be placed.

In the case where the protruding element consists of an elongated element or a protruding protruding element of truncated parallelepipedal shape, the protruding element shall have an upper surface coinciding with the part of the lower surface of the mask from which it emerges and a lower surface separated from the upper surface by at least one slope relative to a horizontal plane, the lowest point of that slope being near the lower opening of each runway, being the point closest to the test support facing it and near which it is brought into the operating position.

The protruding element, comprising a slope emerging from the lower face of the mask, allows the deposit and spread of charged reagents in the mask by moving the mask along an area of the test medium facing the mask track (s) in a horizontal plane relative to the mask track.

According to one example of the application, the mask is provided with one or more tracks, each containing a protruding element emerging from below its lower surface, the protruding element including a hollow sphere whose cavity is intended to receive a reagent, and whose dimensions are adapted to the width of the area of the test medium to be covered by the reagent, the reagent being held by capillarity in the sphere cavity and distributed on the test medium by a hole made in the sphere when the mask is moved.

According to a particular embodiment of the invention, the lower and upper surfaces of the mask are completely parallel to each other, outside the areas constituting the runways or even outside the areas constituting the slopes of the runways.

In the embodiments of the invention described above, the slope at each track of the mask, regardless of the shape of the protruding element, coincides with the track.

Alternatively, the slope may extend over only part of the runway. For example, a runway with the lower opening at the lowest point of its slope may extend beyond the slope, for example in a horizontal plane.

When a mask of the invention is used in combination with a biological sample analytical medium, e.g. an electrophoresis gel, the mask is brought close to the analytical medium: this means that the mask does not come into contact with the areas of the medium on which the reagent (s) must be deposited and spread (also called incubation zones) and that the reagent (s) deposited on the medium are maintained, by the effect of capillary forces, between the tracks of the mask and the analytical medium, which after their spread on specific areas of the analytical medium may result in the following effects of the analysis:

The slope formed at the point of the protruding element of each track of the mask is such that its lowest point is the point closest to the horizontal plane formed by the test support in the position of use. Consequently, the highest point of the slope of the said track is the point furthest from the horizontal plane formed by the test support in this operating situation. The lower face of each track of the mask in relation to the support is therefore inclined with respect to the horizontal.

The position of the tracks in relation to the horizontal plane of the test medium and the position of the lower opening of the opening associated with each track allow the reagents deposited on the test medium to be spread to the lowest point of the slope of the track, since at this point the capillary forces on the reagents are greatest.

The liquid forming the reagent may be spread over the entire runway or only part of the runway.

Thus, the deposit and dispersion of the reagent (s) by means of the mask of the invention can be done in a controlled manner over defined areas of the test medium, without contact being made between the traces of the mask and the test medium.

Outside areas of the test support facing the tracks of the mask during use, contact may be made between the mask and the test support, provided that the mask can slide parallel to the test support along the test support while in motion without damaging it.

According to a particular embodiment of the invention, no point of the mask in use comes into contact with the test medium, except where areas of the mask allow it to be positioned in close proximity to the test medium.

It is preferable to provide that the support of the mask necessary for its positioning near the test medium is made outside the test medium, for example on the plane (or tray) on which the test medium is placed.

When the top and bottom surface of the mask, tracks or slopes are referred to, these are understood to refer to the position of the mask above an analysis support itself in a horizontal position. In other words, the bottom surface of the mask and the bottom surface of each track or slope are in relation to the surface of the analysis support when the mask is in the position of use. The horizontal plane in relation to which the slope of the protruding element is defined may therefore be that of the analysis support when used in a horizontal position.

The invention also concerns a mask, the use of which for the deposition and spread of reagents on an analytical medium uses the principle used in the invention of capillary holding of the reagents between a slope defined at the level of the mask and an area of the analytical medium in relation to that slope, the mask being distinguished from the mask defined above by the fact that the slope at each track of the mask is achieved by the slope of the mask in relation to the analytical medium when used.

In this particular embodiment, the characteristics of the tracks defined for masks with slope integrated into the track are transferable when the slope is formed by using the mask in a sloping position relative to the test medium.

Thus, when the track is elongated, it may be parallelepipedal, with its shape delimiting the capillary area between the track and the test medium for the holding of reagents.

The mask according to the invention shall be of dimensions compatible with the dimensions of the test medium on which the reagents are to be deposited and spread and the traces of this mask shall be of shapes and dimensions compatible with the volume of liquid reagent to be deposited and spread on the test medium and with the shape and size of the delimited areas of the test medium on which the reagents are to be deposited and spread, for example for incubation with the constituents of the biological sample.

To deposit and spread one or more reagents on a biological sample carrier when the mask and the test carrier are placed parallel to each other, the mask shall be moved in a horizontal plane above the plane of the test carrier from the area of the test carrier at which the mask is initially positioned and which corresponds to the initial deposit point of the reagents, so as to spread the reagents on areas of the test carrier facing the tracks of the mask.

To deposit and spread one or more reagents on a sample carrier, when the slope of the masking strips is not due to the structure of the strips but to the slope of the mask relative to the test carrier (or vice versa), the mask shall be moved in a given inclined plane in the same manner as described for the mask with slopes in their structure.

Each movement of the mask over all the identified areas of the test medium to receive the reagents is called scanning . For example, a first scan may be made from the area corresponding to the anode of an electrophoresis medium to the area corresponding to the cathode of such a medium, or in the reverse direction from the cathode to the anode, so as to cover all the defined areas to receive the reagents, these areas corresponding in the case of an electrophoresis medium (such as a gel) to the electrophoretic migration pathways.

The mask according to the invention thus enables, when moving by scanning the determined surface of the test medium after loading the reagents at the opening associated with each track, the deposit and spread of the said reagents over all the determined areas of the test medium, for all the biological samples tested in succession in the direction of the mask movement.

It is therefore unnecessary to multiply the pipetting of each reagent for each sample to be treated. e number of pipetings to be performed corresponds to the number of reagents to be deposited on a row of the test medium and therefore normally corresponds to the number of openings provided in the mask when all the lines of the mask are used.

In addition, the amount of each reagent loaded at the mask level may be significantly reduced from the amount of each reagent normally used when each reagent must be loaded for each sample on the test medium.

For example, if the mask of the invention is intended for the deposition and dispersion of reagents for immunofixation, in order to detect particular constituents of a biological sample previously analysed by electrophoresis, it is observed that for the use of 6 reagents per test sample (usually either a fixer capable of fixing the constituents of the sample so as to achieve a control profile at the electrophoresis medium, and specific anti-serums anti-G, IgA, IgM, κ and λ), the amount of each reagent loaded on the mask is reduced by 4.5 times compared to the amount loaded, for each sample, when using a fixed Ig mask such as the one in patent No. 526 EP 271.

The amount of each reagent loaded at each track of the mask is determined by the size of the incubation area to be covered with the reagent, e.g. by the number of rows of samples in the direction of the mask. When the mask is used for depositing and spreading reagents for immunofixation, the incubation area includes or coincides with the area of the electrophoresis medium which includes the electrophoresis profile of the samples to be analysed.

For example, a mask with three groups of six lines in a row can be used to immunofix nine samples, or even 12 or 18 samples (for example, divided into rows of 3 different samples, each sample occupying 6 lines of the test medium for electrophoresis and therefore the mask).

