MXPA97000254A - Analytical level rod for improved mixing and reagent volume reduc - Google Patents

Abstract

The analytical level rod (2) of the present invention facilitates analyte testing with reduced liquid reagent volumes and provides improved mixing. The level rod (2) includes an elongated member (4) having a fluid displacement member (18) at one end thereof, including a ligand (20) attached to a portion of the elongate member (4) in a region adjacent to the displacement member (18). The displacement member (18) has a configuration substantially conforming to the internal side wall (26) of an analytical wall (3), in which the level rod (2) moves to displace the fluid therein. . When the displacement member (18) is practically centered on the bottom (30) of the wall (3), the portion of the level rod (2) supporting the ligand (20) is preferably separated from the internal side wall ( 26) by a distance (d2) of at least approximately twice the distance (d1) between the displacement member (18) and the internal side wall (26), measured along the line (4 ... 4) which passes through the largest lateral dimension of the displacement member (18). With this configuration, essentially all the liquid in the bottom (30) of the wall (3) is displaced upwards so that the ligand (20) is submerged therein. The relatively large separation (d2) between the portion of the ligand (20) and the side wall (26) of the wall (3) facilitates the flow of turbulence to improve mixing in the ligand region (2).

Description

"ANALYTICAL LEVEL ROD FOR IMPROVED MIXING AND REDUCED REAGENT VOLUME" FIELD OF THE INVENTION This invention is generally related to a dipstick for detection of analysis products. More particularly, this invention relates to a level rod configuration that reduces the * 10 minimum required reagent volume and produces more uniform mixing when it is reciprocated in and out of an analytical well.

BACKGROUND OF THE INVENTION 15 The use of "dipsticks" in the assay for the presence of a test product in a sample is * known in the art. Typical analysis products include materials, eg, drugs, acids nuclei, proteins, contaminants, chemical substances, fines, physiological compounds and the like. In a typical rod-based analytical assay, a coordinating group, which is specifically linked to the product of interest analysis, is bound to a solid support in the level rod. The dipstick is contacted - - with a sample where the presence of the product of interest analysis will be determined. Frequently, steps are taken to assist the removal of non-specifically bonded material from the dipstick. Finally, the dipstick is processed to determine the presence of an analysis product. The level rod generally comprises a solid material that is of flat or columnar geometry. U.S. Patent No. 4,391,904, issued to Lit an et al. (Incorporated herein by reference), discloses test strip kits wherein a number of an immunological pair is ligated to a solid surface. Herzberg et al., In U.S. Patent No. 293,374, discloses a test card with tapered ends. In an extensive format, the dipstick is transferred through one or more analytical wells filled with a liquid reagent. In this format, complete immersion of the solid support requires the filling of the analytical well with a liquid reagent to a depth approaching the distance between the solid support and the distal end of the dipstick. Although this format provides a convenient means for testing a product being analyzed, the cost of the liquid reagent can dramatically increase the cost of the assay and increasing the reagent volume by dilution decreases the inherent sensitivity of the assay. Mixing the liquid by reciprocating the dipstick in and out of the solution is frequently used to accelerate processing. However, the configuration of the level rods of the prior art is not brought to the optimum to carry out this function. In addition, during the removal of the reagent, droplets suspended from the dipstick may contaminate the subsequent processing steps requiring further or additional processing. When developing an assay, it is desirable to minimize the number of processing steps, the time between steps and the volume of reagent used in each step, while simultaneously maximizing assay sensitivity. Even though efforts have been made to optimally carry out the reagent chemistry to achieve these ends, the configuration of the analytical level rod has changed little. Therefore, it is desirable to develop a novel level rod configuration to improve the performance of the assay.

