CA2104780C - Lipid fractionation - Google Patents

Abstract

The method separates a first class of lipoprotein from a second class of lipoprotein in a sample and analyzes the first class of lipoprotein. The second class of lipoprotein is first precipitated. The sample is then contacted with magnetically responsive particles. The sample is then placed in a magnetic field until the magnetically responsive particles have sedimented, thereby caus-ing the precipitated second class of lipoproteins to sediment. The first class of lipoproteins is left in the supernatant of the sample for subsequent analysis.

Description

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LIPID FRACTIONATION
Back round of the Invention This invention relates to the fractionation of lipid mixtures.
Plasma :Lipoproteins are spherical particles which contain varying' amounts of cholesterol, triglycerides, phospholipids, and p~.~oteins. They include an outer surface composed of phospholipid, free cholesterol, and protein, and an inner core containing mostly esterif ied cholesterol and trig:Lycerides. Plasma lipoproteins serve to solubilize and transport cholesterol and triglyceride in the bloodstream.
The relative proportion of protein and lipid in a plasma lipoprotein determines the density of the plasma lipoprotein, Gotto, 1988, Hosp. Pract. 23:4 (Suppl. 1).
Based on their density, particle size, composition, and electrophoretic: mobility, circulating lipoproteins have been categorizcad into four major classes. The classes are: chylomicrons, very-low-density lipoproteins (VLDL), low-density li~~oproteins (LDL), and high-density lipoproteins (IaDL). Some of the characteristics of these classes are shown in. Table 1.

CHARACTERISTICS OF PLASMA LIPOPROTEINS
Diameter Density Origin ~,nQStroms (Q/ml) Chylomicrons 7..°i0-12,000 <0.95 intestine VLDL 300-700 <1.006 liver LDL 180-300 1.019-1.063 catabolism of VLDL

HDL 50-120 1.063-1.21 liver & intestine The major classes of lipoproteins can be further divided into subclasses. LDL includes at least 7 subclasses, as is described in McNamara, 1990, AACC Lipids and Lipoproteins Division Newsletter 4:1, Krauss et al., 1982, J.
Lipid Res. 23:97, and McNamara et al., 1987, Arteriosclerosis 7:483. HDL includes at least two subclasses, HDLz and HDL3, as is described in Whitaker, 1986, Clin. Chem. 32:1274, and Anderson, 1977, Biochim. Biophys. Acta 493:55.
l0 The major risk factors for coronary heart disease are hypercholesterolemia, cigarette smoking, hypertension, diabetes, obesity, and male sex. Although hypercholesterolemia in general is the most prominent of these risk factors, numerous clinical studies have shown that the different lipoprotein classes have very distinct and varied effects on heart disease, Crouse et al., 1985, J. Lipid Res., 26:566-572;
Kannel et al., 1979, Ann. Intern. Med., 90:85-91; Kannel et al., 1984, Circulation 70:157A-205A. In fact the presence of HDL provides a protective effect against coronary heart 2o disease, and therefore relatively low HDL-cholesterol levels may be indicative of greater risk, Miller et al., 1975, Lancet 16:23; Castelli et al., 1977, Circulation 55:767; Cordon et al., 1977, Am. J. Med. 62:707; Heis et al., 1980 Circulation 62:116 (Suppl. 4).
Recognition of the importance of HDL cholesterol as a strong inverse risk factor for coronary artery disease has led to substantial demand for an HDL cholesterol assay suitable for clinical and research use. Various methods, including ultracentrifugation, electrophoresis, and specific precipitation, Havel et al., 1955, J. C.Iin. Invest. 34:1345, have been used to separate various classes of lipoproteins.
Precipitation--based methods have been used widely for routine quantitation of lipoproteins. Several precipitation-b~~sed methods have been described in the recent literature. Most of these methods are based on earlier work by Burstein and colleagues (reviewed in Burstein, M., a:nd Sch.olnick, J.R., Lipoprotein-polyanion-metal interactions, In, Advances in Lipid Research 11, R. Paoletti and D. Kritchevsky, Eds., Academic Press, New York, NY 1973, pp. 67-108). In these methods, the fr~actiomation of lipoproteins in a solution, e.g., serum, is accomplished primarily by selective precipitation followed by centrifugation. The supernatant is then used to determine the cholesterol content remaining, or, by difference, the cholesterol content of that fraction which has been specifically removed from the supernatant. For example, low density lipoprotein (LDL) cholesterol, can be determined in this manner by specifically precipitating only LDL using heparin in citrate buffer at pH 5.04. Others have reported using polyvinyl sulfate or polyethylene glycol to effect specific precipitation of LDL and VLDL. In each case, centrifugation is necessary before measuring the cholesterol content.
Similarly, the measurement of HDL-cholesterol is usually a two-step process in which HDL is first separated from the other apoB-containing plasma lipoproteins, and the: cholesterol content of the HDL-containing fraction measured. The most commonly employed methods are based on those developed by Burstein and his colleagues (Burstein et al., 1988, in Clarkson TB, Kritchevsky D, Pollak OJ (eds): Monographs on Atherosclerosis, New York, Karger) in which the apoB-containing lipoproteins are'removed by precipitation with r~.~Q47~ ~

a polyanion in combination with a divalent cation, Bachorik et al., 1986, Methods Enzymol. 129:78. The two most commonly used precipitants are sodium phosphotungstate-MgCl2 and dextran sulfate (m. wt.
50,000)-MgClZ, Warnick et al., 1982, Clin. Chem. 23:1379.
Other precipitants used include heparin sulfate-MnClZ and heparin sulfate-calcium carbonate. In these assays the precipitating reagent is added to an aliquot of serum or plasma. A heavy white precipitate forms immediately, and the mixture is allowed to stand until precipitation is complete. The precipitate is then sedimented by centrifugation, and an aliquot of the clear supernatant is removed for cholesterol analysis.
The first method used extensively in major population studies employed heparin and Mn++ to precipitate VLDL and LDL, allowing HDL to be measured in terms of the amount of cholesterol remaining in the supernatant solution, Burstein et al., 1960, Clin. Chim.
Acta 5:609, Frederickson et al., 1968, J. Clin. Invest.
47:2446-2457, Manual of Laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, I. National Heart and Lung Institute. DHEW Publications No. (NIH) 75-628, 1974. This method has been extensively studied, Bachorik et al., 1976, Clin. Chem. 22:1828-1834, Warnick et al., 1978a, J. Lipid Res. 19:65-76, and modifications have been described, Warnick et al., 1978a, supra; Warnick et al., 1978b, Clin. Chem. 24:900.

