CA1129806A - Microelectrophoretic apparatus and process - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Disclosed is a cellulose acetate membrane as an article of manufacture, which is divided into at least two strips by at least one slot, each strip having been impregnated with a substrate and color-forming reagent specific to one polymorphic protein system from the group consisting of lactic acid dehydrogenase (LDH), alkaline phosphatase (AP), creatine phosphokinase (CPK), erythrocyte acid phosphatase (EAP), glucose-6-phosphate dehydrogenase (G-6PD), adenylate kinase (AK), group specific component (Gc), lipoprotein (1p), adenosine deaminase (ADA), 6-phosphogluconate dehydrogenase (6-PGD), glutamic pyruvic transaminase (GPT), esterase D (EsD), and others. Also disclosed is a microelectrophoretic process for the simultaneous separation and identification of the components of two or more protein systems.

Description

~%~

This application is a division of Canadian pa-tent application Serial No. 314,426 filed October 26, 1978.

The invention relates to ~el trays and membranes pre-impregnated with developing reagents, which can be used ~n carrying out new clinical and medico-legal processes that have been rendered practical by recently developed microelectrophoretic equipment.

In a co-pending patent application by the present inventor, Canadian application No. 290,310, filed on November 7, 1977, there are disclosed several improvements of microelectrophoretic apparatus which gr~atly simplify and standardize the microelectrophoretic process and which include:
(a) designs that allow the use of either a membrane or a tray with the same basic apparatus; (b) designs which allow the two-dimensional resolution of a p~otein sample placed on a gel-filled tray; and (c) a sample applicator and accessory equipment which permit the simultaneous and precise positioning of, for instance, ten samples.

This apparatus can be used advantageously with various conventional devices and techniques which include compartmentalized gel trays, as disclosed by Siebert et al in United States patent No. 3,616,387 (Figure 5 and column 3, lines 53 to 55) and slotted membranes, as disclosed by Zec in United States patent No. 3,317,418 (Figure 7). Also, when used in conjunction with the further refined articles that are disclosed in the present application, it makes available to research scientists and labora-tory technicians new and improved methodology for separation of spe~ific proteins from microliter quantities of blood. These specific proteins are then identified through use of immunologic techniques or, more commonly, through use of indicator dyes which chemically unite ~lZ98V6i with one specific type of protein and no other. These dyes make contact with the electrophoresed samples by means of an overlay, either cellulose acetate membrane upon gel, or gel upon cellulose acetate membrane. The overlays have been pre-impregnated with the appropriate specific substrate so tha~ a permanent visual record of the pattern i5 produced on the cellulose acetate membrane, whether the membrane is serving as the supporting medium or as the overlay. This technique is described by Grunbaum in 'tAn Automatic One-to-Eight Sample Applicator for Fast ~ualitative and Quantitative Microelectrophoresis of Plasma Proteins ...", Microchem, J. 20, 495 - 510 (1975).

This apparatus is useful in research and application in the fields of Medicine, Immunology, Genetics, Biochemistry, and Forensic Science. Electrophoretic procedures which greatly increase in utility with this apparatus include determinations of significant polymorphic enzyme systems such as lactic acid dehydrogenase (LDH), alkaline phosphatase (AP~, and creatine phosphokinase (CPK). It can be used for diagnostic purposes through determination of specific antibodies of antigens in ihe blood. In the forensic laboratory, it can be used in the pheno-typing of genetic variants of enzymes and other proteins in blood for the purpose of identification or individualization.

A partial list of the factors in blood that can be determined using this apparatus include the following:

lactic acid dehydrogena~e (LDH) alkaline phosphatase (AP) creatine phosphokinase (CP~) erythrocyte acid phosphatase ~EAP) glucose-6-phosphate dehydrogenase (G-6PD) adenylate kinase (AK) 298~

hemoglobin ~Hb) haptoglobin (Hp) group speciEic component (Gc) lipoprotein (I.p) adenosine deaminase (ADA) 6-phosphogluconate dehydrogenase (6-PGD) Çlyoxylase I (GLO I) glutamic pyruvic transaminase (GPT) esterase D (EsD) Glutat~Lione reductase (GsR) Immunoglobulins (Ig) Methodology for the phenotyping of additional systems is being developed.

