WO1993018067A1 - Dosage par immunocapture pour quantification directe de niveaux de cholesterol de lipoproteines specifiques - Google Patents

Dosage par immunocapture pour quantification directe de niveaux de cholesterol de lipoproteines specifiques Download PDF

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WO1993018067A1
WO1993018067A1 PCT/US1993/002011 US9302011W WO9318067A1 WO 1993018067 A1 WO1993018067 A1 WO 1993018067A1 US 9302011 W US9302011 W US 9302011W WO 9318067 A1 WO9318067 A1 WO 9318067A1
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ldl
cholesterol
binding
lipoprotein
agent
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PCT/US1993/002011
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English (en)
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Samar Kundu
Marie Anne La Fleur
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Abbott Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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

  • LDL-cholesterol is actually a measure of LDL-cholesterol, IDL cholesterol and Lp(a)- cholesterol, each of which are thought to be atherogenic markers.
  • Lp(a)-cholesterol concentration in plasma is independent of total cholesterol, HDL-cholesterol or triglycerides.
  • the measurement of Lp(a) should be done independently (Kurchinski et al. (1989) Clin. Chem. 3J>:2156- 2157).
  • a method for estimating Lp(a)-cholesterol concentration involves measuring the total Lp(a) mass in plasma ([Lp(a)]) and calculating the Lp(a)-cholesterol Because the unfractionated plasma layer ([d >1.006 g/mL chol]) also contains IDL and Lp(a) in addition to LDL and HDL, the LDL-cholesterol content of this fraction ([LDL chol]) ? , represents contributions from LDL, IDL, and Lp(a). In most,
  • the plasma LDL-cholesterol concentration is estimated by measuring three separate cholesterol concentrations: total-cholesterol ([Total-chol]), HDL-cholesterol ([HDL-chol]), and triglycerides concentration
  • LDL-cholesterol ([LDL-chol]) plasma concentration is then calculated using Friedewald's equation (Friedewald et al. (1972) Clin. Chem. 1_ ⁇ .:499-502) as follows:
  • the factor [Triglyceride/5] relates to the plasma VLDL- ⁇ cholesterol concentration. It is assumed that all plasma triglycerides are associated with VLDL and that the ratio of 35 triglyceride concentration to cholesterol concentration associated with VLDL is about 5. Thus, the VLDL-cholesterol concentration can be calculated from the triglyceride very low density lipoprotein (VLDL, d ⁇ 1.006 g/mL), low density lipoprotein (LDL, d 1.019-1.063 g/mL) and high density lipoprotein (HDL, d 1.063-1.21 g/mL).
  • VLDL very low density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • LDL is the major contributor to the plasma total cholesterol concentration in man, accounting for the one-half to two-thirds of the plasma cholesterol.
  • Two methods are presently available to determine the quantitative association between LDL-cholesterol and CHD.
  • a first method is based on the use of combined ultracentrifugation and polyanion precipitation procedures (Havel et al. (1955) J. Clin. Invest 24:1345-1353; McNamara et al.
  • Total plasma cholesterol is known to be an unreliable marker for prediction of coronary heart disease (CHD) in many patients.
  • CHD coronary heart disease
  • Epidemiological studies have established several lipoprotein-related risk factors for coronary heart disease. Elevated plasma levels of cholesterol associated with low density lipoprotein (LDL) markedly increase the risk of CHD (Castelli et al. (1986) JAMA 25_6_:2835-2838). Lowering plasma LDL-cholesterol concentrations reduces the risk of CHD, myocardial infarction (Ml) and CHD-related death (Lipid Res. Clinics Program (1984) JAMA 251:351-374; Frick ' et al. (1987) N. Engl. J. Med.
  • NASH National Cholesterol Education Program
  • LDL-cholesterol The recommended ranges for LDL-cholesterol are: ⁇ 130 mg/dL (desirable); 130-159 mg/dL (borderline high); > 60 mg/dL (high). No reliable method for the direct quantitation of LDL is yet available (Friedewald et al (1972) Clin. Chem. 18:499- 502; Warnick et al (1982) Clin. Chem. 2 :1379-1388.
  • Lp(a)-cholesterol concentration is assumed to be about 30% of the total Lp(a) mass concentration (Kostner et al. (1981 ) .f/?erosc/eros s _i:51-61).
  • the calculated LDL-cholesterol concentration then can be corrected for Lp(a)-cholesterol using one of the following equations:
  • the Lp(a) concentration can be calculated from the plasma Lp(a)-protein concentration.
  • the Lp(a)-protein concentration was measured by an ELISA test and the Lp(a) concentration was calculated by multiplying the Lp(a)-protein concentration by 4.21 (Fless et al. (1989) J. Lipid Res. 3__:651-662).
  • Lp(a)-cholesterol correction be made in the LDL-cholesterol concentration because studies have shown that diet and drug treatment will reduce LDL- cholesterol levels but not Lp(a)-cholesterol levels and proper patient monitoring requires an accurate measurement of LDL- cholesterol levels (Albers et al. (1975) Metabolism 24:1047- 1054; Vessby et al. (1982) Atherosclerosis 44:61-71). This is particularly true for patients with elevated levels of Lp(a). For example, the Lp(a) concentration in some patients have been found to be as high as 100 mg/dL (i.e., 30 mg/dL cholesterol) (Fless et al. U.S. Patent (1990) 4,945,040) ' . In such patients, the LDL-cholesterol values will be erroneous if no correction is made for Lp(a)-cholesterol.
  • the LDL-cholesterol concentration is calculated by subtraction of supernatant cholesterol from total cholesterol.
  • the Bio Merieux Kit Bio Merieux, Marcy- PEtoile, France
  • Patent 4,623,628) as a binder for LDL, a water- insoluble anion-exchanger which binds to VLDL and HDL (Maier et al. (1986) U.S. Patent 4,569,917), Ricinus Communis lectin as a agglutinating agent for LDL (Sears (1980) U.S. Patent 4,190,628), surfaces coated with anionic groups which have affinity for LDL (Knox et al. (1988) Eur. Patent 0319250A1), but none of these methods or reagents were evaluated with clinical samples.
  • apolipoprotein B apolipoprotein B (apo B) utilizing apo B-specific antibody
  • RIA competitive fluid phase and solid phase radio-immunoassays
  • ELISA enzyme-linked immunosorbant assay
  • RIDA radial immunodiffusion assay
  • EA electroimmunoassay
  • IPA immunoprecipitation assay
  • the antisera used in these assays lacked sufficient specificity to make the assays useful or reproducible.
  • the methodological problems of each of these assays have been reviewed (Labeur et al. (1990) Clin. Chem. 26_:591-597; Curry et al. (1978) Clin. Chem. 24:280-286).
  • An object of this invention is to develop a method of measuring LDL-cholesterot directly. Another object of this invention is to eliminate the presence of IDL- and Lp(a)- cholesterol in the LDL-cholesterol measurement. Another object of this invention is to directly measure LDL-cholesterol easily, cheaply, quickly, and accurately without the need of highly trained technicians or expensive equipment such as an ultracentrifuge. Still another object of this invention is to reduce the number of analytes and steps that are presently necessary to measure the LDL-cholesterol concentration. Yet another object of this invention is to directly measure LDL- cholesterol without the analytical variability generally associated with LDL-cholesterol measurement.