Summarv of the Invention In general, the invention features a method of separating a first class of lipoprotein, e.g., any of HDL, LDL, or V1~DL, i:n a sample from a second class of lipoprotein, e,.g., a:ny or all of the remaining types of lipoproteins in the sample, e.g., separating HDL from LDL
and/or VLDL, sEaparating LDL from HDL and/or VLDL, or separating VLD~~ from LDL and/or HDL. The method includes: precipitating the second class of lipoprotein, l0 preferably by contacting the sample with a precipitating reagent; contacting the sample with a magnetically responsive particle; and placing the sample in a magnetic field until the magnetically responsive particles have sedimented, thereby causing the precipitated second class of lipoproteins to sediment and leaving the first class of lipoproteins in the supernatant of the sample.
Preferred embodiments include those in which the precipitating reagent is contacted with the sample prior to contacting the sample with the magnetically responsive particles; the precipitating reagent and the magnetically responsive particles are contacted with the sample simultaneously, i.e., they are added simultaneously; the precipitating reagent includes e.g., dextran sulfate and MgCl2, or phosphotungstic acid and MgClZ; the magnetically responsive particles include polyacrolein and a form of iron e.g., iron oxide; the sample contains up to 1,300 mg/dl triglyce:rides; the sample is a biological fluid, e.g., blood, plasma, or serum, from an animal, e.g, a vertebrate, e..g., a mammal, e.g., a human; the sample will not be reaturned to an animal; and the sample will be returned to an animal e.g., the animal from which the sample was talcen.
In another aspect the invention features a method of mea:auring the amount of a constituent, e.g., WO 92/16300 N ~ ~ ~ ~ a PCT/US92/02170 cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, contained in a first class of lipoprotein, e.g., HDL, LDL, or VLDL in a sample. The sample includes a first and a second class of lipoprotein, the second class including any other class of lipoprotein, e.g., if the first class is HDL, the second class can be LDL, VLDL, or both. The method includes: precipitating the second class of lipoprotein, preferably by contacting the sample with a precipitating reagent; contacting the sample with a magnetically responsive particle; placing the sample in a magnetic field until the magnetically responsive particles have sedimented, thereby causing the precipitated second class of lipoproteins to sediment, leaving the first class of lipoproteins in the supernatant of the sample; and determining the amount of cholesterol in the first class of lipoprotein.
Preferred embodiments include those in which the amount of the constituent of interest in the first class is determined by determining the amount of the constituent in the supernatant, and those in which the amount of the constituent in the first class is determined by determining the total amount of the constituent present in the sample, determining the amount of the constituent in the sedimented second class, and subtracting the latter from the former.
Preferred embodiments include those in which: the precipitating reagent is contacted with the sample prior to contacting the sample with the magnetically responsive particles; the precipitating reagent and the magnetically responsive particles are contacted with the sample simultaneously, i.e., they are added simultaneously to the sample; the precipitating reagent includes e.g., dextran sulfate and MgClZ, or phosphotungstic acid and MgCl2; the magnetically responsive particles include ?1~4~~0 polyacrolein and a form of iron, e.g., iron oxide; the sample contain ulp to 1,300 mg/dl triglycerides; the sample is a biol~~gical fluid, e.g., blood, plasma, or serum, from an animal, e.g, a vertebrate, e.g., a mammal, e.g., a human; t:he measurement is performed on an automated device; and the measurement is performed on a manually operated spectrophotometer.
Preferrecl embodiments include those in which the concentration of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the HDL of a sample is determined by precipitating and sedimenting the LDL and VLDL in the sample, then measuring the concentration of the constituent in the supernatant; the concentration of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the LDL of a sample is determined by precipitating and sedimenting the HDL and VLDL in the sample, then measuring the concentration of the constituent in the supernatant; the: concentration of a constituent, e.g., cholesterol, phosphol:ipid, apolipoprotein, triglyceride, or cholesterol eater, in the VLDL of a sample is determined by precipitating and sedimenting the LDL and HDL in the sample, then measuring the concentration of the constituent in the supernatant; the concentration of a constituent, E~.g., cholesterol, phospholipid, apolipoprotein, trigl:yceride, or cholesterol ester, in the HDL of a sample ins determined by precipitating and sedimenting the LDL a:nd VLDL in the sample, determining the amount of the constituent in the sediment, determining the total amount of the constituent in the sample, then subtracting the former from the latter; the concentration o:E a constituent, e.g., cholesterol, phospholipid, alpolipoprotein, triglyceride, or cholesterol ester, in the LDL of a sample is determined WO 92/16300 ?

_ g _ by precipitating and sedimenting the HDL and VLDL in the sample, determining the amount of the constituent in the sediment, determining the total amount of the constituent in the sample, then subtracting the former from the latter; and the concentration of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the VLDL of a sample is determined by precipitating and sedimenting the HDL and LDL in the sample, determining the amount of the constituent in the sediment, determining the total amount of the constituent in the sample, then subtracting the former from the latter.
The invention also includes a method of measuring the amount of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, contained in a first class of lipoprotein, e.g., HDL, or LDL, or VLDL, in a sample.
The sample includes a first and a second class of lipoprotein, wherein the second class of lipoprotein can include any other class of lipoprotein, e.g. if the first class is HDL, the second class can be LDL, or VLDL, or both. The method includes: precipitating the first class of lipoprotein, preferably by contacting the sample with a precipitating reagent; contacting the sample with magnetically responsive particles; placing the sample in a magnetic field until the magnetically responsive particles have sedimented, thereby causing the precipitated first class of lipoproteins to sediment, leaving the second class of lipoproteins in the supernatant of the sample; and determining the amount of cholesterol in the first class of lipoprotein.
Preferred embodiments include those in which the amount of the constituent of interest in the first class is determined by determining the amount of the constituent in the sedimented first class, and those in WO 92/16300 ~~ ~ ~ ~ ~ PCT/US92/02170 _ g _ which the amount of t:he constituent in the first class is determined by determining the total amount of the constituent present in the sample, determining the amount of the constituent ire the supernatant, and subtracting the latter from the l:ormer.
PreferrEad embodiments include those in which the precipitating reagent: is contacted with the sample prior to contacting the sample with the magnetically responsive particle; the precipitating reagent and the magnetically responsive particles are contacted with the sample simultaneously, i.e., they are added to the sample simultaneously; the precipitating reagent includes e.g., dextran sulfate and MgCl2, polyethylene glycol, heparin and citrate, or phosphotungstic acid and MgCl2; the magnetically responsive particles include polyacrolein and a form of iron, e.g., iron oxide; the sample contains up to 1,300 mg/dl triglycerides; the sample is a biological fluid, e.g., blood, plasma, or serum, from an animal, e.g, a vertebrate, e.g., a mammal, e.g., a human;
the measurement is performed on an automated device; and the measurement is performed on a manually operated spectrophotometer.
Preferred embodiments include those in which the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the LDL of a sample is determined by precipitating and sedimenting the. LDL in the sample, then measuring the level of the constituent in the sediment; the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, trig:lyceride, or cholesterol ester, in the VLDL of a sample is determined by precipitating and sedimenting the: VLDL in the sample, then measuring the level of the constituent in the sediment; the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, trig:lyceride, or cholesterol ester, in ~~04?8~