COMPENDIUM OF THE INVENTION The present invention is directed to an analytical level rod that avoids the problems and disadvantages of - - the prior art. In accordance with the present invention, which has specific utility in assays that require processing in a liquid reagent, an analytical level rod comprising an elongate member having a first end portion, a second end portion and an intermediate portion is provided. placed between the first and second end portions. The thickness of the elongated member, which is measured in one direction, increases considerably from the portion intermediate to the second end portion. A coordinating group, which is selected to be specific to the analysis product of interest, is coupled to the intermediate portion in a region adjacent to the second end portion of the elongated member. During the operation, the dipstick is inserted into a well containing a liquid (eg, a reagent containing an analytical product). The second enlarged end portion of the dipstick is configured so that when it remains essentially centered on the base or bottom of the well, essentially displaces all the liquid in the bottom or bottom of the well through a small space or passage formed between the second end portion and the side wall of the well. The displaced liquid then rises along - - * the intermediate portion so that the coordinating group is submerged in the liquid. Since the second enlarged end portion can move essentially all of the liquid in the bottom of the well up to submerge the coordinator group therein, the test of the test product can be carried out with minimal amounts of reagent with the present invention. That is, the second % end portion can be configured so that the The distance between the end portion of the second end portion and the coordinator group may be greater than the depth of the liquid in the analytical well, thereby allowing the test product to be tested with reduced reagent volumes. In the preferred embodiment, the second end portion is configured so as to essentially fill the * space at the base of the well, thus moving essentially all the liquid at the base of the well up. When the second end portion focuses on the base of the well, the space between the second end portion and the internal side wall of the well which is measured along a line extending through the maximum lateral dimension of the second end portion, is preferably from approximately .127 to .762 millimeter, and more preferably, from approximately .254 to .508 millimeter. This configuration provides a virtually complete displacement of the liquid at the base of the well, while allowing the development of a desirable laminar flow adjacent to the second end portion. Another important aspect of the invention is that the portions of the outer surface of the intermediate portion that are closest to the central part of the intermediate portion are separated from the internal side wall of the well by a considerably greater amount than that between the second. end portion and the inner side wall of the well, measured along a line extending through the maximum lateral dimension of the second end portion. More specifically, the site of the points on the outer surface of the intermediate portion that are closest to the central axis of the intermediate portion are separated from the inner side wall by a distance at least twice that distance between the second end portion and the inner side wall of the well, measured along a line extending through the maximum lateral dimension of the second portion. When the second end portion of the dipstick is forced down to the closed end or the bottom of the well, the reagent or liquid at the closed end is forced upward to the intermediate portion. The space between the second end portion and the side wall of the well facilitates a laminar flow therebetween. However, upon entering the enlarged space between the inner side wall and the well and the intermediate portion, the laminar flow of the fluid is interrupted resulting in parasitic currents. Parasitic currents advantageously improve mixing in that region, which accelerates the transfer of the reagent into or out of the surrounding microscopic zone. "f * to the coordinator group, thus improving the reaction kinetics 10. The repetition of this procedure, having reciprocating the dipstick in and out of the liquid reagent continuously interrupts the microscopic zone and reduces the processing time and / or increases the sensitivity 15 The terminal end surface of the second end portion preferably has a curved jßt surface or a projection, which may be in the form of a ridge, to minimize the surface tension between the liquid in the well and the second end portion 20 when that portion of the liquid is removed This construction provides a relatively small interface between the second end portion and the liquid as the second end portion is removed thereby reducing the size of any droplet adhering to the second end portion during the removal of the liquid reagent. Rounded terminal rowing of a second spherical end portion, hemotherphically or in the form of a tear drop provides, for example, the desired results. However, when the terminal end surface of the second end portion is flat it is provided in the projection to extend from that surface in order to reduce the size of any droplet hanging from the terminal end surface when the liquid is removed from the liquid. second end portion. In the preferred embodiment, the coordinator group is fixed to a solid support which is a discrete component which, in turn, is fixed to the intermediate portion of the level rod. Preferably, the face of the first end portion and the intermediate portion is of planar configuration and the solid support is an account that is inserted into a hole in the face by means of a snap fit. Alternatively, the solid support is formed as an integral part of the intermediate portion. The foregoing is a brief description of some deficiencies in the prior art and advantages of the present invention. Other features, advantages and embodiments of the invention will become apparent to those skilled in the art of the following * description, the drawings accompanying the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a front view of a level rod constructed in accordance with the principles of the present invention; Figure 2 shows a dipstick of the Figure 1 that has been rotated 90 ° and placed in an analytical well; Figure 3 is a section of the dipstick taken on line 3-3 in Figure 1; Figure 4 is a section of the dipstick and the well apparatus taken on line 4-4 in Figure 2; and Figure 5 shows a further embodiment of the manner for moving the dipstick according to the present invention. 0 DESCRIPTION OF THE PREFERRED MODALITY Referring to the drawings in detail where like numbers indicate like elements, there is shown, for example, an apparatus 1 for testing the presence - - of the analysis products in accordance with the principles of the present invention. Referring to Figure 2, apparatus 1 generally comprises an analytical level rod 2 which facilitates testing with reduced liquid reagent volumes and improves mixing and a fluid retention container 3. Referring to Figure 1, the level rod 2 generally comprises an elongated member 4 which includes a first or proximal end portion 6, a second or distant end portion 8 and an intermediate or carrier portion 10 between the legs. same. The first end portion 6 includes a fastening member or section 12 and a generally planar section 14 that can serve as a display surface for a label. The second end portion 8 forms a fluid displacement member 18 which will be described in greater detail below. The intermediate or carrier portion 10 carries one or more coordinator groups. In the illustrated mode, the solid supports that are coated covalently or non-covalently with a coordinating group, are coupled with the carrier. The composition of the elongated member and the solid support can be selected from a variety of materials, such as nitrocellulose, plastic, ceramics, metal, polymers or combinations thereof.

