WO1992011939A1

WO1992011939A1 - Device for in vitro testing

Info

Publication number: WO1992011939A1
Application number: PCT/US1992/000090
Authority: WO
Inventors: Emanuel Calenoff
Original assignee: Emanuel Calenoff
Priority date: 1991-01-11
Filing date: 1992-01-10
Publication date: 1992-07-23
Also published as: AU1236392A

Abstract

A device for in vitro assays, which comprises multiple assay wells, wherein each well (1) is an essentially cylindrical, hollow vertical column, said column having an upper end with an opening to said column, and a lower end which is closed so as to provide an assay well reservoir, said lower closed end being concaved (10) inside of said column and essentially planar (11) outside of said column. Additionally, the wells may have one or more vertical grooves (14) that extend to the concave bottom (10) of the well. The present invention also provides caps (2) that one may place on the top of each well. The caps (2) have a bore (21) to the exterior which is sufficiently large to accommodate the addition of solutions with ease; however, the bore (21) is sufficiently small to prevent the disc itself from leaving the well. The device increases the efficiency and economy of performing $(in vitro) assays.

Description

DEVICE FOR IN VITRO TESTING

Technical rigid

The present invention is directed to devices for performing in vitro testing.

Background of the Invention

Devices for performing in vitro testing and similar assays, such as "microtiter plates" and analyt impregnated paper discs are well known in the art. Commonly-used assay methods which employ such devices are, e.g., ELISA assays and RΛST allergy testing.

The devices that are known in the art, howev have several disadvantages. Presently available microtiter wells have flat bottoms. When performing assays which use discs, which often are permeated with e.g., an antigen or other analyte, the discs frequentl adhere to the bottom of the well. This adherence prevents both effective washing of all sides of the di and full surface area exposure to incubating solutions thereby providing unexpected variations in the results the assay.

In addition, presently available microtiter wells do not have caps. Therefore, often in washing procedures, assay discs are inadvertently removed from the well.

Moreover, the materials from which microtite wells are made are usually degraded by organic solvent used in the detection of radioactively labeled analyte Thus, the assay disc is transferred manually from the wel s to other containers for the detection of radioactivity in the assay sample.

In addition, prior art assays have often utilized microtiter dishes coated with a substance to which analyte binds. The coating process is subject to variability often resulting in fluctuating amounts of the substance to which the analyte binds being coated onto the microtiter dish. As a result, quality control is expensive for one producing standardized assays using microtiter dishes since an entire dish must be sacrificed during quality control testing.

The result of the disadvantages listed above is that the performance of in vitro assays in the currently available devices is labor intensive, and often yields erratic results.

It is therefore an object of the present invention to provide an improved device for in vitro assays, which overcomes the disadvantages mentioned above.

Summary of the Invention

The device of the present invention provides a multiple of joined wells, i.e., two or more, in which the bottom of the inside of each well is not flat but is concave, so as to avoid the tendency of assay discs to adhere to the well bottom. Additionally, the wells may have one or more vertical grooves that extend to the concave bottom of the well. These grooves provide an escape vent for air bubbles that often become trapped beneath assay discs thereby ensuring complete access of the disc to incubating fluids and thorough distribution of fluid around the discs in the wells. Complete access of the disc to surrounding fluid is important not only during assays but also when assay results are being determined, e.g., using scintillation counter fluids. The present invention provides caps that one may place on the top of each well. In a preferred embodiment, the caps have an opening which is large enough to allow one to wash assay discs and to administer reagents into each well with ease (and, optionally, automatically) while being small enough to prevent the disc itself from leaving the vial. The top of the caps are sized so as to enable caps to fit on two or more adjacent wells. The caps can be configured so as to fit on adjacent wells either individually or in a contiguous strip or sheet. Contiguous strips of caps have the physical advantage of stabilizing a row of wells and the commercial advantage of being less expensive to produce. The caps may be configured so as to provide an air-tight seal, which is especially important for long-term storage of material within the well. For example, the outside diameter of the cap may be slightly larger than the inside diameter of the well resulting in an air-tight seal. In one embodiment, the present invention is directed to a device for performing in vitro assays, which device is comprised of multiple wells with each well joined to at least one other well, in which each well is an essentially cylindrical, hollow vertical column, said column having an upper end with an opening to said column and a lower end which is closed so as to provide an assay well reservoir, said lower closed end being concave inside of said column and essentially planar outside of said column. In another embodiment, the present invention further comprises a cap, at least a part of said cap having an outside diameter equal to the inside diameter of said cylinder; said cap further comprising an annular upper lip capable of preventing said cap from completely entering said cylinder. In a further embodiment, the cap further comprises a central bore providing communication into and out of said cylinder.