The mask is advantageously loaded with a quantity of reagents such that each defined area of the test medium on which a reagent is to be spread is already covered homogeneously by this reagent in a scan. To determine the loaded volume of each reagent, the scan of the test medium and the width of the area to be covered on the test medium are taken into account.

For example, a volume of 4 μl of reagent may be sufficient to cover a surface of 175 mm2 of analytical medium uniformly, corresponding to a reagent spread of 0.02 μl/mm2 of analytical medium.

The mask according to the invention is preferably a rigid mask, or a mask which has been rigidised in particular by combination with rigidising means which may, for example, contribute to the positioning and/or guidance of the mask.

The choice of material used to make the mask is not limited in principle.

The mask may be made of a material that can be molded to provide a smooth surface, particularly plastic.

The material may be transparent or translucent and examples include polycarbonate, polyethylene polymethylated, crystalline polystyrene, plexiglass.

The mask according to the invention may be disposable after use.

The rigidity of the mask ensures that it moves in a defined plane, horizontal or inclined, in relation to the test support, when combined, for example, with appropriate positioning and guidance devices.

In a preferred embodiment of the invention, a mask meeting the characteristics defined in the preceding pages has several parallel tracks along the length of the mask.

Where, in the most common case, the mask has several parallel tracks, the spacing between the tracks (intertrack spacing) shall be determined according to the number of tracks on the mask, the need to prevent possible interactions between the reagents, in particular between the fixative and the antiserums.

The advantage is that the gap between the different parallel traces is constant, which can be small, for example less than 3 mm, and in particular of the order of 2.5 mm, preferably greater than or equal to 2 mm, especially to prevent possible interaction between the fixative and the antiserum of the adjacent trace.

The area of the test medium at which each reagent is spread shall be at least as wide as the mask track facing that area.

For example, the width of the track and the width of the incubation surface, which may correspond to the spread of the electrophoretic profile on the test medium, are similar and about 2.5 mm. According to another example the width in question is 3.5 mm.

In addition, to prevent interaction between neighbouring reagents, the mask tracks may be arranged to prevent overlap between the different reagent deposition areas or potential diffusion areas of the reagents deposited on the test medium or to load and lower the reagents on the test medium to prevent interaction.

In particular, with respect to the interactions discussed above, it should be ensured that the reagent used to construct the control profile of each sample does not interact with specific reagents (in particular antiserums), which could distort the detection of particular constituents of the sample.

To this end, where specific means are provided for in the mask structure, a first method of making the mask comprising several parallel tracks, spread over the length of the mask with a constant spacing, is to provide a first set of parallel tracks, spread over the length of the mask, and a second set of parallel tracks, parallel to the first set of tracks and with the lower orifices in the same horizontal plane, with the second set of tracks forming an alignment that is out of alignment with the alignment formed by the first set of tracks.

In a variant of the mask, the misalignment of the second set of tracks is replaced by an increase in the gap between the said tracks of the first set and the other tracks of the second set.

The purpose of the second series of traces being shifted or the shifting of this series of traces from the traces of the first series is to prevent the interaction between reagents of the second series of traces and those of the other traces when they are deposited on the test medium.

Where the mask does not have a series of such displaced tracks or has a differential spacing from the other tracks, the interaction between the reagents concerned may be avoided, if necessary, for example by loading and depositing the reagents separately in two steps at the level of the test medium.

For example, the fixer is deposited on the anodic side of the electrophoresis medium and then scanned before the anti-serum is loaded, e.g. in an anodic position but shifted, e.g. about 5 mm towards the cathode from the position of the first load.

However, the use of two-stage loading is not always necessary, particularly if the loading methods do not lead to an unpleasant interaction between the reagents, for example, such interaction can be avoided even if all the reagents are loaded together, for example outside the test surface, and when the simultaneous descent of all the loaded reagents can be achieved on the test medium without causing the unpleasant interaction.

When the mask is designed so that the slope of the tracks is due to the slope of the mask relative to the test medium and not to the structure of the tracks, the shifting of the tracks or groups of tracks is not necessary, and the reagents must then be loaded and unloaded in several steps according to their nature.

The mask according to the invention may be so made that the runways are arranged in several groups, each group being for example one runway positioned out of alignment with other runways aligned with each other and the aligned runways preceding the next out of alignment runway.

In addition, the mask of the invention is such that it allows, by its structure and, where appropriate, by the conditions of its use, the deposition and dispersion of reagents without interaction between the different reagents deposited on the different areas of the analytical medium.

A localized diffusion of the reagents on the analytical medium prior to their application was observed, in particular at the initial deposit area of the reagents on the analytical medium.

To avoid the possible consequences of local diffusion of the reagents at the time of deposition, it is advantageous to deposit the reagents by means of the mask outside the area of the test medium which may contain the constituents of the samples to be detected, for example outside the area containing the electrophoretic profiles.

In the case of immunofixation, for example, the reactants are deposited in an area outside the area corresponding to the electrophoretic profile, for example on the anode side of the medium relative to these profiles.

The advantage of the mask is that the opening through it from side to side is perpendicular to the top and bottom surfaces of the mask.

The shape of the mask openings must allow a sufficient quantity of reagent to be loaded into the mask to allow the reagent to be deposited and spread over the entire area of the test medium without the need to re-load the mask with the reagent.

In addition, this opening must be in a shape and location compatible with the deposition and capillary retention of a given quantity of reagent between the mask track and the test medium until the quantity introduced during the test medium application is exhausted.

When depositing the reagents on the test carrier, when loading the mask over the test carrier with the tracks filled as the reagents are loaded, the seal between the mask and the end of the pipette or any other means used to load the reagents into the mask should be provided.

The volume of the opening may also be provided to allow the loading of the reagents outside the test medium, in which case the reagents must be retained in their entirety by capillarity in the opening until deposition on the test medium. In this case, the mask is then positioned in such a way as to ensure the deposition of the reagents it contains on the test medium.

The advantage is that the opening at each runway can receive excess reagent relative to the amount of reagent needed for the reaction.

The mask of the invention allows for the use of reduced quantities of reagents, e.g. about 15 μl or 10 μl. However, the dimensions of the opening through the mask intended to receive these reagents can be determined to accept a quantity of reagents greater than the quantity actually used.

The geometrical characteristics of the mask, in particular the number of tracks, the spacing between tracks (intertrack spacing) are adapted to the number of deposits, their width and the spacing of these deposits made by rows of deposits on the test medium. The mask according to the invention thus has a sufficient length to accommodate several tracks in an alignment, and a limited (smaller) width in relation to the length of the electrophoretic migration track of the incubation surface of the test medium coming towards this track when the mask is moved to operate.

It is understood and a feature of this device that the same mask can be used to reveal on a single gel several rows of deposits having the same characteristics in number and dimensions.

The geometry of the mask according to the invention is advantageous in that it allows capillary deposition and retention of the reagent between each mask track and the test medium, when the distance between the mask and the test medium is preferably between 0.1 and 1.5 mm, or less than 2 mm. This distance between the mask and the test medium varies according to the point of the mask, in particular this distance is preferably about 0.1 to 0.5 mm at the mask point nearest to the support (corresponding to the lowest point of the track slope or slope of the mask) and this distance is preferably less than 2 mm, preferably 1.5 mm, at the mask point farthest from the test medium (corresponding to the highest point of the mask track slope or slope).