The term "coordinating group" as used herein refers to a variety of different binding agents and molecules that are bound by the binding agents. The coordinator groups are useful in a variety of different in vitro assays such as immunoassays, receptor binding assays and nucleic acid hybridization assays and the like. The coordinator groups can be coupled with solid supports that can be used in a variety of different solid phase assay formats. A variety of different solid phase assay formats are known in the art. For example, immunoassay formats are described in Basic and Clinical Immunology, seventh edition (D. Stites and A. Terr, editors) 1991. Immunoassays can be carried out in many configurations, examples of which have been reviewed in Enzyme Immunoassay, ET Maggio, editors, CRC Press, Boca Raton, Florida (1980); "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Labora tory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V. Amsterda (1985) and Harlow and Lane, Antibodies, A Laboratory Manual, supra, each of which is incorporated herein by reference. A variety of nucleic acid hybridization formats are known to those skilled in the art.

- - * For example, common formats include sandwich trials and competition or displacement trials. Hybridization techniques and formats are usually described in "Nucleic Acid Hybridization A Practical Approach" Editors Hames, B.D. and Higgins, S.J. IRL Press, 1985; Gall and Pardue (1969), Proc. Nati Acad. Sci. U.S. ., 63: 378-383; and John, Burnsteil and Jones (1969) Nature, 223: 582-587. * In the preferred modality, each of the solid supports 20 comprises a member similar to an account which may be spherical, for example. The account-like member has an external surface that is appropriate to be attached to a coordinating group. A variety of coordinating groups can be set to a variety of different solid supports as disclosed in the North American Patent of Liman et al., Number 4,391,904, which is incorporated herein by reference. * A coordinating group, which is specifically linked to the analytical product whose presence has been tested, is fixed to the coating. A variety of different coordinating groups can be used to detect many analytical products of scientific and medical industrial interest. These coordinating groups include but are not limited to nucleic acids, anti-bodies, proteins ^ _ enzymes, receptors, hormones, streptavidin and biotin.

Returning to Figures 1 and 2, more than one solid support can be ligated to a carrier 10 with each solid support coated with identical, different coordinating groups or combinations of coordinating groups. The surface area of the carrier 10 should generally be minimized to avoid non-specific bonding of the material that may interfere with the subsequent processing of the dipstick, and, therefore, for general JB should be textured Smooth and flat configuration.

The cross-sectional thickness of the carrier 10 should be sufficient to maintain the stiffness of the level rod as it is reciprocated in and out of a liquid reagent contained in the container 3. In the preferred embodiment, the carrier 10 has a beam configuration in "I" to add rigidity to the carrier. As shown in Figures 1 and 3, the carrier 10 may comprise a flange 22 and a continuous belt 24, with the flanges 22 * having been constructed to be generally parallel to the inner side wall 26 of the container 3 when the member The elongate is placed in the recess 28 of the container as shown in Figure 2. In the preferred embodiment, the solid support (s) 20 is a discrete component that is coupled with the carrier 10. Preferably, the continuous web 24 It includes one or more openings that can be a depression or - perforation (through hole) to retain one or more of the solid supports. The solid support can be rendered harsh by chemical or physical means before being coated with the coordinator group to maximize the reactive surface area through indentations and microscopic channels. In the preferred embodiment, the solid support comprises a rough spherical score which is inserted into the perforation in the carrier 10 as shown in Figures 1 and 2 and which is held in place by means of a snap fit. A snap fit can be achieved by providing a perforation having a diameter only slightly smaller than that of the bill as will be readily apparent to a person skilled in the art. Even when a specific coordinator group setting arrangement has been described, other arrangements may be used. For example, the solid support can simply be a part of or integrated with the carrier and the coordinator group can be fixed directly to the solid support. See, eg, Litman et al., North American Patent No. 4,391,904 which is incorporated herein by reference. The displacement member 18 acts to displace the liquid reagent in the package 3 from the closed end of the package to immerse the solid support in the liquid reagent. The carrier of preference is - place near the displacement member in relation to the length of the dipstick to help maintain the amount of reagent volume necessary to carry out a test. The displacement member 18 has a configuration and is of sufficient volume to essentially fill the recess 28 in the base of the container 3 so as to displace essentially all of the liquid reagent in the bottom or base 30 of the container 3. Consequently, the The configuration of the outer surface of the lower portion of member 18 essentially conforms to the configuration of the inner side wall 26 and the inner bottom wall 27 of the lower region of the recess 28. Therefore, when the displacement member 18 is hemispherical , has a transverse section that is taken as a plane along the central axis of the recess 28, which is in conformity with the inner side wall 26 in the lower region 30 of the recess 28. Preferably, the displacement member 18 is configured so that the distance d_ between the displacement member and the internal side wall 26 which is measured along a line through the maximum lateral dimension of the member or displacement (as shown in Figure 2) is approximately 0.127 to .762 millimeters and more preferably, from .254 to .508 millimeters. See also Figure 4.