The invention in one aspect pertains to a microelectrophoretic process for the simultaneous separation and identification of the components of two or more protein systems.
The process comprises applying an unknown protein sample of a protein.system different in nature or in origin and a standard protein sample for the system selected to each compartment of a shallow rib-divided tray containing a suitable gel, subjecting the samples so applied to electrophoresis, overlaying upon the sample-carrying gel in the tray a membrane divided into.strips by slots so placed that they accept the dividing ribs of the tray in a manner such that close contact is established and maintained between the surface of the gel and the.surface of the membrane, allowing the proteins distributed on the gel to react with suitable.specific.substrates and color-forming reagents `~ pre-impregnated on each strip of the membrane in an arrangement corresponding to that of the protein systems located in the individual compartments of the gel, and removing and drying the ~l~L2~ 6 membrane to prod~lce a composite electrophoretogram.

The invention also comprehends, as an article of manuacture, a cellulose acetate membrane divided into a-t least two strips by at least one slo-t, each strip having been impregnated with a substrate and color-forming reayent speci~ic to one polymorphic protein system from the group consisting of lactic acid dehydrogenase ~LDH), alkaline phosphatase (AP), creatine phosphokinase (CPK), erythrocyte acid phosphatase (EAP), glucose-6-phosphate dehydrogenase (G-6PD), adenylate kinase (AK), group specific component (Gc), lipoprotein (lp), adenosine deaminase (ADA), 6-phosphogluconate dehydrogenase (6-PGD), glutamic pyruvic transaminase (GPT), esterase D (EsD), and others.

More particularly the slotted membranes are pre-impregnated with specific substrates for the visualization of proteins electrophoresed on gel trays. These membranes may be provided with an underlying coat of tough polyester and thus constitute, after use, a permanent storable electrophoretogram.

~This invention provides specialized gel tray and cover assemblies containing suitable gels for electrophoresis and, in separate embodiments, one or more specific substrates for the visualization of electrophoresed proteins. Said trays are preferably divided into several compartments by parallel ribs rising from their flat bottom surface for use in simultaneous analysis of several parameters.

The trays and membranes described have been designed for use with recently developed improved electrophoretic apparatus disclosed in the above identified co-pending patent application.

By means of such equipment it is now possible, inter _ ~ _ g~

alia, to accurate'y and rapidly carry out simultaneous electro-phoretic separations of several polymorphic protein systems of different origin or of different na~ure, either on a gel or on a membrane, and ~o develop visual records of the patterns produced by contacting the electrophoresed samples with either a membrane or a gel, as the situation requires, said membrane or gel haviny been pre-impregnated with suitable speci~ic substrate for rendering the electrophoresed protein systems visible.
The availability of the new prepackaged gels and membranes, both for elec~rophoresis and, when pre-impregnated with specific substrates, for developing the electrophoresed proteins, will allow a great degree of standardization between widely separated laboratories. Such an improvement in the art, while being certainly welcome in the medical diagnostic field, is obviously invaluable in mass phenotyping studies that can be used in genetic research as well as for identification purposes, forensic or other.
In tAe drawings:

~GURES 1 (A to J, excluding I) is an exploded view in transverse section showing two embodiments of the basic ap-paratus used in this invention: on the left, with a membrane and a membrane bridge, and on the right, with a gel dish and d gel temperature control plate.
FIGURE 2 is a transverse sectional view of an assembled apparatus using a me~brane and a membrane bridge.
FIGURE 3 is a front elevation of the tank of Figure 1 assembled with a gel dish and a tempera~ure control plate.
FIGURES 4A to 4D show an exploded front elevation view of the apparatus in ~igure 2 which comprises a membrane and a membrane bridge.

