ELECTROPHORETIC TECHNIQUE FOR SEPARATING HEMOGLOBIN VARIANTS AND IMPROVED ELECTROPHORETIC GEL FOR USE THEREIN

Abstract: An electrophoretic gel having a pH of from about 5.5 to about 6.5 and comprising (a) a polysaccharide selected from a group consisting of agar, agarose, and mixtures thereof; (b) a buffer; and (c) a composition selected from a group consisting of alginic acid salts and alginic acid esters. An improved electrophoretic technique for assaying the relative distribution of proteins of the type wherein at least one sample is applied to an electrophoretic gel and the electrophoretic gel is electrophoresed. The electrophoretic technique is characterized in that the above described electrophoretic gel is employed therein.

Description: ELECTROPHORETIC TECHNIQUE " FOR SEPARATING HEMOGLOBIN VARIANTS AND IMPROVED ELECTROPHORETIC GEL FOR USE THEREIN Background of the Invention 1. Field of the Invention The instant invention pertains to an electrophoretic technique for separating hemoglobin variants and to an electrophoretic gel for use therein. 2. Description of the Prior Art Electrophoretic techniques for separating hemoglobin variants and electrophoretic gels for use therein are well known to those skilled in the art (1- 5). In general, electrophoretic gels employed for separating hemoglobin variants are of the type comprising an agar and a buffer having a pH of from about 6 to about 6.5, the buffer being selected from a group consisting of citrate and ethylenediaminetetraacetic acid (EDTA). Problems present in prior art electrophoretic techniques for separating hemoglobin variants include (a) chevron band formation of hemoglobin (Hb) A, S, and C; (b) low resolution of HbS and HbC; and (c) dependence of migration distance between HbF and HbA on relative concentration. Accordingly, it would be very desirable to have an electrophoretic technique for the separation of hemoglobin variants wherein one or more of the above mentioned problems are either alleviated or removed. Summary of the Invention In accordance with the present invention there is provided an electrophoretic technique for separating hemoglobin variants wherein one or more of the above mentioned problems are either alleviated or removed. The electrophoretic technique of this invention is of the type wherein (a) at least one sample to be assayed is applied to an electrophoretic gel; (b) the electrophoretic gel of step (a) is electrophoresed; (c) the electrophoresed gel of step (b) is fixed; and (d) the fixed electrophoretic gel of step (c) is dried. The improved electrophoretic technique of the instant invention is characterized in that a novel electrophoretic gel is employed therein. The novel electrophoretic gel has a pH of from about 5.5 to about 6.5 and comprises (a) a polysaccharride selected from a group comprising of agar, agarose, and mixtures thereof; (b) a buffer; and (c) a composition selected from a group consisting of alginic acid salts and alginic acid esters. It is the presence of this composition in the electrophoretic gel which enables one to alleviate or remove at least one of the above-mentioned problems. 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 detailed description of the preferred embodiments. Description of the Preferred Embodiments Salts of alginic acid capable of use in the instant invention preclude, but are not limited to, the lithium, sodium, potassium, and ammonium salts thereof. Preferably, the alginic acid salt is sodium alginate. It is also preferred that the alginic acid salt have at most only a minimal calcium content. With respect to the alginic acid esters, it is preferred that such alginic acid esters be esterified with up to 80% propylene glycol. The polysaccharides which can preferably be employed in the electrophoretic gel of the instant invention is agarose. The agarose can be either low electroendosmosis agarose, medium electroendosmosis, or high electroendosmosis agarose. More preferably, the polysaccharide employed in the electrophoretic gel of the instant invention is low electroendosmosis agarose. Buffers capable of use in the present invention include, but are not limited to, adipic acid, citric acid, ethylenediaminetetraecetic acid, glutaric acid, itaconic acid, maleic acid, malic acid, malonic acid, succinamic acid, succinic acid, and tricarbalyllic acid. The electrophoretic gel of the instant