US 3407133 A
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Oct. 22, 1968 w. E. OLIVA ET AL EXPENDABLE ELECTROPHORESIS APPARATUS 2 Sheets-Sheet l Filed June 18, 1965 lllllllllllll ls INVENTORS WILLIAM E. OLIVA CHARLES A.
HLUTZ ATTORNEY Oct. 22, 1968 Filed June 18, 1965 W. EQOLNA ET AL EXPENDABLE ELECTROPHORESIS APPARATUS 2 Sheets-Sheet 2 INVENTORS HLLIAM E. oLlvA AT TORNEY RLES A. SCH UTZ 3,407,133 lEXPENDABLE ELECTROPHORESIS APPARATUS William E. Uliva, Los Angeles, and Charles A. Schlutz, Glendale, Calif., assignors, by mesne assignments, to Baxter Laboratories, Inc., Morton Grove, Ill.
Filed June 18, 1965, Ser. No. 465,059 12 Claims. (Cl. 204-299) This application relates to the separation of mixtures by differential migration of components through a transport medium in an electric field and more particularly to a new and improved expendable plastic electrophoresis test unit for particle separation in a buffer system to which direct current is applied.
Various types and sizes of electrophoretic test apparatus have been provided in the past for separating cornponents of a mixture wherein each material has its own particular charge which differs from the others in extent and sign. The separation and consequent grouping of materials electrophoretically normally occurs within a buffered support medium wherein those particles of a test sample that have a greater charge will migrate at a faster rate and further in a given time than those of lesser charge. After particle grouping, visual identification of the materials in the test sample is determined by known means such as for instance colorimetric analysis. In the past the procedures described required the use of expensive and relatively complicated apparatus which demanded the presence of technicians possessed of a high degree of skill and available time for detailed attention to the preparation, operation and subsequent clean-up of the test apparatus. All of these steps, together with the cost of the equipment required, have hindered the potential application of electrophoresis as a suitable test procedure, especially in the clinical field where the use of expendable apparatus has been so well accepted for a variety of reasons, economic and otherwise. In the area of activity referred to above, it has become increasingly useful to be able to determine for example the identification of the individual isozymes of lactate dehydrogenase. It will be understood however that this is but one application to which electrophoretic separating is adaptable and is mentioned here for illustrative purposes only. The use of inexpensive, expendable, self-contained electrophoresis equipment will enable the relatively small clinic to use diagnostic procedures which heretofore have been fairly well restricted to use by large commercial and institutional laboratories.
In addition, electrophoresis apparatus presently used is of such design that each setup involves the gathering together and assembly of various pieces of costly and sometimes cumbersome equipment. To accommodate more than one test at a time requires the possession and use of duplicate apparatus, compounding the expense and increasing the probability of error due to improper disassembly and cleaning after use. The present invention makes available for clinical use a kit-type expendable single use apparatus. This quality enables the operator to normally work with a much smaller piece of equipment and further releases him from the problems of assembly and cleanup mentioned above necessitated by reuse of any piece of laboratory equipment. With all necessary comnited States Patent O 3,407,133 Patented Oct. 22, 1968 lCe ponent materials and equipment supplied in a single package including a plurality of lids adapted to contain transport medium many of the above problems are obviated.
It is therefore a primary object of the present invention to provide a new and improved, relatively inexpensive, expendable electrophoresis test apparatus.
A further object of this invention is to provide a means of performing electrophoretic tests whereby all necessary test components for the particle separation procedure are provided in a single kit.
Still another object of this invention is to provide a new and improved means for supporting a pre-poured gel medium for passage of electric current therethrough for particle migration therein.
Another object is to provide a new and novel means for depositing test samples upon a suitable gel support medium for electrophoresis particle migration.
Another object is to provide an improved means of providing current through a buffer in a gel medium in an electrophoresis test apparatus.
A feature of the present invention is the provision of a compact, disposable electrophoresis test apparatus formed of a plastic material and including a base chamber with removable buffer vessels positioned therein and having electrode means and a lid adapted to position a gel support medium between the buffer vessels.
