US3873433A - Method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids - Google Patents

Method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids Download PDF

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Publication number
US3873433A
US3873433A US426202A US42620273A US3873433A US 3873433 A US3873433 A US 3873433A US 426202 A US426202 A US 426202A US 42620273 A US42620273 A US 42620273A US 3873433 A US3873433 A US 3873433A
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United States
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developing solution
lipoprotein
carrier
demonstrating
heparin
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Expired - Lifetime
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US426202A
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English (en)
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Dietrich Seidel
Heinrich Wieland
Johann Eibl
Gerald Eder
Ewald Molinari
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Oesterreichisches Institut fuer Haemoderivate
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Immuno AG
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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
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules

Definitions

  • the invention relates to a method of demonstrating normal and pathological plasm-liporprotein patterns in human body liquids by electrophoretical separation of the lipoproteins in a carrier medium, such as a gel, or on foils, and an apparatus for carrying out the method.
  • the analysis of the serum-plasm-lipoproteins is a prerequisite for an exact diagnosis of different forms of hyperlipoproteinernia and demands have been raised in the past for finding a reliable, easily practicable technique of identifying the normal and abnormal lipoprotein fractions.
  • Demonstration methods are known that are based on the electrophoresis of human body liquids.
  • the lipoproteins of human. serum or the like are subjected to the influence of an electric field in a solid migration medium, mainly a gel, the migration of the lipoproteins causing a separation effect.
  • the lipoprotein bands are made visible in the known method by coloring with lipid coloring agents or by immunological precipitations, e.g. with antiserum.
  • an ultracentrifugation is necessary; the known methods have the disadvantage that they require such a high expenditure of work and such costly apparatus that a complete lipoprotein analysis can be carried out in special laboratories only.
  • the method of the invention which starts from the electrophoretical separation of the lipoproteins in a carrier medium, such as a gel, or on foils, is characterised in that after the required electrophoretical separa tion effect has occurred, the carrier medium is treated with a developing liquid, which comprises one or several of the following substances: polyanions, such as heparin or dextran sulfate, sodium-dodecylsulfate (SOS), sodium-phosphotungstic acid, sodium oleate, sodium salts ofbile acids, suitably in the presence of bivalent cations, such as magnesium and calcium, wherein complex salts of low solubility are formed with the lipoproteins.
  • a developing liquid which comprises one or several of the following substances: polyanions, such as heparin or dextran sulfate, sodium-dodecylsulfate (SOS), sodium-phosphotungstic acid, sodium oleate, sodium salts ofbile acids, suitably in the presence of bivalent
  • the chemical composition of the developing solution or the kind of polyanions to be used, respectively, depends upon the lipoproteins that are to be demonstrated.
  • a developer containing heparin, magnesium ions and sodium chloride, is used.
  • a carrier e.g. a gel plate, is used for demonstrating the LP-X-lipoprotein, the carrier already containing the developing solution or parts of it.
  • the carrier is treated in turns with different developing solutions, the one containing heparin and magnesium ions and the other dextran sulfate and calcium ions.
  • the invention comprises an apparatus for carrying out the method of the invention, which, in a manner known, possesses a carrier in form of a plate bearing a layer of the migration medium, e.g. of the gel, said carrier being immersed into an electrolyte or in a buffer solution.
  • a carrier in form of a plate bearing a layer of the migration medium, e.g. of the gel, said carrier being immersed into an electrolyte or in a buffer solution.
  • the apparatus of the invention comprises a row of slits or holes and possibly a device for bringing into touch the plate-formed carrier with the developing solution.
  • the row of holes or slits is suitably arranged on the side of the anode and for demonstrating the lipoprotein pattern VLDL, LDL, HDL and type III, the row is arranged on the side of the cathode.
  • the row of holes may however also be arranged in the center of the gel carrier having the form of a plate, in which case starting from this row, the lipoprotein LP-X migrates in the direction towards the cathode and the other lipoproteins in the direction towards the anode.
  • the carrier having the form of a plate, is U-shaped and insertable into an electrolyte or into a buffer solution receiving container, respectively, which is divided in longitudinal direction by a separating dam into an anode region and a cathode region.
  • the drawing shows the apparatus of the invention by way of an example.
  • FIG. 1 illustrates a vertical section of the apparatus and FIG. 2 is a top view of the gel carrier.
  • FIG. 3 shows representations of plasm lipoprotein patterns received arrording to the invention.
  • the apparatus comprises a rectangular vessel 1 which may be covered by a lid 2.
  • Platinum sheets 3 and 4 or platinum wires, respectively, serve as cathode K and anode A which continue in the interior of the vessel along the side walls and along part of the bottom.
