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WO1996004556A1 - Dosages du cholesterol de lipoproteines - Google Patents

Dosages du cholesterol de lipoproteines Download PDF

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Publication number
WO1996004556A1
WO1996004556A1 PCT/US1995/009504 US9509504W WO9604556A1 WO 1996004556 A1 WO1996004556 A1 WO 1996004556A1 US 9509504 W US9509504 W US 9509504W WO 9604556 A1 WO9604556 A1 WO 9604556A1
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Prior art keywords
lipoprotein
cholesterol
antibody conjugates
phase
aqueous
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PCT/US1995/009504
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English (en)
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James T. Hsu
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Priority to AU31514/95A priority Critical patent/AU3151495A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates to the clinical assay techniques for lipoprotein cholesterol determination.
  • Plasma lipoproteins serve to solubilize and transport cholesterol and triglyceride in the blood stream, in which aqueous insoluble lipids bind to protein forming lipid-protein complexes that become soluble.
  • a lipoprotein may be visualized as a spherical particle with an outer solubilizing coat of protein, phospholipid and free cholesterol and an inner hydrophilic neutral core of triglyceride and esterified cholesterol.
  • lipoproteins There are four major classes of lipoproteins: chylomicrons, very-low density lipoprotein (VLDL) (density ⁇ 1.006 gm/ml) , low density lipoprotein (LDL) (density, 1.006- 1.063 gm/ml), high density lipoprotein (HDL) (density > 1.063 gm/ml) . These differ in their compositions, protein and lipid ratio, the latter determining the density of lipoprotein.
  • VLDL very-low density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • LDL cholesterol For epidemiologic and clinical purpose, it is convenient to measure their lipoproteins by quantifying the cholesterol moiety rather than their total mass. Increased total blood cholesterol level, especially, an elevated LDL cholesterol correlates with risk of coronary heart disease (CHD) , and HDL cholesterol level is an inverse risk factor. There is another class of lipoprotein Lp (a) which is cholesterol rich for developing premature CHD. Even within the normal range of total cholesterol, higher LDL cholesterol is associated with increased occurrence of CHD. Reduction of elevated LDL cholesterol level is associated with a reduction in the incidence of cardiovascular disease and death in adults.
  • NCEP National Cholesterol Education Program
  • NCEP recommends that the risk of CHD may be classified as: for total cholesterol, desirable, ⁇ 200 mg/dl; borderline-high risk, 200-239 mg/dl, high-risk, >240 mg/dl; for LDL cholesterol, desirable, ⁇ 130 mg/dl, borderline- high risk, 130-159 mg/dl, high risk, >160 mg/dl. That leads to an increased demand for accurate, simple, cost-effective LDL cholesterol and HDL cholesterol measurements.
  • Total cholesterol can serve as a first step for prediction of CHD risk and its measurement is reasonably advanced and reference system is essentially complete.
  • Lipoproteins vary greatly in size, density, relative composition, and biological function. Ultracentrifugation separates lipoproteins according to their density. Plasma is centrifuged at its own density of 1.006 kg/1. The VLDL and chylomicrons float to the top after centrifugation. The second centrifugation is performed after adjustment to increase the density of 1.006 kg/1 to 1.063 kg/1 to float LDL. Then LDL cholesterol is measured. In this method, although LDL cholesterol may be measured directly, it is time-consuming and needs expensive instrument, i.e., ultracentrifuge. It cannot be used in routine work for large population studies.
  • Another ultracentrifugation method for lipoprotein separation is based on discontinuous density-gradient.
  • the lipoprotein may be separated in a single centrifugation step. Different density solutions are carefully placed into each tube along with the sample. After centrifugation to equilibrium, each of the lipoproteins will have migrated into its respective isopycnic density region. That too is time-consuming. It also needs expensive equipment and specialists to run them.
