Background of the Invention 1. Field of the Invention The instant invention pertains to an electro- phoretic tec nigue for separating lactate dehydrogenase isoenzymes and to an electrophoretic gel for use there- in. 2. Description of the Prior Art Electrophoretic techniques for separating lactate dehydrogenase (LD) isoenzymes and electrophor- etic gels for use therein are well known to those skilled in the art. Cawley, Electroporesis and Iπununo- electrophoresis, Little, Brown and Company, Boston, Mass. (1969). In general, electrophoretic gels em- ployed for separating LD isoenzymes are of the type comprising a polysaccharide. A buffer having a basic pH is also commonly present in these electrophoretic gels. Typical polysaccharides employed in prior art electrophoretic gels include, but are not limited to, starch, cellulose acetate, agar, agarose, and co bina- tions thereof.

1 Typical buffers having a basic pH employed in

2 prior art electrophoretic gels include, but are not

3 limited to, the basic pH buffers which are set forth in

4 Table I of Cawley, supra, pp. 331-332.

5 One problem present in a basic prior art

6 electrophoretic technique for separating LD isoenzymes

7 is that, as shown in Figure 1, the symmetry of the D ₁

8 band does not correspond to the other four LD bands in

9 that there is a shoulder or bump at the leading edge of

10 the LD, band.

11 Accordingly, it would be very desirable to 12 _. have an electrophoretic technique for the separation of

13 ^' ID isoenzymes wherein the symmetry of the LD, band is

14 improved to correspond to the other four LD bands.

15 Summary of the Invention

16 In accordance with the present invention ^*

17 there is provided an improved electrophoretic technique

18 for separating LD isoenzymes wherein the symmetry of

19 the LD, band is improved to correspond to the other

20 four LD bands. The electrophoretic- technique of this

21 invention is of the type wherein a sample to be assayed

22 is applied to an electrophoretic gel and the electro-

23 phoretic gel is electrophoresed. The improved electro-

24 phoretic technique of the instant invention is charac-

25 terized in that a novel electrophoretic gel is employed

26 therein. This electrophoretic gel is of the type co -

27 prising a polysaccharide. A buffer having a basic pH

can optionally be present therein. The electrophoretic gel is characterized in that it further comprises either an acid polysaccharide and the salts thereof, wherein the acid moiety thereof comprises at least one carboxyl group and/or a galatomannan polysaccharide. The acid polysaccharide as well as the salts thereof and the galatomannan polysaccharide, when incorporated into the electrophoretic gel either separately or in comination, enables one to obtain an LD, band having improved symmetry. . Still other features and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following de- tailed description of the preferred embodiments. Brief Description of the Drawings Figure 1 is a scan of an LD isoenzyme pattern showing a shoulder or bump at the leading edge of the LD, band. Figure 2 is a scan of an LD isoenzyme pattern showing a greatly reduced shoulder or bump at the lead- ing edge ^' of the LD, band. Figure 3 is a scan of an LD isoenzyme pattern showing a greatly reduced shoulder or bump at the lead- ing edge of the LD, band. Figure 4 is a scan of an LD isoenzyme pattern wherein the shoulder or bump is completely eliminated from the leading edge of the LD, band.

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Description of the Preferred Embodiments Acid polysaccharides capable of use in the instant invention include, but are not limited to, ara- bic acid, tragacanth acid, kahya acid, alginic acid, pectic acid, and linseed acid. The preferred acid polysaccharide is arabic acid. Salts of acid polysaccharides capable of use in the instant invention include, but are not limited to, the sodium, potassium, calcium, and magnesium salts thereof. Examples of such acid polysaccharide salts include, but are not limited to, arabic gum acid, tragacanth gum acid, khaya gum acid, alginic gum acid, pectic gum acid, and linseed gum acid. Galactomannan polysaccharides capable of use in the instant invention include, but are not limited to, guar gum and locust bean gum. The preferred galac- tomannan polysaccharide is guar gum. Polysaccharides which can preferably be em- ployed in the electrophoretic gel of the instant inven- tion are agar and agarose. The agarose can be either low electroendosmosis agarose, medium electroendos- mosis, or high electroendosmosis agarose. More pre- ferably, the polysaccharide employed in the electro- phoretic gel of the instant invention is high electro- endosmosis agarose. Preferably, the buffer employed in the in- stant invention has a pH of about 7 to about 10. More preferably the buffer has a pH of about 8 to about 9.