  • Lp(a) While the role of Lp(a) in CHD has not been fully investigated, a significant correlation between elevated Lp(a) serum or plasma levels in humans, coronary artery disease and the progression of atherosclerotic lesions has been established (Seed et al. (1990) New England J. Med. 222:1494- 1499). Lp(a) and LDL have common structural features. Both have a similar lipid composition and an apo B component.
  • Lp(a) also contains two molecules of apolipoprotein (a) (apo(a)) covalently linked to the apo B molecule by at least one sulfide bond.
  • Lp(a) concentrations in human plasma range from 1 mg/dL to more than 100 mg/dL.
  • Lp(a) is known to be transmitted genetically by an autosomal dominant trait and 20 to 35% of normal individuals have Lp(a) plasma concentrations greater than 30 mg/dL.
  • Lp(a) levels appear to be insensitive to changes in diet (Albers, et al. (1977) J. Lipid Res. 12:331 - 338) and treatment with cholestyramine (Vessby, et al.
  • Neomycin and niacin have been shown to reduce Lp(a) levels by 45% (Gurakar, et al. (1985) Atherosclerosis 5_Z:293-301).
  • Another object of the present invention is to measure the plasma Lp(a)-cholesterol concentration of patients easily and accurately and thus allowing researchers to further investigate the relationship between Lp(a)-cholesterol concentrations and CHD.
  • the assays include radioimmunoassays, enzyme-linked immunosorbent assays, radial immunodiffusion, electroimmunoassays and immunoelectrophoresis.
  • the assays commonly use antibodies directed against the apo(a) components of Lp(a) to measure total Lp(a) or its protein content ⁇ Albers, et al. (1990) Clin. Chem. 22:2019-2026).
  • Another object of the present invention is to directly measure the Lp(a)-cholesterol concentration easily, cheaply, quickly, and ' accurately without the need of highly trained technicians or expensive equipment such as an ultracentrifuge.
  • the present invention relates to a method for directly measuring concentrations of cholesterol associated with specific lipoproteins in a plasma sample.
  • the method involves the specific capture of intact lipoprotein particles containing cholesterol from a plasma sample with a specific lipoprotein binding agent.
  • the quantity of the specific lipoprotein cholesterol present in the sample is then measured by detecting the amount of binding-agent-lipoprotein complexes that have formed in the reaction.
  • the cholesterol contained in the binding-agent-lipoprotein complexes can be detected by a variety of standard methods known in the art such as, enzyme immunoassays, radioimmunoassays, ELISA, EMIT, and the like, or with labeled cholesterol specific binding agents.
  • the specific lipoprotein binding agent can be bound to a solid support.
  • the assay method may also incorporate a further step of separating the solid support from the sample before detecting the presence of cholesterol bound to the solid support.
  • the present invention is also directed to a method for selecting anti-LDL specific antibodies that are useful for detecting the amount of LDL-cholesterol in a sample.
  • Figure 1 Typical antibody titer plots of the monoclonal antibody MB16 obtained by incubating microtiter plates with LDL, VLDL, IDL, HDL and Lp(a) bound to the plates in separate wells and measuring the antibody bound to the lipoproteins by an ELISA assay.
  • Figure 2 Typical competitive binding curves of the monoclonal antibody MB16 obtained by pre-incubating the antibody with a lipoprotein, adding the mixture to a microtiter plate with LDL bound to the plate reaction wells and measuring the antibody bound to the LDL by an ELISA assay.
  • Figure 3 Typical binding curves of the HRPO labeled MAB B06 with lipoproteins LDL, VLDL, IDL, HDL and Lp(a) as described in Example 3.
  • Figure 4 Typical binding curves of MABs 4B5.6, SPL4A5, 8A2.1 , and 465C3D1 bound to microtiter plates for LDL- cholesterol using HRPO labeled MAB B06.
  • Figure 5 Typical binding curves of 1251-LDL to MABs 2D8, 1D1 and MB16 immobilized on Immulon 2 Removawell strips.
  • Figure 6 Typical competitive displacement curves of
  • Figure 7 A is a chart summarizing the composition of the two antigenic epitopes -of apoB.
  • B is a peptide map of the apo B fragments formed by thrombin.
  • Figure 8 Typical peptide fragments of the apo B T2 fragment which are useful in generating LDL-specific MABs.
  • Figure 9 A typical cholesterol standard curve for a specific LDL-cholesterol assay of this invention.
  • Figure 10 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB SPL4A5-Sepharose and the ultracentrifuge method.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB SPL4A5-Sepharose and the Friedewald method.
  • Figure 11 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 8A2.1- Sepharose and the ultracentrifuge method.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 8A2.1-Sepharose and the Friedewald method.
  • Figure 12 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 4B5.6- Sepharose and the ultracentrifuge method.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 4B5.6-Sepharose and the Friedewald method.
  • Figure 13 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 4B5.6- Sepharose and the ultracentrifuge method corrected for Lp(a)- cholesterol.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB 4B5.6- Sepharose and the Friedewald method corrected for Lp(a)- cholesterol.
  • Figure 14 A is a correlation curve for the ratio of LDL- cholesterol concentrations determined by the immunocapture assay using MAB 4B5.6-Sepharose and the ultracentrifugation method to the triglyceride concentration.
  • B is a correlation curve for the ratio of LDL-cholesterol concentrations determined by the immunocapture assay using MAB 4B5.6- Sepharose and the ultracentrifugation method corrected for Lp(a)-cholesterol to the triglyceride concentration.
  • Figure 15 A is a correlation curve for the ratio of LDL- cholesterol concentrations determined by the immunocapture assay using MAB 4B5.6-Sepharose and the ultracentrifugation method to the VLDL-cholesterol concentration.
  • B is a correlation curve for the ratio of LDL-cholesterol concentrations determined by the immunocapture assay using MAB 4B5.6-Sepharose and the ultracentrifugation method corrected for Lp(a)-cholesterol to the VLDL-cholesterol concentration.
  • Figure 16 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB MB16- Sepharose and the ultracentrifuge method.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB MB16-Sepharose and the Friedewald method.
  • Figure 17 A is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB MB16- Sepharose and the ultracentrifuge method corrected for Lp(a)- cholesterol.
  • B is a correlation curve for LDL-cholesterol measurements by the immunocapture assay using MAB MB16- Sepharose and the Friedewald method corrected for Lp(a)- cholesterol.
  • Figure 18 A is a correlation curve for the ratio of LDL- cholesterol concentrations determined by the immunocapture assay using MAB MB16-Sepharose and the ultracentrifugation method to the triglyceride concentration.
  • B is a correlation curve for the ratio of LDL-cholesterol concentrations, determined by the immunocapture assay using MAB MB16- Sepharose and the ultracentrifugation method corrected for Lp(a)-cholesterol to the triglyceride concentration.
  • Figure 19 A is a correlation curve for the ratio of LDL- cholesterol concentrations determined by the immunocapture assay using MAB MB16-Sepharose and the ultracentrifugation method to the VLDL-cholesterol concentration.
