|Publication number||US3317418 A|
|Publication date||2 May 1967|
|Filing date||28 Feb 1963|
|Priority date||28 Feb 1963|
|Publication number||US 3317418 A, US 3317418A, US-A-3317418, US3317418 A, US3317418A|
|Inventors||Juan Zec Zvonimir|
|Original Assignee||Beckman Instruments Inc|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (5), Referenced by (17), Classifications (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
z. J. ZEC 3,317,418
4 Sheets-Sheet l INVENTOR. ZVONIMIR JUAN ZEC 7 w O 1 A w f h I .FIJW M7.
May 2, 1967 ELECTROPHORESIS APPARATUS WITH ADJUSTABLE SAMPLE APPLICATION Filed Feb. 28, 1963 ATTORNEYS F/G M y 2, 1967 2. J. ZEC 3,317,418
ELECTROPHORESIS APPARATUS WITH ADJUSTABLE SAMPLE APPLICATION Filed Feb. 28, 1963 4 Sheets-Sheet 2 v I I I A: :Olto: :Q: :ozzo: :o::o::
ZVONIMIR JUAN ZEC ATTORNEYS z J. ZEC
May 2, 1967 ELECTROPHORESIS APPARATUS WITH ADJUSTABLE SAMPLE APPLICATION Filed Feb. 28, 1963 4 Sheets-Sheet 3 w GU INVENTOR.
ATTORNEYS mm J 4:
ZVONIMIR JUAN ZEC United States Patent o 3,317,418 ELECTROPHORESIS APPARATUS WITH ADJUST- ABLE SAMPLE APPLICATION Zvonimir Juan Zec, Palo Alto, Calif, assignor t Beckman Instruments, Inc., a corporation of California Filed Feb. 28, 1963, Ser. No. 261,762 18 Claims. (Cl. 204-299) This invention relates generally to zone electrophoresis apparatus.
In performing certain electrophoretic separations making use of an absorbent paper strip or other sample carrier, apparatus has been used comprising two electrolyte cells with means for supporting the strip with the ends dipping into an electrolyte. After the strip is saturated with a buffer solution having proper pH, a sample solution is applied to a small area of the strip. An electrical potential is applied to the strip to cause separation of components of the sample having different mobility, for example, of the various protein fractions of a serum sample. After a predetermined time, the strip is removed, dried, and the protein fractions fixed as, for example, by heat denaturation. A suitable dye is then applied to render the fractions visible. Variations in color density along the strip give indication, in general, of the concentration of protein fractions. Sharpness in the separations provides improved resolution. In some apparatus, several strips are disposed side by side so that several samples can be separated at the same time.
In applying sample to the carrier, the manner of presenting the sample to the surface and the accuracy of positioning the sample thereon is highly important to good results. Thus, the boundary definition of the applied sample is delicately controlled as by using apparatus of the kind described in US. Letters Patent 2,868,020. Where a line of samples is applied across several strips laid side by side, the point of application on each is preferably carefully maintained in registry for accurate comparisons. Differences in the speed and force with which the sample reaches the surface may introduce differences in the area of the sample.
Reliable results further require that the carrier be properly supported notwithstanding changing conditions of wetting to which the carrier is subject. in dipping the ends of the carrier into the electrolyte and removing them therefrom, much care is required to prevent uneven and excess wetting as well as contact with the walls of the cells.
Finally, the liquid level of electrolyte ought to be substantially equalized in the cells before a run.
In general, it is an object of this invention to provide improved apparatus of the above character.
A further object of the invention is to provide apparatus capable of precise, repeatable application of samples to a sample carrier.
Another object of the invention is to provide novel means for precision placement of several samples with respect to each other.
An additional object of the invention is to provide means for supporting a sample carrier in tension.
Still another object of the invention is to provide novel means for equalizing the liquid level between the electrolyte cells.
Additional objects and features of the invention will appear from the following description and claims in which a preferred embodiment has been set forth in detail in conjunction with the accompanying drawings.
