FIELD OF INVENTION

The present invention relates to the field of rapid diagnosis by means of a kit, stable at room temperature In particular, the invention refers to a method which can also be performed in environments outside a specialized laboratory such as for example in a pharmacy or other first aid center with immediate response of a bioluminescent feature or with a digital reader for diagnosis and/or for risk prediction. Preferably, the method is applicable to cell extracts of certain markers using immunoassays and the ELISA (enzyme-linked immunosorbent assay) procedure can be implemented.

It is also possible to automate the method using a suitable machine having a chamber to house one or more disposable kits to perform the method and to obtain a quantification of the bioluminescent result.

PRIOR ART

'Patient side' diagnostic kits that exploit chemo colorimetry for the immediate identification of markers present in cell extracts are known. Chemo colorimetry, despite having numerous advantages, results at least in some cases of relatively unrepeatable interpretation and excessively dependent on the person who reads and interprets the chemo chromatic result.

DESCRIPTION OF THE INVENTION

The scope of the present invention is to at least partially solve the disadvantage indicated above. According to the present invention, a disposable kit for testing the presence of an antigen is proposed comprising a base having at least a first and a second recess which can be inferiorly perforated and a cover having at least a third and fourth recess which can be superiorly perforated, the position of said recesses being such that the first and second recesses house the third and fourth recesses when the base is covered by the cover, and wherein the first or third recesses contain first selective biological molecules, such as an antibody, for selecting said antigen and catalyst molecules bound to selective biological molecules, such as an HRP enzyme; and the second and fourth recesses contain corresponding first and second precursors of a chemical reaction, for example hydrogen peroxide and luminol, which, catalysed by said catalyst molecules, generates a bioluminescent effect; the kit further comprising a membrane on which second selective biological molecules are located for selecting and anchoring said antigen to the membrane.

Through the use of a kit configured in this way, it is possible to create a kit that exploits both the advantages of bioluminescence i.e. greater precision and uniqueness of interpretation, in particular in the case of process automation, and the availability of a precise and usable instrument outside the analysis laboratories.

According to a further embodiment of the present invention, the scope of the present invention is achieved through a method comprising the steps of: providing a disposable kit as described above provide a collection tank provided with the membrane pouring into the tank a liquid containing a biological test material containing an antigen performing a superior perforation of the third and fourth recess to allow the generation, in particular by gravity, of a first dosed quantity of liquid containing the selective biological molecules bound to the catalyst molecules in the first recess and starting the chemical reaction by mixing the first and second precursors in the second recess performing in sequence: a first inferior perforation of said first recess to collect the first dosed quantity of liquid in the tank so as to allow the selection and anchoring action when the second selective biological molecules are compatible with the antigen in the tank; a second inferior perforation of said second recess to collect the first and second precursors in the tank to allow the action of the catalyst molecules during the chemical reaction and generate a bioluminescent effect in the tank.

Such method, combining inferior and superior perforation, guarantees to avoid any contamination during the process and, moreover, it is easily automatable.

According to a further embodiment, the scope of the present invention is achieved by a machine for processing the disposable kit as indicated above according to the previously described method comprising, a seat for at least one disposable kit, a closing lid movable between an opening position for loading/unloading the disposable kit from the seat and a closing position wherein the lid is approached above the kit, a mobile slide arranged below said seat to support at least one tank equipped with said membrane and a perforator for the recesses of the base, a mechanism for moving the slide and/or the perforator relative to the seat and allowing in use the inferior perforation of the recesses of the base, and an electronic control unit programmed to control the mechanism according to a predefined sequence of positions of the membrane under the seat.

Such machine is compact and allows precise tests to be performed even outside specialized laboratories, e.g. in pharmacies.

LIST OF THE FIGURES

The present invention is now described by way of non-limiting example by means of the following drawings, wherein:

- Figure 1 is an exploded perspective view of a disposable kit according to the present invention;

- Figure 2 is an enlarged perspective view of Figure 1;

- Figure 3 is an enlarged perspective view of an element of Figure 2;

- Figure 4 is a perspective view of a test machine for processing the kit of Figure 1;

- Figure 5 is a section of the machine of Figure 4;

- Figure 6 is a perspective view of the machine of Figure 4 with elements removed for clarity;

- Figure 7 is a sectioned perspective view of a component of the kit of the present invention assembled in configuration for use;

- Figure 8 is an enlarged view and not to scale of a perforator for the kit of the present invention; and

Figure 9 is a perspective image of a sequence of tanks each comprising a pressure retaining element for a functionalized membrane for bioluminescent antigen detection.