  • B is a correlation curve for the ratio of LDL-cholesterol concentrations determined by the immunocapture assay using MAB MB16-Sepharose and the ultracentrifugation method corrected for Lp(a)-cholesterol to the VLDL-cholesterol concentration.
  • Figure 20 A correlation curve for LDL-cholesterol measurements by the dry immunocapture assay using MAB 4B5.6 as described in Example 12 and the Friedewald method.
  • Figure 21 A correlation curve for LDL-cholesterol measurements by the indirect immunocapture assay using MAB 4B5.6-Sepharose as described in Example 13 and the Friedewald method.
  • Figure 22 Binding curves of the HRPO-digitonin conjugate of Example 16 to Lp(a)-cholesterol, LDL-cholesterol and VLDL-cholesterol particles.
  • Figure 23 A typical calibration curve plot of Lp(a)- cholesterol concentration versus absorbance prepared using the method of Example 18.
  • the present invention provides a method for determining the amount of cholesterol associated with a specific lipoprotein, such as LDL-cholesterol, in a sample.
  • a lipoprotein specific binding agent and a sample are mixed and incubated.
  • the amount of cholesterol associated with the lipoprotein of interest present in the sample is then determined from the amount of cholesterol present in the binding-agent-lipoprotein complexes formed in the reaction.
  • the claimed method utilizes a lipoprotein specific binding agent to form a binding complex with lipoprotein particles in a sample. Following separation of the sample and the binding-agent-lipoprotein complexes, the amount of cholesterol associated with the lipoprotein in the complex is then measured.
  • the lipoprotein particles are captured by a lipoprotein specific binding agent directly or indirectly bound to a solid support.
  • Lipoprotein specific binding agents include lipoprotein specific binding proteins, such as monoclonal and polyclonal antibodies and other lipoprotein specific synthetic or recombinant proteins that specifically bind lipoprotein cholesterol particles.
  • an LDL specific binding agent will include LDL specific binding proteins, such as monoclonal and polyclonal antibodies and other LDL specific synthetic and recombinant proteins, that specifically bind LDL-cholesterol particles.
  • Lipoprotein cholesterol particles are particles composed of a lipoprotein, such as LDL, VLDL, IDL, Lp(a), HDL and the like, containing cholesterol either directly or indirectly bound, coupled (ionically or covalently), absorbed or adsorbed to the lipoprotein particles.
  • a lipoprotein such as LDL, VLDL, IDL, Lp(a), HDL and the like, containing cholesterol either directly or indirectly bound, coupled (ionically or covalently), absorbed or adsorbed to the lipoprotein particles.
  • the specific lipoprotein-cholesterol particles of interest are separated from other lipoprotein cholesterol particles in the sample before the cholesterol determination is made.
  • LDL-cholesterol particles are selectively separated from HDL, Lp(a), IDL and VLDL-cholesterol particles prior to the measurement of the cholesterol associated with the LDL particles.
  • the lipoprotein specific binding agent can be attached directly or indirectly to a solid support, for example, by absorption, adsorption, covalent coupling directly to the support or indirectly through another binding agent (such as an anti-antibody antibody), or the like.
  • the type of attachment or binding will typically be dependent upon the material composition of the solid support and the type of lipoprotein specific binding agent used in the assay.
  • nitrocellulose, polystyrene and similar materials possess chemical properties that permit absorption or adsorption of proteins to a solid phase composed of this material.
  • Other materials such as, latex, nylon and the like contain groups that permit covalent coupling of the lipoprotein specific binding agent to the solid support.
  • the solid support can take the form of a variety of materials, for example, the solid support may be in the form of a bead particle, a magnetic particle, a strip or a layered device.
  • the separation of the binding-agent-lipoprotein complexes from the sample or more specifically from the other lipoprotein particles in the sample can be accomplished in a variety of ways.
  • the solid support can be removed from the sample or the sample can be removed from the solid support.
  • the solid support is a microtiter plate or another type of reaction well device, such as the devices described in US Patents 5,075,077 and 4,883,763 and US Patent Application Serial No. 523,629, incorporated herein by reference
  • the sample can be removed from the wells and the plate washed of any residual sample.
  • the solid support is a particle, such as a latex or magnetic particle
  • the solid support can be separated from the sample by filtration through a fiber matrix, such as the devices described in US Patents 4,552,839 and 5,006,309, US Patent Applications Serial Nos.
  • the binding-agent-lipoprotein complexes can be separated or removed by filtration such as by the Ion Capture Methodology described in co-pending US Patent Application Serial No. 150,278 and US Patent Application Serial No. 375,029, both of which enjoy common ownership and both of which are incorporated herein by reference.
  • These applications describe the use of ion capture separation, in which specific binding members used in an assay are chemically attached to a first charged substance and a porous matrix having bound thereto a second charged substance that binds to the first charged substance.
  • a specific binding pair is formed and separated from the reaction mixture by an electrostatic interaction between the first and second charged substances.
  • the specific binding member is preferably covalently coupled to the first charged substance.
  • charged substances include anionic and cationic monomers or polymers, such as polymeric acids, e.g. polyglutamic acid, polyaspartic acid, polyacrylic acid and polyamino acids; proteins and derivative proteins, such as albumin; anionic saccharides, such as heparin or alginic acid; polycations, such as GafQuatTM, diethylaminoethyl-dextran and cellulose derivatives such as polymeric quaternary ammonium compounds, such as CelquatTM L-200 and CelquatTM H-100.
  • polymeric acids e.g. polyglutamic acid, polyaspartic acid, polyacrylic acid and polyamino acids
  • proteins and derivative proteins such as albumin
  • anionic saccharides such as heparin or alginic acid
  • polycations such as GafQuatTM, diethylaminoethyl-dextran
  • cellulose derivatives such as polymeric quaternary ammonium compounds, such as Celquat
  • the amount of cholesterol in or on a lipoprotein particle can be determined by a variety of methods.
  • lipoprotein cholesterol such as LDL-cholesterol
  • lipoprotein cholesterol can be detected chemically by using the Liebermann-Burchard method or modifications of their method; enzymatically using a cholesterol specific enzyme such as cholesterol oxidase; through the formation of a cholesterol specific binding complex, such as an anti-cholesterol antibody/cholesterol complex; or through the release of the cholesterol from the lipoprotein followed by detecting the amount of cholesterol released using any of the above methods.
  • a cholesterol specific enzyme such as cholesterol oxidase
  • cholesterol specific binding complex such as an anti-cholesterol antibody/cholesterol complex
  • release of the cholesterol from the lipoprotein followed by detecting the amount of cholesterol released using any of the above methods.
  • One skilled-in-the- art may conceive of yet other methods of detection applicable to this method.
  • an LDL-cholesterol measurement can be made as follows. LDL particles present in a plasma sample are specifically captured by an LDL-specific monoclonal antibody immobilized on a solid support. After separating the solid support from the other unbound plasma lipoproteins, the cholesterol content of the bound LDL particles is estimated by releasing the cholesterol and its esters with a detergent solution. A Standard Cholesterol assay reagent comprising of cholesterol ester hydrolase, cholesterol oxidase and horseradish peroxidase is added.