Within these limits, the slope of the slope shall be such that the spacing of the mask from the test medium is compatible with the capillary forces holding the reagent between the runway and the test medium.

For example, a mask according to the invention is made in such a way that the tracks are separated from each other by a gap of not more than 1,5 mm. Preferably the gap between the tracks is 2,5 mm. The width of the tracks is advantageously 2,5 mm.

The mask track for the fastener may also be offset, e.g. may be in a more anodic position than the other track when the mask is in the use position, near an analysis medium consisting of an electrophoresis gel.

A particular mask is characterised by the fact that the track for the fastener is not aligned with the other tracks, being offset from the alignment formed by the others by a distance of 5, preferably 6 to 7 mm.

A particular mask suitable for the implementation of the invention and in particular a mask suitable for use with an electrophoresis gel 10 cm long and about 8 cm wide (between the anode and cathode poles) is such that each track of the mask has the following dimensions: Length: 3 to 15 mm width: 1 to 10 mm slope inclination: 1 to 10° with respect to the horizontal.

A particularly preferred mask, particularly suitable for use with the gel above, is a mask in which each track has the following dimensions: length: 7 mm width: 2.5 mm slope: 5° with respect to the horizontal.

In these particular embodiments, the other mask characteristics described above can naturally be combined with the above specific characteristics, in particular the spacing between traces is advantageously 2.5 mm and/or the gap between the alignment of the traces of the specific reagents and the alignment of the traces of the fixer is 6-7 mm.

The mask is still in a particularly advantageous embodiment of the invention, and the opening through it, which satisfies the characteristics described above, has a conical part forming an angle of about 50°.

In the various embodiments of the invention, the mask thickness is advantageously between 1 and 10 mm.

In a particular embodiment of the invention, the mask has an advantageous width of less than 30 mm, preferably less than 20 mm or 15 mm, still preferably less than 10 mm.

When the mask of the invention is used for deposition and dispersion of reagents on an electrophoresis medium, it may still be characterized as compatible with the location characteristics of the separate biological samples on the electrophoresis medium; in particular, the mask enables: the alignment of the masking line rows perpendicular to the direction of electrophoretic migration; the positioning of the mask near the test medium so that the capillary reagents can be maintained between the masking line and the test medium; the transverse positioning of the mask relative to the direction of electrophoretic migration so that the electrophoretic migration rows on the test medium and the masking line rows can be aligned.

For use with different analytical media, and as an example, a mask according to the invention may have between 1 and 24 tracks, preferably between 6 and 24 tracks, including 6, 9, 12, 15 or 18 tracks.

A mask with 18 tracks, if intended for use in an immuno-fixation reaction following electrophoretic separation, allows for the deposition of three different samples occupying the same row of the electrophoresis medium, and for a given number of rows of samples (e.g. 2 or more, in particular 3 or 4), a fixer to make a profile test and 5 specific reagents such as antiserums, in particular anti-IgG, anti-IgA, anti-IgM, anti-κ and anti-λ antiserums, for each sample.

The invention also relates to a mask as defined above, combined with positioning devices designed to keep the lower surface of the mask traces close to the surface of the test medium to which the mask would be brought for deposit and dispersion of reagents on the test medium.

Appropriate means of positioning may be provided by stops which may rest on the test medium outside the incubation surface containing the biological samples, such stops being of such dimensions that the mask does not come into contact with the test medium in its part corresponding to the incubation surface of the reagents.

The positioning devices may also be combined with means of guiding the mask so as to allow its controlled movement over the test support, as described above.

The invention therefore also concerns a device for depositing and spreading one or more reagents on a biological sample analytical medium comprising: (b) means of positioning and guiding the mask so that the mask is held close to the surface of the test medium and guiding the mask by scanning the surface of the test medium in a horizontal plane parallel to the surface of the test medium to allow the deposition and spread of reagents over each of the specified areas of the test medium facing the traces of the mask.

According to one variant of the invention, the mask used is such that the slope of the tracks results from the slope of the mask relative to the test medium. (b) means of positioning and guiding the mask so that the mask is held close to the surface of the test medium and guiding the mask by scanning the surface of the test medium in a determined inclined plane on the surface of the test medium to allow the deposition and spread of reagents over each of the determined areas of the test medium facing the tracks of the mask.

In one particular embodiment of the invention, the device so defined is such that the means of positioning and guiding the mask allow a distance between the test support and the point of the mask track nearest to the support (corresponding to the lowest point of the slope) of between 0,1 mm and 0,5 mm, and a distance of less than 2 mm, preferably less than or equal to 1,5 mm at the point of greatest distance between the mask and the support (corresponding to the highest point of the slope).

The distance between the test support and the nearest point of the mask track shall preferably be 0,5 mm.

The means of positioning and guiding the mask according to the invention may be any suitable means, if any, present in an electrophoresis device.

The guidance devices include a speed limiter to help define the speed of the mask.

The positioning and guidance devices may, in a particular embodiment of the invention, allow the mask to be moved automatically along the test medium, but the mask according to the invention can be easily moved manually so that the entire area determined, with the reagents contained in the mask, is covered by scanning of the test medium, if necessary by several round trips.

The application also describes a process for depositing and spreading one or more reagents on an analytical medium comprising biological samples, including the steps of: positioning of a mask or device as defined above, in the vicinity of the test medium,loading of the reagent (s) onto the mask so as to allow the deposit of the reagent (s) on the test medium, holding them capillary between the test medium and the test medium or tracks;moving the mask by scanning the test medium so as to allow the dispersion of the reagent (s) on the test medium at the level of the defined areas constitutes a medium, the reagent (s) being dispersed in sufficient quantity to allow their interaction with the biological samples present on the test medium.

When the tracks of the mask have a sloping part, the mask shall be moved in a horizontal plane relative to the plane of the test medium above the medium and parallel to the medium.

Where the masking strips do not have a slope and are therefore inclined relative to the test support to achieve a slope, the mask shall be moved parallel to the plane of the test support itself in a horizontal position if the mask is inclined.

When the mask is placed near the test medium and as soon as the reagents have come into contact with the test medium, it may be moved immediately by sweeping over the test medium.

When the reagents are spread over specified areas of the analytical medium, for example areas corresponding to the electrophoretic migration pathways of biological samples, these areas constitute incubation zones for the reagents with the sample constituents.

One advantage of using the mask according to the invention is that when the spread is complete, the mask can be removed immediately from the defined areas of the incubation areas of the test medium, the amount of reagent loaded in the mask being exhausted.

Another advantage of using the mask according to the invention is that it allows for uniform spread of reagents over incubation areas.

It is preferable to use an excess of reagent compared to the amount needed to cover the areas of the test medium at which incubation must take place between the sample constituents and the reagents.

The amount of reagent left on the test medium per unit of surface swept depends on the surface and in particular on the length of the sweep.

The speed of movement of the mask relative to the test medium is normally between 0.5 and 2 cm/s.