The electrophoretic gel of the instant inven- tion can optionally further comprise a preservative agent. Typical preservative agents include, but are not limited to, antibiotics, halogenated organic com- pounds, and inorganic compounds. One readily available preservative agent capable of use herein is sodium azide. The electrophoretic gel of the instant in- vention can also optionally contain an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. Suitable aklypolyols which can be used herein include, but are not limited to, ethylene glycol, propanediol, butanediol, pentanediol, and glycerol. Preferably, the alkylpolyol has 2 to 4 carbon atoms. The exact concentrations of the various con- stituents employed in the electrophoretic gel of the present invention are not critical. However, in one embodiment of the instant invention, the electrophore- tic gel preferably comprises from about 0.4 to about 1.5 percent weight/volume high electroendosmosis aga- rose; from about 0.01 to about 2 percent weight/volume arabic acid; up to 20 percent volume/volume ethylene glycol; up to 6.5 percent weight/volume sodium azide; and a buffer having a pH of from about 7 to about 10. More preferably, the electrophoretic gel of this em- bodiment of the instant invention comprises from about 0.7 to about 1.2 percent weight/voolume high electro-

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endosmosis agarose; from about 0.5 to about 1 percent weight/volume arabic acid; from about 1 to about 10 percent volume/volume ethylene glycol; from about 0.05 to about 0.15 percent weight/volume sodium azide; and a buffer having a pH of from about 8 to about 9. Opti- mally, the electrophoretic gel of this embodiment of the instant invention comprises about 1 percent weight/volume high electroendosmosis agarose; about , 0.75 percent weight/volume arabic acid; about 5 percent volume/volume ethylene glycol; about 0.1 percent weight/volume sodium azide; and a buffer having a pH of about 8.2 and comprising about 0.3 percent weight/- ^' volume aspartic acid; about 0.4 percent weight/volume bicine; about 0.3 percent weight/volume sodium barbi- tal; and about 0.4 percent weight/volume 2-amino-2- methyl-l,3-propanediol. In another embodiment of the instant inven- tion, the electrophoretic gel preferably comprises from about 0.4 to about 1.5 percent weight/volume high elec- troendosmosis agarose; from about 0.001 to about.2 per- cent weight/volume guar gum; up to 20 percent volume/- volume ethylene glycol; up to 0.5 percent weight/volume sodium azide; and a buffer having a pH of from about 7 to about 10. ^' More preferably, the electrophoretic gel of this embodiment of the instant invention comprises from about 0.7 to about 1.2 percent weight/volume high electroendosmosis agarose; from about 0.01 to about 0.1

percent weight/volume guar gum; from about 1 to about 10 percent volume/volume ethylene glycol; from about 0.05 to about 0.15 percent weight/volume sodium azide; and a buffer having a pH of from about 8 to about 9. Optimally, the electrophoretic gel of this embodiment of the instant invention comprises about 1 percent weight/volume high electroendosmosis agarose; about 0.05 percent weight/volume guar gum; about 5 percent volume/volume ethylene glycol; about 0.1 percent weight/volume sodium azide; and the above described buffer. In a third embodiment of the instant inven- tion,. the electrophoretic gel preferably comprises from about 0.4 to about 1.5 percent weight/volume high elec- troendosmosis agarose; from about 0.01 to about 2 per- cent weight/volume arabic acid; from about 0.001 to about 2 percent weight/volume guar gum; up to 20 per- cent volume/volume ethylene glycol; up to 0.5 percent weight/volume sodium azide; and a buffer having a pH of from about 7 to about 10. More preferably, the elec- trophoretic gel of this embodiment of the instant invention comprises from about 0.7 to.about 1.2 percent weight/volume high electroendosmosis agaroe; from about 0.5 to about 1 percent weight/volume arabic acid; from about 0.01 to about 0.1 percent weight/volume guar gum; from about 1 to about 10 percent volume/volume ethylene glycol; from about 0.05 to about 0.15 percent weight/-

