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WO1982002500A1 - Improved electrophoretic gel for separating isoenzymes - Google Patents

Improved electrophoretic gel for separating isoenzymes Download PDF

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Publication number
WO1982002500A1
WO1982002500A1 PCT/US1982/000023 US8200023W WO8202500A1 WO 1982002500 A1 WO1982002500 A1 WO 1982002500A1 US 8200023 W US8200023 W US 8200023W WO 8202500 A1 WO8202500 A1 WO 8202500A1
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Prior art keywords
volume
alkylpolyol
amount
present
electrophoretic gel
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Ceased
Application number
PCT/US1982/000023
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French (fr)
Inventor
Instruments Inc Beckman
William Allan Gurske
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Beckman Coulter Inc
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Beckman Instruments Inc
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Publication date
Application filed by Beckman Instruments Inc filed Critical Beckman Instruments Inc
Priority to DE823231626T priority Critical patent/DE3231626T1/en
Publication of WO1982002500A1 publication Critical patent/WO1982002500A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/05—Alcohols; Metal alcoholates
    • C08K5/053—Polyhydroxylic alcohols

Definitions

  • the instant invention pertains to an electrophoretic technique for separating isoenzymes and to an electrophoretic gel for use therein.
  • Electrophoretic techniques for separating isoenzymes and electrophoretic gels for use therein are well known to those skilled in the art. Cawley, Electroohoresis and Immunoelectrophoresis, Little, Brown and Company, Boston, Mass. (1969). In general, electrophoretic gels employed for separating isoenzymes are of the type comprising a polysaccharide. A buffer 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 combinations thereof.
  • Typical buffers employed in prior art electrophoretic gels include, but are not limited to, the buffers which are set forth in Table I of Cawley, supra, pp. 331-332.
  • One problem present in a basic prior art electrophoretic technique for separating isoenzymes is that the electrophoretic gel employed therein has a relatively poor shelf life.
  • an improved electrophoretic gel having an improved shelf life.
  • the electrophoretic gel of this invention is of the type comprising a polysaccharide.
  • the improved electrophoretic gel of the instant invention is characterized in that it further comprises a stabilizing amount of an alkylpolyol having 2-6 carbon atoms and 2-4 hydroxyl groups.
  • the alkyl polyol when incorporated into the electrophoretic gel enables one to obtain a resulting product having an improved shelf life.
  • Alkylpolyols capable of use in the instant invention include, but are not limited to, ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3- butanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, erythritol, pentaerythritol, and pinacol.
  • the alkylpolyol is selected from the group consisting of ethylene glycol and propanediol.
  • Polysaccharides which can preferably be employed in the electrophoretic gel of the instant invention are agar and 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 high electroendosmosis agarose.
  • the electrophoretic gel of the instant invention can optionally further comprise an antimicrobial agent.
  • Typical antimicrobial agents include, but are not limited to, antibiotics, halogenated organic compounds, and inorganic compounds.
  • One readily available antimicrobial agent capable of use herein is sodium azide.
  • the electrophoretic gel of the instant invention preferably comprises from about 0.1 to about 15, more preferably from about 1 to about 10, and optimally from about 3 to about 7 percent volume/volume of the alkylpolyol.
  • the electrophoretic gels of the instant invention 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 syringe.
  • 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 abo ⁇ t 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 isoenzymes being assayed therein.
  • the gels can be either fixed or rinsed in an appropriate solution.
  • the gels can optionally be dried at about 80 to about 90°C.
  • Example 1 Electrophoretic gels having the formulations set forth in Table I were cast onto a plastic backing. These electrophoretic gels were then placed in plastic trays, covered with a plastic lid, and sealed in moisture impermeable plastic bags. The sealed gels were next stored at the temperatures noted in Table II and were examined at periodic intervals for weight loss and electrophoretic functionality. The results of the weight loss tests are summarized in Table II.
  • Example 2 Electrophoretic gels having the formulations set forth in Table III were cast onto a plastic backing. These electrophoretic gels were then placed in plastic trays, covered with a plastic lid, and sealed in moisture impermeable plastic bags. The sealed gels were next stored at the temperatures noted in Table IV and were examined at periodic intervals for weight loss and electrophoretic functionality. The results of the weight loss tests are summarized in Table IV.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Cosmetics (AREA)
  • Medicinal Preparation (AREA)
  • Colloid Chemistry (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Gel d'electrophorese du type comprenant un polysaccharide. Le gel d'electrophorese est caracterise en ce qu'il comprend egalement une quantite stabilisante d'un polyol alkylique possedant 2 a 6 atomes de carbone et 2 a 4 groupes hydroxyle. Procede ameliore de stockage d'un gel d'electrophorese du type comprenant un polysaccharide. Le procede de stockage est caracterise en ce que le gel d'electrophorese est stocke en presence d'une quantite stabilisante d'un polyol alkylique possedant 2 a 6 atomes de carbone et 2 a 4 groupes hydroxyle.Electrophoresis gel of the type comprising a polysaccharide. The electrophoresis gel is characterized in that it also comprises a stabilizing amount of an alkyl polyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. Improved method of storing an electrophoresis gel of the type comprising a polysaccharide. The storage process is characterized in that the electrophoresis gel is stored in the presence of a stabilizing amount of an alkyl polyol having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups.