For example, to spread reagents on an electrophoresis medium with a width of 8 cm determined between the anode and the cathode (corresponding to the scanning length), two trips, each one one round trip, can be made, each one taking about 3 seconds, in which case a quantity of reagent per track of 6 μl to 10 μl can be deposited.

At slow speed, i.e. at about 0.5 cm/s and for a track width of 2.5 mm, a reagent of 3-4 μl, for example, is used up after 70 mm of running under the track.

Thus, for 2.5 mm wide traces and a scan stroke of the mask relative to the 70 mm test medium, a volume of reagent of approximately 8 to 10 μl/trace is advantageous.

Thus, when the first scan has been carried out, there will still be some reagent under the runway even if the movement is slow at 0.5 cm/s and even more so if the movement is at an average speed of 2 cm/s.

Further scans will be required to exhaust all the reactants introduced, the number of scans may vary depending on the volume of reagent introduced into each runway.

In practice, the volume of each reagent used is such that 4 sweepings are sufficient to exhaust the reagent.

With 10 μl charged at each opening, the mask is subjected to 2 round trips for a sweep length of 70 mm. Once all the reagents have been distributed on the gel surface after these 4 passes, the mask is removed without any risk of unexpected spread and the incubation phase proper begins.

The method of analysis is particularly advantageous in that two scans (one round trip) are carried out to spread the reagents on the test medium. Even if a small amount of reagent remains on the test medium after scans, it is not necessary to remove it before incubation.

If the scanning length is reduced, the amount of reagent spread per runway can be reduced.

It has been shown above that the mask used for the implementation of the deposit and dispersion process of one or more reagents on an analytical medium is preferably (but not necessarily) a mask in which the traces for the fixator capable of fixing the constituents of biological samples to make a control profile are offset from the other traces as described above.

This lag between the trace of the fixator and, for example, traces for specific antiserums prevents interaction between the reagents when they are deposited on the test medium.

Alternatively, for example, where the tracks for the fastener are not displaced, the mask may be loaded twice, firstly to deposit the antiserum and then the fastener.

Alternatively, loading may be done at once if the conditions for depositing the reagents on the analytical medium are such that they do not lead to interactions between the reagents, in particular between the specific reagents and the fixative if present.

When the mask is such that it must be used in an inclined position to achieve a slope at the level of the tracks, in relation to the test medium, the tracks are not displaced from each other but the loading of the reagents which are intended to prevent their interaction (e.g. fixative and antisera) is done either in two steps: the reagent loaded in the first step is spread by scanning before loading the reagent (e.g. antisera) loaded in the second step, or in conditions which allow to avoid the inconvenient interactions when depositing the reagents on the test medium.

The method of depositing and spreading reagent according to the invention is according to a particular embodiment, such that the loading of the mask with the reagent (s) is carried out outside the surface area of the test medium containing the biological samples.

During this time-consuming filling (30 seconds to 2 minutes), the area of the test medium covered by the reagents at this level is wider than the track itself due to a diffusion phenomenon, which could result in an abnormal profile enlargement, which is revealed after incubation of the reagent with the constituents of the biological sample, if the filling is done vertically from an area containing a profile of the constituents of the samples to be revealed.

If the mask is loaded outside the areas of the test medium containing the constituents of the samples, this disadvantage due to the diffusion of reagents from the storage area does not occur.

Thus, where the size of the surface of the test medium permits, such loading may be carried out in the anode part beyond the area in which the electrophoretic migration profiles of the samples are located.

Where the size of the test medium does not allow such deposition outside the area containing the electrophoretic migration profiles of the samples, the loading of the mask may be carried out outside the medium surface, for example on a thin plastic sheet, this sheet resting on the test medium and in the plane of the surface of the medium but protruding from that surface.

The invention also concerns a process for depositing and spreading one or more reagents on an analytical medium comprising biological samples, which includes the steps of: loading of the reagent (s) on the mask so as to permit deposition of the reagent (s) on the test medium, capillary maintenance between the test medium and the slope of the test mask track (s); positioning of a mask or device as defined above in proximity to the test medium so as to permit deposition of the reagent (s) on the test medium and capillary maintenance between the test medium and the slope of the test mask track (s); moving the mask so as to permit scanning of the surface of the test medium so as to permit analysis of the reagent (s) on the test medium at the incubation level, corresponding to the areas of the test medium or the areas of the test medium, being sufficient to permit interaction with the biological sample (s) on the test medium.

The characteristics indicated above for the implementation of the deposit and spread process are applicable here.

In this way, the mobile mask, loaded before it was placed on top of the test support, was used to introduce all the reagents into the upper openings of the openings associated with each track of the mask, which then acted as a reservoir.

In the configurations described above, when the mask is loaded on top of the test carrier, the mask being already at the required distance (about 0,5 mm at the lowest point) from the test carrier, the reagents are introduced directly between the mask tracks and the test carrier.

This ensures that the pipette is sealed between the tip of the pipette and the upper mask orifice, for example by holding the pipette in a vertical position with the tip slightly pressed against the bottom of the mask opening at the level of the conical part adjacent to the lower opening of the mask. When this lower conical part is extended by a cylindrical part, the latter has a diameter (e.g. 0,8 mm) which does not allow the pipette tip to pass through. This ensures that the forced outlet of the reagent through the lower opening of the mask opening to the upper opening, the tip of the reagent being slightly pressed, does not drop. In this case, the test tube and the test tube are not in contact with the test tube, and the test tube is not in contact with the test tube (0,5 mm) near the test tube.

This is also the case when the sealing is ensured but the surface of the test medium is not near the lower orifice, i.e. when loading is carried out outside the test medium.

In these conditions, the droplet which pearled (but which, because of its very small volume of 10 to 15 μl, did not fall) and remained attached by capillarity to the vicinity of the lower orifice, rises back into the well formed by the upper orifice when the tip-upper orifice contact is removed by spacing the pipette.

This particular mode of use of the mask with pre-loading before placing it on top of the test carrier has the advantage of being automated, for example by the Hydraplus SEBIA automatic, eliminating any manual pipetting, resulting in a further simplification of the filling of the mask.

The mask loaded with the reagents distributed in the upper reservoir openings may be stored in a damp chamber for a period of time ranging from a few minutes to several hours before use.

Various means are possible to get the reagents loaded into the mask to be deposited on the analytical medium.

The mask is covered by a small chamber (Figure 3) which covers all the upper orifices and rests on the periphery of the mask (surface-to-surface airtight support). This chamber has a tip through which a small volume of air between 50 and 200 μl is rapidly injected (for example by means of a syringe). This increase in pressure in the test chamber causes each of the reagents to be dissolved into traces of the reagents and the reagents to come into contact with the surface of the mask.

In another embodiment, the pre-loaded mask assembly and mask door are placed above the test support by attaching the mask door to the guide rail and are brought into the anode position.

The descent of reagents onto the test medium from the upper mask openings acting as reservoirs may be achieved by introducing vertically into each of the upper mask openings a cylindrical rod of a smaller diameter than the lower mask opening (e.g. 0,5 mm) made of a material of a hydrophilic nature (e.g. stainless steel) until this rod comes into contact with the test medium.

