United States Patent
`
`[ 72] Inventors Don P. Metzgar;
`James V. Sorrentino, Stroudsburg, Pa.
`[21] Appl. No. 797,822
`[22] Filed
`Feb. 10, 1969
`[45] Patented Mar. 30,1971
`[73] Assignee
`Richardson-Merrell Inc.
`New York, N .Y.
`
`[54] MULTIPLE WELL TISSUE CULTURE SLIDE
`5 Claims, 3 Drawing Figs.
`
`[52] US. Cl ...................................................... ..
`
`350/95,
`356/244
`[51] Int. Cl ....................................................... .. G02b 21/34
`[50] Field of Search...
`350/92
`--95; 356/244; 206/1 (t); 220/20, 22
`References Cited
`UNITED STATES PATENTS
`1,516,896 11/1924 Turner ....................... ..
`2,302,830 11/1942 Axelrad...
`3,031,924
`5/1962 Lamal ........................ ..
`
`[56]
`
`356/244
`350/95
`350/95
`
`[1113,572,892
`
`
`
`2,281,617 3,432,275
`
`
`
`5/1942 Ramsdell .................... 3/1969 Unger ......................... ..
`
`Primary Examiner-David Schonberg
`Assistant Examiner-T. H. Kusmer
`Attorney-Harvey W. Edelblute
`
`206/ l UX
`350/95
`
`ABSTRACT: A tissue culture microscope slide comprising a
`transparent ?at plate having depressions or wells on the top
`surface thereof. The walls de?ning each well have an opening
`extending from the top to the bottom of the well adjacent to
`an edge of the plate to facilitate the drainage of liquids
`therefrom. The bottom surface of each well is substantially
`parallel with the bottom surface of the plate. The tissue cul
`ture slide after preparation with ?xed, con?uent, viral infected
`tissue culture cells can be used with fluorescent labeled re
`agents and a fluorescent microscope to provide the technolo
`gist or clinician with a tool for rapidly screening the sera of pa
`tients or animals for a variety of viral agents as measured by
`the presence in the patients‘ or animals’ serum of antibody
`against a speci?c viral agent.
`
`Page 1 of 5
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`HOLOGIC EXHIBIT 1009
`Hologic v. Enzo
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`PATENTEUHARBOIHYI
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`3,672,892
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`F|G.I
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`INVENTORS
`O O N P. M E TZGAR
`‘JAM E S V-SORRENTINO
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`Page 2 of 5
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`3,572,892
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`The use of this invention makes available, in one single
`slide, all of the basic materials required to perform the
`fluorescent antibody technique. This invention can reduce the
`total time required for such diagnostic tests, including the
`preparative work, from 7 to 14 days to a maximum time of 2
`hours. With the multiple well tissue culture slide of this inven
`tion, an entire test requiring 6 or even more dilutions can be
`performed on a single slide. This eliminates the processing of
`multiple cultures and the necessity of individual microscopic
`examination of many cultures. The multiple well slide can be
`examined in one microscopic manipulation.
`Any virus agent that can be grown in tissue culture can be
`adapted to use with this invention. By making available a kit
`containing slides having a series of ?xed cells infected with a
`virus or group of viruses, the physician, laboratory or institu~
`tion will have available a rapid and speci?c tool to be used in
`diagnosis of diseases of virus etiology.
`Referring now to the drawings, wherein identical numerals
`refer to identical parts:
`FIG. 1 is a perspective view of the slide showing the top and
`sides thereof;
`FIG. 2 is a perspective view of the slide of FIG. 1 showing
`the top and sides thereof with ,a con?uent sheet of virus-in’
`fected tissue culture cells in each of the wells; and
`FIG. 3 is a perspective view showing the bottom and sides of
`the slide of FIG. I.