FIGURE 5 is a side elevation of a temperature control plate for use with a gel tray.
FIGURE 6 is a detailed view of the applica~or tip shown in Fi~ure 1.
FIGURE 7 is a plan view of a compartmentalized ~ray.
FIGURES 7A and 8 are elevated transverse sections of the compartmentalized gel tray of Figure 7 and of a gel ~ray without dividers, respectively.
FIGURE 9 is a plan view of a snap-on tight-fitting lid lG for the gel trays of Figures 7 and 8.
FIGURES 10 and 11 show front elevation transverse sections of a compartmentalized gel tray and a sliding cover adjacent to it.
FIGURES 12 and 13 show front elevation transverse sections of another type of compartmentalized gel tray and of a sliding cover adapted to it.
FIGURE 14 is a front elevation transverse section of an assembled tray and cover pair, with the tray containing a gel over which a slotted membrane is superimposed.
FIGURE 15 is a plan view of a four-compartment dish containing a gel and a superimposed slotted membrane.
FIGURE 16 is a front elevation transverse section of a dish like that of Figure 15 assembled with a sliding cover.
FIGURE 17 is a plan view of a slotted sheet of filter paper impregnated with various developing systems for electrophoresed proteins.
FIGURE 18 is a plan view of another embodiment of the slotted sheet o Figure 17 consisting of a polyester base coated with cellulose acetate.
FIGURE 18A shows a cross section of the sheet of Figure 17.

9~ 6 FIGURE 19 shows part of a developed electrophorogram on which three protein systems have been separated in the presence of two different concentrations each of appropriate protein standards.
FIGURE 20 is a plan view of a square tray Containing precast gel mixed with ampholines or ~se in electroocusing, appearing with Figures 17, 13, 18A and 21.
FIGURE 21 is a plan view of a square tray filled with a precast gel for use in electrophoresis of haptoglobin-hemoglobin complex, or other proteins, ap~ing with Figs. 17, 18, 18A &20.
FIGURE 22 is an enlargement of the area of the trayof Figure 21 in which are located the sample-receiving cavities in the gel.
FIGURE 22A shows the area of the tray illustrated in Figure 22, but fitted with a removable and disposable plastic slot template.
FIGURE 22B is a front elevation transverse section of the tray-template assembly of Figure 22A.
FIGURE 23 is a plan view of a tray with precast gel, designed for cross-over electrophoresis, appearing with Fig. 19.
The apparatus of the above identified application shall first be described in reference to Figures lA to lF, 2, and 4, for an embodiment involving a membrane system, and to Figures lA, lB, lE to lJ, and 3, for another embodiment involving a gel.
These figures shall be discussed simultaneously in order to avoid repetition and to provide a clearer visualization of the equipment.
With respect to the membrane system, Figures 1 and 2 show transverse sections of the apparatus--in exploded and assembled views, respectively, while Figures 4A to 4D, on the other hand, give an exploded front elevation view of the same 1~2~
components.
The apparatus comprises a tank (lO) which contains an electrolyte solution (not shown). Within the tank are fixed baffles (19), removable baffles (24), septum (23) and electrode frames (26, 28) which extend from slots in inner wall (8) to slots in inner wall t9). A membrane holder (14) is seated on septwn (23) and two of the removable baf~les (24). Channel ~20) of membrane holder (14) straddles the top of septum (23). The membrane holder (14) is provided with tab grips (21, 22) and teeth (18). The teeth (18) engage perforations in a membrane (16) and keep the central portion of the membrane taut. The ends of membrane (16) are immersed in the electrolyte solution (not shown). The membrane must be made from a material that will "wick" the electrolyte solution to all areas of the membrane and keep the membrane saturated. Further, the membrane must have sufficient strength to withstand the force of the teeth when the membrane is wet. The membrane may, for example, be made from cellulose acetate, paper, or Cellogel. A cellulose acetate membrane can be kept and stored as a permanent record of the analysis.
An applicator assembly (30) fits onto the cover ~12).
The applicator assembly is fitted with two feet (33, 34). The foot (34) has a registration pin (38) projecting from it, and the foot (33-) has a runner bar ~35) projection from it. The pin (38) and runner ~35) are adapted to fit into a runner bar slot and registration pinholes on either the coverplate (12) or a sample holder.
The cover (12) includes male and female connectors (ll, 13) which make contact with the electrode wire (2~) through spring interlocks (15 and 17, respectively) on the electrode ~g~
frame (26). When the cover (12) is removed from the top of the tank (10), the electrical connection to the electrode wire ~29) is broken by means of the spring interlocks. The electrode wire (29) is shown in detail in Figure lB. A platinum wire ~29) is run around the slotted periphery of the electrode fr~me ~26).
The wire is connected to metal contacts ~25 and 27), which complete a circuit to spring interlocks ~15 and 17) shown in Figure 2. As shown in Figure 4D, two electrode frames (26 and 28) are placed in the tank ~10) which has slots in its walls to receive the frames.
Referring now to Figures lF and 4A, the applicator assembly comprises a plurality of applicator buttons ~39) which are adapted to hold an applicator shown in detail in Figure 6.
The applicator buttons ~39) are held in place by means of a plurality of leaf springs ~45) which hold each button individually in place. A release button ~40) is provided with a long arm (41) which extends across all of the parallel leaf springs ~45). When the button ~40) is depressed downward, all of the leaf springs are releases such that all of the applicator buttons (39) are free to drop by force of gravity. The release button ~40) is fitted with a groove ~42), such that, when it is in the depressed position, a locking bar ~44) is able to slide into the groove ~42) and hold the release button in the lower position. When the applicator (30~ is not in use, a protective lid ~not shown) is placed over the opening in cover ~12).
In Figure 6, the applicator tip (46) is shown in more detail. The applicator tip includes a capillary opening (47~ for holding the sample fluid. The applicator tip (46) is held in place by two spring-loaded split pins (49), such that the tip is easily removed.

