US 3378481 A
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Description (OCR text may contain errors)
April 16, 1968 c. A. SARAVIS E L 3,378,481
BIOCHEMICAL TEST PLATE Filed Jan. 5, 1966 2 Sheets-Sheet 1 April 1968 c. A SARAVIS ET AL 3,378,481
BIOCHEMICAL TEST PLATE 2 Sheets-Sheet :3
Filed Jan. 5, 1966 United States Patent 3,378,481 BIOCHEMEQAL TEST PLATE Calvm A. Saravis, 110 Evelyn Road, Waban, Mass. 02168, and Herbert Goldsmith, 238 Congressional Lane, Rockville, Md. 20852 Filed Jan. 3, 1966, Ser. No. 518,159 Claims. (Cl. 204-299) The present invention relates to the subject matter of copending application Ser. No. 484,40i1, filed Sept. 1, 1965, by Herbert Goldsmith.
More specifically, the present invention relates to an apparatus for carrying out certain semiqualitative, semiquantitative biochemical electrophoretic reactions, notably, microelectrophoresis and immunoelectrophoresis. The reactions are carried out on preformed sheet media such as microporous films, e.g., microporous cellulose acetate. For general background on electrophoresis, immunoclectrophoresis, and microelectrophoresis, reference is made to the widespread literature available thereon.
For further understanding of the present invention, reference is now made to the attached drawing wherein:
FIG. 1 is an exploded view of the test plate assembly;
FIG. 2 is a perspective view of the underside of template;
FIG. 3 is a diagrammatic view of the assembled test plate ready for use;
FIG. 4 is a side view taken along line 4-4 of FIG. 3; and
PEG. 5 is an enlarged fragmentary section on the lines 55 of FIG. 3.
As shown in the drawing the assembled immuuodiffusion apparatus or plate comprises a face member or template with a smooth planar upper and lower surface 12, 14; an elastomeric pad and a back-up plate or base fill. Conveniently, template 10 and back-up plate or base may be constructed from transparent, preferably autoclavable materials, such as polycarbonate resins. Acrylic resins (Lucite) have proven particularly suitable, even though they are not autoclavable. Still other transparent, (or translucent resins) like polypropylene, polyethylene, styrene, etc. may be employed.
It may be seen from the drawing that lower surface 14 of template 10 is on a boss 11 formed on the underside of template 10 by removal of the lower peripheral margins of template 19 leaving a peripheral cut away portion 13. Correspondingly, back-up plate 39 is provided with a rectangular recess 31 of dimensions corresponding to boss 11, and is just enough larger so that the raised marginal edge portion 33 on back-up plate 30 will clear boss 11.
Referring now to FIGS. 3, 4 wherein is shown the asscmbled apparatus, a microporous film (e.g., microporous cellulose acetate) lies beneath template surface 14 and coextensive therewith. Underlying microporous film St} is a water impermeable film 60, e.g. polyvinylidine chloride (Saran), polyethylene, Paraiilm, etc.
Elastomeric pad 20 dimensioned to fit tightly inside rectangular recess 31 is disposed therein. It may be noted how the height of pad 20 is less than the depth of recess 31 and pad 20 does not, therefore, clear the marginal edge portion 33 of base 30. Suitably, pad 20 is a rubber of 20-30 durometer hardness. Neoprene has been found particularly suitable. Correspondingly the depth of boss 11 is sufficient to enter recess 31, press against pad 2!} as is shown in FIG. 4, and leave a gap at the side margins of template 1t and back-up plate 30.
While electrophoresis reaction tests carried out in microporous films are often more sensitive than those done in conventional semi-solid media such as agar, at the same time there arises need for completely reproducing the physical conditions of the plate from test to test. The desirability of maintaining microporous film 5t) smooth is self-evident. Also desirable is maintenance of film 50' under uniform conditions of compressive stress.