  • the liberated hydrogen peroxide is then quantitated using a Tinder dye reagent comprising of 4-aminoantipyrine and 3,5- dichloro-2-hydroxybenzenesulfonic acid similar to that described (Sidel et al (1983) Clin. Chem. 29:1075-1079).
  • the cholesterol concentration in a given sample is quantitated on the basis of the color generation.
  • a sandwich immunoassay method for the quantitation of LDL-cholesterol in a plasma sample can be used. This involves the specific capture of the LDL particles in the plasma sample by the LDL-specific antibody immobilized on the solid support followed by quantitation of cholesterol in the captured LDL particles by a cholesterol binding agent which is coupled directly or indirectly to a label.
  • the LDL-cholesterol bound cholesterol binding agent is then quantitated by detection and measurement of the label.
  • Another alternative is based on an immunochromatographic assay format (such as described in US Patent 4,954,452 and co-owned and copending U.S. Patent Application Serial No. 072,459, incorporated herein by reference), in which the lipoprotein particles in the test sample bind to a labeled cholesterol binding agent.
  • the resulting complexes then travel along a test strip by capillary action.
  • the labeled LDL complexes are then captured by a high affinity anti-LDL specific antibody immobilized on the test strip followed by detection and measurement of the captured labeled LDL complexes.
  • the test strip is comprised of a porous or bibulous membrane and the result is determined by a visual readout of a detectable signal.
  • label refers to any substance which can be attached to specific binding agents, such as antibodies, antigens, cholesterol binding agents, lipoprotein specific- binding agents and analogs thereof, and which is capable of producing a signal that is detectable by visual or instrumental means.
  • suitable labels for use in the present invention can include chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, radioactive elements, colloidal metallic (such as gold), non- metallic (such as selenium) and dye particles (such as the particles disclosed in U.S. Pat. Nos.
  • Such enzymes include phosphatases and peroxidases, such as alkaline phosphatase and horseradish peroxidase which are used in conjunction with enzyme substrates, such as nitro blue tetrazolium, 3,5',5,5'-tetranitrobenzidine, 4-methoxy-l- naphthol, 4-chloro-l-naphthol, 5-bromo-4-chloro-3-indolyl phosphate, chemiluminescent enzyme substrates such as the dioxetanes described in US Patents 4,857,652 (issued August 15, 1989), 4,931,223 (issued June 5, 1990), 4,931 ,569 (issued June 5, 1990), 4,962,192 (issued October 9, 1990) and 4,978,614 (issued December 18, 1990), incorporated herein by reference, and derivatives and analogs thereof.
  • Fluorescent compounds such as fluorescein, phycobiliprotein, rhodamine and the like, including their derivatives and analogs
  • Cholesterol binding agents bind specifically to cholesterol and include digitonin, tomatine, filipin, amphotericin B and specific binding proteins such as polyclonal and monoclonal antibodies and other synthetic and recombinant proteins that specifically bind cholesterol, cholesterol esters and/or the cholesterol associated with lipoprotein particles.
  • a number of cholesterol binding agents are known in the literature. These include saponins such as digitonin (Berezin et al. (1980) Vopr. Med. Khim. 26_:843-846; TsybuPs kaya et al. (1986) Bioorg. Khim. 1 :1391 -1395), tomatine (Schultz and Sanders (1957) Z. Physiol. Chem.
  • Digitonin, tomatine, amphotericin B and antibodies can be used in the quantitation of cholesterol and its esters in lipoprotein particles. Digitonin and tomatine were chemically modified and then conjugated to horseradish peroxidase (HRPO) and alkaline phosphatase (AP). Amphotericin B and anti-cholesterol antibodies were coupled directly to HRPO and AP. All four HRPO and AP conjugates bind to cholesterol and its esters in lipoproteins which are immobilized on a solid phase. The binding affinity of the enzyme conjugates follow the order digitonin > tomatine > anti-cholesterol antibodies > amphotericin B. Because digitonin conjugates and tomatine conjugates bound effectively to the cholesterol components of lipoproteins, these conjugates are preferred in the present invention.
  • HRPO horseradish peroxidase
  • AP alkaline phosphatase
  • Amphotericin B and anti-cholesterol antibodies were coupled directly to HRPO and AP. All four
  • a method of the present invention is illustrated by the following sandwich assay example.
  • the method involves incubating the sample with a solid phase having an LDL specific binding agent, such as the monoclonal antibody 4B5.6, immobilized on a solid phase and the remaining non-specific binding sites of the solid phase blocked, such as with bovine serum albumin or alkali-treated casein.
  • LDL particles are captured by the antibody on the solid phase.
  • Digitonin or tomatine enzyme conjugates are then incubated with the solid phase. The conjugate binds to the cholesterol associated with the LDL particles on the solid phase.
  • the quantity or presence of enzyme bound to the solid phase or the quantity of unbound conjugate remaining after incubation with the solid phase is determined by incubation of enzyme substrate with the solid phase or the solution containing unbound conjugate.
  • the presence of cholesterol associated with the captured LDL particles is then determined from the presence of enzyme associated with the solid phase or a reduction of enzyme activity in the solution containing unbound conjugate as compared with the original conjugate solution added to the solid phase.
  • the quantity of cholesterol associated with the captured LDL particles is proportional to the quantity of enzyme associated with the solid phase or inversely proportional to the quantity of unbound conjugate. This method is also applicable for any of the other lipoprotein particles or mixtures thereof by substituting the appropriate lipoprotein specific binding agent for the LDL specific binding agent.
  • a critical aspect of this invention is the selection of the lipoprotein specific binding agents.
  • a lipoprotein specific binding agent preferably must selectively bind to the lipoprotein of interest, but not to other lipoproteins.
  • an LDL specific binding agent preferably binds only to LDL and not to other lipoproteins, such as HDL, VLDL, IDL and Lp(a).
  • the cholesterol binding agent is an antibody
  • the lipoprotein specific binding agent and the cholesterol binding antibody must be compatable such that neither binding agent interferes with the binding of the other agent to the lipoprotein particle and cholesterol associated with the particle.
  • the preferred lipoprotein specific binding agent is an antibody which binds specifically to the lipoprotein of interest.
  • the antibody is preferably a monoclonal antibody. Monoclonal antibodies are preferable because production quantities of antibody are readily available and such antibodies generally improve the lot-to-lot reproducibility and consistency in the assay results.
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody and may have less non-specific binding than an intact antibody (Wahl, et aL, J. Nuci. Med. 24:316-325, 1983).
  • Such fragments also may be used for the detection and quantitation of lipoprotein cholesterol particles according to the methods disclosed herein in the same manner as intact antibodies.
  • Such fragments are well known in the art and are typically produced by enzymatic degradation of an antibody, such as with pepsin, papain or trypsin.
  • antibodies and antibody fragments can be prepared using recombinant antibody methods such as those described in US Patent Applications Serial Nos. 513957, 693249, 789619, 776391 , 799770, 799772, and 809083, incorporated herein by reference, wherein antibodies or antibody fragments are produced from the RNA of an antibody producing B-cell from an immunized animal, such as a rat or mouse, using known recombinant techniques.