Referring to the drawing:
FIGURE 1 is an exploded elevation View in section showing apparatus in accordance with the present invention;
FIGURE 1A shows a plastic plug in section for sealing apparatus of FIGURE 1;
FIGURE 2 is a plan view taken along the line 2-2 of FIGURE 1;
FIGURE 3 is an end view taken along the line 33 of FIGURE 2;
FIGURE 4 is a perspective view taken along the line 4--4 of FIGURE 1;
FIGURE 5 is a perspective detail view embraced by line 5-5 of FIGURE 1;
FIGURES 6A and 6B are elevation views of the detail identified by line 6-6 of FIGURE 1 to show two conditions of operation, respectively;
FIGURE 7 is a perspective view of a multi-strip carrier for receiving a plurality of samples; and
FIGURE 8 is a side elevation detail view of a portion of the detail embraced by line 8-8 of FIGURE 1.
The apparatus illustrated in the drawing consists of a container 10 which forms, in effect a base for the apparatus and which forms part of the separate compartments or electrolytic cells 11a and 11b. A central vertical partition wall 12 extends between the end walls 13 and is sealed with respect to both the end walls and the bottom 14 to define a common Wall for the cells 11a and 11b.
Each cell is preferably provided with a plurality of bafiles to isolate the region occupied by the electrodes 15 from the regions where electrical connection is being made to the depending ends of the carrier to be presently described. Thus, each cell is provided with spaced vertical baffles 21, which are sealed with the bottom and end walls at their edges. In addition, each cell is provided with baflles 22 which have their lower edges terminating short of the bottom 14. Bafiles 22 may also have their ends slidably engaged in grooves formed in the end walls 13, thereby permitting their ready removal. Electrodes 15 are connected by suitable means (not shown) to a source of electric potential and are disposed in the cells so as to be normally submerged in electrolyte.
Means for automatically initiating equalization of the liquid level in the cells is provided with respect to container 10. This structure is arranged whereby when a carrier supporting rack, to be presently described, is placed in position on the container 10, a previously established fluid path between the cells 11a and 11b is automatically interrupted.
Referring to FIGURES 6A and 6B, as well as FIG- URE 1, a U-shaped tube 24 is pivotally supported to straddle partition wall 12 with the arms 26 of the tube submerged in the electrolyte. The transverse connection portion 25 of tube 24 extends loosely through a block 27 secured to the top of partition wall 12. Mounted along the outside of arms 26 is a rib member 28 rounded at its upper end. A downward force applied to the rounded upper end of rib members 28 rotates the tube 24 counterclockwise as viewed in FIGURE 6B. In the raised posi tion, openings 29 face upwardly so that liquid is retained in the tube 24. Accordingly, member 24 serves to hold a suflicient amount of liquid so as to act as a siphon when lowered to its depending position with the openings 29 below the liquid level in cells 11.
Carrier support means are provided which may be readily detachable from the container 10 for supporting a sample carrier with extremities of the carrier depending toward the electrolyte cells. A suitable carrier acting as the electrophoretic sample support medium may comprise filter paper, difierent gels, cellulose acetate and/or similar carriers. The latter is believed to combine many of the advantages of both paper and gels and accordingly is preferable. Such a cellulose acetate membrane 31 is shown in FIGURE 7 formed from a single sheet of material. Membrane 31 includes a number of sample receiving strips 32 connected at their ends by laterally extending portions 33 of membrane 31. A plurality of of the detail holes 34a are located in portions 33. Protective guard" strips 32g which normally receive no sample are provided to prevent pattern distortion in the edgemost strips 32.
A carrier support assembly 30 includes a rack having a body portion 35 formed with a rectangular opening 54. Carrier support assembly 30 is dimensioned to be received into the open top of container and supported by the tops of baffies 22. The central portion of the carrier supporting rack is provided with an opening 54 disposed to pass over and thereby receive U-tube 2.4 in its raised condition. However, one edge 54a of opening 54 overhangs the rounded upper ends of rib member 28 so that attachment of the carrier support rack to container 10 automatically raises the distal ends of U- tube 24 out of the electrolyte to interrupt the siphoning operation. Conversely, detachment of the carrier support rack automatically serves to initiate equalization of the liquid level between the cells by returning the U- tube 24 into the electrolyte. It will be observed that as the U-tube 24 pivots in and out of the electrolyte, passages 29 being located on the upper side of U-tube 24, serve to retain sufiicient liquid in U-tube 24 to re-commence siphoning between the cells when the ends are lowered into the electrolyte. Accordingly, fluid transfer between the cells to equalize the level is automatically responsive to attachment and detachment of the carrier support rack.
