Patent Document

CROSS REFERENCE TO RELATED APPLICATION 
     This is a continuation-in-part of U.S. application Ser. No. 11/312,167, filed Dec. 20, 2005, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention is about a method and a device for preserving two or more reagents used in a chemical reaction as well as for a rapid antigen extraction. 
     BACKGROUND OF THE INVENTION 
     Different methods for preserving two or more reagents, in order to put them in contact only when they have to be used for a rapid antigen extraction, are known in the state of the art. For example, in pharmaceutical preparations, a reagent is commonly preserved in a solid state and it is dissolved in a liquid state solvent just before the use, by perforating a mechanical partition wall. 
     Other preservation methods have been described as well, in which two or more containers, each containing a reagent, are inserted into a single main container inside which, after the breakage of the internal containers, the reagent mixing takes place. 
     The traditional methods of extraction of saccharidic antigens of group A streptococcus provide for the use of liquid reagents (Lenneft, E. H., Ed., Manual of Clinical Microbiology, Fourth Edition, American Society of Microbiology, Washington, D.C., 1985, pages 170-171). Typically, two liquid reagents are used in the extraction stage: an acid (acetic, hydrochloric or citric acid) and a sodium nitrite solution. The two reagents are mixed in a test tube in which the wad used to take the biological sample to be examined is inserted. 
     In other known methods, at least one of the two reagents is in a solid state (U.S. Pat. No. 5,536,646), or the reagents are contained in test tubes or flasks with several separate compartments, each containing a single reagents to be mixed immediately before use (U.S. Pat. No. 4,673,639). 
     By mixing the two reagents, nitrous acid, which is a relatively unstable acid, is produced, thus requiring that the reagents are mixed immediately before the extraction process starts. Otherwise, the instability of the resulting nitrous acid solution can reduce the extraction effectiveness. In fact, if the reagents are prematurely mixed with respect to the biological sample addition, according to what described in U.S. Pat. No. 4,851,337, the nitrous acid decomposition takes place and the extraction solution can lose its effectiveness in a very short time. 
     According to the method described in U.S. Pat. No. 5,415,994, the extraction takes place in a well directly obtained in the cartridge containing the immunochromatographic strip. One of the two reagents is contained in a flask, that contains on its turn a phial with the second reagent. The operator mixes the two reagents by breaking the phial and then pours the mixture in the well in which the wad is placed. In this way, the operator does not have to count the drop number of each reagent. However, in this case too the extraction takes place after the two reagents have been mixed. Moreover, the insertion of the wad in the well sometimes causes the even partial occlusion of the liquid drainage conduit and thus the flow is slowed down or even blocked. Otherwise, if the wad is inserted in a manner such that the liquid does not flow therethrough before reaching the immunochromatographic chamber, the reaction liquid can reach the immunochromatographic membrane before the extraction takes place. 
     According to other known methods (U.S. Pat. No. 5,494,801), a third reagent is added to the two default ones in order to neutralize the solution before the chromatographic stage takes place. At present however the use of three reagents is considered too complicated for the operator, and thus the systems providing for the use of two reagents only are preferred. Furthermore, even these methods do not avoid the risk of effectiveness reduction of the extraction due to the time elapsed between the reagent mixing and the sample insertion. 
     Methods that provide for the insertion of the sample, on which the extraction has to be performed, in a device before adding the reagents have been described too (U.S. Pat. No. 6,168,956). In these methods, the operator must apply the reagents according to the optimal time sequence. However, the need to determine the volume of the reagents (for example, by counting the reagent drops) still remains, as well as the possibility to use the same reagents twice. 
     The traditional methods of extraction of lipopolysaccharidic antigens from Chlamydia trachomatis provide for the use of an alkaline reagent able to extract the lipopolysaccharidic antigens, in which an acid or a buffer is inserted to neutralise the extraction. According to a traditional method, the alkaline reagent consists of sodium hydroxide and the aid reagent for neutralizing the extraction solution is hydrochloric acid. 
     The wad through which the biological sample has been taken is inserted into a test tube containing the alkaline reagent for the extraction and it is agitated for a predetermined time, after which the neutralization reagent is added. 
     SUMMARY OF THE INVENTION 
     It is thus an object of the present invention to overcome the problems of the aforementioned prior art method and devices. 
     The present invention can be applied in all the situations in which two or more reagents, that can start a chemical reaction together with a sample to be treated, can not be mixed together before adding the sample. For example, it is possible that the sample has to be put in contact with an unstable reaction product, or with more than one reagent, in a predetermined sequence. 