the HDL of a sample is determined by precipitating and sedimenting the HDL in the sample, then measuring the level of the constituent in the sediment; the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the LDL of a sample is determined by precipitating and sedimenting the LDL in the sample (leaving the HDL and VLDL in the supernatant), determining the amount of the constituent in the supernatant, determining the total amount of the constituent in the sample, then subtracting the former from the latter; the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the HDL of a sample is determined by precipitating and sedimenting the HDL in the sample, (leaving the LDL and VLDL in the supernatant) determining the amount of the constituent in the supernatant, determining the total amount of the constituent in the sample, then subtracting the former from the latter; and the level of a constituent, e.g., cholesterol, phospholipid, apolipoprotein, triglyceride, or cholesterol ester, in the VLDL of a sample is determined by precipitating and sedimenting the VLDL
(leaving the LDL and HDL in the supernatant) in the sample, determining the amount of the constituent in the supernatant, determining the total amount of the constituent in the sample, then subtracting the former from the latter.
A class of lipoprotein, as used herein, can refer to one of the major classes of lipoprotein, e.g., HDL, LDL, or VLDL, or one of the subclasses of the major classes, e.g., one of the seven subclasses of the LDL
major class or one of the two subclasses of the HDL major class.
Methods of the invention use magnetically responsive particles for the rapid removal of V~'O 92/ 16300 21 0 4 7 8 0 lipoproteins that have been selectively precipitated from an aqueous med:W m such as blood, plasma, or serum.
Preferred embodiments provide enhanced fractionation by selective precipitation of lipoproteins of one or more classes (e. g., any of HDL, LDL or VLDL) coupled with magnetically induced sedimentation. Fractionation is an important step in the measurement of pathologically significant lipid components such as HDL cholesterol, LDL
cholesterol, and VLD'L cholesterol.
The use of magnetizable particles eliminates or reduces the need for a centrifugation step in plasma lipoprotein fr<~ctionation. As a result, magnetically based separations are relatively rapid,~do not require expensive or energy consuming equipment, and reduce radiological, biological, and physical hazards.
Furthermore, samples are not exposed to centrifuge-generated heat, which can compromise the integrity of the samples.
Methods. of the invention allow for complete sedimentation of precipitated lipoproteins without additional dil»tion of the sample, even in samples with high concentrations of triglycerides. Thus, the time and expense required for diluting samples, and errors in estimation that arise from dilution, are eliminated.
Magnetic separation methods of the invention can be incorporated into an automated clinical analyzer (of the type commonly used for cholesterol measurements) to allow in-line separation and direct automated measurement of HDL-cholesterol, and/or LDL-cholesterol, and/or VLDL-cholesterol.

-11a-The magnetic separation methods of the invention can be used in the measurement of any lipoprotein component, e.g., phospholipids, triglycerides, apolipoproteins, or cholesterol esters, found in a class of lipoproteins.
Accordingly in one aspect the present invention resides in a combined reagent for separating lipoproteins comprising a selective chemical precipitating reagent and a magnetically responsive particle.
Preferably, the magnetically responsive particle comprises polyacrolein and iron.