`The microscope slide 10 is a rectangular plate of a ?at piece
`of glass or other transparent material such as a synthetic resin,
`e.g., polystyrene. The slide 10 has two long sides or edges
`12-12 parallel to each other; narrow sides or edges 1%14, at
`right angles to the long sides 12-12. Each side 12 is 90 mil
`limeters (mm.) long. Each narrow side 14 has a width of 25
`mm. A longitudinal rib 16 having a width of 5 mm. and trans
`verse ribs 18 having a width of 2 mm. together with raised por_
`tions along the narrow sides 14-14 de?ne square wells or
`depressions 20 therebetween. The top surfaces of ribs 16 and
`18 and the raised portions along narrow sides 14-14 are in the
`same plane and parallel to slide bottom 26. The thickness of
`the slide from the top surfaces of the ribs to the bottom sur
`face 26 thereof is 1 mm. Walls 24 enclose three sides of each
`well 20. These wells are 0.5 mm. deep and each well wall 24
`has a length of 10 mm. Wells 20 are open, from the bottom to
`the bottom thereof, adjacent their respective edge 12. The
`bottom 26 of the microscope slide 10 is ?at and parallel with
`the bottom 22 of each of the wells 20. In FIG. 2 each'of the
`wells 20 contains a fixed ?uent virus-infected tissue culture 23
`attached to the bottom surface 22 thereof. The narrow ends
`1444 form walls, 10 mm. wide, for one side of the wells at
`each end of the slide. The slide has designators A and B for in
`dicating each row of wells 22 and additionally has numerical
`designators 1-6 on ribs 16 between opposed wells so that the
`use of the letter and numerical designators can de?ne each of
`the wells.
`The following examples serve to further illustrate this inven~
`tion.
`
`1
`MULTKPLE WELL TISSUE CULTURE SLIDE
`This invention relates to a multiple well tissue culture slide.
`More particularly, this invention relates to a multiple well tis
`sue culture microscope slide and to such a slide having ?xed,
`con?uent, viral infected tissue culture cells which can be used
`for the rapid diagnosis of viral diseases using ?uorescent an
`tibody technique.
`The use of ?uorescent antibody technique for the detection
`and identi?cation of viruses as well as bacteria, while highly
`reliable, requires a considerable amount of time and effort to
`prepare the reagents and the tissue required for the test.
`Broadly, such tests are carried out by the following procedure.
`Tissue culture cells of the required type and species are placed
`on a culture surface or vessel such as that of a Petri dish,
`microscope slide or microscope coverslip. After a sufficient
`period of incubation in which the cells become attached to the
`culture surface, the cells are infected with a speci?c virus
`agent. After another period of incubation, during which the
`virus agent multiplies, the culture vessel ‘is removed from the
`incubator, the cells are fixed with a suitable ?xative and then
`dried. This then becomes the basic unit for the ?uorescent an
`tibody technique as generally practiced in the prior art. The
`?xed, infected tissue culture vessels are prepared in multiples.
`If serum dilutions are to be used a separate Petri dish,
`microscope slide or coverslip culture must be prepared for
`each dilution. If more than one serum sample is to be used,
`this requires multiples of the culture. Each culture is then
`treated with a dilution of the patient’s or animal’s serum. The
`serum is allowed to remain in contact with the culture for 15
`to 30 minutes. It is then rinsed in a suitable diluent to remove
`the excess serum. The culture is then treated for 15 to 30
`minutes with a ?uorescein conjugated homologous antiserum.
`Usually this antiserum is gamma globulin common to the spe
`cies being tested. Each culture is rinsed to remove the excess
`?uorescein conjugate and then dried.
`After drying, the culture is examined microscopically, with
`a ?uorescent microscope. A positive diagnosis is indicated by
`the presence of speci?c fluorescence in the treated culture.
`The use of the above-described fluorescent technique is
`well known. it is described by (a) Coons, A. 1%., International
`Rev. Cytol, 5:1-23, 1956; (b) Coons, A. H., In General
`Cytological Methods, New York Academic Press, 1958,
`Volume I; (c) Liu, C., Ergebn, Mikrob. lmmunforsch. 332242,
`1960; and (d) Fluorescent Antibody Techniques, Public
`Health Service Publication No. 729 (1966) U.S. Gov't Print
`ing Office.
`Although the ?uorescent antibody technique has many ad
`vantages, it can be a time-consuming and laborious procedure.