DETAILED DESCRIPTION OF THE INVENTION

Figure 1 shows with 1 as a whole a disposable kit comprising a base 2 having a first and a second plurality of recesses 3, 3' a cover 4 having a third and fourth plurality of recesses 5, 5', corresponding shape coupling portions Pl, P2 on the base 2 and on the cover 4 being provided to mount the cover on the base in a unique way and positioned so that the first and second recesses 3, 3' are housed in the corresponding third and fourth recesses 5, 5'.

Preferably, in the illustrated embodiment, the shape coupling portions Pl, P2 are a recess defined for example on the base 2 and a corresponding projection defined on the cover 4 but the inverse is also possible.

Furthermore, in order to reduce production costs and at the same time to maintain high standards of chemical inertness between the reactants and the recesses 3,3', 5, 5' which contain them, the base 2 and the cover 4 are made of a material plastic e.g. a polystyrene film, preferably thermoformed to obtain the desired geometry. As illustrated in the figures, the base 2 and cover 4 made of thermoformed film define a test tray as a whole. It is preferable that the plastic material of the cover 4 is hydrophobic, to allow the complete fall of the liquid reagents into the recesses of the base 2 which contain the respective powder reagent.

According to the invention, at least a pair of first and third recesses 3, 5 as a whole house a selective biological molecule, for example an antibody, which selectively interacts with a target antigen such as to bind even to several parts of the same antigen; and a molecule, e.g. biological as an enzyme HRP peroxidase, catalyst of a chemical reaction that generates a bioluminescent substance bound to the selective biological molecule. Furthermore, another pair of second and fourth recesses 3', 5' house precursors necessary for the generation of a chemical reaction having a bioluminescent effect and catalysed by the catalyst molecule. For example, the precursors of the substance that generates the bioluminescent effect are hydrogen peroxide and 5-amino-2,3- dihydro-l,4-phthalazindione, i.e. luminol. Preferably, in order to increase the effectiveness of the bioluminescent effect after a period of storage at room temperature, at least one of the precursors e.g. the organic precursor such as luminol is biphasic and stored in a biphasic state on board the kit. The phases join following the breaking or perforation of specific recesses on the base 2 or on the cover 4.

In this way, the contents of the first and third recesses 3,5 and that of the second and fourth recesses 3 ’, 5' are both such as to select and couple with a target antigen and such as to produce a chemical reaction to generate a substance with bioluminescent effect bound to the selected antigen. In particular, the catalyst molecule is bound to the selective biological molecule so that the two have the same localization e.g. within a liquid. The substances of the base 2 and cover 4 interact during the test with a functionalized membrane, on which the substances fall by gravity as will be better specified hereinafter. Furthermore, in order to preserve the molecules for long periods and to maintain for as long as possible the selection action of the antigen and the binding which allows to localize the substance which generates the bioluminescent effect, the contents of the first and third recess 3, 5 is bi-phase e.g. the selective biological molecule and the precursor molecule bound to it are dried and a liquid suspension mixture preferably of emulsifiers and saline buffers is provided, e.g. emulsifiers based on polysorbates and phosphate saline buffers. Furthermore, the second and fourth recesses 3', 5' identify two compartments for housing the corresponding precursors.

Therefore, a bottom of the third and fourth recesses 5, 5' is weakened or perforable by hand using a sharp-edged tool (not shown) e.g. of a plastic material; in this way it is possible to make falling by gravity a dosed, in particular pre-dosed, quantity of substance contained in the corresponding first and second recesses 3, 3' below to obtain the desired mixtures.

Furthermore, after the first and second recesses 3, 3' are filled, the functionalised membrane previously exposed to the biological material comprising the antigen is arranged sequentially under each of them, to favour the selection and anchoring of the latter by the antibodies.

Preferably, the bioluminescent effect obtained on the membrane can be captured by an image sensor and subsequently processed by an imaging algorithm e g. to separate a background (dark) from a pattern (brighter) obtained through the bioluminescent effect and counting the pixels of the pattern to obtain a quantitative indication of the antigens coupled to the antibodies. Furthermore, according to a preferred embodiment, the kit comprises a reference membrane 8 wherein third selective biological molecules not indicated for the antigen of interest are present. The corresponding recesses, on the other hand, contain the substances of the recesses corresponding to the test membranes. In this way, the kit includes a substance which allows to generate, after the chemical reaction of the precursors, a substance which generates a bioluminescent effect of comparison whose image allows to define a term of comparison for the images relating to substances with bioluminescent effects generated in the presence of the antigen and useful, as will be explained in greater detail below, for performing image filtering algorithms.