This rod allows a connection between the liquid introduced into the upper orifice and the test medium to be established, and all the liquid introduced then descends capillarly along the rod and is distributed between the track and the test medium.

The simultaneous descent of all reagents onto the test medium can be achieved by simultaneously and vertically introducing a rod into each of the upper masking holes, these rods being made solid by, for example, inserting them into a rectangular plexiglass plate of the same size as the mask and with a geometry which exactly reproduces the arrangement of the masking holes.

The application also describes a process for simultaneously lowering all the reagents on the gel, which consists in placing the anodized mask on top of the test medium and giving it a mechanical impulse, which can be obtained, for example, by gluing the loaded mask to the mask medium.

This impulse allows a drop of reagent, hitherto held capillarly in the mask tank, to be projected by inertia onto the test medium and thus establish a junction between the lowest point of the mask tracks at the lower orifice of the tracks and the test medium so that all the reagents in the tanks are spread capillarly between the mask tracks and the test medium.

In addition, an advantageous method of manufacture is to place the mask, when the mask is loaded outside the test medium, after the reagents have been distributed in the upper openings and the mask has been placed on top of the test medium, briefly in contact with the test medium at the lowest point of the slope of the tracks.

Once the mask is loaded and in the position of use according to one of the above methods of manufacture, i.e. when each track has received a determined amount of reagent, the mask is moved parallel to the test medium by means of guides, in the direction of electrophoretic migration. This movement has the consequence of causing the liquid between the mask and the test medium to settle by scanning the surface of the test medium coming in front of the mask's tracks. The liquid held by capillarity between the mask and the test medium is drawn in particular to the tracks which allows the reagent to be collected to the lowest point during the movement and a certain amount of reagent is then deposited on the test medium and penetrates into the test medium and remains there, and therefore the volume of the liquid is reduced as the scan track is penetrated and displaced.

The invention also concerns a process for depositing and spreading reagents on an analytical medium, in which the loading of the mask with the reagent (s) is automated.

In another preferred embodiment of the invention, the mask movement step for scanning the test medium is automated.

The method according to the invention is advantageously used for the detection of constituents of biological samples previously separated by electrophoretic migration, this detection being particularly capable of involving the technique of immunofixation, the reagents being in this case specific antiserums and preferably a fixative for making a control electrophoretic profile.

Such a process according to the invention can be implemented under the usual conditions of the electrophoresis and immunofixation techniques. Thus, the reagents used are the reagents usually used, although these reagents are advantageously used in a lower quantity in the context of the invention, compared to the quantities usually used.

The advantages of the above methods are that they allow simultaneous analysis of 3n, 2n, n biological samples, respectively, with a mask of 18, 12 or 6 tracks, where n is an integer representing the number of deposition rows.

However, there is no limit in principle to the number of tracks in the mask.

Due to the mask's structure and the way it is used, the amount of each reagent loaded at each track of the mask, i.e. introduced into each opening of each track and maintained by capillarity below each track, can be advantageously reduced to less than 15 μl/track, preferably to less than or equal to 10 μl/track.

When the reagents are deposited on the test medium, they are in liquid form. The purpose of the invention is therefore to use liquid reagents to load the mask. The purpose of the invention is also to use a mask loaded with liquid reagents, the mask being then lyophilized so that the reagents in the mask openings are lyophilized, until the mask is used where, for the deposition step, the reagents are in solution form.

The invention also concerns a kit comprising: at least one of the above-defined masks,at least one analytical medium, including an electrophoresis medium.

Such a kit may also include: reagents for the immuno-fixation of constituents of electrophoresis-separated samples,one fixer for the fixation of all electrophoresis-separated constituents for each sample.

The kit may also include at least one comb for depositing samples on the test medium.

If the mask is to be loaded outside the electrophoresis support, the kit may include means, for example as described above, to lower the reagents from the openings in the mask onto the test support.

Such a kit is preferably suitable for the simultaneous separation of 9 or even 12 samples on each electrophoresis medium by means of an 18-track mask.

Preferably such a kit allows the simultaneous separation of 18 samples on each electrophoresis medium.

A kit of the invention may also contain instructions for use of the mask of the invention, for example in the form of a package leaflet containing information on the quantities of reagents to be loaded onto the mask and/or on the conditions of movement of the mask such as the speed of scanning or the recommended number of scans.

If applicable, in a particular embodiment of the kit of the invention, the mask is not accompanied by the analytical medium, which can be obtained independently.

The mask according to the invention is advantageously loaded with reagents.

In addition, the invention concerns an electrophoresis support for the separation of at least 9 biological samples arranged in 3 rows of 3 samples for their immunofixation, comprising at least 18 migration pathways, the migration pathways being spaced 2 mm apart and 3 mm wide, and the total length of the migration pathways not exceeding 63 mm.

Further features, details and advantages of the invention will be apparent from the following description, made by way of example with reference to the attached drawings, in which: Figure 1 is a schematic view from below, in perspective, of a mask-wearing device according to the invention;Figure 2 is a schematic view from above, in perspective, of this device;Figure 3 is a schematic view from above, in perspective, of this device and an associated lid;Figures 4 and 5 are views from below and to the side of the mask according to the invention;Figure 6 is a cut-out view according to line VI-VI of Figure 4;Figures 7 and 8 are schematic views from above, in perspective of two embodiments;Figures .9 to 17 illustrate a mask embodiment in which the tracks are aligned according to a single row.

The Commission has

View from the top of the guide to position the mask on top of the electrophoresis gel and allow the mask to be swept over the gel in a given direction and stroke.

The Commission has

The carriage is attached to the rail (14) by means of the slide (49). The carriage has four feet (30) which rest on the plane on which the electrophoresis gel is positioned on either side of the gel.

The Commission has

View from above and below the mask door which receives and holds the mask by means of notches (43) and springs (28).

The mask cover has a support zone (46) where pressure is applied to bring the mask into contact with the gel.

The Commission has

The mask carrier and the mask holder are joined together by means of two spring steel blades (47) held by rivets (48). This assembly, when hung on the guide rail (14) by the slide (49), allows the mask to be held parallel to and near the gel without contact.

To lower the reagents that have been loaded into the wells of the mask, pressure is applied to (46) the mask door, causing the spring blades (47) to bend and the lower part of the runways where the lower opening of the filling wells (34) is located to come into contact with the gel (a stopper system allows the flow of this bend to be limited so as not to damage the gel). The mask is released by releasing the pressure in (46) and returning to its original position, and all the reagents in the wells (36) are distributed capillarly between the bottom of the tracks (32) and the gel.

The Commission has

Shows an upper view of the whole cart + mask door + mask.

Figures 14 to 17

Showing the top and bottom views of various types of masks.

The Commission has

Show the guide assembly + mask fixed to the positioning bar (50) on the electrophoresis tray in extreme scanning positions.

The device shown in Figures 1 to 3 comprises a mask 10 according to the invention, mounted removably on a supporting arm 12, substantially C-shaped, which is hung and guided in translation on a rail 14 of a positioning bar 16 and fixed on a tray (not shown) which carries the test support (such as an agarose gel).