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Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
Description of the Preferred Embodiments Reagents A "magnetically responsive particle" or "magnetic particle", as used herein, is any particle dispersable or suspendable in aqueous media and separable from suspension by application of a magnetic field. This includes particles that are intrinsically magnetically responsive or particles that have been rendered magnetically responsive, e.g., by attachment to a magnetically responsive substance or by incorporation of such substance into the particles. The magnetic component can be incorporated into a particle e.g., by impregnating the substance in a polymeric matrix such as cellulose or polyacrolein. The magnetic particles can be ferromagnetic, superparamagnetic, or more preferably paramagnetic.
The magnetic component of magnetically responsive particles can be any component that is susceptible to a magnetic field, e.g., salts, oxides, borides, or sulfides of iron, cobalt, or nickel; rare earth elements having high magnetic susceptibility, e.g., hematite or ferrite;
or pure magnetically responsive metals or alloys thereof.
A wide variety of particles e.g., cellulose/iron oxide or polyacrolein/iron oxide, are suitable for use in the invention.
Magnetic particles can be obtained from Cortex Biochem., Inc., (San Leandro, CA). Several types of magnetic particles were tested. They are, in order of descending preference, polyacrolein/iron oxide (e. g., Cortex ,#CM1001), cellulose/iron oxide (e. g., Cortex ~CM1000), low density cellulose/iron oxide (e. g., Cortex #CM1003), and magnetizable charcoal (e. g., Cortex #CM5002).
Methods for the fabrication of magnetically responsive particles are known to those skilled in the art, see e.g., U.S. Patent 4,628,037, which discloses the production of particles with magnetic metal oxide cores; U.S.
Patent No. 4,687,748, which discloses the preparation of spheres composed of magnetic particles included in a polymer matrix, e.g., in a carbohydrate matrix; U.S. Patent No.
l0 4,795,698, which discloses the production of magnetic-polymer particles by the coprecipitation of transition metals in the presence of a polymer having available coordination sites; and U.S. Patent No. 4,661,408, which discloses the preparation of magnetically responsive particles with a core of Cr02.
Magnetically responsive particles are also commercially available, as described herein.
The magnetically responsive particles may be added sequentially or simultaneously with the precipitating reagent.
If added simultaneously, (using a single 2o precipitation/magnetically responsive particle reagent) the size and surface characteristics should be chosen to allow the particles to remain in suspension until the precipitation is complete - at which time magnetic separation is applied. For this type of "simultaneous addition" procedure, particles in the range of 1-10 microns have been used for selective lipoprotein precipitation assays.
Magnetically responsive particles smaller than 1 micron will also work well since they are likely to remain in suspension until the magnetic field is applied. The upper limit of particle size depends on factors which, in addition to size, determine sedimentation rate in the absence of a magnetic field e.g., particle density, the nature of the functional groups on the surface of the particle (e. g., hydrophilic groups would result in relatively slower sedimentation while hydrophobic groups would result in more rapid sedimentation), and on the rate of reaction for the specific lipoprotein precipitation reaction being used. The particles must remain in suspension long enough to allow precipitation of the desired lipoprotein.
Factors affecting the choice of size of a magnetically responsive particle are known to those skilled in the art, see e.g., E.P. Patent No. 230768; U.S.
Patent No. 4,628,037; U.S. Patent No. 4,687,748.
1o The reagent used for the specific lipoprotein precipitation will vary with the lipoprotein to be precipitated and can be chosen by methods known to those skilled in the art. Heparin and citrate, heparin-Mn++, dextran sulfate-Mg++, polyethyleneglycol, polyethylene glycol-polyvinyl sulfate, and phosphotungstate-Mg++ have all been used successfully for selective precipitation of one or more class of lipoproteins.
Precipitating reagents useful in the analysis of HDL-cholesterol are known to those skilled in the art and include the following: heparin/Mn++ (available e.g., from WAKO
Chemicals, Dallas, TX) (Burstein et al., 1960, supra, Warnick et al., 1978, J. Lipid. Res. 19:65); phosphotungstic acid/MgCl2 (available e.g., from Sigma Chemical or Roche Diagnostics) (Drager et al., 1982 Lab. Med. 6:198, Burstein et al., 1969, Life Sci. 8:345 ); dextran-S04/MgCl2, (available e.g., from DMA) (Warnick et al., 1982, Clin. Chem. 28:1379);
heparin/MgCl2/sucrose; (Burstein, 1962, C.R. Acad. Sci.
225:605); Heparin/Ca++ (Noma et al., 1978, Clin. Chem. 24:150);
heparin/Ca++/Ni++ (Noma et al., 1979, Clin. Chem. 25:1480); or polyethylene glycol, (available e.g., from Diagnostic Chemicals Ltd., Monroe, CT)(Vikari, 1976, J. Clin. Lab.
Invest. 36:265) Subclasses of HDL can be separated from one another and from other classes of lipoprotein, see e.g., Whitaker et al., 1986, Clin. Chem. 32:1274, or Gidez et al., 1982, J.
Lipid. Res. 23:1206.
Precipitating reagents useful in the analysis of LDL-cholesterol are known to those skilled in the art and include the following: heparin and citrate (available from Genzyme, One Kendall Square, Cambridge, Massachusetts or E. Merck, A.G.
(Germany) as LDL cholesterin Cat. No. 14992) (Wieland et al., 1983, J. Lipid Res. 24:904); polyvinylsulfate, (available from Boehringer, Mannheim (FRG) as LDL-cholesterol Cat. No. 726290) (Assmann et al., 1984, Clin. Chem. Acta 140:77, Maier et al., 1983, Clin. Chem. 29:1173, Kerscher et al., 1985, Clin.
Biochem. 18:118); PVS/PEGME (polyvinyl sulfate polyethyleneglycol methyl ether) (available from BioMerieux, Cat. No. 61532, 69260 Charbonnieres, France) (Wehmeyer et al., Abstract Presented at 1983 national meeting of Am. Assoc. for Clin. Chemistry, Boehringer Mannheim, GmbH, Research Center, Tutzing, Germany); heparin/Ca++/EDTA/lipase (Bartl et al., 1983, Clin. Chem. Acta 128:199); dextran So4/Ca++ (available from Immuno A.G. Vienna, Austria as Quantolip LDL-cholesterol) (Walton et al., 1964, J. Clin. Path. 17:627, Cornwell et al., 1961, J. Lipid Res. 2:110);heparin/resin (Noma et al., 1978, Clin. Chem. 24:1504).
For each specific precipitation reagent one or more types of magnetically responsive particle can be applied over a wide range of particle concentrations, e.g., phosphotungstate or dextran sulfate can be used to precipitate LDL and VLDL, leaving HDL in the supernatant solution. With either of these reagent systems many magnetically responsive 3o particles work well in a "sequential addition" mode, i.e., wherein the precipitating reagent is added first and the magnetically responsive particles are added after allowing precipitation to take place. Several also perform satisfactorily in the "simultaneous addition" mode, i.e., wherein the precipitating reagent and the magnetic particles are added simultaneously. Reagents, however, may be added in any sequence which results in effective precipitation and sedimentation.
The concentration of magnetically responsive particle needed to effect rapid complete separation will vary, e.g., with the concentration of lipid in the sample. The concentration for a given application can be determined by methods known to those skilled in the art and as described to herein. In many cases the concentration of magnetically responsive particles in the reagent mixture will vary from 5 to 50 mg/ml and more preferably the concentration will vary from 15-25 mg/ml.
The precipitating reagent can be bound, by methods known to those skilled in the art, to the surface of the magnetically responsive particles, to enhance stability, lot-to-lot consistency, or to assure even faster separation, but the use of magnetically responsive particles to provide rapid, efficient separation usually does not require that the 2o precipitating reagent be bound to the magnetic particles.
Example 1: Comparison of Magnetic Particle-Based Sedimentation with a Centrifuge-Based Method for the Determination of HDL
Cholesterol Content of Serum In this example magnetic particle-based separation methods of the invention were compared with a method in which centrifugation was used to sediment the precipitated lipoproteins. In all cases lipoproteins were precipitated with dextran S04/MgCl2.
Magnetic particles and precipitating reagent were 3o combined to form a combined magnetic particle/precipitating reagent. Magnetic particles were added to the combined reagent as a 50 mg/ml slurry of particles in water. 100 ml of combined magnetic particle precipitating reagent included between 10 and 50 ml of slurry, 50 mls. of dextran sulfate-MgCl2, and water (if needed) to make 100 ml. The dextran sulfate-MgCl2 solution was purchased from DMA or was formulated according to the method of Warnick et al., 1982, Clin. Chem. 28:1374, from commercially available material. The data in Table 2 was obtained with dextran sulfate-MgCl2 obtained form DMA. In the magnetic separations shown in Table 2, 0.1 mls of the combined precipitation reagent/magnetic particle reagent (50 ml magnetic particle slurry/100 ml combined reagent) was added to a 0.50 ml serum sample and allowed to incubate for 10 min. (Later experiments show that 1 min. of incubation is sufficient.) Samples to be magnetically sedimented were placed in a Serono Q

Diagnostics magnetic rack for 1-10 min. (Usually 1-3 min. is sufficient.) Magnetic particles were obtained from Cortex Biochem., Inc. (San Leandro, CA). Four types of particles were tested: M-1, M-2, M-3, and M-4.
M-1 refers to Low Density Cellulose/iron oxide (#CM1004) (1-10 ~ in diameter); M-2 refers to Cellulose/iron oxide (#CM1000) (1-10 a in diameter); M-3 refers to Polyacrolein/iron oxide (#CM1001) (1-10 ~u in diameter);
M-4 refers to Magnetizable charcoal (#CM5002) (1-25 ~ in diameter).
For centrifuged samples, 0.05 ml of precipitating reagent was added to a 0.50 ml sample, allowed to stand for 10 minutes and then spun at 2000 rpm for 15 min. in a standard laboratory centrifuge. Total cholesterol in the supernatants (which is equivalent to HDL cholesterol) was analyzed by standard methods, and results expressed as mg/dl of cholesterol.
The results are shown in Table 2.