Another feature is the provision of an integrally formed multi-reservoir electrophoresis chamber wherein a pair of spaced buffer vessels are provided with a buffer in contact with a gel medium provided in a plastic lid for imposition of direct current upon the system with consequent passage of the current through the gel medium.
Still another feature is the provision of an integrally formed multi-reservoir electrophoresis chamber wherein a pair of spaced electrode containing buffer vessels are provided with a wick to maintain a buffer in contact with a gel medium poured and set in a plastic lid for imposition of direct current upon the system with consequent passage of the current through the gel medium.
A further feature is the provision of a plastic lid for an electrophoresis chamber, the lid being adapted to contain a gel support medium and formed to receive a further lid thereon in stacked relationship for compact shipping of a plurality of lids with a single base chamber.
Still another feature of the invention is the provision of a template for forming predetermined spaced test sample wells in a solid gel medium.
Another feature of the invention is the provision of foil electrodes formed to substantially fit a pair of buffer vessels and having means for connection to a source of current.
The above and other objects and features of the present invention will become apparent from the following description and accompanying drawings forming part of this application.
In the drawings:
FIGURE l is a plan view of the electrophoresis kit of the present invention;
FIGURE 2 is an enlarged plan view of the apparatus of FIGURE l with the lid removed;
FIGURE 3 is a horizontal sectional view taken along lines 3--3 of FIGURE l;
FIGURE 4 is a perspective illustration of the lid and template of the present invention;
FIGURE 5 is a typical partial sectional view as taken generally along, and in the direction of any of the lines 5--5 of FIGURE 1;
FIGURE 6 is a plan view of a further embodiment of the present invention;
FIGURE 7 is a plan view of the device of FIGURE 6 with the lid removed;
FIGURE 8 is a horizontal sectional View taken generally along, and in the direction of lines 8-8 of FIG- URE 6;
FIGURE 9 is a side view of the apparatus of FIGURE 6; and
FIGURE 10 is a partial sectional view identical t0 FIGURE 5 showing the foil electrode in the operable position.
Referring now particularly to the drawings wherein like reference characters indicate like parts, the electrophoresis kit of the present invention shown in FIGURE 1 is indicated generally by the reference numeral 10 and includes a substantially rectangular electrophoresis chamber 12 having sidewalls 14 and end walls 16 surrounding, and extending upwardly from, a flat base portion 18 (see FIGURE 3). Each of the walls 14 and 16 is provided with an area of reduced thickness at the upper edge portion defining an L-shaped lid rest in the walls 14 and 16. The chamber 12 is shown with a lid 20 resting upon the top portion thereof. The lid 20, as illustrated in more detail in FIGURE 3, is formed with an upper portion of reduced outer peripheral dimension defining an outer ledge 22 to accommodate stacking additional lids one upon the other for shipment as a compact unit. An inner ledge 23 is likewise formed on the lid 20 to rest upon the chamber walls upon assembly of the kit. The top portion of the walls 14 and 16 together with the inner ledge 23 thus provide complementary engageable surfaces as shown in FIGURES 3 and 5.
FIGURE 2, an enlarged view of the unit 10 of FIG- URE 1, shows the chamber 12 with lid 20 removed. Positioned within the chamber 12 are a pair of substantially rectangular buffer vessels 24 and 26. The vessels 24 and 26 are open -at the top and positioned parallel to the end walls 16 of the chamber 12. Vessel support pads 28 are provided upon the base 18 and extend upwardly therefrom to provide support for the vessels 24 and 26 within chamber 12 maintaining the vessels 24 and 26 in predetermined vertical position within the chamber 12. The support pads 28 for the vessels 24 and 26 further hold them in spaced longitudinal relationship within the chamber 12 until removed by lifting from the chamber 12. Recesses 32 are formed in the outer surface of the side walls 14 to receive the nubs 33 formed on the lid 20 to hold the lid in its closed position upon the chamber 12 during electrophoresis. The vessels 24 and 26 may 'be provided with integrally formed outwardly projecting hanger tabs (not shown) to allow suspension of the buffer vessels within the chamber 12 from the walls thereof. A foil electrode 34 of suitable electrical conducting properties is provided for retention within each of the buffer vessels 24 and 26 and is shaped to substantially fit within the vessels 24 and 26 as seen in FIGURES 2 and 3. Each foil electrode 34 has an outwardly extending tab portion 36 which is adapted to protrude from the chamber 12. The tabs 36 are connected to a suitable source of direct current 38 through switch 39. The tabs 36 are illustrated in their extended outfolded operable position in FIGURE 2. It will be understood that although foil electrodes are illustrated, other known electrodes such as nichrome or platinum wire (as seen in FIGURE 8) may be used. Normally a pair of electrodes are provided with each lid 20 supplied with the kit and may be contained within the vacant section of the chamber 12 between the buffer vessels. The electrode may in fact be painted or printed in the buffer vessels 24 and 26 without departing from the spirit and scope of the present invention.