Description

IMPROVED ELECTROPHORETIC GEL FOR SEPARATING ISOENZYMES.
Background of the Invention
1. Field of the Invention
The instant invention pertains to an electrophoretic technique for separating isoenzymes and to an electrophoretic gel for use therein.
2. Description of the Prior Art
Electrophoretic techniques for separating isoenzymes and electrophoretic gels for use therein are well known to those skilled in the art. Cawley, Electroohoresis and Immunoelectrophoresis, Little, Brown and Company, Boston, Mass. (1969). In general, electrophoretic gels employed for separating isoenzymes are of the type comprising a polysaccharide. A buffer 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 combinations thereof.
Typical buffers employed in prior art electrophoretic gels include, but are not limited to, the buffers which are set forth in Table I of Cawley, supra, pp. 331-332. One problem present in a basic prior art electrophoretic technique for separating isoenzymes is that the electrophoretic gel employed therein has a relatively poor shelf life.
Accordingly, it would be very desirable to have an electrophoretic gel for use in the electrophoretic separation of isoenzymes which possesses an improved shelf life.
Summary of the Invention
In accordance with the present invention there is provided an improved electrophoretic gel having an improved shelf life. The electrophoretic gel of this invention is of the type comprising a polysaccharide. The improved electrophoretic gel of the instant invention is characterized in that it further comprises a stabilizing amount of an alkylpolyol having 2-6 carbon atoms and 2-4 hydroxyl groups. The alkyl polyol when incorporated into the electrophoretic gel enables one to obtain a resulting product having an improved shelf life. 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
Alkylpolyols capable of use in the instant invention include, but are not limited to, ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3- butanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, erythritol, pentaerythritol, and pinacol. Preferably, the alkylpolyol is selected from the group consisting of ethylene glycol and propanediol.
Polysaccharides which can preferably be employed in the electrophoretic gel of the instant invention are agar and 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 high electroendosmosis agarose.
The electrophoretic gel of the instant invention can optionally further comprise an antimicrobial agent. Typical antimicrobial agents include, but are not limited to, antibiotics, halogenated organic compounds, and inorganic compounds. One readily available antimicrobial agent capable of use herein is sodium azide.
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 0.1 to about 15, more preferably from about 1 to about 10, and optimally from about 3 to about 7 percent volume/volume of the alkylpolyol. The electrophoretic gels of the instant invention 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 syringe. 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 aboμt 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 isoenzymes being assayed therein. If desired, the gels can be either fixed or rinsed in an appropriate solution. 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 illustraton only and are not intended to be limitations on the disclosed invention. Example 1 Electrophoretic gels having the formulations set forth in Table I were cast onto a plastic backing. These electrophoretic gels were then placed in plastic trays, covered with a plastic lid, and sealed in moisture impermeable plastic bags. The sealed gels were next stored at the temperatures noted in Table II and were examined at periodic intervals for weight loss and electrophoretic functionality. The results of the weight loss tests are summarized in Table II.
TABLE I
Gel Gel
Within Scope Outside Scope
Ingredient of Invention of Invention HE Agarose, X X
1% w/v Arabic Acid, X X
1% w/v Sodium Azide, X X
0.1% w/v Ethylene Glycol, X
5% v/v
0.05M Barbital Buffer X X
*Each formulated electrophoretic gel had an initial weight of from about 4.45 to about 4.70g. TABLE II
Weight Loss
Gel Gels Within Scope of Invention Gels Outside Scope of Invention
Age, Temperature, °C. Temperature, °C.
Days 4 25 37 50 .70 4 25 37 50 70
1 0/2* 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
8 0/2 0/2 0/2 0/2 1/2 2/2 0/2 0/2 4/4 2/2
15 0/2 0/2 0/2 0/4 0/2 2/3 0/2 0/2 5/6 2/2
22 0/2 0/2 0/2 1/2 2/2 0/2 0/2 0/2 2/2 3/3
23 N/R** N/R N/R 0/2 N/R N/R N/R N/R 1/2 N/R
29 0/2 0/2 0/2 2/2 N/R 0/2 0/2 0/2 N/R N/R
30 N/R N/R N/R 0/2 N/R N/R N/R N/R 2/2 N/R
37 N/R N/R N/R 0/2 N/R N/R N/R N/R N/R N/R
A3 0/2 0/2 0/2 N/R N/R 1/2 0/2 0/2 N/R N/R
50 0/2 0/2 0/2 N/R N/R N/R N/R N/R N/R N/R
51 N/R N/R N/R N/R N/R 0/2 0/2 0/2 N/R N/R
57 0/2 0/2 0/2 N/R N/R 1/3 0/2 0/2 N/R N/R
64 1/2 0/2 0/2 N/R N/R N/R N/R N/R N/R N/R
Failures/ 1/18 0/18 0/18 3/18 3/8 6/18 0/16 0/16 14/18 7/9
Total
Tested
%Failures 6 0 0 16.7 37.5 33.3 0 0 77.7 77.8
*Failures/Number Tested **N/R denotes "Not Run"
Example 2 Electrophoretic gels having the formulations set forth in Table III were cast onto a plastic backing. These electrophoretic gels were then placed in plastic trays, covered with a plastic lid, and sealed in moisture impermeable plastic bags. The sealed gels were next stored at the temperatures noted in Table IV and were examined at periodic intervals for weight loss and electrophoretic functionality. The results of the weight loss tests are summarized in Table IV.