~12~6 The applicator tip (46~ may be modified in lenyth or width; for example, to vary the amount of sample held, or to cover more than one applicator position.
Referring to Figures lD and 4C, the membrane holder (14) is shown in detail. The membrane holder is made of one piece o~
mclded flexible plastic. The holder is i-tted with teekh (18) and can be bent inwardly, such that the teeth (18) grip corresponding perforations in a membrane (16). When released, holder (14) applies tensile force to the membrane and main-tains it taut. To avoid tearing the membrane, teeth (18) are perferably semicylindrical projections or cylindrical projections, and membrane performations (113), Figure 19, are circular.
The gel electrophoretic system can be visualized by reference to Figures lA, lB, lF to lJ, 3 and 5, which together show an exploded transverse section view and a front elevation view of the assembled components (Figure 3). In this system membrane (16) and membrane holder (14) have been replaced by gel tray or dish (103), gel temperature control plate (80) and blotter paper strip (107).
Temperature control plate (80) is fitted with four retainers, two of which are shown (82, 84) for receiving and holding in place a square dish or tray (103) containing the gel material (104), such as agarose gel, that may be employed for lipoprotein separation. A temperature control liquid is circulated in the plate (80) from liquid supply (99) by means of inlet (100) and outlet (102), as shown in Figure 5.
In Figure 3, the temperature control plate (80) is shown in place within the tank (10). The square tray (103) holding gel (104) is placed on plate (80) and is kept in fixed position by retainers (82, 84). Tray (103) is preerably made . .=. . . ~