When assembled as a kit for distribution to the user the foil electrode tabs 36 may be folded as seen in FIG- URE 3 over the top of a sponge wick 40 provided in each of the buffer vessels 24 and 26 of the unit 10. The sponge wicks 40 of suitable porosity carry and convey a buffer solution poured into vessels 24 and 26 prior to electrophoresis to provide the buffer communication 4between the vessels 24 and 26 and the gel support medium, which will be discussed in the description of FIGURE 3. The sponge wicks 40 are shown formed to tit the vessels 24 and 26 conforming substantially to the internal dirnensions of the vessels and upon saturation with buffer solution contact the gel within lid 20 upon closure of the chamber 12.
In FIGURE 3 a lid 20 is illustrated in position upon the chamber 12 to effectively enclose the chamber 12 to aid in prevention of evaporation therefrom. The lid 20 is further formed to receive and contain a suitable gel medium 46 by providing a gel reservoir 42 when positioned for the receipt of gel therein. The gel reservoir 42 within the lid 20 is defined by the vertically extending peripheral wall 44 of lid 20. It will be understood that the gel medium may be pre-poured and thus packaged and provided by the manufacturer with the lid 20; or alternatively it may be poured by the user. With the lid -20 in position upon the chamber 12, the gel medium at the end portions of lid 20 rests in electrical contact upon the sponge wicks 40. The lid, buffer vessels and Sponges are dimensionally proportioned so that the gel may adequately contact but not unduly compress the sponges. Vertically extending gel retainers 48 may be formed upon the inside surface of the lid 20 extending laterally thereacross in spaced relationship thereon to aid in holding the semi-solid support medium 46 in position within the lid 20 when inverted and locked in position upon charnber 12 for operation of the apparatus. A second lid 50 is illustrated in phantom in FIGURE 3 to show the stacked relationship between the lids. The lids 20 and 50 are of like construction and are thus adapted to form a compact package for shipment and storage. In this manner several lids may be provided with a single chamber 12.
FIGURE 4 shows a lid 20 in position to receive a gel pour therein. Nubs 33 are integrally formed on the inner walls of the lid 20 and are provided to engage the depressions 32 in the outer surface of chamber 12 when the lid is placed in position thereon. In this manner a relatively tight fit is accomplished to hold the gel medium 46 on Sponges 40 and further to inhibit evaporation during electrophoresis.
A template 52 as seen in FIGURE 4 is used to locate test sample well formation in the gel support medium within lid 20. Template 52 is illustrated with a plurality of openings 53 through which a suitable well drilling tool may be passed to form the wells.
FIGURE 5 shows, in an enlarged fragmentary manner, a portion of the lid 20 in place upon the chamber 12. The foil electrode 34 is shown folded over the sponge wick 40 with foil tab 36 in its non-operable position. The foil electrode 34 as provided with the unit 10 lines the inner side wall and bottom of each of the buffer vessels 24 and 26 and is adapted to be folded outwardly and pass between the top surface of the wall 44 of lid 20 and top of the wall of chamber 12 (as shown in FIGURE l0). Upon folding the foil 34 to operable position the sponge wick 40 contacts the buffered gel medium 46.