The present apparatus provides a structure which maintains films 5t], 6t) smooth between elastorneric pad 2t"; and the lower surface 14 of template 1% under uniform compressive stress. After assembly (as shown in FIG. 3) the various components are secured together by a multiplicity of threaded bolts 40, eight being illustrated. Appropriate threaded openings 42 are provided in marginal side portions 33 of base plate 30 with matching smooth bore apertures being provided in template it above the cut-away side marginal portion 13. Desirably, the bolts are formed of autoclavable material such as nylon, etc.
Upon assembly the bolts 4!) are tightened to a predetermined torque level (with conventional metered wrenches), 4 /2 in.-bs. being recommended. As can be readily appreciated, the compressive stress applied by the tightened bolts is transmitted through the rigid template material and through the rigid base plate material across the area of contact between these members. Compression of compliant pad 20 ensures uniform distribution of this compressive stress to films 50, 60.
Since the bolts 49 are always tightened to the same predetermined torque level, electrophoretic tests (using the same reagents and microporous film) are reproducible. Such reproducibility is important. Frequently the biochemical reactions between an antigen and an antibody are so sensitive and complex that they are affected materially by variations in the stress level across film 50 even to the point where it becomes diflicult to distinguish substantive differences in test results from run of the mill expected deviations attributable to experimental technique and film condition.
A plurality of spaced apart apertures 49 penetrate through template 10 from the upper planar surface 12 to lower planar surface 14. Each of the apertures 49 comprises a cup shaped depression 61 extending partly through the material of template 10 and a bore 63 extending from the base of cup shaped depression 61 axial ly thereof through the remainder of template 10 to lower planar surface 14. Also present are reactant Wells 27 which may also be a cup shaped depression, or alternatively, cylindrical and terminating in a cone and a bore 63 extending axially thereof through to a trough 25 on the underside of template 10 connecting two reactant wells 27.
The apertures 49 form a column of apertures which extends parallel to sponges '75, 76 and the liquid electrode slots 57, 59 hereinafter described. The column of apertures is displaced from axial symmetry toward the cathode side of the apparatus. Reactant wells 27 are disposed in pairs one on each side of the aperture column with trough '25 connecting each 27 of a pair. The troughs 25 extend perpendicular to the aperture column, and in each instance passes between individual apertures 49. As shown in FIG. 1, there are therefore six apertures and five troughs 25.
Experience has shown that in :all events the sample apertures 49 should be off center so to speak as shown in the drawing, being displaced toward the cathode.
Troughs 25 extend perpendicular to side marginal edges 53, of template 10 almost but not quite to a pair of liquid electrode slots 57, 59 (FIGS. 2, 4). These liquid electrode .slots 57, 59 are cut or otherwise formed in the underside of template 10 just inside from the marginal edges of boss 11 and parallel thereto, and extend up past boss 11 into the main body of template 10. At their upper terminus each slot 57, 59 is in open communication with a buffer port 65, 67 on the upper surface of template 10. Removable vial caps 77 seal elf the bufi'er ports 65, 67 and the liquid in electrode slots 59, 57. Extending laterally inward from side marginal edges 53, 55 of template '10 are wick slits 69, 71. A single elongated wick slit may be used at each side edge or, as in the preferred embodiment shown, several small wick slits pierce the side edge. In any event, Wick slit 69 is in open communication with liquid electrode slot 59 and Wick slit 71 is in open communication with liquid electrode slot 57. A wick 73 of suitable construction is disposed in each wick slit. Sintered hydrophilic polyethylene serves well for the wick material.
Wicks 73 extend past the side marginal edges 53, 55 of template so that they may be embedded into sponges 75, '76 disposed adjacent the assembled apparatus and in contact with the side edges 53, 55 thereof. Thereby a good electrical path is provided from sponge 75 through wick 73 in slit 71 into the liquid electrode in slot 57. Conversely, the same path exists for current from the liquid electrode in slot 59 through the wick 73 in slit 69 into sponge 76.
When template 10, films 50, 60, pad 2t) and a back-up plate 30 are assembled with film 50 in contact with lower surface 14 of template 10 in the manner shown in FIG. 4 of the drawing, and the entire assembly secured with bolts 49 (torqued up to a predetermined stress, e.g., 4 /2 in.-lbs.), film 60 serves to insulate and protect resilient pad from contact with the carrier and the reactants.