Two groups of pins 34 are carried on the upper surface of the carrier rack body portion 35. One group of pins is formed to extend vertically from a stationary pin support member 36 screwed to body portion 35. The other group of pins is formed to extend vertically from a pin support member 37 pivotally mounted on an axis 38. Member 37 is spring biased to move to the right as viewed in FIGURE 1. Biasing is supplied by a spring 39 which is supported around a stud 40 and provided with arms 41 and 43. The end of arm 41 is bent downwardly to extend selectively into one of three holes 42 so as to adjust the tension in the spring. The other arm 43 of spring 39 is bent and adapted to be received in notch 44 formed in the side of member 37. As thus arranged, a sample carrier of the type such as shown in FIGURE 7 can be supported upon spaced pins 34 with a constant tension notwithstanding changes in the wetting and heating conditions thereof.
Upwardly extending handles 45 are provided along two sides of the carrier supporting rack. The other two sides carry substantially parallel side members 46 pivotally secured by studs 49 to body portion 35. Side members 46 are spaced from the body portion to form elongated slots 47 to receive the extremities of a carrier.
Means for guiding the carrier ends include pins 4% distributed along slot 47 and supported by the body portion 35. Outer guides 50 are carried by side members 46 and distributed therealong so that the ends of pins 48 and guides 50 nearly meet in a common plane. Thus, pins 48 and guides 50 are disposed to extend into slots 47 and are adapted to press against the end portions of the carrier when disposed therein. The distal end 51 of each side member 46 is provided with a recess 52 adapted to receive the head of a position limiting stud 53 so that the side members can first be pivoted upwardly as in FIGURE 3 to accommodate insertion of the end portions of the carrier and then to be moved to a closed position whereby the end portions of the carrier are firmly engaged by the pins and guided downwardly toward the electrolyte cells.
Applicator means 80 for transferring liquid sample to the carrier is supported in position above strips 32. The applicator means 80 includes a reciprocable member 64 having a sample retaining tip portion, described below. Placement of member 64 can be positively indexed to any one of several positions distributed either laterally or lengthwise of the carrier surface. The indexing means is 4 formed in a cover member 58 (FIGURE 4) of generally rectangular construction. It includes a top 59 and four depending sides 60. Cover member 58 is dimensioned to slip readily into a retaining rim 19 secured to the top of container 10. A rectangular recess 61 is formed in top 59 to provide an access passage permitting a liquid sample applicator to be manipulated with respect .to carrier 31. Recess 61 can be closed by a rectangular plug 18 (FIGURE 1A) whenever the applicator is not in active use. Plug 18, therefore, serves to prevent evaporation during an electrophoretic run. Three transverse slots 62 are defined between grates 63 which serve to prevent an attendant from inadvertently reaching in and touching the electrically energized carrier. The spacing between grates 63, therefore, is minimal commensurate with permitting access of a member 64, FIGURES l and 5.
Member 64 is arranged to be movable between retracted and advanced positions, whereby in its advanced position sample is transferred to carrier 31. Member 64 is arranged to reciprocate freely in a vertically disposed guide member 65. Guide member 65 is secured upright upon a base member 66 by a brace 67. Base member 66 has an upper surface 68 upon which is mounted a bracket 69 which pivotally supports a cocking lever 70 for movement about an axis 71. The operative end 72 of lever 70 engages a notch 73 formed in the side edge of member 64. Thus, lever 70 is adapted to raise member 64 vertically in its guides by downward movement applied to the righthand end of the lever as viewed in FIGURE 1.
A release lever 74 is also pivotally supported for move ment about axis 71 and provided with an operative end 75 constructed to engage a notch 76 in member 64. End 75 is urged counter-clockwise by a spring 77 retained in a recess formed in surface 68. Thus, end 75 serves as a detent for locking the member 64 in its retracted position as lever 70 is moved clockwise.
Means are provided for selectively adjusting the distance between advanced and retracted positions of member 64. As best shown in FIGURE 1, a U-shaped clamp member 78 embraces the sides of member 64 and the arms of the U are adapted to be pinched together by a knurled screw 79, threadedly engaged therebetween. Accordingly, member 78 is adapted to be selectively positioned vertically along member 64 whereby its downward travel is limited by contact with the upper end of guide 65.