  • Lipoprotein specific binding agents according to the present invention also include bacteriophage described in US Patent 4,797,363, incorporated herein by reference. Bacteriophage tail or head segments are capable of selectively binding antigens. By mutation and selection processes, bacteriophage having the necessary binding characteristics to selectively bind lipoprotein cholesterol particles can be obtained.
  • Lipoprotein specific binding agents according to the present invention also include nucleic acid sequences, such as DNA and RNA, which selectively bind to lipoprotein cholesterol particles.
  • a library of nucleic acid sequences are tested for the desired binding characteristics and the sequences that are specific for lipoprotein cholesterol particles are isolated and replicated.
  • the lipoprotein specific binding agent is selective for only one lipoprotein, but some recognition of or binding to other lipoproteins can be tolerated.
  • an antibody selected for its ability to bind only to LDL particles present in a sample can minimally capture other lipoproteins and still be utilized in this invention.
  • the present invention can tolerate an LDL specific monoclonal antibody that does not cross-react with Lp(a) or HDL, but does cross-react with VLDL up to 20% and with IDL up to 20%.
  • the LDL specific monoclonal antibody should not cross-react with other lipoprotein and non-lipoprotein materials present in a sample.
  • the measurement of a specific lipoprotein cholesterol level can also be accomplished indirectly by removing all the other lipoproteins from the sample.
  • the selective binding agents of a group of selected lipoproteins can be used to remove these lipoproteins from a sample, leaving behind substantially only one lipoprotein in the sample. Measurement of the cholesterol in the sample after this group of lipoproteins have been removed gives an indication of the amount of cholesterol present in the remaining lipoprotein. For example, selectively removing HDL, VLDL, IDL and Lp(a) will essentially leave behind LDL in the sample. Measurement of the cholesterol in the remainder of the sample gives an indication of the LDL-cholesterol present in the sample.
  • lipoprotein specific binding agents such as antibodies, that are not selective for only one lipoprotein, such as an antibody that binds to both VLDL and LDL but not Lp(a)
  • VLDL and LDL antibody cross-reacting lipoproteins
  • Lp(a) non- cross reacting lipoprotein
  • the present invention has the capability of quantitating the amount of cholesterol associated with lipoproteins. This advancement in technology will more clearly define the correlation between lipoprotein cholesterol levels and CHD. For example, by specifically detecting LDL-cholesterol and not the cholesterol associated with other lipoproteins present in a plasma sample, the present invention improves the precision, accuracy and reproducibility of LDL-cholesterol measurement of a sample and thereby a better indication of the association of the LDL-cholesterol level with CHD. Thus, the dietary or therapeutic drug treatment of a patient can now be more carefully monitored for improved results in lowering the risk of CHD in the patient. Moreover, the simplicity of performing LDL-cholesterol measurements will also be improved.
  • MABs Monoclonal antibodies
  • SPL4A5, 8A2.1 , 8B3.5, and 8A6.6 (IgGi ), and 4B5.6 (lgG2b) were obtained from Alexander Karu, University of California at Berkeley as ascites precipitated by 50% ammonium sulfate and dialyzed against TRIS buffer pH
  • the MAB SPL4A5 is described in U.S. Patent 4,619,895 for use in the diagnosis of patients suffering from Type IV hypertriglyceridemia.
  • the blood of such patients contains LDL particles of size between 215-230 A which is not present in normal individuals.
  • the antigenic epitope of these LDL particles which is recognized by the MAB SPL4A5 was not disclosed.
  • the MABs 8A2.1, 8B3.5, 8A6.6 and 4B5.6 were described in LaBelle, et al. (1990) Clin. Chimica. Acta. 191 :153-160. These four MABs reportedly reacted with LDL and apo B, but not with HDL.
  • the antigenic epitopes of the LDL and apo B which were recognized by the four MABs were determined by Western blot using apo B fragments cleaved from apo B by thrombin.
  • the MABs 8A2.1 , 8A6.6 and 8B3.5 recognized thrombolytic fragment T3 and the MAB 4B5.6 recognized only thrombolytic fragment T2.
  • the MABs Prior to use in the experiments herein, the MABs were purified on a Protein A Sepharose column as described in Example 1 ⁇ infra).
  • the MABs SPL4A5, 8A2.1 , 8A6.6 and 8B3.5 were eluted from the column with pH 6.0 citrate buffer and the MAB 4B5.6 was eluted from the column with pH 4.0 citrate buffer.
  • MABs BI9, B18, B06, B05, B04 and B02 were obtained from Jean-Charles Fruchart, SERLIA Institut Pasteur, , Lille Celdex, France, as purified IgG fractions.
  • MABs 457C4DI, 465C3DI, 464BIB3, and 464BIB6 were obtained from Gustav Schonfeld, Washington University Medical Center, St. Louis, MO. These MABs are all IgGi and were described in Tikkanen, et al. (1982) J. Lipid Res. 23:1032- 1038; and Tikkanen, et al. (1983) J. Lipid Res. 24:1494-1499.
  • the MABs differed in reactivity toward LDL obtained from different individuals and were suggested to recognize different antigenic epitopes of LDL.
  • the MABs were supplied in ascites fluid and were purified on Protein A Sepharose column as described in Example 1 (infra). All four antibodies were eluted from the column with citrate buffer at pH 6.0.
  • 16 (MB16), 2D8 and IDI (IgGi), and 5EII (lgG2a) were obtained from Yves Marcel, Clinical Research Institute of Montreal, Canada.
  • the MABs were characterized and the antigenic epitopes of apo B recognized by these MABs was determined using apo B fragments cleaved from apo B by thrombin, synthetic oligopeptides and recombinant proteins (Marcel, et al. (1987) Mol. Immunol. 24:435-447; Marcel, et al. Arteriosclerosis (1987) 7:166-175; Chen, et al. (1988) Eur. Biochem.
  • MAB MB16 recognized the peptide residues 4154-4189 of thrombolytic fragment T2.
  • MAB IDI recognized the peptide residues 1-1297 of thrombolytic fragment T4.
  • MAB 2D8 recognized the peptide residues 1297-2177 of thrombolytic fragment T3.
  • MAB 5EII recognized the peptide residues 2488-3636 in the T3/T2 region of LDL (B,E) receptors. All four MABs were reported to react with LDL and solubilized apo B.
  • MABs IDI, 2D8 and 5EII required the presence of lipids in order to react with VLDL, whereas MAB MB16's reactivity was found to be lipid independent.
  • the MABs were obtained as 50% ammonium sulfate precipitates of the ascites fluid. Prior to use herein, the MABs were purified on Protein A Sepharose columns as described in Example 1 (infra). MAB 5EII was eluted from the column with pH 5 citrate buffer and the others were eluted from the column with pH 6 citrate buffer.
  • Three purified anti-LDL monoclonal antibodies A016-08, A016-09, and A016-10 were obtained from Medix Biotech, Foster City, CA. All three purified anti-LDL MABs were characterized in terms of their cross-reactivities with other lipoproteins in order to select LDL-specific antibodies.