3L~2~

from a material that is an electrical insulator and a good thermal conductor. Addi-tionally, the tray must be inert to the electrolyte. As the plate is adapted to recei~e a square tray, 90 rotation of -the gel media is possible. Greater resolution can be obtained by performing two migrations on the gel (104).
First, the sample is pulled apart in a linear path by the electric current. The gel tray is then rotated 90 and the first migration is pulled apart from an orthogonal direction.
Contact with the electrolyte solution is made by means of wicks (106, 108) which rest on edge of the gel and pass down through wick recesses (not shown) in plate (80) and into the electrolyte solution. The tank ~10) is provided with wick-retaining members (110 and 111) for receiving the lower end of each wick and holding the ends in place within the electrolyte solution. The wick-retaining members prevent the wicks from sliding off the gel and align the wicks so that they contact the gel (104) evenly across the surface. The wick alignment prevents a contact gradient from occurring. Wicks (106, 108) may be made, for example, from filter paper or plastic sponge.
Du~ing the electrophoresis process, the electric current flowing through the gel (104) causes generation of heat in the ~el. The thermal convection in the gel tends to broaden the bands and cause errors due to poor resolution. This band broadening is alleviated by passing a liquid through plate (80) which has a temperature lower than the ambient temperature. For some measurememts, for example, a plate temperature of 4C has been found suitable. Blotter strips (i~7) made of the same material as the wicks (106, 108) and impregnated with electrolyte solution are placed along the edges of the surface of the gel (104) to facilitate electrical contact between the ~lZ~ 6 gel and the wicks during electrophoresis.
The remaining figures illustrate various tray and lid embodiments (Figures 7 to 16 and 20 ko 21A) and membrane embodiments (Figures 17 to 19) which are the subject of the present invention and which can be used with the equipment already described to perform the diagnostic and idenki~ica~ion processes that shall be disclosed below.
In Figure 7, there can be seen a plan view of a novel electrophoresis tray or dish (120) which is essentially a shallow square container comprising a flat surface (128) surrounded by a peripheral wall (122). A number of parallel straight dividing ribs (121) separate the dish surface area into compartments which are permanently labelled by having one letter printed in each compartment t160). The peripheral wall may be provided with a top ridge (123) which forms a shoulder (129) for accommodating a similar peripheral ridge on a lid. The compartmentalized dish (120) just described can be seen in elevated cross section in Figure 7A. In Figure 8, on the other hand, there is shown again in elevated cross section, an embodiment of a conventioral gel tray or dish (103), filled with a layer of gel (104).
Figure 9 illustrates one type of lid for gel trays (103, 120), consisting essentially of a thin flat sheet (127) provided with a peripheral ridge (126) adapted to fit tightly within peripheral ridge (123) on shoulder (129) o gel trays (103, 120). An assembled tray and lid system is shown in Figure 14, which comprises tray (120), lid (125), gel layer (104~ and slotted membrane (95) shown in elevated cross section.
The tray and snap-on lid embodiments described in Figures 7 to 9 and 14 may further be provided with various conventional means for fastening the assembly more tightly and for stacking.
Figures 10 to 13 show further embodiments of the trays and lids of the present invention wherein either -the lid (135) slides into a track ~ormed by the walls of the tray (liO), or the tray tl40) slides into a track ormed by the walls o~ the lid ~145). Effective sealing of the assembled systems can be achieved in a number of ways. For example, both the tray (140) and the lid (145) can be provided with one end wall (not shown), perpendicular to the track walls (131, 132) and at opposite ends, so that the ends of the assembly be closed. Also, flat horizontal surfaces (not shown), or shoulders, may be provided in the end wall areas of the trays which can fit closely with the lid surfaces and form additional seals.
Figure 15 shows a tray in plan view which is similar to that of Figure 14, except that the tray has only three dividing ribs (121). Circular holes (113) are designed to engage teeth (18) of membrane holder (14) shown in Figures lD and 4C. In -~