     More specifically, the present invention can be applied to the bacterial antigens extraction processes performed with wad in human or animal samples. In particular, the present invention can be used for the extraction of saccharidic antigens from group A and B streptococcus, as well as of lipopolysaccharidic antigens from Chlamydia. 
     The main object of the method and the device of rapid antigen extraction according to the present invention is thus to simplify the aforementioned prior art extraction processes, in particular:
         a) the operator does not have to predetermine and check the reagent volume, for example by counting the dispensed reagent drops;   b) the time needed to dissolve one or more solid state reagents is not required;   c) the time elapsed between the reagent mixing and the insertion of the wad with the biological sample does not reduce the extraction effectiveness, for example when the highly unstable nitrous acid is used.       

     More in particular, the present invention relates to a method and a device for a diagnostic test employing an immunochromatographic strip, wherein the said strip is dipped in the vial containing the antigen extracted by the reagents provided for in the device&#39;s compartments according to the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further characteristics and advantages of the method of rapid antigen extraction according to the present invention, and the device for performing said method, will be better highlighted in the following description of preferred embodiments, given in the explanatory but not limiting way with reference to the annexed figures of drawings, in which: 
         FIG. 1  is a schematic sectional view of a first embodiment of a device for performing the method of rapid antigen extraction according to the present invention; 
         FIG. 2  is a schematic sectional view of a second embodiment of a device for performing the method of rapid antigen extraction according to the present invention; 
         FIG. 3  is a schematic sectional view of an embodiment of a flexible extraction device inserted in a rigid tube for the breakage of the mechanical barrier between the two reagents; 
         FIG. 4  is a schematic sectional view of a further embodiment of the device of the present invention; 
         FIG. 5  is a schematic sectional view of a particular of the device of  FIG. 4 ; 
         FIG. 6  is a schematic view of the device of  FIG. 4  during use. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     With reference to  FIG. 1 , according to the method of the invention, the two reagents are poured in predetermined volumes in a preferably but not necessarily cylindrical test tube  1  during the manufacturing stage of the device for performing said method, said test tube  1  comprising a first inner container  2  able to be inserted in a second outer container  3  that forms the main body of the test tube  1 . The container  2  is configurated to form a cap for the container  3 . The two containers  2  and  3  are then assembled so that the wad A ( FIG. 3 ) with the sample to be tested sequentially passes through the reagent R 2 , placed inside the container  2 , and the reagent R 3 , placed inside the container  3 , respectively, by breaking the mechanical barrier  4  that forms the bottom wall of said container  2  and separates said two reagents R 2  and R 3 . The containers  2  and  3  can be manufactured with any kind of material compatible with the reagents R 2  and R 3  contained therein, and can have a proper shape and a sufficient volume to contain the wad A, said reagents R 2  and R 3  being in turn either in liquid or in solid state. The test tube  1  is sealed at its top using any known sealing system, for example with a metallic sheet (not shown). 
     Another embodiment of the device for performing the method according to the present invention is shown in  FIG. 2 . After pouring the reagent R 3  in the test tube  1 , it is possible to form a partition wall  14  in said test tube  1  having the same function of the container  2  mechanical barrier  4 , for example by adding solid paraffin, heating it up to its melting and thus letting it cool down until it forms a proper physical partition element  14  similar to said mechanical barrier  4 , thus being able to define two separate containers, an upper one  12  and a lower one  13 , inside the same test tube  1 . At this point it is possible to add the second reagent R 2  into the so formed upper container  12  of the test tube  1 . In this case too the test tube  1  is subsequently sealed at its top using a known sealing system. 
     The device consisting of the test tube  1  for performing the method according to the invention is then able to ensure that the two reagents R 2  and R 3  are put in contact only when the wad A bearing the sample is present. Therefore, according to the method of the invention, the transportation of the first reagent to the second reagent is performed by the wad A itself. 
     Another most preferred embodiment of the invention is shown in  FIGS. 4 and 5 . A test tube  101  comprises an inner container  102  shaped and configurated to be inserted in an outer container  103  and to form a cap for this latter. 
     To this end, the outer container  103  comprises a body  104  and an open portion  105 . The body  104  is substantially cylindrical in shape and is closed at the bottom, preferably with a rounded profile. Advantageously, the body  104  is tapered downwardly. 
     The inner surface of the body  104  comprises an annular rib  107  that is situated in a position wherein it can interfere with the outer surface of the inner container  102 , while this latter is fitted in the outer container  103 , and thus can act as a gasket ring. 