FIGURE 6 is a plan view of a modification of the electrophoresis test unit of FIGURE l and shows a lid 20 provided upon a chamber 56 having side walls 58 and end walls 60. The chamber 56 has integrally formed divider walls 62 and 64, as seen in FIGURES 7 and 8, which detine with walls 58 and 60 a pair of buffer vessels 66 and 68 in longitudinal spaced relationship therein. The walls 62 and 64 are vertically coextensive with the outer walls 58 and 60 of the chamber 56. Sponge wicks 40 are positioned within the buier vessels 66 and 68 to convey buier to a gel medium 46 which is contained in the lid 20. A Wire electrode 70 is provided through each opening 72 in the side walls 58. The lid 20, which is adapted for complementary interlocking engagement with the upper peripheral edge of the side walls 58 of chamber 56, is of similar construction to that described in reference to unit of FIGURE 3.
FIGURE 8 shows the lid 20 in position upon the chamber 56 and is a further view of integrally formed dividers 62 and 64 which separate chamber 56 into three compartments including the buffer vessels 66 and 68 and a vacant chamber 74 therebetween.
FIGURE 9 is a side view of the electrophoresis kit of the present invention showing an ice pack 76 positioned upon the top of the lid to serve as a coolant during electrophoresis.
FIGURE 10 is a view similar to FIGURE 5 and shows the foil electrode 34 in its folded out operable position whereby it resides between the complementary engaging wall surfaces of the lid 20 and chamber 56.
In order to electrophoretically separate materials by differential migration in an electrically conductive buffer system subjected to flow of current therethrough utilizing the disposable test apparatus of the present invention, the test sample, which may contain proteins, peptides, and amino acids, or like materials subject to electrophoretic particle separation, is deposited within a buffered support medium. The support medium described and illustrated herein is gel, although other known medias such as lter paper or cellulose acetate may be utilized. The gel, which may have been pre-poured by the supplier or alternatively prepared and poured by the user is contained within a lid 20 provided with the test kit of the present invention. The test sample is placed at predetermined positions in contact with the solid gel. If it is desired to place the sample in a predetermined location in the gel, a template 52 may be used to locate a well position wherein a suitable removal device is used to take gel from the lid 20 through openings 53 formed in the template 52. The bulered gel, as discussed above in the description of FIGURE 5 is maintained within the lid 20 by wall 44 and further by gel retainers 48. In this manner, when the test sample perfuses the solid gel, the lid is ready to be inverted and positioned upon the chamber 12. Since the butter vessels 24 and 26 are provided with wicks 40 which rest with their top portions substantially in vertical alignment with the upper edges of the chamber 12, the gel medium will rest in electrical contact with the buier saturated sponge wicks 40 with the lid 20 in place for operation. Prior to lid placement, a buier solution is poured over the wicks 40 in vessels 24 and 26. Since the gel medium in lid 20 is prebuffered, with the lid in place there exists a system or path of continuous buffer from one vessel 24 through the gel medium 46 to the second vessel 26, electively completing a circuit through switch 39. To initiate electrophoresis direct current is applied through the components of the system. This is accomplished by connecting the foil electrodes 34 which are provided within the buffer vessels 24 and 26 to a suitable current source 38. It will be understood that passage of current causes flow of buffer ion salts to migrate through the buffer-gel system. In operation heat is generated within the system as an incident to the imposition of direct current on the system. Such heat, if not adequate dissipated, would interfere with the proper function ofthe system. In FIGURE 9 ice pack 76 is positioned upon the unit 10 to dissipate heat generated within the chamber. It will be understood that any suitable coolant means may be employed to accomplish the above stated heat exchange objective. The coolant could be positioned in the vacant center chamber between the vessels.
The potential is applied through the system for a predetermined length of time, after which a suitable method of analysis is employed to locate and identify the electrophoretically separated constituents of the test sample. It will be understood that the direction and rate of particle migration are functions of the magnitude of the field, the charge on the particles, the pH of the buffer, and othei factors such as particle size, viscosity and the like. The term particles is used to denote material of extremely small dimension, e.g. molecules such as amino acids.
Thus is provided a relatively inexpensive, expendable electrophoresis apparatus which includes all of the necessary parameters for testing and is so constructed as to oier a compact unit which may include several gel lids with a single disposable electrophoresis chamber.