this drawing and in the elevated cross section of the tray shown in Figure 16, there can be seen a four-compartment tray (130) comprising three dividing ribs ~121), which has been covered in Figure 16 by sliding lid (135) between tracks (131). The compartments are filled with a gel (104) on which electrophoresis of various blood enzyme and protein systems has been run and a slotted membrane (95) comprising four strips marked by letters A, B, C, and D (160) separated by slots (96), has been super-imposed upon the gel in order to contact the prot~ins on the gel with the enzyme substrates or color reagents with which the membrane has previously been impregnated. In this manner, a blood serum sample containing the isoenzymes of the lactic acid dehydrogenase (LDH) system has been applied to the gel (104) in 8~
compartment A of tray (130), and, after electrophoretic system separation the gel has been contacted with strip A of membrane (95) previously impregnated wit.h the tetrazolium dye and other conventional components used to develop visible electrophoretograms from this particular enzyme sys-tem.
Similarly, the other compartments and strips (B, C, D) were used to detect and identify the components of different blood polymorphic enzyme or other protein systems either from the same blood sample or -from samples of different origins. In this manner, as shown in the drawing of Figure 15, creatine phosphokinase (CP~), alkaline phosphatase (AP) and total protein were run and developed simultaneously in the remaining compartments an~ strips (8, C, and D), respectively.
Figures 17 and 18 show plan views of parts of two different slotted membranes preimpregnated with the substrates or other color producing reagents required by the protein systems indicate~ on the drawings.
Thus, Figure 17 shown five strips labelled, A, B, C, D, and E (160) of a membrane (115) separated by slots (96) which extend to continuous border surfaces (116). The entire membrane (115) can be made of ~ilter paper with border areas (116) impregnated with paraffin and the strips each impregnated with the substrates or other developing reagents required by the enzyme and protein systems indicated, namely LDH(A~, CPK(B), plasma protein(C~, hemoglobin(D), and any other system--XYZ(E).
After development, the dried membrane can be preserved as a permanent replica of the electrophoretogram.
An impermeable type of membrane is shown in Figures 18 and 18A. Again, the membrane (136~ is divided into strips by slots (96) with each cellulose acetate strip ~Ar B, C, D) 298(36 impregnated with a particular enzyme substrate and/or color developing system as indicated, namely CPK, LDH, phosphogl~comu-tase (PG~), and any other system (XYZ~ on strips A, B, C, and D, respectively. One feature o~ this particular membrane, however, is that the cellulose acetate strips ~138) are carried by an inert, resistan-t and non-porous sheet of polyester ~137) such a~
Myla ~ polyester, as can be clearly seen in Figure 18A, a cross-sectional elevation view of the membrane o Figure 18.
Other polymeric substances such as polyamides, polyimides, polyethylene, halogenated polyethylenes and the like, can be used as inert non~porous support instead of polyester, if desired.
Figure 19 illustrates the type of pattern that may be seen on a membrane after separation and color development.
Three strips (200, 201, 202~ of a membrane (95) are shown, separated by slots (96). Each resulting compartment of strip has three columns labelled A, B, and C, (160). On each strip has been applied a different polymorphic blood en~yme system sample which was subsequen~ly caused to electrophoretically migrate away from the original application point, i.e., the first bar(I) below position A, to yield the bars appearing at distances II, III, IV, V, and VI. Standard preparations of the polymorphic systems are run with each unknown sample in two different concentrations (B, C~ to assist in the identification and quantification of the unknown samples. In comparing the components of the unknown in column A of section 200 with its neighboring B a.ld C columns, it becomes evident that the level IV
component of the sample appears in higher concentration than normal (B, C). So does the level VI component of the sample in section 202~ For the sample in 201, on ~he other hand, it ~2~
can be se~n that the level V component is missing, while a level VI component appears which is not present in the standard preparation (B, C). This then is the type of one-sheet simultaneously prepared record that can be made with the equipment and processes of this invention, identifying and quantifying up to 10 different or similar protein s~stems from one blood sample or from up to ten blood samples. With such profiles, the task of diagnosing several clinical conditions or identifying classes of indivi~uals, for example, is greatly simplified due to the quantity of information which can be prepared and observed simultaneously on one electrophoretogram.