Furthermore, it is possible that the kit comprises initial recesses 10, 11 on the corresponding base 2 and cover 4, containing as a whole initialisation substances of the biological test sample containing the target antigen, for example biological molecules from an oropharyngeal swab. For example, the initialisation substances comprise a dried protease inhibitor contained in one of the initial recesses and a lysis solution preferably based on saline buffers. The initial recess 10 can also be perforated to allow mixing of the two substances after perforation and preferably, the lysis mixture is arranged above the protease inhibitor.

Preferably, for the scope of washing molecules not bound to the antigens, the base 2 comprises washing wells 12 containing a substance capable of removing any non-specific bonds on the membranes by the substances coming from the recesses and not bound to the antigens. For example, the washing substance comprises emulsifiers and salt buffers, e.g. emulsifiers based on polysorbates and phosphate salt buffers, wherein the percentage of salt buffers is greater than that in the suspension solution. For example, the percentage of salt buffers is 1% in the washing substance and is 0.1% in the suspension substance.

According to a preferred embodiment, the disposable kit 1 is configured to perform an ELISA procedure in sequence and further comprises at least a first and a second recess of primary antibody, preferably monoclonal, 13, 14 on the corresponding base 2 and cover 4 containing an antibody dried, e.g. in recess 13, and a suspension solution e.g. in the recess 14. Preferably at least one washing well 12, in the example of Figure 1 two wells 12, are interposed between the primary antibody recesses 13, 14 and the first and third recesses 3, 5. Furthermore, in the first or third recess 3, 5 are contained primary antibodies. In addition, the substance collecting membrane of the recesses carries antigen-specific antibodies of the corresponding primary monoclonal antibodies. Such antibodies are in particular polyclonal. Preferably at least one washing well 12, in the example of Figure 1 two wells 12, are interposed between the first and third recesses 3, 5 and the second and fourth recesses 3', 5'. Therefore, as illustrated in the figure, the base 2 and the cover 4 have an elongated shape so as to be able to arrange the recesses and wells in longitudinal sequence as described above. This also makes possible to optimize space when automatic processing machines capable of processing several disposable kits 1 in parallel are provided.

According to the embodiment shown in the figure, the initialization recesses 10, 11 are arranged in an initial position of the sequence, which develops longitudinally along the disposable kit 1 . According to a preferred embodiment of the present invention, at least one disposable kit 1 is used in a machine comprising a longitudinally movable shuttle and provided with a inferior perforator 16 arranged in use under the base 2 and having a material stiffness and/or shape such as to perforate (Figure 2), when the shuttle reaches the suitable position, a corresponding bottom of the recesses 3, 3', 10, 13 and of the wells 12, 10 in order to make the dosed liquid, in particular pre-dosed, contained in each recess or well, flow out by gravity. Preferably, the perforator has an elongated shape, e.g. vertical, so as to guide the liquid present in the recesses and/or wells downwards by surface tension before perforation by said perforator. Furthermore, the shuttle is shaped and includes a tank 17 from which the perforator 16 projects and which receives the incoming fluid by gravity. The tank 17 comprises e.g. on the bottom or other wet surface by the dosed quantity of liquid, the membrane 18 e.g. based on Poly VinylDenFloride or other material used in the sector to support antibodies, on which the non-antigen-specific conjugated polyclonal antibodies are applied in localized positions so as to bind to the antigens present in the initial solution containing the biological test material. According to the example of Figure 1, the initial solution containing the biological test material, taken by means of a brush or swab 19, is obtained by perforating the first initial recess 11 and mixing the dried protease inhibitor with the lysis solution. The dosed quantity of dried inhibitor and lysis solution, e.g. 800 microlitres, falls by gravity into the tank 17 after the perforation performed with a relative from bottom to top movement of the perforator 16 with respect to the base 2.

The shuttle continues perforating in sequence on the opposite side of the cover 4 with respect to the base 2 for each recess 13 (Figure 2), 3, 5 and wells 12 and the various quantities of liquid are all deposited in the tank 17 until the chemical reaction is reached which generates the bioluminescent effect.