  • lipoproteins are separated based on their size. It is known that there is a high correlation between lipoprotein density and particle size, owing to their chemical composition and structure. Lipoprotein separated by chromatography is correlated with those separated by ultracentrifugation. Agarose column and high performance liquid chromatography (HPLC) with gel permeation column have been successfully used to separate LDL, VLDL, HDL, and recovery of each lipoprotein is high. In case of incomplete separation with one agarose column, multiple columns may be used. Nevertheless, the procedure is time-consuming and cumbersome. Compared to agarose chromatography, HPLC is relatively simple and rapid. However, expensive equipment is needed. Chromatography technique is only restricted to laboratory research and cannot be used for diagnosis purpose because of the complexity and the length of the procedure, and the need for special instruments.
  • This method is recommended by the Centers for Disease Control (CDC) as a reference for LDL cholesterol measurement.
  • the procedure involves combination of ultracentrifugation and chemical precipitation. It begins with the separation of VLDL by ultracentrifugation at 1.006 gm/ml. HDL and LDL are then separated. Instead of by ultracentrifugation, heparin- manganese precipitation of density larger than 1.006 gm/ml fraction is applied, where LDL is precipitated and HDL left in the supernatant. After sedimentation of LDL, HDL cholesterol is measured. LDL is calculated as the difference in cholesterol between the density >1.006 gm/ml fraction and the HDL. This procedure still needs one step of ultracentrifugation.
  • LDL cholesterol is mostly derived by Friedewald estimation. It requires three separate measurements to determine the total cholesterol, HDL cholesterol and total triglycerides. LDL cholesterol is estimated from the Friedewald formula as follows:
  • LDL Cholesterol Total Cholesterol - (HDL Cholesterol +
  • Triglyceride/5) Each of these measurements could introduce a certain degree of distortion and could lead to imprecision and inaccuracies in LDL cholesterol determination. LDL cholesterol value is increasingly inaccurate at triglyceride levels above 200 mg/dl. The procedure is not reliable in hypertriglyceridemic states (>400 mg/dl) and requires fasting samples as well.
  • LDL is precipitated by heparin at pH-5.12, achieved by including sodium citrate buffer.
  • LDL is precipitated from serum by polyvinyl sulfate in the presence of EDTA and polyethylene glycol methylether.
  • LDL may be precipitated from serum by unspecified amphipathic polymers in imidazole buffer at pH 6.10.
  • LDL cholesterol is calculated as the difference between total cholesterol and cholesterol in the supernate, or is measured directly after dissolving the precipitate.
  • polyclonal antibodies are coated to latex beads which are used to adsorb HDL and VLDL. After filtration to remove the latex beads, LDL is left in the supernatant and its cholesterol content is measured. That procedure requires several expensive antibodies to adsorb HDL and VLDL.
  • LDL may be isolated from other lipoproteins by agglutinating the LDL with a lectin. Agglutin.ation is a clumping together of LDL particles which causes them to precipitate. Therefore, the cholesterol content of isolated LDL may be determined (Sears, U.S. Pat. No. 4,190,628).
  • the present invention provides a method for direct quantitative determination of VLDL, LDL, HDL as well as apolipoproteins cholesterol in a sample of blood plasma comprising the following steps in which: a) An antibody against the lipoprotein or apolipoprotein is partitioned in one of the phases of aqueous two-phase system with or without the help of partitioning enhancer by conjugation; b) A sample of blood plasma is applied to an aqueous two-phase system; c) After mixing and incubation for a short period of time, the phases are separated by gravity or centrifugation within several minutes; d) The phase containing the antibody and the targeted lipoprotein or apolipoprotein is removed; e) The targeted lipoprotein cholesterol is directly determined with an enzymatic procedure.
  • This invention is based on an aqueous two phase system in which plasma cholesterols are partitioned in the bottom phase while polyethylene glycol (PEG) conjugated anti-LDL monoclonal antibody (PEG-Mab) is partitioned in the top phase.