`Thus, a test requiring 6 serum dilutions requires a total of 12
`different Petri dishes, microscope coverslip or microscope
`slide cultures. Each one must be prepared, treated, rinsed,
`dried and examined separately.
`It is a primary object of this invention to provide a multiple
`well tissue culture microscope slide which is easy to use and
`which decreases the time and manipulative steps required for
`performing the diagnosis of viral disease using ?uorescent an
`tibody technique.
`it is another object of this invention to provide such a tissue
`culture slide in which the wells contain a fixed, con?uent sheet
`of cells infected with virus. Such a slide can be supplied to a
`technologist or clinician performing antiviral tests, and thus
`further decrease the time and manipulative steps required for
`making tests on patients or animals.
`Other objects will appear from the description hereinafter.
`Briefly, the multiple well tissue culture slide of this inven
`tion comprises a transparent ?at plate having depressions or
`wells on the top surface thereof. The walls de?ning each of the
`wells have an opening extending from the top to the bottom of
`the well adjacent to an edge'of the plate in order to facilitate
`the drainage of liquids therefrom. The bottom surface of the
`plate is substantially flat and preferably the bottom surface of
`each well is also ?at and substantially parallel with the bottom
`surface of the plate.
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`EXAMPLE 1
`
`This example shows the use of the multiple well tissue cul
`ture slide of FIG. 1.
`‘
`A. A single multiple well slide, as shown in FIG. 1, is
`prepared for the patient’s serum to be examined. The slide is
`completely immersed in a culture vessel containing nutrient
`medium for the cells to be used. In this case human Wi-38
`diploid cells were used. Wi-38 cells are added to the medium
`covering the slide. After a stationary incubation period of 4 to
`5 days at 35° C. to 37° C. the multiple well culture is infected
`by adding an inoculum of adenovirus type 3 to the culture
`medium.
`E. The virus infected multiple well slide is removed from the
`nutrient medium and ?xed with acetone for 10 minutes and
`then dried. The culture is attached to all the surfaces of the
`plate. The culture is wiped off of the plate, except for that in
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`3,572,892
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`3
`the bottom of the wells 22 as appears as 28 in FIG. 2. Each of
`the wells now contain a ?xed con?uent sheet of Wi-38 cells
`infected with adenovirus type 3.
`C. Serum is withdrawn from the convalescing patient
`wherein adenovirus type 3 is suspected as a cause of the ill
`ness. Dilutions of the patient’s serum are prepared and each
`well is covered with about 0.1 milliliter of a single dilution.
`One row of wells, e.g., Row A of FIG. 2, containing the control
`serum (normal serum) dilutions and one row, e.g., Row B of
`FIG. 2, of wells containing the patient’s serum, are prepared.
`Dilutions are prepared in two-fold serial dilutions of 1:2, 4, 8,
`l6 and 32.
`D. The multiple well culture is then placed in a moist
`chamber for 15 to 30 minutes at 35° C. to 37° C. and then
`rinsed with 0.02 Molar phosphate buffered saline solution at
`pH 7.2 to remove the excess serum.
`E. Each well is then overlayed with a ?uorescein labeled
`conjugated antiserum for the gamma globulin contained in the
`patient’s serum. Such labeled conjugated antiserum is availa
`ble commercially. This antiserum is absorbed on the con?uent
`?lm in a moist chamber for 30 minutes at 35° C. to 37° C. and
`rinsed as above with the phosphate buffer.
`I
`F. The multiple well culture is then examined microscopi
`cally in a ?uorescent microscope, for the presence of speci?c
`?uorescence. lf positive, the preparation will ?uoresce green,
`if negative, no ?uorescence will be seen, as in the case of the
`normal serum controls.
`The above test is carried out with a single slide and is ob
`served microscopically in a single operation. The normal
`serum and suspected serum are side by side, allowing for
`simultaneous observation.
`
`4
`tions are considered to be within the purview and scope of the
`appended claims.