Other advantages of the present invention will be apparent to those of skill in the art based upon the following detailed description and claims.

Brief Description of the Drawings

FIG. 1 is an elevation showing a side view of multiple wells of the device of the present invention, in a linear array.

FIG. 2 is an elevation showing a longitudinal cross-sectional view of an assay well with a cap.

FIG. 3 is an elevation showing a longitudinal cross-sectional view of two joined assay wells.

FIG. 4 is an elevation showing a horizontal cross-sectional view on line 4 - 4 of FIG. 1 showing two joined assay wells.

Detailed Description

As used herein, the term "assay well" means a reservoir of any convenient size for performing a desired assay. The type of assay performed in the device is not critical, since the device may be designed to accommodate many different assays. Examples of assays that can be performed in the device include ELISA, RAST, DNA hybridization, RNA hybridization, DNA blotting, RNA blotting, protein blotting.

As used herein, a column is "essentially cylindrical" when its sides are generally parallel to one another; certain tolerances are permissible as the side wall arrangement is not critical. One may deviate from the "essentially cylindrical" configuration described herein and still be within the general rubric of "reservoir" as intended by the present invention. As used herein, "multiple wells" are at least two wells, may be 3 or more wells, are preferably at least 8 wells, and may be at least 96 wells. The multiples of wells may be in any convenient array, for example, linear, or circular, or in the form of a plate, for example, a 96-well plate. Each well in the multiple wells is joined to at least one other well.

The device of the present invention provides wells in which the bottom of the inside of the well is not flat but is concave, so as to avoid the tendency of assay discs to adhere to the well bottom. This non- adherence provides for effective washing of all sides of the disc and equal exposure of both disc surfaces to incubating solutions/analytes thereby increasing the sensitivity and precision of the assay. The outside dimensions of the well have a flat bottom so as to allow the wells to stand upright.

The present invention provides a cap that one may place on the top of the well. In a preferred embodiment, the cap has an opening through it, which is large enough to allow one to wash assay discs with ease while being small enough to prevent the disc itself from leaving the vial. The caps are sized so as to enable caps to fit on two or more adjacent wells. Other advantages of the present invention will be apparent to those of skill in the art based upon the following detailed description and claims.

The wells of the present invention are ideally non-adsorptive to prevent analytes to be assayed from adhering nonspecifically to the well. The wells may be made out of non-adsorptive materials, for example, poorl adsorptive plastics such as polypropylene, polyethylene, etc. In addition or alternatively, the wells may be mad non-adsorptive by coating. Non-adsorptive treatments an materials are well known in the art and include silicon, and bovine serum albumin (BSA) . In addition, the wells of the present invention may be made of materials that are not degraded by the solvents used in the assay procedure, e.g., in scintillation counter fluids. An example of a scintillation-counter-fluid-resistant plastic is "Barax" which is manufactured by British Petroleum. The wells may be made of material that allows one to separate the individual wells so that an assay may be placed in a desired array in a well holder or other suitable support means. The wells may be used as storage containers or may be used as incubation containers during a particular assay run. When used as storage containers, any convenient means may be employed to seal the wells. For example, caps can be designed so as to provide an essentially air tight seal by, for example, having the diameter of the cap sufficiently large so that it abuts completely the inside surface of the well at the point that the well contacts the cap. The wells may be sealed from above with adhesive strips or sheets of material such as transparent acetate.

In commercial production, the wells may be pre¬ loaded with assay discs, wetting solution, and/or other reagents designed for use in a particular assay. The wells and/or caps of the present invention may be color- coded to enable the laboratory technician or other individual using the device to color key samples or assays.

Referring now to the drawings a device for in vitro assays, 1 as shown in FIG. 1, contains multiples of a well 1, which may be aligned at juncture 30 in a linear array. Several linear arrays may be placed within a convenient holder. The wells 1 may also be precast into a block in a format which may contain, for example, 96 wells. As shown in FIG. 2, which is an enlarged detai of FIG. 1, one can see a longitdudinal cross-sectional view of the well 1. As seen in cross-section, well 1 ha essentially parallel vertical walls forming a cylinder, having an outer wall 13 and an inner wall 12. One end o the cylinder is open and the other end is closed. The closed end has a concave bottom 10 on the interior of th well 1 and optionally has a planar base 11 on the exterior of the well 1. Because assay discs typically have a size in the range of 5 to 6 mm, the bottom 10 diameter at its widest point will be about 0.2 mm larger than the size of the assay disc. The concave bottom 10 prevents assay discs from adhering to the bottom of the well reservoir. The planar base 11 permits one to stand a well upright.