  • Fillets 5 extend in upward direction from the bottom of the vessel; they represent a separating dam and divide the vessel 1 in longitudinal direction into the cathode and the anode region.
  • the gel plate 7 rests on the fillets 5, thus bridging the separating dam and immerses into the buffer solution with its limbs 8 extending downwardly.
  • the gel plate is cast into an about U-shaped stencil 9.
  • the stencil is suitably made of plastics.
  • a row of slits 10 which serves for receiving the human body liquid to be examined, e.g. the human serum.
  • the lipoproteins migrate in the direction of the anode and are thereby separated into the individual fractions depending upon their migration speed.
  • the stencil is removed from the apparatus and is immersed into a container (not shown) which holds the developing solution. It is however also possible to drop or spray the developing solution onto the stencil, thus making the individual lipoprotein bands visible after an appropriate period of influence.
  • EXAMPLE 1 For demonstrating the lipoprotein bands in the human serum, the electrophoresis was carried out in an agarose migration medium as described by R. I. Noble, I. Lipid Res. 9.693 (1968). A veronal buffer with a pH of 8.6 and with an ionic strength of 0.05 was used. After the electrophoresis had continued for an hour, the stencil was removed from the apparatus and put into a developing bath which consisted of 0.55 dextran-sulfate 2,000 and of a 2.2 aqueous calciumchloride solution. The carrier was allowed to stay in the bath for 15 to 20 minutes at room temperature. During that period a complete precipitation of the following lipoprotein bands occurred:
  • chylomicrons B-lipoproteins pre-[i-lipoproteins OZ-llPOPI'OIGlHS, which in FIG. 3a are denominated in turn with 1,2,3 and 4.
  • EXAMPLE 2 In accordance with example 1, type III human serum on an agar stencil was subjected to the electrophoresis in a veronal buffer having a pH of 8.6 and an ionic strength of 0.05 for the purpose of determining the hyperlipoproteinemia. After the electrophoresis had continued for an hour, the stencil was removed and put into a developing bath consisting of 0.25 heparin and 0.95 70 magnesium chloride in a 0.95 aqueous sodium-chloride solution and was allowed to stay in the bath for 20 minutes. The precipitation for the field VLDL, characteristic of type III was clearly to be recognised and is denominated with 5 in FIG. 3b. The field extends from the B-position 2 as far as the pre-B- position 3.
  • EXAMPLE 3 For demonstrating LPX-lipoprotein, four samples of human serum were inserted into the four holes of an object holder carrying an agar plate and by means of filter paper the object holder was connected with a veronal buffer solution, having a pH of 8.6 and an ionic strength of 0.05. The samples thus prepared were subjected to the electrophoresis. After the electrophoresis had continued for half an hour, the object holder was removed and put into a developing bath consisting of 0.25 heparin and 0.95 magnesium chloride in 0.9 aqueous sodium-chloride solution, and was allowed to stay in the bath for minutes. Two samples which are denoted with N in FIG. 3c were negative, in the other two positive samples, denoted with P, the LPX- band was easily recognisable and was denoted with 6.
  • EXAMPLE Human full plasm was subjected to a polyacrylamide-electrophoresis, according to the method by B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964), wherein a tris-glycin buffer [tris-(hydroxymethyl)- aminomethane-buffer] with a pH of 8.8 and an acrylamide-monomer concentration of 3.5/7 was used.
  • a tris-glycin buffer [tris-(hydroxymethyl)- aminomethane-buffer] with a pH of 8.8 and an acrylamide-monomer concentration of 3.5/7 was used.
  • the glass tube was removed from the apparatus, the polyacryl-amide gel was removed and treated with a developing solution of the following composition: 0.55 dextran sulfate 2,000 and 2.2 aqueous calcium-chloride solution.
  • pre-B-lipoproteins B-lipoproteins a-lipoproteins which are denoted with 3,2 and 4 in FIG. 3d.
  • a method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids which comprises electrophoretically separating lipoproteins in a carrier medium or on foils,
  • a developing solution comprising at least one of the following substances: polyanions, selected from the group consisting of heparin and dextran sulfate, sodiumdodcyl -sulfate (SOS), sodium phosphotung stic acid, sodium oleate, sodium salts of bile acids, lowsoluble complex salts being formed with the lipo- 1 proteins.
  • polyanions selected from the group consisting of heparin and dextran sulfate, sodiumdodcyl -sulfate (SOS), sodium phosphotung stic acid, sodium oleate, sodium salts of bile acids, lowsoluble complex salts being formed with the lipo- 1 proteins.
  • bivalent cations selected from the group consisting of magnesium and calcium, are additionally present in the developing solution.
  • a method according toclaim 1 wherein for selectively demonstrating lipoprotein type III and lipoproteins VLDL, LDL and HDL the carrier is successively treated with a first developing solution and a second developing solution, the first developing solution comprising heparin and magnesium ions and the second developing solution comprising dextran sulfate and calcium ions.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
US426202A 1973-02-12 1973-12-19 Method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids Expired - Lifetime US3873433A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
AT121873A AT320865B (de) 1973-02-12 1973-02-12 Verfahren zum Bestimmen von normalen und pathologischen Plasma-Lipoproteinen in menschlichen Körperflüssigkeiten und Apparat zur Durchführung des Verfahrens