Figure 20 is a top plan view of the basic square tray or dish ~103) earlier disclosed, filled with a polyacrylamide gel (104~ into which there are dispersed ampholines, i.e., the type o~ isomers of polyaminopolycarboxylic acids conventionally used for the high resolution electrofucusing process. Upon application of an electric field by means of special electrodes (not shown) placed directly on the surface of the gel, a linear pH gradient is formed and protein molecules electrophoresed in such a gradient concentrate in very narrow zones in which the net electrical charge on a given molecule is zero. In such a system, the only variable affecting the separation is the isoelectric point of a given protein. No buffer need be placed in the tank ~10) of the basic apparatus ~Fig. lA). Cooling of the gel with plate 80 (Fig. 3 and 5) is essential since a very high voltage is applied which creates a high amperage and consequently a large quantity of heat which must be dissipated instantaneously. The present apparatus allows the electrodes to be applied in any direction. Furthermore, it offers a simple 30 easily standardized alternative to the difficult preparation of ~2~8~
gels immediately before use, an alternative that is superior in terms of handling, transport ancl storage, to the fle~ible sheets heretofore available commercially.
Figure 21 is a top view o a tray (103) containing a precast gel (104) in which a line of ten rectangular sample-receiving cavities (148) have been made. The par~ of -the -tray containing the sample-receiving cavities ~148) is illustrated on a large scale in Figure 22 in order to show sufficient detail to facilitate visualization of the tray-template assembly shown in top view of Figure 22A and in cross section, in Figure 22B.
In these figures, it can be seen that peripheral wall (122) of tray (103) is provided with two cylindrical recesses (149) designed to accept pins ~151) which serve to fasten a removable and disposable plastic insert or template ~150) to the tray The template shown ~15Q) is provided with ten tongues (154) which serve to shape gel cavities (148) when the gel (104) is first poured into the tray (103), and to keep the cavities free of liquid between the time of fabrication and use. In other words, the tray with precast gel shown in Figure 21 is prepared by affixing insert or template ~150) to an empty tray ~103) by inserting the pins ~151) in recess (149). A liquid gel making preparation is then poured into the tray to form gel tlO41 upon cooling, and the resulting assembly closed by a suitable cover (not shown), can be shipped or stored without deterioration until needed for electrophoresis. At that time, the plastic insert or template (150) is removed, and protein samples and standards can be placed in the gel cavities (148) for electrophoresis. The type of tray just described is used mainly for the genetic typing of polymorphic proteins such as those from the haptoglobin-hemoglobin complex, but can be used for the separation o~ many protein mixtures requiring the relative].y high resolution medlurn of acrylamide gel. The polyacrylamide gel in the tray can have either a fixed cencentration of polymer throughout, or the concentration may var~ to produce either linear gradient or a step gradient.
The polyacrylamide molecules act as a mechanical sieve, aiding both separation and resolution.
Figure 23 shows a square tray with precast gel designed to employ specific antisera in cross-over electro-phoresis for the analysis of Australian antigen (infectioushepatitis), syphilis, and other infections diseases. The tray and the method can also be applied to species identification in the investigation of fresh or dried blood or other physiological fluids. As can be seen from the drawings, the precast gel tray (103) is basically similar to that of Figure 21, except that the gel sheet (104) is provided with three pairs of lines of ten cavities (148) each, said lines being labelled 1, 2, 3, 4, 5, and 6 ~160). The trays are prepared in the same manner as that of Figure 21, exGept of course that six plastic templates are used instead of the single one (150) employed for the tray of Figures 21 to 22B. The template pins (151, Figures 22A and 22B) snap into cylindrical recesses ~149) and the tray is then filled with gel (104) as earlier described. Before use, the templates are removed, leaving open cavities (148~ for receiving antigen and antiserum samples. The gel cavities are used in pairs, with the antigen sample being pl~.ced in the cavity located on the electrically negative side of the pair (rows 1, 3, and 5), as indicated by the minus sign (-) printed on the le~thand portion of the tra~'s peripheral wall (122), while the specific antisexum sample is 8~6 placed in the cavity located in the positive side of the pair (rows 2, 4, and 6). When voltage is appl.ied to the up to 30 samples in the tray, the antigen and antiserum proteins migrate toward each other and react upon crossing-over or meeting to create a visible line of precipitin. Nine such lines (205) ca~ be seen in the drawing, having formed between certain pairs only. The absence of a precipitin line betweerl a pair of cavities indicates the absence of the specific antigen in the sample that was placed in the lefthand c~vity of that pair.