According to the alternative embodiment shown in Figure 1, on the same disposable kit 1 there are side-by-side and preferably aligned sequences of recesses containing test substances based on primary antibodies different from sequence to sequence and, moreover, there is a sequence of reference recesses which, in the embodiment, have the same secondary antibody as the other sequences and one of the primary antibodies of the other sequences. In Figure 1, there are two quadruplets of recesses 13, 3; the first with the corresponding preferably monoclonal primary antibody e.g. COVID- 19-specific antibody; influenza A-specific and influenza B-specific and one of the above antibody, the second quadruplet with the same secondary antibody conjugated to the catalyst for each of the primary antibodies. Correspondingly, the shuttle carries three membranes 18 each having preferably polyclonal antigen-specific antibodies in combination with the corresponding primary antibodies and the reference membrane 8 having preferably polyclonal antibodies specific for a different antigen, e.g. the melanoma antigen, from that of the other three membranes. Furthermore, the shuttle comprises a plurality of perforators 16 aligned with the corresponding quadruplets of recesses (three recesses with test substances and a fourth recess with reference substances) so that, in a stop position of the shuttle, each perforator 16 is under the corresponding recess 13, 3, 3' and, during a single relative perforation movement, all said recesses are perforated and the liquid contained is released by gravity reaching the corresponding tanks 17. In the example of Figure 1, both the quadruplets and the perforators 16 are arranged along respective lines parallel to each other and superimposed during the relative perforation movement, e.g. a relative vertical movement.

Preferably, the wells 12 and/or the first initial recess 10 each contain a dosed, in particular predosed, volume of liquid sufficient to supply suitable quantities of substances into all the tanks 17 of the shuttle and, therefore, during the perforation movement, can be perforated by one or at least two perforators 16. Preferably, the wells 12 and the first initial recess 10 are aligned along an axis of symmetry of the quadruplets of recesses 13, 3, 3' and 14, 5, 5'.

According to a preferred embodiment (Figure 3), the tanks 17 are fluidly connected to each other through one or more transversal channels 22 preferably parallel to said line defined by the perforators 16. Furthermore, to prevent the substances present in the recesses 13, 3, 3' are directed towards the adjacent tank i.e. incorrect, a bottom of each tank 17 has a lower height than that of the channels 22. Furthermore, to guide the liquid into the correct tank 17, each perforator 16 extends projecting from the bottom of the corresponding tank 17 and is surrounded by an annular recess 23 having at least a lower portion of the bottom than that of the channels 22 and preferably at the same height as the bottom of the corresponding tank 17. When one or more perforators 16 perforate the well 12, on the other hand, the large quantity of liquid e.g. greater than the volume of the annular recess 23 and of the tank 17 below the channels 22, causes a transversal distribution of liquid thanks to the latter. Preferably (Figure 8) one or more perforators 16 has a cross section having a concave section defining an elongated cavity 16a to facilitate the channelling also by capillarity of the fluid from the base 2 to the tank 17. According to the embodiment of the figure, the elongated cavity 16a is positioned side by side by a flat or convex wall 16b of the perforator 16 which tends to favour the channelling of the fluid into the elongated cavity 16a.

Figure 9 also illustrates a further preferred but non-limiting embodiment wherein an arm 60 presses on the membrane (not shown in Figure 9) and has a contact head 61 with the latter spaced from the side walls of the tank for the purpose of preventing phenomena of capillarity and therefore a mixing of the biological material taken or biological material dosed coming from the base 2 between adjacent tanks during the test. For the same purpose, the arm 60 is made or is covered with a layer of a smooth and/or hydrophobic material or other so as to prevent adhesion by capillarity or surface tension on the arm 60 itself and increase the risk of mixing of biological material during the test. In fact, each perforator 16 is associated with its own tank wherein the biological substances must mix without losses of biological material from the adjacent tanks, in particular with reference to the reference membrane 8. The arm 60 can exert pressure on the membrane both elastic e.g. the arm is elastic e.g. flexionally elastic, or in a rigid way, for example by means of a releasable mechanism for constraining the vertical position, the arm being sliding in the vertical direction when the constraining mechanism is released.

Figure 4 illustrates a machine 30 for the automatic processing of biological material using the disposable kit 1.

The machine 30 defines a housing seat for one or more disposable kits 1 arranged in suspension, i.e. with a free space below the base 2, a perforation cover 31 movable between an open position (shown in the figure) and a closed position wherein, being superimposed and approached to the kits 1, perforates the recesses of the cover 3 by means of suitable perforators (not illustrated), and a slide 33 movable below the base 2 along guides G in the longitudinal direction of the latter and carrying a perforation shuttle for each kit 1 arranged in the machine 30. It should be noted that the slide receives, at each base 2 and cover 4, a disposable mask M bearing the membranes 8, 18 and possibly also including the perforators 16 and the tanks 17. Alternatively, the perforators 16 and the tanks 17 are on board the machine 30 even in the absence of the membranes 8, 18 and are treated appropriately between tests.