TABLE III*
Gels Gels Within Scope Outside Scope
Ingredient of Invention of Invention
ME Agarose, X X
1% w/v
Sodium Azide, X X
0.1% w/v
Ethylene Glycol, X
5% v/v
Tis, 0.765% v/v X X
EDTA, 0.045% w/v X X
Boric Acid, X X
0.240% w/v
*Each formulated electrophoretic gel had an initial weight of from about 4.35 to.about 4.65g. TABLE IV
Weight Loss
Gel. Gels Within Scope of Invention Gels Outside : Scope of Invention
Age, Tempisrature, °C. Temperature, °C.
Days 4 25 37 4 25 37
1 N/R* N/R N/R 0/2 0/2 0/2
3 0/1** 0/1 0/1 N/R N/R N/R
5 0/1 0/1 0/1 N/R N/R N/R
8 0/1 0/1 0/1 0/2 0/2 0/2
10 0/1 0/1 0/1 N/R N/R N/R
12 0/1 0/1 0/1 N/R N/R N/R
15 0/1 0/1 0/1 0/2 0/2 0/2
17 0/1 0/1 0/1 N/R N/R N/R
19 0/1 0/1 0/1 N/R N/R N/R
22 0/1 0/1 0/2 1/2 1/2 2/2
24 0/1 0/1 0/1 N/R N/R N/R
26 0/1 0/1 N/R N/R N/R N/R
29 0/1 0/1 0/1 0/2 0/2 0/2
32 0/1 0/1 N/R N/R N/R N/R
36 0/1 0/1 N/R 1/2 0/2 0/2
39 0/1 0/1 N/R N/R N/R N/R
43 0/1 0/1 N/R 2/2 0/2 0/2
46 1/1 1/1 N/R N/R N/R N/R
50 1/1 0/1 N/R 2/2 0/2 0/2
53 0/1 0/1 N/R N/R N/R N/R
57 0/1 0/1 N/R 2/2 0/2 1/2
64 0/1 0/1 N/R 2/2 0/2 0/2
Failures/
Total 2/21 1/21 0/12 10/20 1/20 3/20
Tested
%Faillures 9.5 4.8 0 50 5 15
*N/R denotes "Not Run" **Fai Llures/Number Tested
The data set forth in Tables II and IV show that gels within the scope of the instant invention, i.e., electrophoretic gels comprising a polysaccharide and a stabilizing amount of an alkylpolyol having 2-6 carbon atoms and 2-4 hydroxyl groups, are able to better maintain their weight over a period of time at various storage temperatures than corresponding gels devoid of the alkylpolyol. In other words, the data set forth in Tables II and IV indicate that the incorporation of an alkylpolyol into a polysaccharide matrix tends to prevent the collapse of the polysaccharidewater structure and the resulting weight loss due to the exodus of the water therefrom.
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.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. An electrophoretic gel of the type comprising a polysaccharide, characterized in that said electrophoretic gel further comprises a stabilizing amount of an alkylpolyol having 2-6 carbon atoms and 2-4 hydroxyl groups.
2. The electrophoretic gel of claim 1 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
3. The electrophoretic gel of claim 1 wherein said alkylpolyol is present in an amount of from about 1 to about 10% volume/volume.
4. The electrophoretic gel of claim 1 wherein said alkylpolyol is present in an amount of from about 3 to about 7% volume/volume.
5. The electrophoretic gel of claim 1 wherein said alkylpolyol is selected from a group consisting of ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1,4,5-butanetriol, erythritol, pentaerythritol, and pinacol¬
6. The electrophoretic gel of claim 5 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
7. The electrophoretic gel of claim 5 wherein said alkylpolyol is present in an amount of from about 1 to about 10% volume/volume.
8. The electrophoretic gel of claim 5 wherein said alkylpolyol is present in an amount of from about 3 to about 7 volume/volume.
9. The electrophoretic gel of claim 1 wherein said alkylpolyol is selected from the group consisting of ethylene glycol and propanediol.
10. The electrophoretic gel of claim 9 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
11. The electrophoretic gel of claim 9 wherein said alkylpolyol is present: in an amount of from about 1 to about 10% volume/volume.
12. The electrophoretic gel of claim 9 wherein said alkylpolyol is present in an amount of from about 3 to about 7% volume/volume.
13. An improved method of storing an electrophoretic gel of the type comprising a polysaccharide characterized in that said electrophoretic gel is stored in the presence of a stabilizing amount of an alkylpolyol having 2-6 carbon atoms and 2-4 hydroxyl groups.
14. The method of claim 13 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
15. The method of claim 13 wherein said alkylpolyol is present in an amount of from about 1 to about 10% volume/volume.
16. The method of claim 13 wherein said alkylpolyol is present in an amount of from about 3 to about 7% volume/volume.
17. The method of claim 13 wherein said alkylpolyol is selected from a group consisting of ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2, 3-butanetriol, 1, 2,4-butanetriol, erythritol, pentaerythritol, and pinacol.
IS. The method of claim 17 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
19. The method of claim 17 wherein said alkylpolyol is present in an amount of from about 1 to about 10% volume/volume.
20. The method of claim 17 wherein said alkylpolyol is present in an amount of from about 3 to about 7%. volume/volume.
21. The method of claim 13 wherein said alkylpolyol is selected from the group consisting of ethylene glycol and propanediol.
22. The method of claim 21 wherein said alkylpolyol is present in an amount of from about 0.1 to about 15% volume/volume.
23. The method of claim 21 wherein said alkylpolyol is present in an amount of from about 1 to about 15% volume/volume.
24. The method of claim 21 wherein said alkylpolyol is present in an amount of from about 3 to about 7% volume/volume.
PCT/US1982/000023 1981-01-19 1982-01-11 Improved electrophoretic gel for separating isoenzymes Ceased WO1982002500A1 (en)