The entire assembly can then be placed into a split chamber 97 of any suitable construction such as is shown in FIG. 3 wherein the assembly rests on a stand and sponges 75, 76 dip down respectively into the liquid of the cathode compartment and the anode compartment. Appropriate electrical connections 98, 99, as usually provided for electrophoretic apparatus, are present (i.e. rnicroelectrophoresis, immunoelectrophoresis, zone electrophoresis). Wicks '73 are pressed into the liquid saturated sponges 75, 76.
Thereafter the buffer, e.g., barbital buffer, ionic strength 0.05, pH 8.2 is charged into each reactant well 27 and into each sample aperture 49. To draw liquid buffer throughout the system in an air free manner, an ordinary syringe fitted with a needle punctures the rubber cap 77, and suction applied until liquid buffer is drawn up into the buffer port. The buffer thereby fills slots 57 and 59. The syringe application of suction is repeated at the other buffer port. Then lambda quantities of the test samples (e.g. blood protein) may be placed into sample apertures 49, and the samples forced down into the microporous film, by a rubber bulb placed over aperture 49. The current is applied (e.g. 150 volts) for about 20 minutes, then turned off.
Following electrophoresis, reactants (e.g. antiserum) are placed in troughs through apertures 27, and the two materials are allowed to diffuse laterally through the membrane or film 50. The immunoprecipitin reactions which occur between the samples and the antiserum are revealed subsequently by staining the film in known manner.
Exemplary materials of construction are: Plexiglas (acrylic resin) for the templates 10 and back-up plate 30; neoprene for pad 20; cellulose acetate microporous film (Millipore Celotate) for film (Parafilm) for film L 60; nylon bolts; sintered polyethylene wicks; and cellulose sponges.
It will be obvious to those skilled in the art that various changes may be made without departing from the spirit of the invention and therefore the invention is not limited to what is shown in the drawings and described in the specification, but only as indicated in the appended claims.
What is claimed is:
1. A biochemical electrophoresis test plate comprising: a planar template containing an ordered plurality of spaced apart apertures and reactant wells therethrough, said template having a central boss on the underside thereof; a microporous film underlying said template at the central boss thereof; a resilient pad essentially coextensive with the boss; a back-up plate containing a central recess into which said resilient pad and said central boss fit closely, the height of said pad being less than the recess depth; and attachment means associated with the marginal edge portions of the back-up plate surrounding said recess and with the marginal edge portions of the template surrounding the boss for securing together the test plate, the microporous film then being under a substantially uniform state of stress, said template further having an elongated liquid elect-rode slot adjacent each of two opposing side marginal edges and parallel thereto, the slot extending up through the central boss into the main body of said template, at least one port extending from the upper surface of the template through to each slot for filling the slots with liquid electrode solution: at least one side slit extending from the slot through to the side marginal edge of the template adjacent thereto; a wick in each slit, the wick being dimensioned overlength for the slit and extending outward of the side marginal edge of the template, whereby an electrically conductive path exists for current flow from one side marginal edge of the template successively through a wick, a liquid electrode slot, the microporous film, the other liquid electrode slot and wick associated therewith to the opposing side marginal edge of the template.
2. The apparatus of claim 1 wherein on each side a sponge is pressed against the wick and the side of the template, the wick being thereby forced into the sponge to create a good electrical connection therewith.
3. The apparatus of claim 1 wherein caps are provided to cover the port openings in the template surface and thereby the liquid electrode slots.
4. The apparatus of claim 1 wherein the ordered plurality of spaced apart apertures are disposed as a column of apertures parallel to the liquid electrode slots, said column being displaced from axial symmetry toward the cathode side of the apparatus.
5. The apparatus of claim 4 wherein the reactant wells are disposed in pairs one on each side of the aperture column, each pair being connected by a reactant trough on the underside of the template, the reactant trough extending perpendicular to the aperture column.
No references cited.
MORRIS O. WOLK, Primary Examiner.
R. M. REESE, Examiner.