  • the selected antibodies bound to LDL particles of blood samples from normal individuals and from individuals with high triglycerides blood levels (300-400 mg/dL), did not bind to Lp(a) or HDL, and reacted with VLDL and IDL at less than about 10% of the LDL reactivity.
  • Suitable antibodies for use in the present invention bind to (1 ) Lp(a) preferably at less than about 5% of the LDL reactivity and more preferably at less than about 2% of the LDL reactivity; (2) HDL preferably at less than about 2% of the LDL reactivity and more preferably at less than about 1% of the LDL reactivity; and (3) VLDL and IDL, preferably at less than about 20% of the LDL reactivity and more preferably at less than about 10% of the LDL reactivity.
  • Lipoprotein fractions (LDL, HDL, VLDL, IDL and Lp(a)) purified by ultracentrifugation (see Example 2, infra) were coated on separate wells of a Maxisorb Nunc Immuno Plate as follows: one hundred microliters (100 ⁇ L) of each lipoprotein fraction at a lipoprotein-cholesterol concentration of about 1 ⁇ g/ml in 20 mM phosphate buffered saline at pH 7.0 (PBS) was dispensed into separated wells of the microtiter plate, the plate was incubated at 37°C for one hour, and the plate was then washed five times with PBS containing 0.05% (v/v) Tween 20 (PBS-Tween 20).
  • PBS-Tween 20 PBS-Tween 20
  • the non-specific binding sites were blocked with 200 ⁇ L of 10% (v/v) fetal bovine serum (FBS) in PBS at 37°C for one hour and then were washed five times with PBS-Tween 20.
  • FBS fetal bovine serum
  • Each MAB was diluted in 3% (v/v) FBS in PBS to a final antibody concentration of about 2 ⁇ g/ml and the diluted MAB solutions were then serially diluted on the plates. After incubation at 37°C for one-half hour, the plate was washed five times with PBS-Tween 20.
  • HRPO horseradish peroxidase
  • the specificities of the MABs were determined by competitive binding of the MABs to the other lipoproteins in microplate wells coated with LDL.
  • the LDL-coated plates were prepared as described previously (see section 2a above).
  • Each MAB was diluted with 3% (v/v) FBS in PBS to a concentration that was two times the MAB concentration at 50% LDL-binding as determined from the binding curves prepared in section 2a above. Examples of such curves are shown in Figure 1.
  • Purified lipoprotein samples were diluted in PBS starting at the following cholesterol concentrations: LDL-cholesterol concentration of 45 mg/dL; VLDL-cholesterol concentration of 45 mg/dL; IDL-cholesterol concentration of 22.5 mg/dL; HDL-cholesterol concentration of 22.5 mg/dL; and Lp(a) at a total mass of Lp(a) of 22.5 mg/dL.
  • Fifty microliters (50 ⁇ L) of each lipoprotein solution were then serially diluted with PBS in reaction wells blocked by 10% (v/v) FBS in PBS. To each of these wells were added 50 ⁇ L of the diluted MAB solutions.
  • the MAB-lipoprotein mixtures were incubated at room temperature for one-half hour on a rotator at 100 ⁇ m. The contents from each well were then transferred to LDL- coated reaction wells and the plates were incubated at 37°C for one-half hour. The amount of MAB bound to the LDL-coated reaction wells were measured according to the method described in section 2a above. Typical competitive binding curves are shown in Figure 2. A summary, of the test results are presented in Table 2. The cross-reactivities were determined at 50% inhibition of binding by a competing lipoprotein using the following equation:
  • MAB B18 did not show any inhibition of binding even by LDL.
  • the results also indicate extensive binding of all MABs, particularly with IDL. This is in contrast to the binding curves obtained in section 2a above.
  • MB16 in Figure 1 shows much less affinity towards VLDL and IDL
  • MB16 in Figure 2 shows almost equal affinity for both LDL and IDL. This indicates that the affinity of the MABs toward lipoproteins differs depending on whether the reaction
  • MABs were coated onto the reaction wells of microtiter plates as follows. MABs were diluted in PBS as follows: SPL4A5 (20 ⁇ g/mL), 8A2.1 (20 ⁇ g/mL), 8A6.6 (2 ⁇ g/mL), 4B5.6 (3 ⁇ g/mL), 8B3.5 (5 ⁇ g/mL), 465C3D1 (2 ⁇ g/mL), 457C4DI (2 ⁇ g/mL), 464B1 B3 (1.5 ⁇ g/ml, 464BIB6 (1.5 ⁇ g/mL), B19 (2 ⁇ g/mL), B18 (2.5 ⁇ g/mL), B06 (10 ⁇ g/mL), B04 (10 ⁇ g/mL), B02 (4 ⁇ g/mL), MB16 (5 ⁇ g/mL), IDI (5 ⁇ g/mL), 2D8 (5 ⁇ g/mL), 5EII (5 ⁇ g/mL), A016-08 (10 ⁇ g/mL), A01
  • Figure 4 shows typical binding curves for 4B5.6, SPL4A5, 8A2.1 , and 465C3D1. Based on these results, it is likely that the binding affinity of the MABs are dependent on the orientation of the lipoprotein particles, i.e. the MABs are LDL conformation dependent.
  • RIA competitive radioimmunoassays
  • lodine-125 labeled LDL (1251- LDL) was prepared enzymatically using immobilized lactoperoxidase and glucose oxidase (Enzymobeads, Bio-Rad) according to the standard procedure described in Tsao, et al. (1987) J. Biol. Chem. 25J7:15222-15228.
  • the specific activity ranged between 1.87-2.60 ⁇ Ci/ ⁇ g.
  • the 1251-LDL sample was stored at 4°C in 0.1 M TRIS-saline pH 7.5 containing 10 mg/ml lipid-free bovine serum albumin (BSA) (Armour CRG-7) and was used within 18 days after preparation.
  • BSA bovine serum albumin
  • Typical binding curves of 1251-LDL using MABs 2D8, 1D1 and MB16 are shown in Figure 5.
  • the Removawell plates were prepared as follows. The concentrations of the MABs used in the preparation of the Removawell plates were exactly the same as described in section 2c above. One hundred microliters (100 ⁇ L) of each MAB solution in PBS were added to each well and incubated at 37°C for one hour.
  • the plates were washed five times with PBS and then blocked with 200 ⁇ L of 10% FBS in PBS. The plates were then washed five times with PBS.
  • the lipoproteins were diluted in PBS to the following lipoprotein-cholesterol concentrations: LDL- cholesterol, VLDL-cholesterol and HDL-cholesterol concentrations of 20 mg/dL; IDL-cholesterol concentration of 10 mg/dL; and Lp(a) at a Lp(a) total mass of 20 ⁇ g/dL.
  • microliters (50 ⁇ L) of each lipoprotein solution were serially diluted in PBS in microtiter reaction wells blocked by 10% FBS in PBS.
  • Fifty microliters (50 ⁇ L) of 1251-LDL (100,000 cpm) diluted in 3% (w/v) BSA in PBS were added to each well.
  • the contents from each well were transferred completely to respective MAB Removawell plates.
  • the plates were incubated at room temperature on a rotator at 100 rpm for 20 hours. The plates were then washed eight times with PBS.