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Application Number Priority Date Filing Date Title
DE823231626T DE3231626T1 (en) 1981-01-19 1982-01-11 ELECTROPHORETIC GEL FOR SEPARATING ISOENZYMS

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US22657281A 1981-01-19 1981-01-19
US226572810119 1981-01-19

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DE (1) DE3231626T1 (en)
IT (1) IT1149707B (en)
SE (1) SE8205387L (en)
WO (1) WO1982002500A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004252A1 (en) * 1984-03-12 1985-09-26 Beckman Instruments, Inc. Electrophoretic technique for separating hemoglobin variants and electrophoretic gel for use therein

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3497437A (en) * 1967-06-21 1970-02-24 Baxter Laboratories Inc Method of electrophoresis
US3578499A (en) * 1968-08-02 1971-05-11 Grace W R & Co Gelling composition for general purpose cleaning and sanitizing
US3808118A (en) * 1969-03-13 1974-04-30 Helena Lab Corp Microporous support medium
US3873514A (en) * 1974-05-17 1975-03-25 Bio Rad Laboratories Preparation of gel for affinity chromatography
US4222836A (en) * 1979-06-01 1980-09-16 Corning Glass Works Acidic agar gel electrochromatography of glycohemoglobins

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3497437A (en) * 1967-06-21 1970-02-24 Baxter Laboratories Inc Method of electrophoresis
US3578499A (en) * 1968-08-02 1971-05-11 Grace W R & Co Gelling composition for general purpose cleaning and sanitizing
US3808118A (en) * 1969-03-13 1974-04-30 Helena Lab Corp Microporous support medium
US3873514A (en) * 1974-05-17 1975-03-25 Bio Rad Laboratories Preparation of gel for affinity chromatography
US4222836A (en) * 1979-06-01 1980-09-16 Corning Glass Works Acidic agar gel electrochromatography of glycohemoglobins

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004252A1 (en) * 1984-03-12 1985-09-26 Beckman Instruments, Inc. Electrophoretic technique for separating hemoglobin variants and electrophoretic gel for use therein

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DE3231626T1 (en) 1983-12-01
IT1149707B (en) 1986-12-10
IT8219192A0 (en) 1982-01-19
SE8205387D0 (en) 1982-09-20
SE8205387L (en) 1982-09-20

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