- The average distance between either the face 24a of the front continuous belt and the inner side wall 26 or the rear face (which is hidden from the view) of the continuous belt 24 and the inner side wall 26 is preferably considerably larger than that of the belt. to facilitate turbulent flow in the solid support region of the carrier 10. Preferably, the distance d2 between the face of the continuous belt and the side wall 26 which is measured along a line passing through the central axis of the carrier 10 and which essentially remains perpendicular to the face of the respective continuous tape is at least about twice as great as when the displacement member is centered at the bottom of the recess 28. Preferably, the voids such as the depressions or perforations within the displacement member that would trap the liquid during the insertion of the displacement means towards the liquid. In addition, the displacement member, in general, is configured in such a way that its terminal end surface which is directly opposite the closed end of the well has a minimum surface area. A minimal surface area reduces the size of any droplet that hangs from or adheres to the displacement member when the dipstick is removed from the liquid in the container 3. This results in a smaller amount being carried toward the subsequent containers or wells Accordingly, the remote end surface of the member 18 is preferably pointed or curved. Preferred configurations of the displacement member having a minimum remote area are spherical, hemispheres or tear drop configurations. When a triangular solid configuration (tetrahedron) is used for the displacement member as shown with the displacement member 18 'in Figure 5, a projection 32 which may be in the form of a tetrahedron or pyramid, for example, is preferably provides on the lower surface 34 of the member 18 'to remove the otherwise flat surface and therefore to reduce the size of any hanging droplet. Similarly, if the conical shape is attached to the sliding member, its lower surface can be configured to end at a point. The transition from the carrier 10 to the displacement member is preferably gradual, preferably through a curve (as illustrated in Figure 2) and is generally designated with the reference number 16 (or a positive angle line for avoid level or hollow areas that would trap the liquid). In the container used together with the dipstick, it can be of a variety of sizes and shapes in cross section, for example, square, round or rectangular and - therefore, it includes tubes and wells of icrovaloración. The shape of the cross section of the closed end of the recess will be in the same manner as that of the cross section of the base region of the displacement member, not taking into account the small or hollow indentations within the displacement member. In the preferred embodiment, the recess or well of the container is circular in cross section with a rounded bottom and the displacement member is hemispherical to conform to the well configuration and displace a maximum amount of fluid. The present invention provides the advantages of a lower and mixed or improved reagent volume when vertically reciprocating in and out of a liquid reagent in a container 3. The displacement member must be immersed sufficiently deep in the liquid and used enough liquid in such a way that the displacement member displaces the liquid upwards and causes the solid support to be immersed. The relatively large space between the side wall and the faces of the continuous belt interrupts the laminar flow that leaves from the passage formed between the displacement member and the side wall 26. This produces turbulent eddy currents that result in improved mixing, which is particularly important - when the liquid reagents comprising a colloidal suspension are mixed. Since the reaction kinetics is limited by the diffusion rate to and from the microscopic zone surrounding the solid support, the increased mixing intensifies the concentration gradient that extends from the solid support and limits the reactive zone surrounding the support solid. The reduction of the reagent depletion zone accelerates the processing time and / or increases the sensitivity. Removal of the dipstick to allow the reagent to settle on the closed end of the recess allows the cycle to be repeated. The foregoing is a detailed description of a specific embodiment of the invention. It is recognized that deviations from the disclosed modality can be made within the scope of the invention and that obvious modifications will occur to those skilled in the art. The full scope of the invention is set forth in the claims set forth below and their equivalents. Accordingly, the claims and specification should not be construed to unduly limit the full scope of protection to which the invention is entitled.

Claims (22)

- R E I V I N D I C A C I O N S

1. An analytical level rod comprising an elongated member having a first end portion, a second end portion and an intermediate portion positioned between the first and second end portions, the thickness of the elongated member measured in one direction increases considerably from the portion > intermediate to the second end portion, and a group 10 coordinator coupled to the intermediate portion in the region adjacent to the second end portion.