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volume sodium azide; and a buffer having a pH of from about 8 to about 9. Optimally, the electrophoretic gel of this embodiment of the instant invention comprises about 1 percent weight/volume high electroendosmosis agarose; about 0.75 percent weight/volume arabic acid; about 0.05 percent weight/volume guar gum; about 5 percent volume/volume ethylene glycol; about 0.1 per- cent weight/volume sodium- azide; and the above de- scribed buffer. The electrophoretic gels of the instant in- vention can be prepared via any technique well known to those skilled in the art. See, for example, Cawley, supra. In general, the gel solution is prepared by mixing the various ingredients present therein while heating the mixture to a temperature of about 80 to about 100°C. The electrophoretic gel can be prepared by either standard molding or casting techniques. The gels can be stored at any convenient temperature, for example from about 2° to about 40°C, preferably from about 15° to about 26°C. It is preferred to store the electrophoretic gels in sealed, plastic trays until ready for use. Samples can be applied to the electrophoretic gels of the instant invention via any technique used in the prior art, e.g., via -a microliter syrings. The electrophoretic gels can be electrophoresed at 100 volts for 20 minutes. The gels are next incubated at

an appropriate temperature, e.g., room temperature to about 50°C, for a convenient period of time, e.g., for up to about two hours, with any known substrate capable of reacting with the LD enzymes present therein. If • desired, the gels can be rinsed in an acetic acid solu- tion (5%). In addition, the gels can optionally be dried at about 80 to about 90°C. The following examples are provided for the purpose of further illustration only and are not in- tended to be limitations on the disclosed invention. Example 1 The four electrophoretic gel formulations set forth in Table I were each employed in the following protocol in order to demonstrate the improved electro- phoretic technique of the instant invention for separ- ating LD isoenzymes and the improved electrophoretic gel for use therein. The sole difference between the four electrophoretic gels employed in this comparative experiment was that the electrophoretic gels within the scope of this invention contained either arabic acid, i.e., a specific acid polysaccharide, and/or guar gum whereas the electrophoretic gel outside of the scope of this invention was devoid of both an acid polysac- charide and a galactomannan polysaccharide. Protocol Electrophoretic Procedure 1. A control serum was applied to each gel via a template technique.

2. Gels were electrophoresed at 100 volts for 20 minutes. 3. A standard colorimetric LD substrate was applied to the gels and each gel was incu- bated at 45°C. for 30 minutes. 4. Gels were soaked in 5% acetic acid and dried at 80-90°C. 5. Gels were scanned in a densitometer at 600nm. The results obtained from the above protocol for each gel formulation of Table I are shown in Figures 1-4, respectively.

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TABLE I .

Gel Outside Electrophoretic Gels Scope of Invention Within Scope of Invention

Formulation

Ingredients

1% wt/v HE Agarose X X X

0.75% wt/v Arabic Acid X X

0.05% wt/v Guar Gum X X

0.1% wt/v Sodium Azide X X X X

0.3% wt/v Aspartic Acid X X X X

0.4% wt/v Bicine X X X X

0.3% wt/v Sodium Barbital X X X X

0.4% wt/v 2-Λmino-2- methyl-l ,3-propanediol X X X

\

I

As shown in Figure 1, an electrophoretic technique for separating LD isoenzymes employing a prior art electrophoretic gel devoid of both an acid polysaccharide (wherein the acid moiety thereof com- prises at least one carboxyl group) and a galactomannan polysaccharide yields an LD isoenzyme pattern having a significant shoulder or bump at the leading edge of the LD, band. In contrast, an electrophoretic technique for separating LD isoenzymes employing any one of three different embodiments of this invention's improved electrophoretic gel greatly reduces this shoulder or bump on the leading edge of the LD, band. Based on this disclosure, many other modifi- cations and ramifications will naturally suggest them- selves to those skilled in the- art. These are intended to be comprehended as within the scope of this inven- tion.