  • Each well was then transferred to Falcon polystyrene tubes (12 x 75 mm) and the bound radioactivity was counted on an APEX automatic Y-counter. The background was subtracted to calculate the net binding.
  • the competitive displacement curves for 4B5.6 are shown in Figure 6.
  • the evaluation of the monoclonal antibodies described above indicates that at least two types of antigenic epitopes exist: one dependent mostly on the peptide sequence of the apo B fragments and the other dependent on the conformation dictated by the presence of associated lipids in the lipoproteins and also on the sizes of the particles.
  • the results are summarized in Figure 7A.
  • the antigenic epitopes of apo B fragments formed by thrombin which are known are shown in Figure 7B and the antigenic epitopes of apo B are known to bind the following MABs
  • MAB 1 D1 binds to the T4 fragment 1-1297.
  • MAB 2D8 binds to the T3 fragment 1297-2177.
  • MABs 8A2.1 , 8A6.6 and 8B3.5 bind to the T3 fragment 1297-3249.
  • MAB 5EII binds to the T3/T2 fragment 2488-3636.
  • MAB MB47 binds to the LDL (B.E) receptor binding fragment 3350-3506 (Weisgraber et al (1988) Proc. Natl. Acad. Sci., USA 25.: 9758-9762).
  • We found that all these MABs are highly cross-reactive with other apo B containing lipoproteins, such as VLDL and IDL.
  • the MAB SPL4A5 for which the binding epitope of apo B is not known, appears to have higher affinity for LDL with smaller particle size as outlined in the U.S.
  • MABs would be useful in selectively binding LDL in the presence of other lipoproteins.
  • MABs which are specific for the apo B T3 fragment or sub-fragments thereof, can also selectively bind to LDL, with low (less than 20%) cross-reactivity with VLDL, IDL and Lp(a), as illustrated by MAB 8A2.1 in Table 3.
  • MAB 8A2.1's LDL selectivity may be due to particle size because MAB 8A2.1 failed to bind to LDL particles from every plasma sample tested.
  • MABs specific for apo B fragments T4, T3/T2 and sub-fragments thereof will produce MABs which are cross-reactive with other lipoproteins.
  • FIG. 8 shows some typical peptide fragments of apo B T2 fragment useful in generating LDL-specific MABs.
  • CNBr-activated Sepharose 4B Pulacia LKB
  • carbolink hydrazide agarose beads Pieris Chemicals
  • Sulfolink coupling agarose beads Pieris Chemicals
  • any other hydrophilic solid phase such as Trisacryl (IBF), HEMA-epoxy Bio Gel, HEMA vinylsulfone Bio Gel (Altech Associates), glycosylated silica gel or control porous glass, hydrophilic latex beads, other cellulosic materials etc. can also be used.
  • Examples 4, 5, and 6 show the methods used in the covalent attachment of the monoclonals antibody to CNBr-activated Sepharose 4B, carbolink hydrazide gel and sulfolink gel, respectively. 4. EVALUATION OF ANTIBODY IMMOBILIZED SOLID PHASES
  • the antibody immobilized solid phases were evaluated in terms of their binding efficiencies by incubating the solid phases with purified LDL fractions from normal subjects and then determining the amount of LDL bound by measuring the amount of cholesterol in the bound solid phases.
  • the cholesterol assay was performed using the reagents as described in Example 7. A typical cholesterol standard curve is shown in Figure 9.
  • Example 8 The protocol for the lipoprotein capture assays is described in Example 8.
  • the efficiency of LDL capture on a Sepharose 4B matrix having MAB SPL4A5 bound thereto is shown in Table 4. The result shows that 90% or more LDL particles are being captured on the antibody matrix.
  • the LDL capture efficiencies were between 92%-98% for LDL having LDL-cholesterol concentrations ranging from about 6 mg/dL to about 24 mg/dL.
  • the other two antibody- matrices namely hydrazide gel and sulfolink gel, are better than the CNBr-activated Sepharose 4B matrix, a comparative study was undertaken using approximately the same amount of
  • MAB Matrix concentration 2.8 ⁇ g/ ⁇ L. 100 ⁇ L sample size. * LDL concentration is the amount of LDL having the listed LDL-cholesterol concentration. TABLE 5 LDL CAPTURE EFFICIENCY OF SPL4A5 MATRICES
  • ** LDL concentration is the amount of LDL having the listed LDL-cholesterol concentration.
  • the measured LDL-cholesterol concentrations increased with increasing VLDL concentrations and the LDL-cholesterol measurements were particularly affected when the VLDL-cholesterol concentration exceed 50% of the LDL-cholesterol concentration.
  • concentration of VLDL-cholesterol in plasma in most plasma samples is usually less than 50% of the LDL-cholesterol concentration except in a few rare pathological states (such as type III and type IV hypertriglycerimic patients).
  • an LDL-cholesterol measurement can be affected by VLDL-cholesterol up to ⁇ 10% without affecting the clinical significance of the LDL- cholesterol measurement.
  • SPL4A5-Sepharose 4B Matrix Thirty-four subjects were used in this study (See Table 7 for lipid profiles). The LDL-cholesterol concentrations determined by the two reference methods and the immunocapture assay are presented in Table 8. The correlations between the immunocapture assay and the two reference methods are shown in Figure 10 (A and B). The results indicated that several samples (such as Nos. 15-19) had much lower LDL-cholesterol concentrations as measured by MAB SPL4A5-Sepharose as compared to both reference methods. This suggests that the MAB SPL4A5 alone cannot capture all LDL particles of heterogeneous sizes present in these samples.
  • LDL-cholesterol concentrations measured by both reference methods are actually not a true measurement of LDL-cholesterol but instead are a mixture of LDL-, IDL-, and Lp(a)-cholesterol concentrations.
  • these three lipoprotein particles are considered to be potential atherogenic markers and all previous data base on LDL- cholesterol values are based on these two reference methods, a correction for IDL and Lp(a) should be made in order to obtain a better correlation between the disease state and the true LDL-cholesterol concentrations.
  • No reliable method of IDL quantitation is presently available except through the lengthy ultracentrifugation techniques.
  • Lp(a) mass A number of methods for quantitation of Lp(a) mass are known. We have used a commercial ELISA kit (TEMUMO Medial Corporation, Elkton, MD) to estimate the Lp(a) mass and the concentrations of cholesterol are then calculated by multiplying the total Lp(a) mass with 0.3, because 30% is generally assumed to be the cholesterol content of Lp(a).
  • the Lp(a)-subtracted LDL-cholesterol concentration of the two reference methods are presented in Table 8.
  • the LDL- cholesterol concentrations minus the Lp(a)-cholesteroI contributions were correlated with the LDL-cholesterol concentrations obtained by the present immunocapture assay using MAB 4B5.6-Sepharose and are shown in Figure 13 (A and B).
  • the correlation between immunocapture assay using MAB 4B5.6-Sepharose and ultracentrifugation method has an intercept of 0.96 a slope of 0.96.
  • the correlation between immunocapture assay using MAB 4B5.6-Sepharose and Friedewald method has a correlation coefficient (r) of 0.95 and a slope of 0.89.