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WO 92/16300 ~~ ~ ~ ~ PCT/US92/02170 Example 2: Amount of Magnetic Particles Reauired.
The amount of magnetic particles needed for reliable reaults was determined by comparing the HDL-cholesterol values obtained in a centrifugation based assay with values obt=ained in magnetic separation using differing amounts of polyacrolein/iron oxide particles.
The amount of ~~articles was varied by varying the amount of slurry addedl to 100 ml of combined reagent, as described above:. O.:LO ml of combined reagent was added to 0.50 ml of sample" All other materials and methods are described i.n Example 1. The results are shown in Tables 3 and 4. The results are expressed as mg/dl HDL
cholesterol.

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01 N M 00 sf' e-1 d' ~D M e-I l0 Gl O ~ I~ In d' d' ~Y d' 01 d' d' M
U ~
.,.r w zl '"~ N r, w ~c ~ co rn o ~.l ~~~ ~o N 00 (~ d' N n-1 N 01 O 00 N N
M tn M 00 00 111 tI1 'd' lf1 M If1 ll1 N ~D tn N 01 ~-I d' O1 O1 O O 1 M tf1 M CO l~ If1 !11 si' d' d' d' 1 I I~ CO M O I O 10 O I~ O 00 I !f1 M CO 00 1 II1 d' In M tn d' 00 d' M O CO 01 01 ~D I~ 00 01 01 NInM00I~~'d'd~d'M~'d' O I'~ t~ tf1 N ~-I O 00 ri CO wl O
M tn M 00 00 U.1 tf1 cr In M In In N M V' In t0 h CO p1 O rwi N M
r-1 r-i ri rl r-1 r~ r~l ri N N N N

M 01 M 00 H I~ d' rl N N M M M N 111 (~ H
N
G
z ~ °' oo,~
H -r1 d' CO N f~ N CO 10 r-1 ~ N N M M M N ~ ~ H
W ~
O
O w U
r-r O ~rl w o ro o v ,~
'.~' ~ 10 ~' d' 01 N N N f~ lf1 N
N M M d' N Ifl I~ H
W~ H
dP
If1 M I~ rl I~ r-1 t~ N O
ri N M M M N tn t~ H
N
tT O
~ ~I-1 d' O O c0 H OW11 r-I
N d' M M N 111 I~ H
~r-1 fU
' . ' ' . ' . ' M O d' O N CO d' O
N d' M d' N lfI I~ H
U cn ., ~OI ,~ N M
mz z.1~4~~~~

Example 3: Further Comparison Studies.
In this example a magnetically based separation method was compared with a centrifugation based method. In Table 5, magnetic separation was performed with dextran sulfate/MgClZ (obtained from DMA) as the precipitating reagent (RDI HDL-M). In magnetically sedimented samples, 0.10 ml combined magnetic particle/pre~cipitating reagent (20 ml magnetic particle slurry/100 ml combined reagent) was added to 0.50 ml of sample and the magnetic field applied 10 minutes later. Total. sample cholesterol and total sample triglycerides were determined by standard methods and are shown in mg/dl. All other methods and materials are as described in Example 1.

~~ s~
+~ O
I
:T m--I a m ~r t~ mn M N w o0 ftl S-1 D C~ M M M M M M si' d' M
z ~ ac x w a~ ~ ~ >~
rl as o w~ ~a ~r1 ~f Ca N lf1 G~ O 00 N d' I~ N
L.r 0 x M M M d' M M d' d' d' N Gl dY
'Lf U
CO 1 I 1 I d' 1 1 I
M 1 I I I t~ 1 I I
O
v b ~
o °~ ~ o, M I ~ ~ o, ~ I
E-~ O ~ 01 10 I rl 'd' N lf1 I
e-i rl r-1 I N N N r-i I
U
N
.l.l td O rl N M d' If7 10 t~ 00 01 wz WO 92/16300 Pte: ~ ' US92/02170 d' d' ~O O cr CO tn In 01 N 1O CO lf1 O lf1 t~
t0 ttl M Ifl !' M tC) M M 10 d' d' d' In d' d' id vG o0 1 CO Ov d' 1C Ov M CO O t0 ~-~1 I~ C1 1C lf1 M I n M L!1 M M 10 d' If1 d' lf~ d' d' I 01 C1 1 t~ d' d' tC1 r-1 r-1 In d' ( n 1 I~ O 1 In ri \D M O 01 01 O I O t~ 10 I N 1 r-I N ri N rl N I e-I ltl O
ri N
1!1 CO 10 1 01 I~ M N CO I~ M lf1 lf1 01 t0 I~
d' d' N I 00 to I~ r-I M O N t0 l~ M M 00 N N N I N N N N N N N N r1 N N N
O r-I N M d' In to t~ 00 a1 O ~-I N M d' 111 -~ r-I r-I ,--1 rl e-i e-I r-I r-1 r1 N N N N N N