`We claim:
`1. A multiple well tissue culture microscope slide compris
`mg:
`a. a ?at plate of transparent material;
`b. a plurality of wells or depressions on the top surface of
`' the plate;
`c. said wells having an opening on one side thereof adjacent
`an edge of said plate, said opening extending from top to
`bottom of the well to facilitate drainage of liquids
`therefrom; and wherein
`d. the bottom surface of said plate opposite each well is ?at.
`2. A multiple well tissue culture microscope slide compris
`mg:
`a. a ?at plate of transparent material;
`b. a series of depressions or wells spaced from each other on
`the top surface of said plate;‘
`_
`‘
`c. the bottoms of said depressions being ?at and m a plane
`parallel with the bottom of the slide;
`d. each of said depressions de?ned by sidewalls and a bot
`tom;
`e. said sidewalls having an opening adjacent one of the plate
`edges, said opening extending from the top to the bottom
`of said wells to facilitate drainage of liquids therefrom.
`3. A multiple well tissue culture microscope slide compris
`
`a. a ?at plate of transparent material;
`b. ribs projecting upwardly on the top surface of said plate
`as an integral part thereof defining a series of wells spaced
`from each other;
`c. said ribs enclosing a major portion of the wells and having
`an opening extending from the top to the bottom of- the
`wells adjacent a plate edge to permit ?uid communication
`between the well and said plate edge to facilitate drainage
`of liquids therefrom; and
`d. wherein the bottom surface of said wells is substantially
`?at and parallel to the bottom surface of said plate.
`4. A multiple well tissue culture microscope slide compris
`mg:
`a. a transparent, ?at rectangular plate;
`b. a first row of depressions or wells on the top surface of
`said plate adjacent one of the plate long sides;
`c. a second row of depressions or wells on the top surface of
`said plate adjacent the other long side of the plate;
`(1. the bottom surface of the wells being substantially ?at
`and parallel with the plate bottom; and
`e. each row of wells having an opening extending from the
`bottom surface thereof to the top facing the adjacent
`plate edge, said openings facilitating the drainage of ?uid
`out of said wells.
`5. A multiple well tissue culture slide of claim 4 having at
`tached to the bottom surface of said wells a ?xed, con?uent
`sheet of cells infected with virus.
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`EXAMPLE 2
`
`This example shows a test to determine the presence of
`adenovirus antibody in a patient’s serum.
`A kit containing 8 slides such as shown in FIG. 2 is supplied
`to the physician or laboratory. Each slide has a con?uent cell
`tissue culture infected with a different adenovirus type which
`has been ?xed, as described in Example 1, paragraphs A and
`B. in this instance, however, the adenovirus is that of types I,
`2, 5, 3, 4, 7, l4 and 2 l. The physician treats each slide with the
`human suspected serum and with the normal serum as
`described in paragraph C of Example 1 and, by following the
`remaining procedure of Example 1, can determine within
`about 2 hours if one of the adenovirus types being screened
`was involved in the patient‘s illness.
`Although the present invention has been described in con
`junction with a preferred embodiment, it is to be understood
`that modi?cations and variations may be resorted to without
`departing from the spirit and scope of the invention, as those
`skilled in the art will readily understand. Thus, it will be ob
`served that in place of diagnosing for viral antibodies, the
`novel slide of this invention may be used for other diagnostic
`tests with a ?uorescent microscope, such as, for instance, in
`diagnosing for bacterial illness. Such variations and modi?ca
`
`Page 4 of 5
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`mg?
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`UNITED STATES PATENT OFFICE
`CERTIFICATE OF CORRECTION
`
`Patent No.
`
`3,572,892
`
`Dated
`
`March 30, 1971
`
`Inventor(8)
`
`Don P. Metzgar and James V. Sor rent ino
`
`It is certified that error appears in the above-identified patent
`and that said Letters Patent are hereby corrected as shown below:
`
`Column 2, I ine 1%, "bottom’I should read --top--; column 2.
`l ine 48, "fluent" should read --conf1 uent—-;
`
`Signed and sealed this 5th day of December 1972.
`
`(SEAL)
`Attest:
`
`EDWARD M.FLETCHER,JR.
`Attesting Officer
`
`ROBERT GOT‘I‘SCHALK
`Commissioner of Peter
`
`Page 5 of 5