The lower end of member 64, as shown in FIGURE 5, is constructed with an interchangeable tip portion of sample retaining element to retain a liquid sample with precise selected boundary definition defined by a pair of fine parallel strips 81 supported at their ends by rods 82. Rods 82 are releasably secured in vertically disposed notches 83 formed in the side edges of member 64. Liquid sample is deposited between strips 81 and retained therein by capillary forces. Other tip arrangements giving precise boundary definition are shown and described in the aboveidentified patent. Accordingly, it is to be understood that various tip arrangements can be substituted without departing from the invention.
Means are provided for positively indexing the reciprocable member (and the sample retaining tip portion) to transfer a sample to the carrier at any one of a plurality of positions distributed laterally of the surface of the elongated carrier. Accordingly, the undersurface 84 of base member 66 carries a depending tooth or detent 85 adapted to engage one of several grooves 86 formed in the top surface of cover member 58. Grooves 86 run in the direction of carrier strips 32 and are spaced apart in a direction running laterally of the carrier. Base member 66 is, therefore, adapted to rest upon cover member 58 with detent 85 engaged to retain the base member against relative sliding lateral movement.
Surface 84 further carries a cylindrically shaped fulcrum 87. To the right of fulcrum 87, surface 84 slopes upwardly toward surface 68. Fulcrum 87 is adapted to engage any of three transverse grooves 88 which are spaced apart in a direction running lengthwise of the carrier. It is to be noted that grooves 88 are spaced apart the same distances as slots 62 whereby disposition of fulcrum 87in one of slots 88 serves to locate element 64 in a positionally related one of slots 62.
Thus, if it is desired to release detent 85 from one of grooves 86 and slide the applicator assembly in a direction across the carrier, the right-hand edge of base member 66 can be depressed toward the cover member thereby raising detent 85 upwardly out of its groove. The fulcrum thus serves to permit release of detent 85, guiding of member 64 along slot 62, and by disposing the fulcrum in a selected one of grooves 88, provides indexing of member 64 to transfer a sample to the carrier at any one of three positions distributed lengthwise along the surface of the carrier.
Operation of the apparatus is as follows: Container is filled with a suitable electrolyte. At the time of initially filling container 10, U-tube 24 is also filled with electrolyte. The arms of U-tube 24 are permitted to hang downwardly below the liquid level in container 10 thereby serving to equalize electrolyte in cells 11 by siphon action. With side members 46 raised, carrier 31 is positioned over carrier support assembly 30. One column of holes engages their respective pins 34. The other column of holes can then be slipped easily over the second column of pins. Side members 46 are then lowered into position to form .slots 47 and firmly retain and properly direct the end portions of the carrier for dipping into the electrolyte.
The support assembly is then lowered onto container 10 and as edge 54a engages rib members 28, U-tube 24 is raised out of the electrolyte and received into opening 54 thereby automatically removing the fluid path between cells 11. Cover member 58 is then placed in position within rim 19 on container 10. Liquid sample is applied between strips 81 of member 64 and the latter cocked to its retracted position. The applicator assembly is then indexed to the selected position defined by positioning fulcrum 87 in one of grooves 88, and by locating detent 85 in a desired groove 86. "If it is assumed that a line of liquid samples is to be applied. across carrier 31, detent 85 will be located in one of the slots 86 nearest an edge of cover member 58. To release member 64, the righthand end of lever 74 is pressed downwardly, raising the other end 75 clockwise out of notch 76. As end 75 clears notch 76, member 64 falls by gravity to transfer sample to the carrier. Cooking lever 70 is then depressed to raise member 64 again to its retracted position, and base member 66 is rocked about fulcrum 87 and slidably moved to the next adjacent groove 86 where the operation is repeated. Thus, sample is applied to all strips 32 with the exception of guard strips 32g.
Should it be desired in a particular application, additional spring means can be provided so as to cause member 64 to be projected more rapidly toward the carrier. For instance, a spring can be disposed between a retaining ring on the bottom end of member 64 and undersurface 84.
It will be evident from the foregoing that there has been provided apparatus having many advantages over the conventional equipment previously described. The
manner in which a sample is presented to the surface of' the carrier is under a predictable control. The positioning of a sample on the carrier is quite precise, both for locating samples transversely of the carrier as well as lengthwise thereon. Accordingly, comparisons made between samples applied side by side are more reliable.