WO 92/16300 r'CT/US92/02170 C1 M e-I M I~ r"1 I~ N G1 CO 00 01 G~ 00 00 N tt1 d' M d' M tf1 M v-1 lf1 M M lf1 In M d' 01 1C d' 10 ri O O~ d' O~ l~ l~ 00 00 d' t~ I~
N to d' M In N to M r-I lf1 M M In ~0 M d' O

d' M

t0 O d' O
t0 M 00 M rl I 1 I I I I 1 1 I o0 p~ M r-1~ 1 I 1 I I 1 I 1 1 d' co 1 I I 1 i 1 1 I I M
N

lft tD I~ I~ d' l~ M l~ 10 t11 d' O r1 M O l~
e-i O ri O~ Lf1 t~ 01 d1 lf1 N O I~ O O d' 01 N N N ~ N M ~ ri rl N N N N N N
I~ CO 01 O e-i N M d' ltl l0 t~ 00 01 O
N N N N M M M M M M M M M M d' d' 21D~~~~

lfl I~ U7 d1 I~ 01 ~0 CO d' O ewl In I~ d' v-I M
M lf1 ti' N In N ~i' ~C d' M I~ M d' d' In d' M 1~ tf1 00 CO 10 tW -1 d' G1 O tW0 In N d' MlI1'd'NInMd'f~d'MI~Md'd'lnd' 1 1 1 I 1 tD I I 1 1 1 1 1 1 I
I I i I I 00 I I I I 1 1 1 1 I

r-1 M M I~ O CO 01 O 10 CO t0 I M In O Q1 ~D d' O 10 CO O Q1 CO In N r-1 I e-I O O 1~
N N rl e-I e-1 N N ri M N I N N N ~-I
N M d' t!1 ~D t~ CO 01 O ri N M d' lf1 ~D I~
d' d' d' d' d' d' C' d' In tn tf1 t~1 lf1 tn In In The experiments described in Table 6 are similar to those described in Table 5 except that the dextran sulfate precipitating reagent used in the magnetically based method was not purchased from DMA but was formulated from commEarcially available dextran sulfate, as described alr~ove.

U
~r1 O
N +~ t H a N mn o vo r, ~r oo .~r roro ~x a~
x H~
wro~
~ ~o .a w +a N
N ov lI1 r1 c~ M d' ri vD
o x ~r ~c ~n ~ ~, ~r M ~n ~n a~ a~
U tn dl d sr O o r-1 rl 01 1P o0 l~ lf1 e-I N d' N l~ 10 t~
-/ r/ e-i e-i M M
r~
O
N
N
r-1 Gl ,~ 10 10 rl d' 01 C7 O t0 f~ rl ~ d' I~ O C1 10 ri 00 l0 N N N N N N N ~-I N
E-~ U
O
.N
r-~1 N f'~ ~!' lfl t~ I~ CO
wz ~10~?8~

ri O O C1 d' f~ 10 N d' ~D C11 01 M ri l~ In !17 ~p 1p cr M M M lfl In M M M In M !~ \O
M M N 00 In O~ d' O 10 0~ 1~ O t0 tf1 C~ 00 tWp 1p cr M M M In In M d' d' tC1 M IWG
tn 00 Cn O M M I~ M M I~ C1 1 O u1 N If1 ri M 00 cf O N l~ e-I d' I~ I~ 1 01 M t0 lf1 r-1 ri ri e-1 M ri ri N N1 N d' I~ 00 tn tf1 00 lf1 00 M I~ 1 r-1 d1 ri I~
In In r-i O CO cp M d~ 10 Q1 l~ 1 01 O M 01 N N N f~ r-I N r-I N N r"1 ri 1 r-I N N ri O rl N C1 d' 111 l0 t~ 00 Q1 O e-1 N M d' lfl r-I r-1 r-1 r~ r-i ri r-i r-1 e-I r-1 N N N N N N

2 ~. ~~7~U' N v-1 00 d' 00 d' r-1 I~ lf1 ri lf1 N ~ d' O~ rl 111 M d' In d' lt1 to M to d' tl1 d' d' CO d' 1p tn M O t~ rl d' N 1G 1C In 1D e-i N t0 ~ M
tf1 M In lf1 In In lf1 M In d' In sr V' GO si' t0 O In I~ I~ O O I~ I~ 00 10 01 01 ri t0 C~ O
C1 M rl v-1 a1 01 In 01 ri 01 lf1 1~ N 01 10 01 M ri rl e-I M v-1 ri ~-I e-I
lf1 tf1 N O 1p O 00 O N N 00 ~ d' 00 l~
O~ O CO t!1 N 10 d' Q1 10 O O I~ l~ CO ~ 10 rl N ~ N N N N ~ N '-I N ~ ~ N N e-1 ~0 t~ 00 01 O v-1 N M d' In ~ I~ CO Q1 O r-1 N N N N M M M M M M M M M M d' d' ~.1~~1~ ~
00 N M 01 C~ ~i G1 lf1 N 01 N rl t~ CO O ri M M In M C7 d' M M M d' In 10 N ll~ M
lf1 N N 00 00 ~i !~ d' O O M N d' 10 In rl N M M tI1 M M d' M M d' s1' tf1 d' N lf1 M
d' 00 00 N O l'~ In r-I O 1D 01 I~ 01 O N ~O
O ~i O CO d' t'~ l~ 10 !~ ri 00 10 M O In 00 ri e-1 v-1 ~i N rl N e-1 r-i N l~ t0 1D d' l'~ O 00 01 t~ N N d' tn sr tf1 sr ~O M d1 C~ lLl t0 O ~i' r-1 tl) r-I M ~d' 00 O
e-I ri r-I e-I ri ri ri ri N ri N N v-1 rl e-1 N M d' In ~O l'~ 00 01 O ri N M d' In tD f~
~ ~r ~ ~ ~r ~r m mw wm ~n z~o~~~o ,..i o co Q, m o ~ o ~ ~ ~ o w ~ h o tn In ~p M d' h M M lf1 M M lf1 M
d' 01 h h d' 01 d' G1 1D e-I !f1 O tt1 d' 00 v-1 N .~ ~ ~ In M M h M M In M M lt1 M
Cp Q1 rl C1 d' h N N ~O lC1 M d' M O N d' d' 01 h !f1 01 1p d' d' 1~ h N M d' CO CO M
rl e-i r-I h N N r-1 e~i M M ri rl 0p h d' N h ~ h '~f' CO ~ N O e-1 M ri N
O d' O h d' In d' h ~O CO r-i 01 h M h N
N v-~I e-~ N N N M N ~ N N r-1 N N N N
CO 01 O rl N M d' t~l ~O h 00 G1 O ~ N M
lf1 tf1 10 ~O ~O ~O t~ ~G ~O ~O 10 tp h h h h .~ ~4~SQ

~r t~~~ r ~ ~ ~~o ~ o, In t~ o In o~ 00 r, r~ r~ cn r~ in ~ mn wo In c~ er lI1 d' !~ O ~O IT t0 01 1C O d' cy7 N rl r~ cn c~ r~ r~ In ~r ~r wwo vo ~r In v-1 tn I~ d~ 0p 170 I~ 10 N d' O 01 N C11 t~ CO e-i In In O N t'~ 10 N 00 ~O e-I O
N N N N r-1 W rl r1 e-1 N ~-~I r-I
N O 00 O t0 tV 00 r-I O ri e-1 C11 d' rl lf1 d' C11 N CO f'~ I~ CO d' M 00 10 e-1 In NNNMr-ItVNNNNNNNN
d' tn t0 I~ C~0 Cn O ri N c 1 d' !n ~O f~
t~ I~ !~ I~ I~ h CO 00 CO t0 00 00 00 00 Table 7 compares the effect of waiting 1 minute or minutes after addition of the combined magnetic particle/precipitating reagent before applying the magnetic field. Other conditions were as described for Table 6.