Having thus described the invention, what is claimed is:
1. An expendable electrophoresis test kit comprising:
a plastic chamber having means defining a pair of spaced buffer solution vessels therein,
means to position a buffered gel support medium between said buifer solution vessels and in electrical contact with the buffer solution in each vessel,
said last named means including a generally dish shaped removable plastic lid portion positioned upon said chamber,
means on the under surface of said lid for retaining said gel support medium,
means including electrodes within the buier solution vessels to provide passage of current through said buffer solution builered gel medium to permit charged particle migration within said gel medium for particle group identification therein.
2. An expendable electrophoresis test kit comprising:
a plastic chamber having a pair of spaced inner walls defining a pair of spaced buter solution vessels therein,
means to support a buffered gel medium bridge between said bui'rer solution vessels in electrical contact with the buffer solution in each vessel,
said last named means including a dish shaped plastic lid portion,
means including electrodes within the buffer solution vessels for passage of current through said buffer solution and buffered gel medium to provide charged particle migration within said gel medium, and a ledge formed upon said lid to provide for stacking an identical lid in nested relationship thereon to provide compact plurality of lids with a single base chamber.
3. An expendable electrophoretic test kit for particle migration within a buffered gel medium comprising:
a substantially rectangular open top plastic chamber,
a pair of longitudinally spaced plastic vessels removably positioned within the tank, each of said vessels provided to receive a butler solution therein,
an electrode within each of said vessels to afford electrical potential to the butler solution upon connection to a source of direct current,
a removable lid positioned in said chamber and adapted to hold a buffered gel support medium on its under surface, over and between said vessels in electrolytic contact with the buffer solution therein whereby passage of current within the system promotes electrophoretic particle migration of a test sample within the buiered gel support medium.
4. An expendable electrophoresis kit for particle 1migration within a bulered gel medium comprising: current ow therethrough comprising:
an open top plastic base chamber,
means within the chamber defining a pair of spaced plastic buffer vessels therein,
an electrode within each of the butter vessels and adapted for connection to a source of direct current,
buier carrying wick means provided within said vessels,
and a plastic lid removably positioned upon the chamber and including gel medium support means 0n its under surface for providing a buffered gel bridge between the wicks whereby imposition of potential between the vessels through the gel medium induces particle migration therein.
5. An expendable electrophoretic test kit for particle migration within a buffered gel medium comprising:
a substantially rectangular open top plastic chamber, a pair of longitudinally spaced plastic vessels removably positioned within said chamber and open at the upper portion thereof, said vessels provided to conhaving means for connection to a source of potenpotential to pass electrical current through the buffered solution in said buffer vessels,
.a plastic lid portion removably positioned upon said chamber,
a buffered solid gel support medium provided within said lid,
means formed within said lid portion to hold the buffered gel medium on its under surface over said vessels in electrical contact therewith whereby flow of current within the system promotes particle migration tain a buffered solution therein, 10 of a test sample within the buffered gel support meda foil electrode positioned within each of said vessels ium to provide electrical potential to the buffer upon con- 9. An expendable electrophoresis kit comprising: upon connection to a source of direct Current, a substantially rectangular open top plastic chamber, abuffered gel support medium, a pair of vertically extending lateral walls integrally a lid removably positioned to enclose said chamber formed adjacent each end of said chamber and lonand to receive and maintain said gel support medgitudinally spaced to define a pair of buffer vessels ium on its under surface over said buffer vessels in therein, said walls being vertically co-extensive with current passing relationship whereby passage of the outer walls of said chamber whereby said vescurrent through the system prOmOtCS ClCrOpllOresels are adapted to receive a buffer solution therein, tic particle migration of a test sample Within the a wick within each of said 'buffer vessels to hold the gel. buer solution, 6. An expendable electrophoretic test kit fOr particle a foil electrode provided within each of said vessels migration Within a buffered gel medium Comprising having means for connection to a source of potential a substantially rectangular open top plastic Chamber, to pass electrical current through the buffered solua pair of longitudinally spaced buffer vesels removably tion in said vessels,