     The open portion  105  has a larger diameter than the body  104  and presents a stepped internal profile. 
     The inner container  102  comprises a body  108  and an open portion  109 , both having a diameter substantially equal to or slightly less than the internal diameter of the corresponding parts of the outer container  103 , so that the inner container  102  can be inserted in the outer container  103  without substantial clearance between the two surfaces. To this end, the body  108  of the inner container  102  is substantially cylindrical in shape and slightly tapered downwardly, in order to follow the profile of the outer container  103 . 
     The body  108  has a closed beveled bottom, so that a proximal connecting portion  110   a  and a distal connecting portion  110   b , with respect to the open portion  109 , are defined. The closure wall  110  is substantially planar and has a thickness that decreases from the proximal connecting portion  110   a  to the distal connecting portion  110   b  to the body  108 . This feature is very important while the wad is inserted in the inner container  102  and, after having absorbed the reagent contained therein, is then forced against the closure wall  110  in order to puncture it. In fact, the beveled shape of the bottom of the inner container  102  together with the smaller thickness of the distal connecting portion  110   b  allow to concentrate the force in that point and to make easier the puncturing of the wall. Thus, thanks to this particular shape of the closure wall  110 , the inner container  102  is made of one piece, so that the barrier to be punctured by the wad can be made of a material, such as polyethylene, that is thicker and more resistant that a paraffin film. This provides for enhanced impermeability and tightness to leakage with respect to the paraffin barriers as normally used in the prior art devices, wherein the paraffin (or similar weak materials) is employed to improve the puncturing action. 
     Another advantage deriving from the use of a harder, thicker material for the closure wall  110  of the inner container  102  is that, when the wad has punctured it and is then removed, it sticks against the stiff edge of the closure wall  110 . In such a way, as shown in  FIG. 6 , the removing action of the wad also brings the inner container  102  out, thus leaving the outer container  103  ready for the subsequent analysis. 
     The body  108  of the inner container  102  extends for a length that is less than the length of the outer container  103 , in order to create in this latter a bottom chamber  111 , wherein a reagent R 3  can be kept. The length of the inner container  102  is such as to allow the annular rib  107  of the outer container  103  to interfere with the surface of the substantially cylindrical portion of the body  108  above the bottom portion thereof. 
     The open portion  109  of the inner container  102  has an external profile that can substantially fit with the internal profile of the corresponding portion of the outer container  103 . To this end, the outer surface of the open portion  109  of the inner container  102  is stepped. 
     In a particularly preferred embodiment of the invention, the upper rims  113 ,  114  of the outer and inner containers  103 ,  102 , respectively, have tooth-shaped annular corrugations  113   a ,  114   a . As shown in  FIG. 5 , these corrugations  113   a ,  114   a  serve the function of allowing a secure welding of a closure sheet  115 , such as a conventional peelable metallic sheet. In fact, in absence of such corrugations, it may happen that the upper rims  113 ,  114  of the inner and outer containers  102 ,  103  are not perfectly levelled, so that the closure sheet can be welded on one rim only. As a consequence, leakage can occur or the internal reagents can be contaminated by outside. 
     Conversely, the tooth-shaped corrugations  113   a ,  114   a  are sufficiently thin to melt during the thermal welding of the closure sheet  115 , so that they auto-level themselves to give a complete and efficient welding. 
     Very preferably, the outer container  103  is made of a flexible material, such as polyethylene. This allows the body  104  to be squeezed when the wad, after having put into contact, the two reagents R 2  and R 3 , is removed from the test tube  101 , thus assuring that the whole antigen solution is released from the wad. 
     Preferably, both the inner container  102  and the outer container  103  are made of the same material such as polyethylene. 
     As in the previous embodiments, in this case too the two reagents R 2  and R 3  are poured in predetermined amounts in the inner and in the outer containers  102 ,  103 , respectively, during the manufacturing stage of the device, which is then sealed on the top openings with conventional peelable films. 