,_ s' ~ ~ri i ~UOH~7E'Nwn~~Irovo~~o~
I ~ri ~rl O O ~'i ~ d' M sr d' d' d' M M M
x x ~~
~ s~ ,_ > tr ~a c:
(d ~, ~r~
1 ,'F'.., O H I
> tl~ O a ri N d' N 10 G1 sf' e-1 10 I~
I,Y" 0 O tf7 d~ M ~d' d' M V' M M M
x ~~
d r~i ri ft7 ~"., tT O
1~ ''~ ~rl ,~'1", N N d' N tn O lt1 e-I I~ 00 tlld'Md'ehd'd'MMM
N O
U tl1 O
'O
.,i O
I U d' 00 ri l~ d' N tn M !~ N
~ri ?~ N 00 01 In 10 In 111 d' I~ O
f..1 r-1 r-/ rl d' rl wl r-I M N N
N D~
O
fa O
N
ri 4) M In e-1 d' C1 d' tf1 10 CO I~
1~ r-I GO e-I N lfl tn CO N I~ N O
N N N N N N N N N N
O .O
Ei U
O
O
y1 . pp ~ O wl N M d' tl1 10 I~
Id O 00 00 0~ O~ a1 01 01 01 01 01 C4 x 210~~8U
00 t0 N O N M I~ O O d' ~C ~f1 M M rl N
d' M d' M M N N d' d' M d' ~ M M d' M
tp tn In n e-1 ,-1 1p 01 00 N lf1 M N N rl N
d' M d' N M N N M M M d' I~ M M d' M
CO 1C t0 CO O N t0 I~ 00 M l~ IW-I N v-1 M
d'Md'NMNNMMMtfII~MMd'M
In d' M l!1 O ri tn M d' M CO CO C7 10 M N
Op 1C O t~ O O O In lf1 O 01 r-1 N O I~ N
N N to r'1 ~ e-I e-1 N N e-I N rl N
10 O1 01 O1 C1 d' CO M rl d' ~ In d' I~ CO 00 tl1 O O t0 rl N ~-I 01 ~-1 01 M O CO N 1n O
N N N ~ ~ ~ ~ ~ ~ N N ri rl r-1 N
00 O~ O ri N M d' tIl t0 I~ 00 Ql O r-I N M
01 01 O O O O O O O O O O r1 .-I r-1 ri r-I ri r~ ~-I e--I r-I ri r-I ri ri e-1 r~ ri r~

2.~a~z~

r' M u' d' oo u~ M
I N M M cr ~r o~
w .~r ~ cr co ~r N
N N M M cr d' vo M w ~ v~ u~ M
N N M d' M cr d' lf1 N d' ~ CV d' N ~0 M tI1 M 00 l~
M ri r-I ri ~i r-1 M C1 O~ r' 10 u~ 00 ~ M d' V7 ~i ~G
N ri N N N N N
cr lf1 lp t~ CO C~1 O
ri e-1 e-~I rl r-I ~i N
rl r-1 ri e--I e-1 ~i rl ~1.0~~8 0 Example 4~ Magnetically Responsive Particle-Based _Clinical Assay for HDL Cholesterol The precipitating reagents dextran sulfate and MgCl, together, precipitate LDL and VLDL in serum. In this assay magnetically responsive particles, preferably polyacrolein-iron particles (polyacrolein:iron oxide (Fe304) - 40:60) 1-10 ~C in diameter and the precipitating reagents are added to the sample simultaneously. After precipitation, the LDL and VLDL are pelleted by the application of a magnetic field to the sample. HDL
remains in the supernatant. The amount of HDL
cholesterol is then assayed using an enzymatic reagent that measures total cholesterol. The intensity of color produced in the reaction is proportional to the concentration of HDL cholesterol. The assay is described in detail below.
The preferred sample is serum, though EDTA plasma may be used. The sample need not be fasting. Plasma (or serum) should be separated from the erythrocytes as soon as possible after collection since various changes can occur during storage. HDL cholesterol in plasma samples is stable for at least four days at 4°-8°C or up to 2 weeks at -20°C.
Precipitation and fractionation are performed as follows:
a. Dispense 0.50 mL of each serum sample and control into an appropriately labeled test tube;
b. Add 0.10 mL of combined magnetic particles/precipitating reagent (dextran sulfate (0.1 mmol/1)), MgCl2 (250 mmol/1), and magnetically responsive particles (lOg/1) to the sample and vortex immediately for 10 seconds;
c. Incubate 1-10 minutes at 15°-30°C;

d. Place the tubes on a magnetic surface and wait approximately 3 minutes for complete sedimentation of the: magnetically responsive particles.
Longer sedimentation times may be necessary if the sample has a high level of triglycerides (see below). In any case, the sedivmentat.ion time can be established by methods known to those skilled in the art. The assays are performed in 10 x 75 mm or. l2 x 75 mm round bottom test tubes. Round bottom tubes are preferable to tubes with pointed or tapered bottoms because round bottom tubes result in a larger proportion of the sample being held more closely to the source of magnetism. The tubes are placed in racks that contain magnets in the base of the rack. Magnetic racks suitable for use in methods of the invention are available through: Serono Diagnostics, Allentown, PA; Gen-Probe, Inc., San Diego, CA; Ciba-Corning Diagnostics, E. Walpole, MA; Advanced Magnetics, Inc., Cambridge, MA; and Amersham Corp., Arlington Hts., IL. A suitable rack. exerts a magnetic flux density of approximately 175 tc~ 265 gauss 0.5 inch from the surface upon which the bottom of the sample tube rests.
e. Obtain an aliquot of clear supernatant for the cholesterol assay by transferring the supernatant solution to a second labeled test tube for analysis. HDL
cholesterol in the supernatant is stable for at least 72 hours when stored at. 2°-8°C.
f. Determine cholesterol content with a cholesterol identifying agent, e.g., with the DMA
Enzymatic Cholesterol Reagent Set (DMA Cat. No. 2340).
Measurements can be converted to cholesterol concentration by comparison to known calibrators, using e.g., the DMA HDL cholesterol standard (DMA Cat. No.
2331-153).