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US (1) US3873433A (de)
AT (1) AT320865B (de)
BE (1) BE810865A (de)
CH (1) CH598594A5 (de)
DE (1) DE2314964C3 (de)
FR (1) FR2217692B1 (de)
GB (1) GB1425545A (de)
NL (1) NL7317413A (de)
NO (1) NO146038C (de)
SE (1) SE408968B (de)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4105521A (en) * 1977-09-21 1978-08-08 Helena Laboratories Corporation Clinical procedure for measuring lipoprotein cholesterols
US4151065A (en) * 1978-01-30 1979-04-24 The Regents Of The University Of California Horizontal slab gel electrophoresis
US4167467A (en) * 1977-09-21 1979-09-11 Helena Laboratories Corporation Clinical procedure for measuring lipoprotein free cholesterols
US4234400A (en) * 1979-01-26 1980-11-18 The Regents Of The University Of California Horizontal slab gel electrophoresis
US4297198A (en) * 1978-05-29 1981-10-27 Mochihiko Ohashi Concentrating electrophoresis apparatus
US4298441A (en) * 1979-06-27 1981-11-03 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method of preserving the electrophoretic properties of lipoproteins
US4306956A (en) * 1980-11-10 1981-12-22 Miles Laboratories, Inc. Discontinuous gel electrophoresis process
US4321120A (en) * 1980-03-19 1982-03-23 Nardi Ronald V Process for detecting proteins specific to hypertension in mammals
DE3234565A1 (de) * 1981-09-18 1983-05-05 Toa Medical Electronic Co., Ltd., Kobe, Hyogo Stuetzkoerperzelle in einem elektrophoretischen apparat
US4405720A (en) * 1981-03-04 1983-09-20 The United States Of America As Represented By The Department Of Health And Human Services Silver stains for protein in gels
US4521519A (en) * 1981-05-02 1985-06-04 Boehringer Mannheim Gmbh Reagent for the precipitation of apo-B-containing lipoproteins
FR2560220A1 (fr) * 1984-02-27 1985-08-30 Int Biotechnologies In Dispositif d'electrophorese a gel horizontal
US4709810A (en) * 1986-11-14 1987-12-01 Helena Laboratories Corporation Container for an electrophoretic support medium
US4782027A (en) * 1987-01-22 1988-11-01 President And Fellows Of Harvard College Protein detection by negative staining
US4911816A (en) * 1986-02-04 1990-03-27 Oncor, Inc. Process for conducting electrophoresis and transfer
US4954237A (en) * 1987-03-16 1990-09-04 Helena Laboratories Automatic electrophoresis apparatus
US5074981A (en) * 1989-04-26 1991-12-24 The University Of Tennessee Research Corporation High speed gel electrophoresis
US5168067A (en) * 1990-05-25 1992-12-01 Miller Michael A Method and kit for screening for apolipoprotein b or calculated ldl cholesterol
US5364793A (en) * 1990-11-30 1994-11-15 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US5405520A (en) * 1994-01-27 1995-04-11 Eastman Kodak Company Connectors for electrophoresis device
US5844097A (en) * 1990-11-30 1998-12-01 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US6063250A (en) * 1998-05-15 2000-05-16 C.C. Imex Running tank assembly for electrophoresis
WO2002037093A2 (en) * 2000-11-02 2002-05-10 Gene Bio-Application Ltd. Gel trap for electrophoresis
US6402915B1 (en) 1998-05-15 2002-06-11 C.C. Imex Running tank assembly for electrophoresis
WO2006082575A1 (en) * 2005-02-07 2006-08-10 Gene Bio-Application Ltd. Double chamber tank for horizontal gel electrophoresis
US20090071830A1 (en) * 2007-07-24 2009-03-19 Applied Biosystems Inc. Systems and Methods for Isolating Nucleic Acids