  • PEG-Mab helps specifically the LDL transportation to the top from the bottom phase.
  • Plasma LDL cholesterol value is obtained by measuring the top phase cholesterol.
  • the aqueous two-phase systems are formed when two polymers, such as PEG and dextran, or one polymer and one low molecular weight component (e.g. salt) , such as PEG and potassium phosphate, are mixed at appropriate concentrations in the presence of water.
  • the polymers are generally water- soluble.
  • the distribution of a protein between the phases depends on the properties of respective phases and the protein.
  • the conditions of the system is adjustable so that lipoprotein or apolipoprotein is partitioned in one phase (for example the bottom phase) .
  • the addition of polyethylene glycol conjugated with an antibody against LDL will bring only LDL to another phase (i.e. the top phase) .
  • the measurement of cholesterol of that phase (top phase) will therefore give the plasma LDL cholesterol value.
  • the components of the first phase of the aqueous two-phase system may be selected from polyethylene glycol, polyvinyl alcohol, polypropylene glycol, dextran, etc. and components for the second phase may be selected from dextran, methyl cellulose, potassium phosphate, etc.
  • the specific reagents used to adsorb the lipoproteins or apolipoproteins are preferable monoclonal antibodies of the lipoproteins or apolipoproteins. But certainly polyclonal antibodies may also be used wherever possible for purely economical reasons. By standard procedures, such as those developed by Harris, et al. in J. Polymer Science, 22, 341 (1984), these antibodies may be readily conjugated to the selected components of the first phase. Because of the specific functions of these conjugate components, they are termed as the partitioning enhancers or anchoring agents in the present invention.
  • the present invention is simple, rapid, inexpensive, and easy to perform.
  • One of the great advantages is that only one antibody is used. It is also easily applicable to other lipoprotein cholesterol measurements such as lipoprotein (a) [Lp(a)], HDL and VLDL. It can even be applied to measure the cholesterol of denatured lipoproteins, such as malondialdehyde- reacted LDL (Kondo, et. al. E. P.
  • glycated LDL Cohen, WO 94/00592
  • cholesterol of apolipoproteins such as A-I, A-II, A-IV, B-48, B-100, C-I, C-II, C-III, D and E (Albers, et al., Clinics in Laboratory Medicine, 137, 1989). All of these lipoprotein cholesterols are measurable with the same approach as the present invention, but with different antibodies according to the targeted lipoprotein or apolipoprotein.
  • PEG 3,400 and phosphate phase system was prepared according to the following composition:
  • Ultracentrifuge prepared LDL was used to examine the partitioning of LDL in the two-phase system. In the PEG ' 3,400/phosphate system described in Example 1, LDL was found to partition in the bottom phase, as measured by the cholesterol contents. Ultracentrifuge prepared HDL and VLDL were also virtually partitioned in the bottom phase.
  • Example 4 Partitioning of Human Plasma
  • a monoclonal antibody against T2 fragments of LDL apo B (Caltag, So. San Francisco, CA) binding specifically to LDL and not cross-reacting with VLDL and Lp(a) was used in this preparation. 600 ⁇ l of monoclonal antibody solution with 1 mg/ml in 0.1 M NaHC0 3 was added to 100 ⁇ l of methoxy- polyethylene glycol nitrophenyl carbonate (Sigma chemical, St. Louis, MO). The mixture was incubated for 24 hours at 4*C. The conjugated material was stored at 4 * C for further use.
  • the antibody of LDL itself is hydrophilic and partitions in the phosphate phase of PEG/phosphate system.
  • the conjugated molecules, PEG-antibody partitions in the PEG phase.
  • the conjugated PEG is called partitioning enhancer or anchoring agent in the present invention.
  • PEG-anti-LDL-apo B conjugate or simply PEG-Mab was partitioned in the two-phase system described in Example 1.
  • Plasma samples were ultracentrifuged at 50,000 RPM for 18 hours at 10*C.
  • the top VLDL fraction was removed and the bottom HDL-LDL infranant was collected.
  • the bottom of the centrifuge tube was washed with 0.15 M NaCl and the solution and infranant were combined. 40 ⁇ l of 5,000 unit/ml heparin
  • Example 8 Partitioning of LDL and Plasma in the