Positioned Within said ehsrnher and oPen at the top a plastic lid portion removably positioned upon said and provded to contain a buffered solution therein, ehamber, a foil electrode positioned within each of said vessels a Ibuffered gel support medium provided within said to provide electrical potential to the buffer `upon conlid, nection to a source of current, means formed within said lid portion to hold the bufapOrOuS Wick Within each of Said Vessels, fered gel medium on its under surface over said abuffered gel support medium, vessels in electrical contact therewith whereby ow a lid removably positioned to enclose said chamber of current within the system promotes particle mi and t0 receive and maintain Said gel Support med- 3'; gration of a test sample Within a buffered gel Supium on its under surface over said buffer vessels in port medium and a template for predetermined test Passing relationship whereby Passage of Current sample well location within the gel support medium. through the system Promotes electrophoretic Per' 10. An expendable gel 'support retaining lid for an ticle migration of a test sample Within the gelelectrophoresis test chamber having a pair of spaced 7- An eXPendable electrophoresis kit Comprising: 40 buier vessels with electrodes therein comprising: a substantially rectangular open top plastic chamber, a substantially flat base portion, a Pair of vertically extending lateral Walls integrally a vertically extending wall formed at the periphery formed adjacent each end of said chamber and lon- Of the base portion, gitndinally sP' leed to detlne a Pair of butter Vessels gel support medium retaining means provided within therein, said walls being vertically co-extensive with the base te hold the gel within a lid upon inversion the outer walls of Said Chamber whereby said Ves' thereof for positioning the gel in electrical contact sels are adapted to receive a buffer solution therein, with the bulfer Vessels, and locking means on the a wick within each of said buffer vessels to hold the lid adapted to engage the Chamber to effectively enbuffer solution, close the chamber against evaporation therefrom a foil electrode provided within each of said vessels .during electrophoresis 11. A gel medium support retaining lid for an electrotial to pass electrical current through the buffered solution in said vessels,
a lid portion removably positioned upon said chamber providing a cover therefor to prevent evaporaphoress test chamber having a pair of spaced buffer vessels with electrodes therein to position the buffered gel support medium over land between the spaced buffer vessel comprising:
tion therefrom and means formed within said lid portion to hold a buffered gel medium over said vessels in electrical contact with said butter solution whereby ow of current through the system a substantially at base portion,
a peripheral wall extending vertically from said base portion to provide a generally dish shaped member,
gel retaining means integrally formed within said member, fastening means upon said wall to lock the lid upon the test chamber during electrophoresis and further seal the chamber minimizing evaporation therefrom. 12. In combination with an expendable electrophoresis chamber including a chamber having a pair of spaced buffer vessels and including electrode means within the vessels adapted for connection to a source of direct current, a gel medium support retaining lid to hold a buffered gel between the vessels in electrical contact therewith comprising:
a base portion having an upwardly extending peripheral wall, and of a configuration to cover the chamber, locking means formed upon said peripheral wall to hold the lid in position upon the chamber during electrophoresis to inhibit evaporation therefrom.
promotes particle migration of a test sample within 60 the gel support medium.
`8. An expendable electrophoresis kit comprising:
a substantially rectangular open top plastic chamber,
a pair of vertically extending lateral walls integrally formed adjacent each end of said chamber and longitudinally spaced therein to dene a pair of buffer vessels, said walls being vertically co-extensive with the outer walls of said chamber, said vessels further adapted to receive a buffer solution therein,
a wick within each of said buffer vessels to hold the buffer solution,
a foil electrode provided Within each of said buier vessels having means for connection to a source of means provided within the base portion to maintain solid gel in position thereon when said `base portion is in its inverted operable position upon the unit, said base portion having means formed upon the Wall defining a peripheral for receiving in stacked relationship an additional lid thereon.
References Cited UNITED STATES PATENTS 3,047,489 7/ 1962 Raymond 204--299 3,129,158 4/ 1964 Raymond et al 204-299 3,317,417 5/1967 Raymond 204-299 10 FOREIGN PATENTS 1,272,055 8/1961 France,
OTHER REFERENCES Smithies, An Improved Procedure for Starch-gel Electrophoresis: Further Variations in the Serum Proteins of Normal Individuals, in Biochemical Journal, vol. 71, No. 3, 1959, pp. 585-587.
10 JOHN H. MACK, Primary Examiner.
E. ZAGARELLA, Assistant Examiner.
Citations de brevets