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK159837C (da) * 1986-12-03 1991-04-29 Radiometer As Fremgangsmaade og apparat til afsloering af en proteinforurening paa en ph elektrode

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3766047A (en) * 1972-09-11 1973-10-16 F Elevitch Gel for electrophoresis
US3773647A (en) * 1972-05-12 1973-11-20 Electro Nucleonics Low voltage electrophoretic testing system
US3783118A (en) * 1971-12-13 1974-01-01 Pharmacia Fine Chemicals Ab Method for the electrophoretic separation of substance mixtures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3783118A (en) * 1971-12-13 1974-01-01 Pharmacia Fine Chemicals Ab Method for the electrophoretic separation of substance mixtures
US3773647A (en) * 1972-05-12 1973-11-20 Electro Nucleonics Low voltage electrophoretic testing system
US3766047A (en) * 1972-09-11 1973-10-16 F Elevitch Gel for electrophoresis

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4147606A (en) * 1977-09-21 1979-04-03 Helena Laboratories Corporation Clinical procedure for measuring lipoprotein triglycerides
FR2404222A1 (fr) * 1977-09-21 1979-04-20 Helena Lab Corp Procede de determination clinique des lipoproteines a cholesterol
US4167467A (en) * 1977-09-21 1979-09-11 Helena Laboratories Corporation Clinical procedure for measuring lipoprotein free cholesterols
US4105521A (en) * 1977-09-21 1978-08-08 Helena Laboratories Corporation Clinical procedure for measuring lipoprotein cholesterols
US4151065A (en) * 1978-01-30 1979-04-24 The Regents Of The University Of California Horizontal slab gel electrophoresis
US4297198A (en) * 1978-05-29 1981-10-27 Mochihiko Ohashi Concentrating electrophoresis apparatus
US4234400A (en) * 1979-01-26 1980-11-18 The Regents Of The University Of California Horizontal slab gel electrophoresis
US4298441A (en) * 1979-06-27 1981-11-03 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method of preserving the electrophoretic properties of lipoproteins
US4321120A (en) * 1980-03-19 1982-03-23 Nardi Ronald V Process for detecting proteins specific to hypertension in mammals
US4306956A (en) * 1980-11-10 1981-12-22 Miles Laboratories, Inc. Discontinuous gel electrophoresis process
US4405720A (en) * 1981-03-04 1983-09-20 The United States Of America As Represented By The Department Of Health And Human Services Silver stains for protein in gels
US4521519A (en) * 1981-05-02 1985-06-04 Boehringer Mannheim Gmbh Reagent for the precipitation of apo-B-containing lipoproteins
DE3234565A1 (de) * 1981-09-18 1983-05-05 Toa Medical Electronic Co., Ltd., Kobe, Hyogo Stuetzkoerperzelle in einem elektrophoretischen apparat
US4414073A (en) * 1981-09-18 1983-11-08 Toa Medical Electronic Co., Ltd. Supporting body cell in electrophoretic apparatus
FR2560220A1 (fr) * 1984-02-27 1985-08-30 Int Biotechnologies In Dispositif d'electrophorese a gel horizontal
US4588491A (en) * 1984-02-27 1986-05-13 International Biotechnologies, Inc. Horizontal gel electrophoresis device