invention can optionally further comprise a preservative agent. Typical preservative agents include, but are not limited to, antibiotics, halogenated organic compounds, and inorganic compounds. Readily available preservative agents capable of use herein are sodium azide and ethylmerurithiosalicylic acid, sodium salt. The electrophoretic gel of the instant invention can also optionally contain an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. Suitable alkylpolyols 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 constituents employed in the electrophoretic gel of the present invention are not critical. However, the electrophoretic gel of the instant invention preferably comprises from about 0.5 to about 1.2 percent low electroendosmosis agarose; from about 0.1 to about 1 percent sodium alginate; an effective amount of the preservative agent; and a stabilizing amount of the alkyl polyol. More preferably, the electrophoretic gel of the instant invention comprises from about 0.7 to about 1 percent low electroendosmosis agarose; and from about 0.5 to about 0.7 percent sodium alginates. Optimally, the electrophoretic gel of the instant invention comprises about 0.8 percent low electroendosmosis agarose; about 0.6 percent sodium alginate; about 0.01 percent ethylmereurithiosalicylic acid, sodium salt; 5 percent 1,2-propanediol and about 0.08 M buffer comprising 0.2 percent maleic acid and about 1 percent maleic acid, disodium salt. The electrophoretic gels of the instant invention can be prepared via any technique well known to those skilled in the art. 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 18° 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 syringe. The electrophoretic gels can be electrophoresed at 50 volts • for 30 minutes. If desired, the gels can be fixed in an alcohol.acetic acid mixture such as 60 percent reagent alcohol, 30 percent deionized water, and 10 percent glacial acetic acid. 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 intended to be limitations on the disclosed invention. Examples 1-6 Gels having the formulations set forth in Table I were prepared. Each gel also employed 0.05 M citrate and 5% 1,2-propanediol (a stabilizer). The following electrophoretic procedure was employed. 1. Fill each side of an electrophoretic cell with 45 ml of 0.05 M citrate buffer. 2. Gently blot surface of gel with a gel blotter. Align template on gel surface. Pipet 5 1 of sample hemolysate having a concentration shown in Table I onto each sample slot. Wait 5 minutes after last sample is applied, and remove excess sample with template blotter. Discard blotter. Remove and discard template. NOTE: To get comparable amounts of Hb to soak into gel, the Hb concentration must be increased in the agar containing gels due to the gels' increased viscosity. 3. Place gel onto gel bridge and into electrophoresis cell, matching + and - on bridge with those on the gel. Cover and guide into power supply. 4. Electrophoresis at 50 volts for 35 minutes. 5. Upon completion of electrophoresis, remove gel from the electrophoretic cell and place in gel frame, then in gel holder. 6. Place in fixative solution for 5 minutes. 7. Remove from fixative solution and dry at 70°C. for about 20 minutes. 8. Place dry gel in stain for about 3 minutes. Note: For very high viscosity gels (i.e., gels employed in Examples 4 and 6), a more sensitive stain is required. Violet and Blue stains are comparable. 9. Destain for about 3 nutes. 10. Blot gel firmly with a gel blotter. 11. Dry for about 5 minutes. 12. Visually observe patterns ' . A visual comparison of each of the gels of Examples 1-6 showed that in each case wherein alginate was employed, the HbA, HbS, and HbC bands consistently exhibited less chevron formation when compared to the HbA, HbB, and HbC bands on corresponding gels devoid of alginate. Based on this disclosure, many other modifications and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be comprehended as within the scope of this invention. Bibliography 1. Gratzer et al., J. of Chromatography, _5_:315-329 (1961). 2. Robinson et al., J. Lab & Clin. Med., 50:645-752 (1957). 3. Barwick et al., Biochimica et Biophysica Acta, 668:485-429 (1981). 4. Determination of Hemoglobin Variants (Citrate Agar), Corning Medical, Medfield, Massachusetts 02052. 5. Titan IV Citrate Hemoglobin Procedure, Helena Laboratories.