Furthermore, the carrier is properly supported at all times notwithstanding variations in conditions of wetting. The support of the carrier is further enhanced by the manner of engaging the extremities of the carrier whereby they are properly dipped into the electrolyte. And, finally it'will be evident that there is a complete and automatic termination of an equalizing fluid path between the electrolyte cells by merely attaching the carrier support rack to the container.
Although the apparatus has been described with reference to electrophoresis apparatus, it will be apparent that it may be used in other applications, for example, such as in chromatographic analysis.
Furthermore, while a preferred embodiment has been described, it will be understood that various omissions and substitutions by those skilled in the art may be made without departing from the spirit of the invention. The intention, therefore, is to be limited only as indicated by the scope of the following claims.
1. In zone electrophoresis apparatus having container means forming two cells for electrolyte, sample carrier support means carried by said container means and adapted to support an elongated sample carrier in position to receive liquid sample applied to same, a liquid sample applicator comprising a sample retaining element for transferring liquid sample to said carrier, said element being movable between retracted and advanced positions, said element serving in its advanced position to transfer said sample to said carrier, said element being disposed to be normally under forces acting to move same toward said advanced position, means for moving said element to said retracted position, detenting means for locking said element at said retracted position, and means for releasing said detenting means to free said element for movement to said advanced position to transfer liquid sample to said carrier.
2. Zone electrophoresis apparatus as defined in claim 1 further including means for selectively adjusting the distance between said advanced and retracted positions.
3. In zone electrophoresis apparatus having container means forming two cells for electrolyte, sample carrier support means carried by the container means and adapted to support an elongated sample carrier in position to receive liquid sample applied to same, a cover member supported by the container means and disposed generally to overlie the sample carrier, the cover member including an access passage therethrough generally overlying a sample receiving region of the carrier, a liquid sample applicator supported above said cover member for manipulation through the access passage to apply liquid sample to said carrier, said applicator including a member movable between retracted and advanced positions, a sample retaining element carried by the last named member for transferring liquid sample to the carrier, said element in its advanced position serving to transfer sample to said carrier, and means for positively i11- dexing said element to transfer sample to the carrier at any one of a plurality of positions distributed laterally of the surface of said elongated carrier.
4. Zone electrophoresis apparatus as defined in claim 3 wherein said applicator includes a base member adapted to rest upon the cover member, and further wherein the last said means serves to retain said base member and said cover member against relative sliding movement therebetween in a direction running laterally of the carrier, the last said means including spaced apart grooves formed in one of said base or cover members and a detent carried by the other for engaging said grooves.
5. Zone electrophoresis apparatus as defined in claim 4 further including means for releasing the detent to permit said base member to be slidably moved to another of said grooves.
6. Zone electrophoresis apparatus as defined in claim 5 wherein said detent releasing means comprises a fulcrum interposed between said base member and said cover member, said fulcrum extending in a direction leading laterally of said carrier, said applicator being supported to rock about the axis of said fulcrum to withdraw said detent from said grooves, whereby said sample retaining element can be indexed to transfer a sample to the carrier at any one of a plurality of laterally distributed positions.
7. Zone electrophoresis apparatus as defined in claim 3 further including means for positively indexing said sample retaining element to transfer a sample to the carrier at any one of a plurality of positions distributed lengthwise along the surface of said elongated carrier.
8. Zone electrophoresis apparatus as defined in claim 6 wherein one of said base or cover members carries said fulcrum and the other includes a plurality of grooves disposed and formed to selectively receive said fulcrum, said grooves being spaced apart in a direction running lengthwise of said carrier whereby said fulcrum serves to register said element in position to transfer a sample to one of a plurality of positions distributed lengthwise of said carrier.
9. Zone electrophoresis apparatus as defined in claim 8 wherein said access passage includes a grate having a plurality of elongated slots extending in a direction laterally of the carrier and spaced apart in a direction longitudinally of the carrier, said slots being respectively dimensioned and positioned to accommodate manipulation of said element through one of same with said fulcrum disposed in a positionally related one of said grooves whereby said grate is adapted to guard agansti inadvertent entry of the fingers of an attendant into electrically charged electrolyte.