The bar 16 extends on one edge of this plate parallel to the direction of electrophoretic migration and has two transverse lights 18 in which are engaged hollow plots 20 carried by another bar 21 solidary to the edge of the plate, the hollows of the plots 20 fit on nipples emerging from the migration plate (not shown) and associated with screws 22 to adjust the transverse position of the bar 16 and therefore the mask 10 in relation to the plate and the test medium.

The rail 14 runs parallel to the direction of electrophoretic migration and is engaged in a corresponding groove at one end of the arm 16 which carries a handle or rod 26 of translational displacement on the rail 14 in either direction.

Alternatively, motorised means may be provided on bar 16 for the automatic movement of arm 12, e.g. an electric micromotor with a sprocket or pulley on the output shaft, cooperating with a chain or belt, respectively, connected to arm 12.

The mask 10 shall be attached to the arm 12 by any appropriate means, e.g. by elastic enclosure as shown in figure 28 and shall consist of a flat, elongated, substantially rectangular plate, which extends transversely, i.e. perpendicularly, to the direction of electrophoretic migration.

This plate is held above the test support at a predetermined distance from it by plots or wedges 30 formed or fixed protruding on the lower face of the arm 12 and intended to rest on the edges of the above-mentioned plate.

The lower face of the mask 10 has a series of 32 oblique tracks (or ramps), parallel to each other and arranged in two transverse rows in which they are offset in a quinquantic, in the example shown. These tracks (or ramps) 32 extend parallel to the direction of electrophoretic migration and are all inclined in the same direction. Their lower end has a hole 34 for depositing reagent on the test medium. This hole 34 is the lower hole of a conduit or passage through the mask 10 for its entire thickness and opening onto the top face of the mask through an opening 36 with a significantly larger diameter than the lower hole 34.

In one embodiment of the invention shown in Figures 4 to 6, these holes 34 and 36 are the ends of small cylindrical ducts with a circular section connected to each other by a trunk duct 38.

The mask holder 12 with the mask 10 attached is suspended on the guide rail 14 and a transverse adjustment by means of the screws 22 allows the mask ramps to be brought to the vertical of the sample migration tracks which can be viewed by adding to the samples deposited an appropriate dye such as bromophenol blue.

The mask above the test carrier shall be scanned manually by means of the handle 26 or the motorised means mentioned above.

As shown schematically in Figure 3, a lid 40 may be placed on and attached to the mask 10 to close the ducts 34, 36, 38 formed in the mask 10 in a significantly watertight manner. A tube 42 of the lid 40 protrudes over the upper orifices 36 of these ducts and allows a small amount of air to be injected between the mask and the lid to apply pressure to the reagents in the mask ducts and to bring them down into the ducts to contact the test medium.

The reagents may be deposited in the masking tubes 10, closing these tubes with the lid 40 in a significantly watertight manner, transporting the mask 10 - lid 40 assembly and placing it on the test carrier before the reagents are used.

Two variants of the mask 10 have been shown schematically in Figures 7 and 8: The one in Figure 7 comprises 18 through pipes arranged in two parallel rows and displaced in pentagons from one row to the other. The one shown in Figure 8 comprises two groups of six passing conduits, each group being aligned along the length of the mask and comprising five aligned conduits and one displaced conduit.

- Examples of use of the mask according to the invention Example 1 Simultaneous immunofixation of 9 samples with a mobile mask with 18 tracks (Figure 4).

The manipulation was performed using the SEBIA Hydrasys® electrophoresis machine on a gel for immunofixation, measuring 0.7 x 83 x 101 mm.

The applicators (French Patent No. 2 671 290 and European Patent No. 0 493 996) were used to deposit the samples on the electrophoresis medium, with 18 teeth of 3 mm spaced 2 mm apart. Each sample was deposited on 6 consecutive teeth, each applicator thus allowing the deposition of 3 different samples on the electrophoresis gel and 3 applicators were used for the 9 samples to be analysed. The deposits were made on the gel by the Hydrasys® automaton, in 3 parallel rows located respectively at 18, 38 and 58 mm from the cathodic edge of the gel.

Once the migration was completed, the invention's mobile mask was installed with 18 tracks, the geometrical characteristics of which are as follows: track width 2.5 mm, track length 7 mm, track spacing 2.5 mm, slope of the tracks 5°. A row of 3 tracks (for loading the fastener) is offset by 5.5 mm from the row of 15 tracks (for loading the antiserums).

The 20 guide rail slots were positioned on the two nipples carried by the Hydrasys® automatic migration tray.

The 18-track mask was embedded in the mask holder 12 which was itself attached to the guide rail. The mask and its mask holder were brought into the high position, i.e. the anodic side of the gel. A dye, bromophenol blue, incorporated into the samples deposited on the gel, allowed to visualize the position of the electrophoretic tracks on the test medium.

The mask was then loaded by introducing the various reagents needed for immunofixation through the upper openings of the tracks (36) at a rate of 10 μl of reagent per track in the usual order: fixator, anti-IgG, anti-IgA, anti-IgM, anti-kappa and anti-lambda.

Once introduced between the tracks and the gel, these 10 μl of reagent spread under each track for about 5-6 mm beyond the areas on the anode side to be revealed.

Once the loading of the mask was completed, it was moved by means of the handle (26) by sliding along the guide rail. This sweep was performed without a hit, at a nearly constant speed from the high position (anode side) to the low position (cathode side) of the gel, achieving a stroke of 63 mm. This sweep was completed in about 3 seconds.

The mask was then scanned in the opposite direction under the same conditions, and the two scans were repeated once. All the reagents initially introduced under the runways were then deposited on the gel above the electrophoretic migration zones. The mask could be removed before the incubation phase of the reagents on the gel was carried out for 5 minutes at 20°C.

The steps of pumping, drying, washing, staining, discoloration, drying were then carried out according to the usual protocols used for immunofixation.

Example 2 Simultaneous immunofixation of 12 samples with a mobile mask with 18 tracks (Figure 4).

The procedure was the same as in the previous example, but four applicators with 18 teeth were loaded at a rate of 3 samples per applicator. By means of the Hydrasys® automaton, deposits were made on the gel in 4 parallel rows located respectively at 18, 33, 48 and 63 mm from the cathodic edge of the gel. The migration was carried out for 28 volts per hour at a constant power of 20 W at 20 °C. The procedure was then followed as in the previous example.

Example 3 Simultaneous immunofixation of 4 samples with a 12 track mobile mask (Figure 8)

Applicators with 15 teeth of 4 mm width, spaced 2 mm apart, were used to make the sample deposits on the gel, the applicators being loaded at a rate of 2 samples per applicator (sample No 1 teeth 2 to 7; sample No 2, teeth 9 to 14).

The Hydrasys® machine was used to deposit the gel in 2 parallel rows 23 mm and 53 mm from the cathodic edge of the gel respectively.

The temperature controlled migration of 20°C at constant power of 20 W was continued until 42 Volt hours were accumulated.

Once the migration was complete, the mobile mask was installed with 12 tracks with the following geometrical characteristics: track width 3.5 mm, track length 7 mm, track spacing 2.5 mm, slope of the tracks 5°. A row of 2 tracks (for loading the fastener) is offset by 5.5 mm from the row of 10 tracks (for loading the antiserums).

The 20 guide rail plots were positioned on the two nipples in the Hydrasys® migration plane. The 12 track mask was attached to the mask door which was itself attached to the guide rail groove, the whole being brought into the high position.