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Abstract

La présente invention concerne une procédure de dosage et/ou un appareil permettant de mesurer directement le cholestérol des VLDL, LDL, HDL, et des apolipoprotéines dans le plasma sanguin et dans les liquides organiques. L'invention porte sur un procédé de détermination quantitative directe du cholestérol des VLDL, LDL HDL et apolipoprotéines dans un échantillon de liquide organique dans lequel un anticorps agissant contre la lipoprotéine ou l'apolipoprotéine est fractionné en une ou deux phases d'un système aqueux à deux phases, avec ou sans l'aide d'un activateur de fractionnement par conjugaison. Un échantillon de liquide organique, tel que du plasma sanguin est appliqué au système aqueux à deux phases. Après mélange et incubation de courte durée, les phases subissent une séparation par gravité ou centrifugation pendant plusieurs minutes. La phase contenant l'anticorps et la lipoprotéine ou l'apolipoprotéine ciblées est retirée. Le cholestérol de lipoprotéines ciblé est alors directement déterminé au moyen d'une procédure enzymatique.
PCT/US1995/009504 1994-08-01 1995-07-31 Dosages du cholesterol de lipoproteines Ceased WO1996004556A1 (fr)

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Application Number Priority Date Filing Date Title
AU31514/95A AU3151495A (en) 1994-08-01 1995-07-31 Lipoprotein cholesterol assays

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Application Number Priority Date Filing Date Title
US28248994A 1994-08-01 1994-08-01
US08/282,489 1994-08-01

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WO1996004556A1 true WO1996004556A1 (fr) 1996-02-15

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011140A1 (fr) * 1996-09-11 1998-03-19 Pharmacia & Upjohn Ab Technique de purification d'alipoproteine et composition a utiliser dans le cadre de cette technique
WO1999036785A1 (fr) * 1998-01-16 1999-07-22 Abbott Laboratories Dosage immunologique utilise pour detecter des lipoproteines a tres basse densite et anticorps appropries
US5990081A (en) * 1995-03-03 1999-11-23 Esperion Therapeutics, Inc. Purified APO A and APO E compounds and methods for using them
US6090921A (en) * 1996-08-23 2000-07-18 Esperion Therapeutics, Inc. Process for purifying apolipoprotein a or apolipoprotein e
US6107467A (en) * 1996-09-11 2000-08-22 Pharmacia & Upjohn Ab Process for purifying a compound
EP1029928A3 (fr) * 1999-01-27 2002-09-18 Matsushita Electric Industrial Co., Ltd. Procede de determination du cholesterol et capteur utilisable pour sa mise en oeuvre
US6559284B1 (en) 1996-09-11 2003-05-06 Esperion Therapeutics, Inc. Process for purifying a protein
US6737275B2 (en) 2001-02-05 2004-05-18 The Board Of Regents For Oklahoma State University Direct serum lipids assays for evaluation of disease states
WO2008086019A1 (fr) * 2007-01-09 2008-07-17 Cholestech Corporation Dispositif et procédé de mesure du cholestérol associé au ldl
US7582484B2 (en) 2002-01-18 2009-09-01 Cholestech Corporation High-density lipoprotein assay device and method
US7772007B2 (en) 2004-04-02 2010-08-10 Cholestech Corporation Assay device for direct measurement of LDL cholesterol
US7795038B2 (en) 2002-04-09 2010-09-14 Cholestech Corporation High-density lipoprotein assay device and method
WO2012024691A1 (fr) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Systèmes multiphasiques pour analyse de matériaux solides

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746605A (en) * 1983-10-26 1988-05-24 Boehringer Mannheim Gmbh Process and a reagent for the determination of low density lipoproteins (LDL)
US4945040A (en) * 1988-02-29 1990-07-31 Arch Development Corporation Immunoassay for lipoprotein(a)
US4980065A (en) * 1989-10-18 1990-12-25 Lehigh University Separation of mixtures by aqueous two-phase systems
US5078886A (en) * 1989-10-18 1992-01-07 Lehigh University Separation of mixtures by two-phase systems
US5093254A (en) * 1990-01-23 1992-03-03 The United States Of America, As Represented By The Secretary Of Commerce Aqueous two-phase protein extraction
EP0484863A1 (fr) * 1990-11-07 1992-05-13 Daiichi Pure Chemicals Co. Ltd. Anticorps monoclonal et procédé pour mesurer des lipoprotéines à faible densité ayant réagi avec la malondialdehyde
US5407810A (en) * 1993-08-20 1995-04-18 Genentech, Inc. Aqueous multiple-phase isolation of polypeptide

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746605A (en) * 1983-10-26 1988-05-24 Boehringer Mannheim Gmbh Process and a reagent for the determination of low density lipoproteins (LDL)
US4945040A (en) * 1988-02-29 1990-07-31 Arch Development Corporation Immunoassay for lipoprotein(a)
US4980065A (en) * 1989-10-18 1990-12-25 Lehigh University Separation of mixtures by aqueous two-phase systems
US5078886A (en) * 1989-10-18 1992-01-07 Lehigh University Separation of mixtures by two-phase systems
US5093254A (en) * 1990-01-23 1992-03-03 The United States Of America, As Represented By The Secretary Of Commerce Aqueous two-phase protein extraction
EP0484863A1 (fr) * 1990-11-07 1992-05-13 Daiichi Pure Chemicals Co. Ltd. Anticorps monoclonal et procédé pour mesurer des lipoprotéines à faible densité ayant réagi avec la malondialdehyde
US5407810A (en) * 1993-08-20 1995-04-18 Genentech, Inc. Aqueous multiple-phase isolation of polypeptide