As seen in FIG. 2, the well 1 may be fitted with a cap 2. The cap 2 has essentially cylindrical parallel sides forming an essentially cylindrical column with an outer wall 26 having a diameter dimensioned so a to fit within the inner wall 12 of well 1. The cap 2 ha at one end of the column an annular lip 20 which has a diameter greater than the inner wall 12 of well 1 thereb preventing the cap 2 from completely entering the well 1 In a preferred embodiment, the lip 20 does not extend beyond the outer wall 13 of the well 1 so as to permit adjacent wells, as shown in FIG. 1 and FIG. 3, to each have a cap 2. In another preferred embodiment, the cap is slightly tapered beneath the annular lip 20 so as to provide a void 22 between the cap 2 and the internal wal 12 of the well 1 at the cap's upper end and fit snugly against the internal wall 12 of well 1 at the cap's lowe end 23.

The cap 2 also may have a central bore 21 which, when the cap 2 is placed into well 1, permits communication between the outside of well 1 and the inside of well 1. The bore 21 may be preferably about 0.2 mm smaller in diameter than an assay disc present in well 1 thereby preventing the assay disc in the well 1 from passing through the bore 21 and out of the well 1 during assay wash steps. As stated above, the standard assay disc has a size in the range of between about 5 mm and about 6 mm. Thus, as shown in FIG. 2, the bore 21 diameter in the cap 2 will preferably be a minimum size of about 4 mm. Additionally, the upper end 24 of the bore 21 can be tapered at an angle that is about 23° relative to the inside wall 25 of the lower end of the bore 21.

As also shown in FIG. 2, the inner wall 12 of well 1 may have a vertical groove 14. It is preferable that the bottom of the groove 14 be slightly above and sloping toward the bottom and center of the concave bottom 10 so that fluids may fully drain from the groove 14. One or more grooves 14 may be present in well 1. The primary function of the vertical groove 14 is to permit air bubbles to escape from beneath discs that may be placed in the well l. The groove 14 provides an escape vent for air bubbles thereby ensuring complete access of the disc to incubating fluids and thorough distribution of fluid around the discs in the well l. As shown further in FIG. 3, which is an elevation showing a longitudinal cross-sectional view showing two joined assay wells, the well 1 is attached to an adjacent well 1 at a juncture 30. The wells 1 may be constructed of a material, such as polystyrene, that allows the user of the wells to snap off one or more wells at juncture 30 thereby enabling one to rearrange the wells and thereby vary the array. Exemplarv Assay Using the Device of the Present Inventio

Disc Loading Procedure

1. Place allergen disc into round-bottom well. Wells can be individual or in sequence as part of a strip of wells each well containing a paper disc with different covalently coupled allergen from the next.

2. Add 50 μl of Incubation Buffer to each dis loaded well to keep disc moist. (Incubation Buffer - 50 mM PBS + 0.3% HSA + 0.1% NaN-_j) .

3. Cap each loaded well snugly with an open aperture cap.

4. If not used immediately, seal open aperture of each cap by applying adhesive strips of clea acetate on top of cap thereby conferring an air-tight closure. Store at 4°C until use.

Assay Procedure

1. Use freshly loaded wells or strips of well or stored, preloaded wells or strips. In case of the latter, peel off and discard adhesive strip.

2. Load wells or strip(s) into strip holder in a documented sequence where the order of allergens being tested is known and maintained. 3. As a system calibrator, load PRIST discs

(Pharmacia Diagnostics, Piscataway, NJ) into duplicate wells and pipette, through the cap aperture, 100 μL of controlled serum containing 25 IU IgE/ l into each well.

4. Pipette, through the cap aperture, 100 μl of serum from each patient being tested into each well o the strip of wells corresponding to the profile of allergens being tested.

5. Seal tops of caps with the adhesive acetate strip. 6. Incubate at room temperature for 18 to 24 hours.

7. Remove and discard adhesive strip.

8. Wash discs, employing 250 μl of Wash Buffer per well, three times. Do not aspirate last wash but allow discs to soak in Wash Buffer for 10 minutes. (Wash Buffer = 50 mM PBS + 0.1% Tween 20.)

9. Repeat Step No. 8 twice. After third soaking step, wash discs twice and aspirate. 10. Pipette 50 μl of ¹²⁵I-labeled, affinity- purified anti-IgE through the cap aperture into each well. (Anti-IgE is diluted with solution containing 50 mM PBS + 5% Horse SErum + 0.1% NaN3.)