By means of transverse adjustment, the mask traces were brought as described in Example 1 to the apex of the alignment of the electrophoretic migration traces of the samples.

The mask was then loaded by introducing 14 μl of reagent per track.

The reagents were spread as in the previous examples by having the mask swept 4 times.

All the reagents were then deposited on the surface of the gel and the mask could be removed.

The incubation was then continued and the pumping, drying, washing, dyeing, discoloring, drying steps were carried out as usual.

Example 4 The technique of 36 IF painting with a moving mask has 18 tracks (Figure 4).

The penta-IF technique is routinely used to detect the presence of paraproteins in the samples analysed in the form of monoclonal or oligoclonal bands of immunoglobulin nature.

This technique is performed by revealing side by side for each analyzed sample, the total protein profile and the profile of all immunoglobulins by performing immunofixation with a pentavalent antiserum i.e. having both anti IgG anti IgA anti IgM anti kapa and anti lambda specificities.

The manipulation was performed in this example on an agarose gel for immunofixation of 0.7 x 83 x 101 mm using the Hydrasys® SEBIA electrophoresis machine. 18 3 mm toothed combs were used, spaced 2 mm apart. Each sample to be analysed was deposited in double side by side, i.e. 9 samples per applicator.

For the 36 samples, four applicators were used.

The deposition on the gel was carried out by the Hydrasys® automaton in 4 parallel rows situated respectively 18, 33, 48 and 63 mm from the cathodic edge of the gel. The migration was then carried out at a controlled temperature of 20 °C at a constant power of 20 W until accumulation of 28 Volts per hour. The invention's 18-track mobile mask corresponding to Figure 4 was then installed.

The mask consists of two rows of 9 tracks, each 5.5 mm apart. The 9 most anodic tracks are intended to receive the fixer, the other 9 tracks, the pentavalent antiserum. Each track has a width of 2.5 mm, a length of 7 mm, a spacing of 2.5 mm and a slope of 5°.

The mask was encased in the mask door and brought in a high anodic side-up position.

The reagents were introduced at 10 μl/track.

The steps described in examples 1 to 3 were then followed.

Claims (51)

1. A mask, suitable for depositing and distributing reagents on an analytical support for biological samples, comprising:

   • a lower surface and an upper surface that are at least partially mutually parallel, separated by a distance constituting the thickness of the mask;

   • one or more lane(s), corresponding to delimited zones, localized at the level of the lower surface of the mask and comprising a projecting element (32) that projects from the lower surface of the mask, each projecting element comprising a portion constituting a slope with respect to a horizontal plane;

   • characterized in that the mask comprises, associated with each lane, an opening for loading and depositing reagents, said opening traversing the mask over the whole of its thickness from an upper loading orifice (36) on the upper surface of the mask to a lower deposit orifice (34), said lower deposit orifice being located in the slope, at proximity of the lowest point of the slope of the lane;

   the mask being such that the lane or lanes it comprises can hold, by capillary action between the lane and the corresponding facing surface of the analytical support against which the mask is to be placed, reagents loaded into each opening and deposited on the analytical support.
2. A mask according to claim 1, suitable for distributing reagents on an analytical support for biological samples, comprising:

   • a lower surface and an upper surface that are at least partially mutually parallel, separated by a distance constituting the thickness of the mask;

   • one or more lane(s) each comprising a projecting element (32) of elongate shape emerging beneath the lower surface of the mask, said projecting element comprising a portion constituting a slope with respect to a horizontal plane;

   • associated with each lane, an opening for loading and depositing reagents, said openingtraversing the mask over the whole of its thickness from an upper loading orifice (36) on the upper surface of the mask to a lower deposit orifice (34), said lower deposit orifice being located in the slope at proximity of the lowest point of the slope of the lane;

   the mask being such that the lane or lanes it comprises can hold, by capillary action between the lane and the corresponding facing surface of the analytical support against which the mask is to be placed, reagents loaded into each opening and deposited on the analytical support.
3. A mask according to claim 1 or 2, suitable for distributing reagents on an analytical support for biological samples, comprising:

   • a lower surface and an upper surface that are at least partially mutually parallel, separated by a distance constituting the thickness of the mask;

   • one or more lane(s) each comprising a projecting element (32) emerging beneath the lower surface of the mask, constituted by a protuberance in the shape of a truncated parallelepiped, said projecting element comprising a portion constituting a slope with respect to a horizontal plane;

   • associated with each lane, an opening for loading and depositing reagents, said opening traversing the mask over the whole of its thickness from an upper loading orifice (36) on the upper surface of the mask to a lower deposit orifice (34), said lower deposit orifice being located in the slope at the proximity of the lowest point of the slope of the lane;

   the mask being such that the lane or lanes it comprises can hold, by capillary action between the lane and the corresponding facing surface of the analytical support against which the mask is to be placed, reagents loaded into each opening and deposited on the analytical support.
4. A mask according to claim 2 or 3, suitable for distributing reagents on an analytical support for biological samples, wherein the projecting element (32) has an upper surface that is coextensive with the portion of the lower surface of the mask beneath which said projecting element emerges, and a lower surface separated from the upper surface by at least one slope with respect to a horizontal plane.
5. A mask according to claim 1, wherein each projecting element (32) comprises a lower surface and an upper surface that are mutually parallel and parallel to the lower and upper surface of the mask, the slope of the lane being produced by inclining the mask with respect to the analytical support in the position of use.
6. A mask according to claim 5, wherein the projecting element (32) is of elongate shape, in particular is of parallelepiped shape.
7. A mask according to claim 4 or 6, characterized in that the lowest point of the slope of the lane, located at proximity of the lower orifice (34) of each lane opening, is the point that is the closest to the analytical support when brought in the position of use.
8. A mask according to any one of claims 1 to 7, characterized in that it is rigid or stiffened.
9. A mask according to any one of claims 1 to 8, in which the volume of the opening is such that it can constitute a reservoir for the loaded reagents.
10. A mask according to any one of claims 1 to 9, in which the opening for each lane traverses the thickness of the mask including the thickness of the projecting element (32) in a perpendicular manner, the opening comprising a portion in the shape of a truncated cone (38) terminated by a lower orifice (34) that is cylindrical in shape.
11. A mask according to any one of claims 1 to 10, comprising a plurality of mutually parallel lanes, distributed over the length of the mask.
12. A mask according to claim 11, comprising:

    • a first series of mutually parallel lanes disposed in a first alignment;