     During use of the device, the sealing film is removed and the wad—which was previously used to contact a body fluid or mucuous membrane of a patient—is dipped in the inner container  102  in order to contact the reagent R 2  contained therein. After the required time is elapsed, the wad is pushed to puncture the closure wall  110  of the inner container  102  and is then put into contact with the reagent R 3 . At this time the reaction takes place and the extraction of the antigen from the wad is performed. After the prescribed time is elapsed, the wad is removed by simultaneously squeezing the body  104  of the outer container  103  in order to squeeze also the wad to release all the antigen solution imbibed thereon. After squeezing the wad, the removal of the wad is completed and this action allows the inner container  102  to be removed together with the wad, as explained above. At this stage, the outer container  103 , containing the whole antigen solution to be tested, is ready for the subsequent analysis. The analysis is typically an immunochromatographic test performed by means of a strip that is dipped in the antigen solution directly in the outer container  103  of the test tube  101 . In this connection, the fact that the removal of the inner container  102  does not leave any residue of the barrier separating the two containers in the outer container  103  (as this barrier is associated with the removed inner container  102 ), avoids the risk that the strip test is altered by the presence of solid material that could interfere with the capillarity movement of the liquid solution on the strip. 
     According to a preferred aspect of the invention, the sample is taken with a pharyngeal wad A following well known procedure. The wad A is then inserted into the first container  2  or  12  of the test tube  1 , after the removal of the seal, and it is then driven in the second container  3  or  13 , by breaking the barrier  4  between the containers  2  and  3  or the partition wall  14  between the containers  12  and  13 . The extraction of the antigen by means of the so formed nitrous acid is thus started. When the expected extraction time is lapsed, the wad A is removed, preferably with the first container  2  if present, from the test tube  1  and the liquid can be poured directly from said test tube  1  into the immunochromatographic device for the antigen detection. 
     For example, according to the method of the invention, the reagent R 2  contained in the first container  2 ,  12  can be a 0.4 M acetic acid. The operator inserts the wad A into the first container  2 ,  12  of the test tube  1 . The reagent R 2  is almost fully absorbed by the wad A. By pushing the wad A against the bottom  4 ,  14  of the container  2 ,  12 , said bottom  4 ,  14  breaks, allowing said wad A to pass through and thus to reach the reagent R 3 , for instance a 2 M sodium nitrite, in the second container  3 ,  13 . At this point, the nitrous acid formation reaction takes place. Therefore, if the antigen is present, the antigen extraction takes place in the best conditions for the effectiveness of said extraction. Once the expected time for the extraction of the bacterial antigens from the wad A is lapsed, the wad A is removed from the test tube  1  and the liquid can be poured in the immunochromatographic strip cartridge well. 
     Another example of the method according to the present invention allows to extract Chlamydia antigens from cervical or urethral wads. The sample is taken with a cervical or urethral wad according to well known procedures. The wad is then inserted into the first container  2 ,  12  of the test tube  1 , after the removal of the said test tube seal, and it is left in contact with the reagent R 2  for the required extraction time. Once the extraction is finished, the wad is pushed into the second container  3  by breaking the barrier  4 , or into the second container  13  by breaking the partition wall  14 , and it is put in contact with the neutralization reagent R 3 . 
     In this case, the reagents comprise an alkaline reagent (R 2 ) and an acidic neutralization reagent (R 3 ). 
     According to a traditional method, the cervical o urethral wad is inserted into a test tube containing 5 drops of 0.2 N sodium hydroxide, and it is left in the solution for 2 minutes. After shaking the wad, a predetermined volume of 0.1 N hydrochloric acid is added to neutralize the extraction solution. After shaking the wad again, said wad is then removed and a certain volume of the extraction solution is added to the test cartridge. 
     In order to take biological samples from particular sites, for example from the nasal cavities or the urethra, devices having a flexible and thin structure are available on the market, thus being difficult to break the partition wall between the two reagents with said devices. In this case, the sampling device can be inserted in advance into an assembly provided with the proper stiffness and resistance features for breaking the partition wall. For example, after the insertion of the sampling wad A into the test tube  1 ,  101 , it is possible to surround said wad A with a tube B having a proper diameter. By pushing the tube B, that breaks the barrier  4  or the closure wall  110 , the wad A is transported into the container  3 ,  13 ,  103  (see  FIG. 3 ). 
     It should be understood that several modifications could be made to the device, formed by the test tube  1 ,  101 , that performs the method of rapid antigen extraction according to the present invention, as it is also defined in the appended claims. For example, the sealing of the test tube  1  can be obtained by using a cap, or by thermal sealing with an aluminum sheet coupled with polyethylene. Furthermore, although in the description the sample collection system is indicated as a “wad”, this denomination is merely used for convenience, since the most common systems for taking the A group streptococcus are the pharyngeal wads, while the most common systems for taking the Chlamydia trachomatis are the cervical or urethral wads. Therefore, it should be obvious for a man skilled in the art that it is possible to use any sampling system compatible with the immunological array format.

Technology Category: 7