US4911816A (en) * 1986-02-04 1990-03-27 Oncor, Inc. Process for conducting electrophoresis and transfer
US4709810A (en) * 1986-11-14 1987-12-01 Helena Laboratories Corporation Container for an electrophoretic support medium
US4782027A (en) * 1987-01-22 1988-11-01 President And Fellows Of Harvard College Protein detection by negative staining
US4954237A (en) * 1987-03-16 1990-09-04 Helena Laboratories Automatic electrophoresis apparatus
US5074981A (en) * 1989-04-26 1991-12-24 The University Of Tennessee Research Corporation High speed gel electrophoresis
US5168067A (en) * 1990-05-25 1992-12-01 Miller Michael A Method and kit for screening for apolipoprotein b or calculated ldl cholesterol
US5364793A (en) * 1990-11-30 1994-11-15 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US5583201A (en) * 1990-11-30 1996-12-10 Monoclonetics International, Inc. Methods for diagnosis of peripheral nerve damage
US5844097A (en) * 1990-11-30 1998-12-01 Monoclonetics International, Inc. Methods for the diagnosis of peripheral nerve damage
US5405520A (en) * 1994-01-27 1995-04-11 Eastman Kodak Company Connectors for electrophoresis device
US6402915B1 (en) 1998-05-15 2002-06-11 C.C. Imex Running tank assembly for electrophoresis
US6063250A (en) * 1998-05-15 2000-05-16 C.C. Imex Running tank assembly for electrophoresis
WO2002037093A3 (en) * 2000-11-02 2002-07-25 Gene Bio Applic Ltd Gel trap for electrophoresis
WO2002037093A2 (en) * 2000-11-02 2002-05-10 Gene Bio-Application Ltd. Gel trap for electrophoresis
US20040020775A1 (en) * 2000-11-02 2004-02-05 Yitzhak Ben-Asouli Gel for electrophoresis
US20040020776A1 (en) * 2000-11-02 2004-02-05 Yitzhak Ben-Asouli Gel trap for electrophoresis
US20060207882A1 (en) * 2000-11-02 2006-09-21 Gene Bio-Application Ltd. Gel for electrophoresis
US7264703B2 (en) 2000-11-02 2007-09-04 Gene Bio-Application Ltd. Gel trap for electrophoresis
US7320747B2 (en) 2000-11-02 2008-01-22 Gene Bio-Application Ltd. Gel for electrophoresis
WO2006082575A1 (en) * 2005-02-07 2006-08-10 Gene Bio-Application Ltd. Double chamber tank for horizontal gel electrophoresis
US20080217178A1 (en) * 2005-02-07 2008-09-11 Yitzhak Ben-Asouli Double Chamber Tank for Horizontal Gel Electrophoresis
US8241477B2 (en) * 2005-02-07 2012-08-14 Gene Bio-Application Ltd. Double chamber tank for horizontal gel electrophoresis
US20090071830A1 (en) * 2007-07-24 2009-03-19 Applied Biosystems Inc. Systems and Methods for Isolating Nucleic Acids
US8568580B2 (en) * 2007-07-24 2013-10-29 Applied Biosystems, Llc Systems and methods for isolating nucleic acids

Also Published As

Publication number Publication date
DE2314964A1 (de) 1974-09-05
AT320865B (de) 1975-03-10
NO146038C (no) 1982-07-14
DE2314964C3 (de) 1975-11-13
FR2217692B1 (de) 1978-02-17
NO146038B (no) 1982-04-05
NL7317413A (de) 1974-08-14
GB1425545A (en) 1976-02-18
CH598594A5 (de) 1978-05-12
DE2314964B2 (de) 1975-03-27
SE408968B (sv) 1979-07-16
BE810865A (fr) 1974-05-29
FR2217692A1 (de) 1974-09-06

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