    • a second series of mutually parallel lanes that are parallel to the lanes of the first series, and forming a second alignment offset with respect to the first alignment.
13. A mask according to claim 11, in which the lanes are organised in several groups along the mask's length, each group being constituted by a lane disposed offset with respect to the other lanes aligned in-between themselves, the lanes being mutually parallel lanes.
14. A mask according to any one of claims 1 to 4 and 8 to 13, in which the length of the slope of each lane coincides with the length of that lane.
15. A mask according to any one of claims 1 to 4 and 8 to 13, in which the length of the slope of each lane extends over the length of that lane.
16. A mask according to any one of claims 1 to 15, wherein the lanes allow depositing each reagent in a quantity equal to or in the range 4 to 15 µl in each opening of the mask and holding said reagents between the lanes of the mask and the analytical support by capillary action, when the mask is brought at proximity of the analytical support.
17. A mask according to claim 16, for depositing each reagent in a quantity equal to or in the range 4 to 15 µl in each opening of the mask and holding said reagents between the lanes of the mask and the analytical support by capillary action, when the mask is brought to a distance from the analytical support of 2 mm or less from the point of the mask that is furthest from the analytical support.
18. A mask according to any one of claims 1 to 17, for depositing each reagent in a quantity equal to or in the range 4 to 15 µl in each opening of the mask and holding said reagents between the lanes of the mask and the analytical support by capillary action, when the mask is brought to a distance from the analytical support equal to or in the range 0.1 to 0.5 mm from the point of the mask that is closest to the analytical support.
19. A mask according to any one of claims 1 to 18, in which the lanes are separated from each other by a distance of 1.5 mm or more.
20. A mask according to claim 19, in which the distance between the lanes is of 2.5 mm.
21. A mask according to any one of claims 1 to 20, in which the dimensions of the lanes and their spacing are such that the reagents held by them between the mask and the analytical support by capillary action do not interact during deposition or distribution onto said analytical support.
22. A mask according to any one of claims 1 to 21, in which the lane width is 2.5 mm.
23. A mask according to any one of claims 1 to 22, in which the length of each lane is in the range 6 to 7 mm.
24. A mask according to any one of claims 1 to 23, in which the lane intended for the fixative is offset with respect to the neighbouring first lane by a distance of 5 to 7 mm.
25. A mask according to any one of claims 1 to 24, in which the slope of each lane forms an angle in the range 1 ° to 10° to a horizontal plane.
26. A mask according to any one of claims 1 to 25, in which each lane has the following dimensions:

    • length: 3 to 15 mm;

    • width: 1 to 10 mm;

    • inclination of the slope: 1° to 10° to the horizontal.
27. A mask according to any one of claims 1 to 26, in which each lane has the following dimensions:

    • length: 7 mm;

    • width: 2,5 mm;

    • inclination of the slope: 5° to the horizontal.
28. A mask according to any one of claims 1 to 27 in which the upper orifice of the opening has an angle of 50°.
29. A mask according to any one of claims 1 to 28 in which the thickness of the mask is comprised between 1 and 10 mm.
30. A mask according to any one of claims 1 to 29, comprising 6, 12 or 18 lanes.
31. A mask according to any one of claims 1 to 30, made in a material which is proper to be moulded, and allowing the making of a smooth surface.
32. A mask according to any one of claims 1 to 31, associated with positioning means intended to hold the mask in the proximity of the surface of the analytical support close to which the mask will be brought for deposition and distribution of reagents on the analytical support.
33. A device for depositing and distributing one or more reagents on an analytical support for biological samples, comprising:

    a) a mask (10) according to any one of claims 1 to 32;

    b) means (12, 14, 16) for positioning and guiding the mask allowing the mask to be positioned so that the mask is held in the proximity of the surface of the analytical support and allowing the mask to be guided by sweeping the surface of the analytical support in a horizontal plane parallel to the surface of said support to allow deposition and distribution of the reagents over each of the predetermined zones of the analytical support coming into line with the lanes of the mask.
34. A device according to claim 33, in which the means for positioning and guiding the mask (10) can establish a distance between the analytical support and the point on the mask that is closest to said support in the range 0.1 mm to 0.5 mm.
35. A device according to any one of claims 33 or 34, in which the positioning and guiding means allow automatic displacement of the mask (10) along the analytical support.
36. A method for depositing and distributing one or more reagents on an analytical support comprising biological samples, comprising the steps of:

    • loading the reagent or reagents onto a mask (10) to allow the reagent or reagents to be deposited on the analytical support, and being held between said support and the slope or the lane or lanes of said mask by capillary action;

    • positioning a mask (10) according to any one of claims 1 to 32 or a device according to any one of claims 33 to 35, at the proximity of the analytical support to deposit the reagent or reagents on the analytical support and hold them between said support and the slope of the lane or lanes of said mask by capillary action;

    • displacing the mask by sweeping the surface of the analytical support to distribute the reagent or reagents on the analytical support in incubation zones, corresponding to delimited zones of said support, the reagent or reagents being distributed in a quantity sufficient to allow their interaction with the constituents of the biological samples present on said analytical support.
37. A method according to claim 36, in which sweeping of the mask allow the deposit of a quantity of each reagent that is comprised between 4 µl and 10µl by lane on the delimited zones of the analytical support.
38. A method according to any one of claim 36 or claim 37, in which the mask is loaded with the reagent or reagents away from the zone of the surface of the analytical support comprising the constituents of the biological samples.
39. A method according to claim 36, in which the mask is loaded with the reagent or reagents prior to the step for positioning the mask in the proximity of the analytical support and in that the reagents are deposited on the analytical support following an impulse resulting from air pressure exerted on the mask, or by a mechanical junction between the reagents and the analytical support, or by projecting the reagents onto the support, or by brief contact between the mask and the analytical support at the lowest point of the slope of the lane.
40. A method according to claim 39, in which loading of the mask (10) with the reagent or reagents is in an automated manner.
41. A method according to any one of claims 36 to 40, in which the displacement of the mask (10) is in an automated manner.
42. A method according to any one of claims 36 to 41, in which the analytical support is an electrophoresis support on which the constituents of one or more biological samples have been separated by electrophoretic migration.
43. A method according to claim 42, in which the reagent or reagents are intended to allow immunofixation of the constituents of biological samples separated by electrophoresis.
44. A method according to claim 43, in which one of the reagents is a fixative allowing fixing all the constituents of biological samples separated by electrophoresis.
45. A method according to any one of claims 43 or 44, in which the immunofixation reaction encompasses a step of revelation of the constituents of the immunofixated biological samples.
46. A method according to claim 36, in which the fixative is loaded into the appropriate lane or lanes of the mask, then deposited and distributed over the predetermined zone(s) of the electrophoretic support after controlled sweeping of the reagent(s) on the analytical support within the delimited zones of said support, the fixative and the reagents being distributed in a sufficient quantity to allow their reaction with the constituents of the biological samples found on said analytical support.
47. A method according to any one of claims 36 to 46, in which the quantity of reagent loaded onto each lane of the mask is equal to or in the range 4 to 15 µl in each lane.
48. A method according to any one of claims 36 to 47, in which the mask suitable for depositing and distributing reagents is constituted so that the quantity of reagent loaded onto each of its lanes is in excess by comparison with the quantity of said reagent distributed onto the incubation zone of the electrophorectic support when the mask is swept from the anodic position to the cathodic position of the electrophorectic support, corresponding to the cathode.
49. A kit comprising:

    • at least one mask according to any one of claims 1 to 32;

    • at least one analytical support.
50. A kit according to claim 49, further comprising:

    • reagents for immunofixation of the constituents of samples separated by electrophoresis;

    • a fixative for fixing each sample of the assembly of constituents separated by electrophoresis.
51. A mask according to any one of claims 1 to 32 or kit according to any one of claims 49 or 50, in which the reagents are loaded into the mask and are in the freeze dried form.