10. In zone electrophoresis apparatus having container means forming two cells for electrolyte, and carrier support means carried by said container means for supporting a sample carrier above said container means with the extremities of the carrier depending toward the cells, said carrier support means including means continuously applying resilient longitudinal tension forces to said carrier tending to extend same in a predetermined direction whereby changes in the physical condition of said carrier are accommodated.
11. Zone electrophoresis apparatus as defined in claim 10 wherein the last said means comprises a group of fixed pins adapted to engage holes in one region of said carrier, and another group of pins adapted to engage holes in another region of said carrier, resilient means urging the last said group away from the first said group with a predetermined force to apply tension to a carrier engaged by both said groups of pins whereby changes in the physical condition of the carrier are accommodated.
12. Zone electrophoresis apparatus as defined in claim 11 wherein said resilient means includes means for selectively adjusting the predetermined force to vary the applied tension.
13. In zone electrophoresis apparatus having container means forming two cells for electrolyte, and carrier support means carried by said container means for supporting a sample carrier of the strip type above said container means with the extremities of the carrier depending toward the cells, said carrier support means comprising a rack having a body portion and two substantially parallel :side members carried by same and spaced therefrom to form elongated slots between said body portion and said :side members, said slots being adapted to receive the extremities of said carrier, pins disposed to extend into said slots and adapted to press against the end portion of a carrier supported by said rack, said side members being pivotally connected to said body portion for movement to a first position to accommodate insertion of said end portions, and to a second position whereby the end portions of a carrier are firmly engaged by said pins and guided downwardly toward the cells.
14. Zone electrophoresis apparatus as defined in claim 13 wherein a plurality of said pins are carried by said side members to engage one face of said carrier and a plurality of pins are carried in opposed relation thereto by said body portion to engage the reverse face of said carrier.
15. In zone electrophoresis apparatus having container means forming two cells for electrolyte, and carrier supportmeans readily detachable from and carried by said container means for supporting a sample carrier above said container means with the extremities of the carrier depending toward the cells, fluid level adjusting means automatically responsive to detachment of the carrier support means to initiate equalization of the liquid level between said cells, and automatically responsive to attachment of said support means to discontinue said equalization.
16. Zone electrophoresis apparatus as defined in claim 15 wherein said fluid level adjusting means includes siphon means automatically responsive to detachment of the carrier support means to form a fluid path initiating fluid transfer between said cells to equalize the liquid level therebetween, and automatically responsive to attachment of said support means to remove said fluid path.
17. In zone electrophoresis apparatus having container means forming two cells for electrolyte, a sample carrier comprising a single sheet of electrophoresis carrier material, said sheet being formed to include a laterally extending portion at each extremity and a plurality of elongated strips carried between said portions in laterally spaced relation, the ends of said strips being integral with said portions, the edgemost strips being disposed and adapted to receive no sample thereby serving as guard strips to prevent pattern distortion in the edgemost sample receiving strips, and carrier support means carried by said container means for supporting said sample carrier above said container means with the extremities of the carrier depending toward the cells, said carrier support means including means for applying constant tension to said carrier tending to extend same in a predetermined direction whereby changes in the physical condition of said carrier are accommodated.
18. In zone electrophoresis apparatus having container means forming two cells for electrolyte, carrier support means carried by the container means for supporting an elongate sample carrier above the container means with the extremities of the carrier depending downwardly toward the cells, wherein the carrier support means includes means for resiliently tensioning the carrier to extend the same in a predetermined direction whereby changes in the wetness of the carrier are accommodated, a sample carrier comprising a single sheet of electrophoresis carrier material, said sheet being formed to include a laterally extending portion at each extremity and a plurality of elongated strips carried between said portions in laterally spaced relation, the ends of said strips being integral with said portions, and the edgemost strips being disposed and adapted to receive no sample'thereby serving as guard strips to prevent pattern distortion in the edgemost samplereceiving strips.
References Cited by the Examiner UNITED STATES PATENTS 2,768,948 10/1956 McDonald 204-18O 2,834,729 5/1958 Pickles et al. 204- 2,868,020 1/1959 Williams 23-259 3,047,489 7/1962 Raymond 204299 FOREIGN PATENTS 525,981 3/1955 Italy.
G. BATTIST, E. ZAGARELLA, Assistant Examiners.
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