Preferably, the lid 31 is operated by hand via a tilting handle 34 and the slide 33 is motorized to bring the perforators 16 in sequence to the positions suitable for perforating the recesses and wells of the base 2, waiting a predetermined time for the recesses to be emptied and wells after perforation and advancing to the next position. The movement and stop of the slide 33, with the relative times, speeds and/or other functional parameters is controlled by an electronic control unit (not shown) preferably on board the machine 30.

In the embodiment of Figures 3, 4, the stroke of the slide exceeds the size of the disposable kit 1 so as to be uncovered with respect to the base in a final station 35 wherein an image acquisition device (not shown) is arranged for acquire an image of the tanks 17 with the relative bioluminescent effect. Through the image acquired by the image acquisition device, preferably an electronic device, it is possible to perform a quantitative analysis of the bioluminescent effect. Preferably, by means of a boundary detection or binarization algorithm it is possible to divide the pixels of the digital image of the tank 17 with a bioluminescent effect into background pixels (darker) and bioluminescent pixels (lighter) and the latter can be counted. Furthermore, after being identified, the bioluminescent pixels of the initial image can be classified on the basis of the luminous intensity. Furthermore, if the reference membrane 8 is present in kit 1, the relative image is processed and the relative data are considered false positives. In particular, it is possible that the voiding after washing has not completely disposed of the unbound primary antibody and/or that the secondary antibody has also bound to biological material not containing the target antigen. In both cases, the bioluminescence chemical reaction is activated without the relative effect being attributable to the presence of antigens.

Furthermore, preferably, the electronic control unit is connected in data exchange to the image acquisition device and is programmed to perform at least one of the imaging operations described above. Furthermore, preferably when the lid 31 is closed, through suitable casing panels 36 (some of which have been removed for clarity), at least the final station 35 is shielded from ambient light to increase the contrast of the bioluminescent effect.

For example, according to the embodiment of Figure 6, the machine further comprises a darkroom 40, e.g. a box-shaped body which, placed on the shuttle, defines an environment closed to light, on which the image acquisition device is applied. Preferably, the darkroom 40 is movable along a guide 41 via a motor 42 connected to the electronic control unit in order to be able to darken the area around several shuttles arranged in series.

Furthermore, the machine 30 comprises a tilting support 43 carried by the slide to command, by means of a motor 44 connected to the electronic control unit, the emptying of the tanks 17 at the end of the timed period and controlled by the electronic control unit to allow the substances to interact. For example, the tilting support 43 comprises a bar transversal to the guides G on which are applied e g. four shuttles which each receive the mask M and each carry the perforators 16. To empty the tanks 17 without interfering with the base 2, the motor 44 commands a rotation around an axis A transverse to the guides G in such a way that the contents of the tanks fall into a tray 46 which is also preferably carried by the slide 33 above the guides G. In this way, the guides themselves are not contaminated by the test substances.

The tilting support 44 can also be actuated by means of suitable motors 47 to perform a vertical upward and downward stroke in order to perforate the recesses of the base 2 following a command signal coming from the central control unit. Meanwhile the recesses of the base 2 are inferiorly perforated, the action of the perforators 16 is opposed by the lid 31. Furthermore, according to the embodiment of Figure 6, a belt drive actuation with at least one motor 48 controls, thanks to the electronic control unit, the position of the slide 33 along the guides G. Figure 7 illustrates an embodiment of what can be mounted on the slide 33 : the mask M carries the membranes 8, 18 and rests on a hollow body 50 defining the tanks 17 and the perforators 16. The body 50, preferably, is coupled on a specific projection provided on the slide 33 to objectify its position with respect to the base 2. The disposable kit comprises e.g. in one package, either the base 2, the cover 4 and the mask M with the membranes, or even the hollow body 50.

According to a preferred embodiment, the slide 33 comprises one or more vibration actuators, e.g. rotary motors to which an eccentric weight is connected, controlled by the control unit to favour the action of the test substances by stirring the liquid in the tanks 17. Preferably, such vibrating or alternating action e.g. in the vertical direction of the perforators 16 can also be activated after perforation and while the perforators 16 are inside the base 2 to favour the complete emptying of the recesses towards the tanks 17.

According to a preferred embodiment, the dosed fluid in the recesses of the cover 4 of the disposable kit is closed in the cover by various closures e.g. a peelable film preferably peelable by hand, caps closed at the time of making the kit in the factory. Such closures are perforable and lacerated to perforate the recesses of the cover 4 from top, e.g. by means of the machine 30 or other manual tool to evacuate the fluid by gravity towards the recesses of the base 2.

Based on the embodiment, the arm 60 can be carried by the shuttle 33 or be carried by the hollow body 50 and can also preferably be disposable.