Claims: The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. An electrophoretic gel having a pH of from about 5.5 to about 6.5 and comprising: (a) a polysaccharide selected from a group consisting of agar, agarose, and mixtures thereof; (b) a buffer; (c) a composition selected from a group consisting of alginic acid salts and alginic acid esters. 2. The electrophoretic gel of claim 1 wherein said salt is selected from a group consisting of lithium, sodium, potassium, and ammonium, and wherein said alginic acid esters are esterified with up to 80% propylene glycol. 3. The electrophoretic gel of claim 1 wherein said gel is agarose. 4. The electrophoretic gel of claim 3 wherein said buffer is selected from a group consisting of adipic acid, citric acid, ethylenediaminetetraecetic acid, glutaric acid, itaconic acid, maleic acid, malic acid, malonic acid, succinamic acid, succinic acid, and tricarbalyllic acid. 5. The electrophoretic gel of claim 4 wherein said salt is selected from a group consisting of lithium, sodium, potassium, and ammonium, and wherein said alginic acid esters are esterified with up to 80% propylene glycol. 6. The electrophoretic gel of claim 5 wherein salt is sodium. 7. The electrophoretic gel of claim 6 further comprising an effective amount of a preservative agent. 8. The electrophoretic gel of claim 6 further comprising a stabilizing amount of an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. 9. The electrophoretic gel of claim 6 comprising: (a) from about 0.5 to about 1.2% low endoelectroosmosis agarose; (b) from about 0.1 to about 1% sodium alginate; (c) an effective amount of a preservative agent; (d) a stabilizing amount of an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. 10. The electrophoretic gel of claim 9 comprising: (a) from about 0.7 to about 1% low endoelectroosmosis agarose; and (b) from about 0.5 to about 0.7% sodium alginate. -lO- ll. An electrophoretic procedure of the type comprising: (a) applying at least one sample to a gel; (b) electrophoresing said gel of step (a); (c) fixing said electrophoresed gel of step (b); and (d) drying the fixed gel of step (c); characterized in that the gel is said electrophoretic gel of claim 1. 12. The procedure of claim 11 wherein said salt is selected from a group consisting of lithium, sodium, potassium, and ammonium, and wherein said alginic acid esters are esterified with up to 80% propylene glycol. 13. The procedure of claim 11 wherein said gel is agarose. 14. The procedure of claim 13 wherein said buffer is selected from a group consisting of adipic acid, citric acid, ethylenediaminetetraecetic acid, glutaric acid, itaconic acid, maleic acid, malic acid, malonic acid, succinamic acid, succinic acid, and tricarbalyllic acid. 15. The procedure of claim 14 wherein said salt is selected from a group consisting of lithium, sodium, potassium, and ammonium, and wherein said alginic acid esters are esterified with up to 80% propylene glycol. 16. The procedure of claim 15 wherein said salt is sodium. 17. The procedure of claim 16 wherein said electrophoretic gel further comprises an effective amount of a preserviate agent. 18. The procedure of claim 16 wherein said electrophoretic gel further comprises a stabilizing amount of an alkylpolyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. 19. The procedure of claim 16 wherein said electrophoretic gel comprises: * (a) from about 0.5 to about 1.2% low endoelectroosmosis agarose; and (b) from about 0.1 to about 1% sodium alginate. 20. The procedure of claim 16 wherein said electrophoretic gel comprises: (a) from about 0.7 to about 1% low endoelectroosmosis agarose; and (b) from about 0.5 to about 0.7% sodium alginate.

Inventors: GURSKE WILLIAM A, ULFELDER KATHI J, GURSKE, WILLIAM, A., ULFELDER, KATHI, J.
Applicants: BECKMAN INSTRUMENTS INC, BECKMAN INSTRUMENTS, INC.
Application year: 1985
Main classification: G01N27
IPC codes: G01N27, G01N33