WO 92/16300 ~ PCT/US92/02170 g. Due to dilution of the sample during the precipitation step, multiply the HDL cholesterol concentration by 1.2 to obtain the final result.
Expected values for HDL are typically in the range of 30-70 mg/dL in males and 30-85 mg/dL in females. Each laboratory, however, should establish its own range of, expected values.
Determination of HDL-Cholesterol in Samples With Hicth Levels of Triglycerides Many precipitating reagents and sedimentation methods are not suitable for use with samples which contain high levels of triglycerides (greater than about 300-500 mg/dl) when centrifugation is used for sedimentation in that the supernatant is cloudy or turbid after precipitation and centrifugation. In these cases, samples with high levels of triglycerides must be diluted prior to precipitation to avoid erroneous cholesterol determinations. Methods of the invention, however, can be used on samples with triglyceride levels as high as 1000-1300 mg/dl, without dilution of the samples, although high triglyceride samples may require slightly longer sedimentation times than are used with normal samples.
Other embodiments are within the claims.
What is claimed is:

Claims (42)

Claims

1. A method of separating a first class of lipoprotein in a sample from a second class of lipoprotein in said sample comprising, precipitating said second class of lipoprotein by contacting the sample with a selective chemical precipitating reagent, contacting said sample with a magnetically responsive particle, and placing said sample in a magnetic field until said magnetically responsive particle has sedimented, causing said precipitated second class of lipoproteins to sediment, leaving said first class of lipoproteins in the supernatant of said sample.

2. The method of claim 1, wherein said second class of lipoprotein is precipitated by contacting said sample with a precipitating reagent.

3. The method of claim 2, wherein said precipitating reagent is contacted with said sample prior to contacting said sample with said magnetically responsive particle.

4. The method of claim 2, wherein said precipitating reagent and said magnetically responsive particles are contacted with said sample simultaneously.

5. The method of claim 2, wherein said precipitating reagent comprises dextran sulfate and MgC12.

6. The method of claim 2, wherein said precipitating reagent comprises phosphotungstic acid and MgC12.

7. The method of claim 1, wherein said magnetically responsive particle comprises polyacrolein and iron.

8. The method of claim 1, wherein said first class of lipoprotein comprises HDL.

9. The method of claim 1, wherein said sample comprises up to 1,300 mg/dl triglycerides.

10. A method of measuring the amount of a constituent of a first class of lipoprotein in a sample, said sample comprising a first and a second class of lipoprotein comprising, precipitating said second class of lipoprotein by contacting the sample with a selective chemical precipitating reagent, contacting said sample with a magnetically responsive particle, placing said sample in a magnetic field until said magnetically responsive particle has sedimented, thereby causing said precipitated second class of lipoproteins to sediment leaving said first class of lipoproteins in the supernatant of said sample, and determining the amount of said constituent in said first class of lipoprotein.

11. The method of claim 10, wherein said determination of said amount of said constituent in said first class is made by determining the amount of said constituent present in said supernatant.

12. The method of claim 10, wherein said determination of the said amount of said constituent in said first class is made by determining the total amount of said constituent in said sample, determining the amount of said constituent in said sedimented class, and subtracting said amount in said sedimented class from said total amount.

13. The method of claim 10, wherein said constituent is cholesterol.

14. The method of claim 10, wherein said second class of lipoprotein is precipitated by contacting said sample with a precipitating reagent.

15. The method of claim 14, wherein said precipitating reagent is contacted with said sample prior to contacting raid sample with said magnetically responsive particle.

16. The method of claim 14, wherein said precipitating reagent and said magnetically responsive particles are contacted with said sample simultaneously.

17. The method of claim 14, wherein said precipitating reagent comprises dextran sulfate and MgCl2

18. The method of claim 14, wherein said precipitating :reagent comprises phosphotungstic acid and MgCl2.

19. The method of claim 10, wherein said magnetically responsive particle comprises polyacrolein and iron.

20. The method of claim 10, wherein said first class of lipoprotein comprises HDL.

21. The method of claim 10, wherein said sample comprises up to 1,300 mg/dl triglyceride.

22. The method of claim 10, wherein said measurement is performed by an automated device.

23. A method of measuring the amount of a constituent of a first class of lipoprotein in a sample, said sample comprising a first and a second class of lipoprotein comprising, precipitating said first class of lipoprotein by contacting the sample with a selective chemical precipitating reagent, contacting said sample with a magnetically responsive particle, placing said sample in a magnetic field until said magnetic responsive particle has sedimented, thereby causing said precipitated first class of lipoproteins to sediment, leaving said second class of lipoproteins in the supernatant of said sample, and determining the amount of said constituent in said first class of lipoprotein.

24. The method of claim 23, wherein said determination of said amount of said constituent in said first class is made by determining the amount of said constituent present in said precipitated first class of lipoprotein.

25. The method of claim 23, wherein said determination of said amount of said constituent in said first class is made by determining the total amount of said constituent in said sample, determining the amount of said constituent in said supernatant and subtracting said amount in said supernatant from said total amount.

26. The method of claim 23, wherein said constituent is cholesterol.

27. The method of claim 23, wherein said first class of lipoprotein is precipitated by contacting said sample with a precipitating reagent.

28. The method of claim 27, wherein said precipitating reagent is contacted with said sample prior to contacting said sample with said magnetically responsive particle.

29. The method of claim 27, wherein said precipitating reagent and said magnetically responsive particles are contacted with said sample simultaneously.

30. The method of claim 27, wherein said precipitating reagent comprises dextran sulfate and MgCl2.

31. The method of claim 27, wherein said precipitating reagent comprises heparin and citrate.

32. The method of claim 23, wherein said magnetically responsive particles comprises polyacrolein and iron.

33. The method of claim 23, wherein said first class of lipoprotein comprises LDL.

34. The method of claim 23, wherein said sample comprises up to 1,300 mg/dl triglyceride.

35. The method of claim 23, wherein said measurement is performed by an automated device.

36. A combined reagent for separating lipoproteins comprising a selective chemical precipitating reagent and a magnetically responsive particle.

37. The combined reagent of claim 36, wherein said selective chemical precipitating reagent comprises dextran sulfate.

38. The combined reagent of claim 36, wherein said selective chemical precipitating reagent comprises phosphotungstic acid.

39. The combined reagent of claim 36, wherein said selective chemical precipitating reagent comprises heparin.

40. The combined reagent of claim 36, wherein said selective chemical precipitating reagent comprises polyethyleneglycol.

41. The combined reagent of claim 36, wherein said selective chemical precipitating reagent comprises polyvinyl sulfate.

42. The combined reagent of claim 36, wherein the magnetically responsive particle comprises polyacrolein and iron.