It should be noted that in the preparation of the various precast gel trays and membrane described so far, it is possible to delay addition of any unstable substrate and reagents to the precast gel or membrane until the moment of use.
In the case of membrane, however, some of the unstable ingredients may be safely incorporated at the time of membrane manufacture by resorting to the technique of freeze drying.
A better realization of the possibilities of the equipment and processes of the present invention may be obtained by reference to the medical, genetic and forensic literature for review of the polymorphic protein systems already mentioned and assessment of the information -that they can reveal.

Claims (8)

1. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
   
   l A microelectrophoretic process for the simultaneous separation and identification of the components of two or more protein systems, comprising:
   (a) applying an unknown protein sample of a protein system different in nature or in origin and a standard protein sample for the system selected to each compartment of a shallow rib-divided tray containing a suitable gel;
   (b) subjecting the samples so applied to electrophoresis;
   (c) overlaying upon the sample-carrying gel in the tray a membrane divided into strips by slots so placed that they accept the dividing ribs of the tray in a manner such that close contact is established and maintained between the surface of the gel and the surface of the membrane;
   (d) allowing the proteins distributed on the gel to react with suitable specific substrates and color-forming reagents preimpregnated on each strip of the membrane in an arrangement corresponding to that of the protein systems located in the individual compartments of the gel; and (e) removing and drying the membrane to produce a composite electrophoretogram.
2. 2. The process of Claim 1 wherein the electrophoresis is carried out on the slotted membrane and the specific substrates and color-forming reagents are in the gel.
3. 3. The process of Claim 1 wherein the gel compartments carry identical protein systems of different origin and the membrane is impregnated with the one substrate and color-forming reagent combination required by said system.
4. 1. The process of Claim 1 wherein at least two protein systems to be determined are selected from the group of polymorphic enzyme systems consisting of lactic acid dehydrogenase, alkaline phosphatase and creatine phosphokinase.
5. 5. The process of Claim 4 wherein at least one additional protein system is determined, said additional system being selected from the group consisting of hemoglobin and total blood protein.
6. 6. The process of Claim 5 wherein further additional polymorphic protein systems to be determined fill all the compartments of a ten-compartment tray, said additional system being selected from the group consisting of phosphoglucomutase, erythrocyte acid phosphatase, glucose-6-phosphate dehydrogenase, adenylate kinase, esterase D and adenosine deaminase.
7. 7. As an article of manufacture, a cellulose acetate membrane divided into at least two strips by at least one slot, each strip having been impregnated with a substrate and color-forming reagent specific to one polymorphic protein system from the group consisting of lactic acid dehydrogenase (LDH), alkaline phosphatase (AP), creatine phosphokinase (CPK), erythrocyte acid phosphatase (EAP), glucose-6-phosphate dehydrogenase (G-6PD), adenylate kinase (AK), group specific component (Gc), lipoprotein (lp), adenosine deaminase (ADA), 6-phosphogluconate dehydrogenase (6-PGD), glutamic pyruvic transaminase (GPT), esterase D (EsD), and others.
8. 8. The slotted membrane of Claim 7 consisting of a polyester base layer coated with cellulose acetate in the reagent-containing strip areas.