2. The level rod according to claim 1, wherein the elongate member is rigid.

3. The apparatus according to claim 2, wherein the intermediate portion is an "I" beam configuration. *

4. The level rod according to claim 1, wherein the change in thickness 20 between the intermediate and second end portions is gradual.

5. The level rod according to claim 1, wherein the intermediate portion is generally planar. - -

6. The level rod according to claim 1, wherein the first end portion is generally planar and includes a label receiving surface.

The level rod according to claim 1, wherein the second end portion has an end surface configured generally in hemotherphically.

The level rod according to claim 1, wherein the second end portion has a conical and tetrahedra configuration with a pointed bottom end surface.

9. The level rod according to claim 1, wherein the coordinating group is selected from the group consisting of an antibody, a protein, an enzyme, nucleic acid, streptavidin and a biotin.

10. A test apparatus of an analytical product comprising: a package having a recess formed therein, the recess being defined by at least one side wall and a bottom wall; and an elongate member capable of slidable receipt in the recess, the elongated member has a first end portion, a second end portion - - forming a displacement member and an intermediate portion positioned between the first and second end portions, the intermediate portion has a coordinating group coupled thereto in a region adjacent to the displacement member, the displacement member essentially fills the portion of the recess which is adjacent to the lower wall when the displacement member is placed against it.

The apparatus according to claim 10, wherein the configuration of the displacement member is essentially conformed to the configuration of the corresponding limit of the recess of the container adjacent to the bottom wall.

The apparatus according to claim 10, wherein the intermediate portion includes an outer surface having first and second end regions, the first region being closer to the central axis of the intermediate portion than the second region, the distance between any point in the first region and at least one side wall being at least approximately twice the distance between the displacement member and the side wall, which is measured along a line passing through the digestion lateral maximum of the displacement member, when the - Scroll member focuses on the bottom of the recess.

The apparatus according to claim 10, wherein the distance between the displacement member and at least one internal side wall that is measured along a line through the maximum lateral dimension of the second end portion. , is approximately .127 to .762 millimeters when the displacement member is centered in the lower part of the recess.

The apparatus according to claim 13, wherein the intermediate portion includes an external surface area that is separated from at least one side wall by a distance that is considerably greater than the distance between the displacement member and at least a side wall.

15. The apparatus according to claim 10, wherein the displacement member is in the form of a tear drop.

16. The apparatus according to claim 10, wherein the displacement member is generally spherical.

17. The apparatus according to claim 10, wherein the displacement member is generally hemispherical.

18. The apparatus according to claim 10, wherein the displacement member is generally configured in the form of a tetrahedron.

19. The apparatus according to claim 10, further comprising a solid support in the form of an account, the intermediate portion includes an opening, the coordinator group is fixed to the solid support and the solid support is placed in the opening.

20. The apparatus according to claim 10, wherein the coordinating group is selected from the group consisting of an anti-body, a protein, an enzyme, nucleic acid, estraptavidin and biotin.

21. The apparatus according to claim 10, wherein the configuration of the cross-section of the adjacent recess * to the bottom wall is selected from the group consisting of a rectangular and circular box.

22. The apparatus according to claim 21, wherein the configuration of a transverse section of the displacement member is essentially identical to the configuration of the cross section of the recess. - SUMMARY OF THE INVENTION The analytical level rod (2) of the present invention facilitates testing of the test product with reduced liquid reagent volumes and provides improved mixing. The level rod (2) includes an elongate member (4) having a fluid displacement member (18) at one end thereof and a coordinator group (20) attached to a portion of the elongate member (4) in a region adjacent to the displacement member (18). The displacement member (18) has a configuration that essentially conforms to the internal side wall (26) of an analytical well (3) wherein the level rod (2) is positioned to displace the fluid therein. When the displacement member (18) is essentially centered on the lower part (30) of the well (3), the portion of the level rod (2) that supports a coordinating group (20) is preferably separated from the wall ( 26) by a distance (d2) of at least about twice the distance (di) between the displacement member (18) and the internal side wall (26), which is measured along a line (4 ^ ^ 4) which passes through the largest lateral dimension of the displacement member (18). With this configuration, essentially all the liquid in the part - - bottom (30) of the well (3) moves upwards so that the coordinating group (20) is immersed in it. The relatively large space (d2) between the portion of the coordinator group (20) and the side wall (26) of the well (3) facilitates the turbulent flow to improve mixing in the region of the coordinator group (20). * ^^^