11. Seal wells as before and incubate for 18 to 24 hours at room temperature.

12. Remove adhesive strips and wash discs as per Steps 8 and 9.

Counting Discs and Analyzing Data l. Remove strips or individual wells containing processed discs from strip holder.

2. Drop each well into a 12 x 75 mm polyethylene or polystyrene test tube and load onto counting racks in known sequence. The wells attached in strips can be snapped off and dropped into respective test tubes, one by one.

3. Load counting racks onto calibrated gamma counter and count each sample for 2 minutes to obtain the counts per minute of each sample. 4. Multiply the registered counts of each unknown sample by the formula: 25,000 counts/count average of the 25 IU IgE calibrators.

5. Determine the result for each allergen tested by employing the table below: -11-

Ranoe of Counts

18,001-40,000 8,001-18,000 3,601- 8,000 1,601- 3,600

751- 1,600

500- 750

Less than 500 0 Undetectable

The above protocol may be automated to increas the efficiency further. Those of skill in the art will recognize, however, that the present invention vastly increases the efficiency of performing the RAST test by decreasing the time for setting up the assay and washing the discs, and pipetting assay reagents into the wells. Furthermore, scintillation counting of the assay^' results can be done in the same container in which the assay is performed. This increased efficiency compares favorably to the commercially available ELISA assays which, unlike the RAST test, often have the disadvantage of being unable to low levels of specific IgE (which fall in modified RAST class I sensitivities) . In addition, this device allows a saving in the amount of reagent required to wash assay discs. For example, in the RAST assay, it requires approximately 60% less WASH buffer to achieve acceptable background levels and precision in the assay, thereby increasing the economy in the performance of th assay. The foregoing description is for illustration purposes only and is not intended to limit the invention as set forth in the following claims.

Claims

1. A device for performing in vitro assays, said device comprised of multiple wells, wherein each well is an essentially cylindrical, hollow vertical column, said column having an upper end with an opening to said column and a lower end which is closed so as to provide an assay well reservoir, and said lower closed end being concave inside of said column.

2. The device of claim 1 wherein the lower closed end of the well is essentially planar outside of said column.

3. The device of claim 1, wherein the column of the well further comprises at least one lateral groove.

4. The device of claim 1 wherein said well i comprised of a non-adsorptive material.

5. The device of claim 1 wherein said well is comprised a material resistant to scintillation counter fluid solvents.

6. The device of claim 1 further comprising an assay disc in a well.

7. The device of claim 6 further comprising wetting solution in a well.

8. A device for performing in vitro assays said device comprised of multiple wells, wherein each well is an essentially cylindrical, hollow vertical column, said column having an upper end with an opening to said column and a lower end which is closed so as to provide an assay well reservoir, and said lower closed end being concave inside of said column, said device further comprising a cap, at least a part of said cap having an outside diameter of sufficient size to create an air-tight seal with a rim at the upper end of said well; said cap further comprising an annular upper lip capable of preventing said cap from completely entering said cylinder; and said lip being of sufficiently small size to allow the placement of a second cap on an adjacent well, so that the second cap is capable of forming an airtight seal with the adjoining well's rim.

9. The device of claim 8 wherein said cap further comprises a central bore providing communication into and out of said cylinder.

10. The device of claim 9 wherein said central bore is smaller than the diameter width of an assay disc.

11. The device of claim 9 wherein the column inside the well further comprises at least one lateral groove; and wherein the well is comprised of a non- adsorptive material.

12. The device of claim 11 further comprising a cap, at least a part of said cap having an outside diameter of sufficient size to create an air-tight seal with a rim said well; said cap further comprising an annular upper lip capable of preventing said cap from completely entering said cylinder; and said annular lip of said cap being of sufficiently small size to allow the placement of a second cap on an adjoining well so that said second cap forms an airtight seal with said adjoining well's rim, wherein said well is comprised a material resistant to scintillation counter fluid solvents.

13. The device of claim 12 wherein said cap further comprises a central bore providing communication into and out of said cylinder.

14. The device of claim 13 wherein said cap further comprises a central bore providing communication into and out of said cylinder.

15. The device of claim 1 wherein the wells are in a plate.

16. The device of claim 8 wherein the wells are in a plate.

17. The device of claim 12 wherein the wells are in a plate.

18. The device of claim 13 wherein the wells are in a plate.

19. The device of claim 14 wherein the wells are in a plate.