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CLINICS IN LABORATORY MEDICINE, Volume 9, Number 1, issued March 1989, BACHORIK, "Measurement of Total Cholesterol, HDL-Cholesterol and LDL-Cholesterol", pages 61-72. *
JOURNAL OF CHROMATOGRAPHY, Volume 513, issued 1990, DIAMOND et al., "Correlation of Protein Partitioning in Aqueous Polymer Two-Phase Systems", pages 137-143. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 38, issued 1980, MATTIASSON et al., "Partition Affinity Ligand Assay (PALA) A New Approach to Binding Assays", pages 217-223. *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5990081A (en) * 1995-03-03 1999-11-23 Esperion Therapeutics, Inc. Purified APO A and APO E compounds and methods for using them
US6506879B1 (en) 1995-03-03 2003-01-14 Esperion Therapeutics, Inc. Purified Apo A and Apo E compounds and methods for using them
US6090921A (en) * 1996-08-23 2000-07-18 Esperion Therapeutics, Inc. Process for purifying apolipoprotein a or apolipoprotein e
US6423830B1 (en) 1996-08-23 2002-07-23 Esperion Therapeutics, Inc. Process for purifying apolipoprotein A or apolipoprotein E
US6767994B1 (en) * 1996-09-11 2004-07-27 Pharmacia Ab Process for purifying a compound
US6107467A (en) * 1996-09-11 2000-08-22 Pharmacia & Upjohn Ab Process for purifying a compound
WO1998011140A1 (fr) * 1996-09-11 1998-03-19 Pharmacia & Upjohn Ab Technique de purification d'alipoproteine et composition a utiliser dans le cadre de cette technique
US6559284B1 (en) 1996-09-11 2003-05-06 Esperion Therapeutics, Inc. Process for purifying a protein
WO1999036785A1 (fr) * 1998-01-16 1999-07-22 Abbott Laboratories Dosage immunologique utilise pour detecter des lipoproteines a tres basse densite et anticorps appropries
EP1029928A3 (fr) * 1999-01-27 2002-09-18 Matsushita Electric Industrial Co., Ltd. Procede de determination du cholesterol et capteur utilisable pour sa mise en oeuvre
US6762062B2 (en) 1999-01-27 2004-07-13 Matsushita Electric Industrial Co., Ltd. Method of determining cholesterol and sensor applicable to the same
US6737275B2 (en) 2001-02-05 2004-05-18 The Board Of Regents For Oklahoma State University Direct serum lipids assays for evaluation of disease states
US7582484B2 (en) 2002-01-18 2009-09-01 Cholestech Corporation High-density lipoprotein assay device and method
US7795038B2 (en) 2002-04-09 2010-09-14 Cholestech Corporation High-density lipoprotein assay device and method
US7772007B2 (en) 2004-04-02 2010-08-10 Cholestech Corporation Assay device for direct measurement of LDL cholesterol
WO2008086019A1 (fr) * 2007-01-09 2008-07-17 Cholestech Corporation Dispositif et procédé de mesure du cholestérol associé au ldl
US7824879B2 (en) 2007-01-09 2010-11-02 Cholestech Corporation Device and method for measuring LDL-associated cholesterol
WO2012024691A1 (fr) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Systèmes multiphasiques pour analyse de matériaux solides
WO2012024688A1 (fr) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Systèmes multiphases et leurs utilisations
WO2012024693A1 (fr) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Séparation d'analytes biologiques en fonction de leur densité à l'aide de systèmes multiphasiques
WO2012024690A1 (fr) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Systèmes à phases multiples ayant des propriétés de phases multiples
US20130280693A1 (en) * 2010-08-20 2013-10-24 President and Fellows of Harvard College a University Density-based separation of biological analytes using multiphase systems
US9176105B2 (en) * 2010-08-20 2015-11-03 President And Fellows Of Harvard College Density-based separation of biological analytes using multiphase systems
US9714934B2 (en) 2010-08-20 2017-07-25 President And Fellows Of Harvard College Multiphase systems and uses thereof
US9857353B2 (en) 2010-08-20 2018-01-02 President And Fellows Of Harvard College Kit for density-based separation of biological analytes using multiphase systems
US10436768B2 (en) 2010-08-20 2019-10-08 President And Fellows Of Harvard College Density-based separation of biological analytes using mutliphase systems
US10732167B2 (en) 2010-08-20 2020-08-04 President And Fellows Of Harvard College Multiphase systems and uses thereof

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