  • the correlation of the present assay method becomes better after correcting for Lp(a)-cholesterol in the LDL-cholesterol concentrations determined by the two reference methods (see Figures 12 and 13).
  • the present assay method is virtually independent of the triglyceride concentration (tested up to 470 mg/dL, see Table 7) and VLDL-cholesterol (tested up to 88 mg/dL, see Table 8).
  • the results are shown in Figures 14 (A and B) and 15 (A and B).
  • the Y-axes in Figures 14A and 15A represent the ratio of LDL- cholesterol concentrations determined by the present method and ultracentrifugation method.
  • the Y-axes in Figures 14B and 15B represent the ratio of LDL-cholesterol concentrations determined by the present method and ultracentrifugation method corrected for the Lp(a)-cholesterol contribution.
  • N/A F.E. LDL-cholesterol cannot be used for individuals with high TRIG levels (greater than about 400 mg/dL).
  • X-axes in Figures 14 and 15 represent triglyceride and VLDL concentration, respectively.
  • MB16-Sepharose 4B Matrix The LDL-cholesterol concentrations measured by the immunocapture assay using MAB MB16-Sepharose and by the two reference methods are presented in Table 8.
  • Figure 16 (A and B) shows the correlations between the immunocapture assay using MAB MB16-Sepharose and ultracentrifugation and Friedewald methods.
  • the correlation between immunocapture assay using MAB MB16-Sepharose and ultracentrifugation method has a correlation coefficient (r) of 0.91 a slope of 0.96.
  • the correlation between immunocapture assay using MAB MB16- Sepharose and Friedewald method has a correlation coefficient (r) of 0.93 and a slope of 0.90.
  • Figure 17 shows the correlations after the Lp(a)-cholesterol contribution has been subtracted from the two reference methods. In this study also, the correlations become better after Lp(a)-cholesterol correction similar to that observed for the MAB 4B5.6-Sepharose immunocapture assay.
  • the immunocapture assay using MAB MB16-Sepharose showed no dependency on the triglyceride concentration (tested up to 333 mg/dL; see Table 7). The results are shown in Figure 18 (A and B). However, the immunocapture assay using MAB MB16-Sepharose showed some dependency on VLDL- cholesterol (see Table 7). The results are shown in Figure 19 (A and B). This dependency is not unexpected because MAB MB16 showed about 19% cross-reactivity with VLDL compared to only 7% for MAB 4B5.6 in the competitive RIA (see Table 3).
  • the Y-axes in Figures 18A and 19A represent the ratio of LDL-cholesterol concentrations determined by the present method and ultracentrifugation method.
  • the Y-axes in Figures 18B and 19B represent the ratio of LDL-cholesterol concentrations determined by the present method and ultracentrifugation method corrected for the Lp(a)- cholesterol contribution.
  • the X-axes in Figures 18 and 19 represent triglyceride and VLDL concentration, respectively.
  • Plasma samples of known LDL-cholesterol concentrations as determined by reference methods were used to generate standard curves.
  • Standards with LDL-cholesterol concentrations of 74, 101 , 135 and 207 mg/mL were prepared by diluting the plasma samples with 1% alkali-treated casein in 20 mM phosphate buffered saline (PBS) at pH 7.4.
  • PBS phosphate buffered saline
  • Digitonin-Peroxidase Conjugates Digitonin (2.5 mg/mL in water) (water soluble containing 50% digitonin and sodium deoxycholate commercially available from Sigma Chemical Company, St. Louis, MO) was oxidized with sodium meta-periodate (a solution of 1.68% w/v periodate in water was added to the digitonin solution to a final concentration of 0.02 M periodate) (Tschesche and Wulff (1963) Tetrahedron 1 ⁇ :621-634). The mixture was stirred at 4°C for one hour and then dialyzed against 20 mM phosphate buffered saline (PBS), pH 8.0, at 4°C overnight.
  • PBS phosphate buffered saline
  • the oxidized digitionin was then mixed with ethylenediamine (a solution of 0.25 M ethylenediamine in 20 mM PBS, at pH 8.0, was added to the oxidized digitonin solution to a final concentration of 0.05 M ethylenediamine) and incubated at 4°C.
  • the mixture was then reduced by two additions of 100 ⁇ L of 4 mg/mL sodium borohydride in 0.1 N sodium hydroxide (i.e. 100 ⁇ L of the sodium borohydride solution per 30 mg of digitonin), after 30 minutes and after 60 minutes. After incubating at 4°C for two hours, the mixture was dialyzed against 0.01 M carbonate buffer, pH 9.5, at 4°C overnight. Five milligrams (5 mg) of horseradish peroxidase (HRPO)
  • HRPO 155 Ku/mg, commercially available from Amano International
  • HRPO was oxidized by adding 50 ⁇ L of a freshly prepared solution of 0.2 M sodium meta-periodate per milligram of HRPO to the HRPO solution and incubating the mixture in the dark at room temperature for 20 minutes. The mixture was then dialyzed against 2 liters of 1 mM acetate buffer, pH 4.5, at 4°C for 4 hours.
  • the ethylenediamine derivatized digitonin solution and the oxidized HRPO solution were mixed in digitonin:HRPO weight ratios of 1:5 and 1:10.
  • 0.2 M carbonate buffer, pH 9.5 50 ⁇ L buffer/mg digitonin
  • the reactions were stirred in the dark at room temperature for two hours and 100 ⁇ L of sodium borohydride solution (4 mg/mL in water) was added to each reaction. After incubating for two hours at 4°C, the reactions were dialyzed against 20 mM PBS, pH 7.4, at 4 °C overnight.
  • 3-Hydroxycholan-5-en-24-oic acid (commercially available from Steraloids Inc., Wilton, NH) was reacted with N-hydroxysuccinimide and 1 ,3-dicyclohexylcarbodiimide to form an active ester under typical reaction conditions.
  • the active ester was reacted with 6-aminohexanoic acid to form 6-(3-hydroxycholan-5-en-24-carbonylimino)hexanoic acid.
  • This carboxylic acid was reacted with N-hydroxysuccinimide and 1 ,3-dicyclohexylcarbodiimide to form a second active ester which was reacted with bovine serum albumin to form the immunogen.
  • the immunogen was purified on a Sephadex G- 25 column using standard purification techniques.
  • 3-Hydroxycholan-5-en-24-oic acid may be coupled directly to natural or synthetic proteins to form immunogens useful in the preparation of both polyclonal and monoclonal anti-cholesterol antibodies.
  • Other amino acid linking groups like aminohexanoic acid, such as aminoacetic acid, aminopropanoic acid, aminoheptanoic acid and the like, may be used to prepare useful immunogens.
  • diamino linking groups such as ethylenediamine and the like, can be used to prepare useful immunogens.
  • the steroid with such a linking group can be coupled to natural or synthetic proteins to form immunogens useful in the preparation of both polyclonal and monoclonal anti-cholesterol antibodies.
  • the preparation of polyclonal and monoclonal antibodies using immunogens is well known in the art (Tijssen, "Laboratory Techniques In Biochemistry And Molecular Biology: Practice and Theory of Enzyme Immunoassays", Vol. 15, Elsevier, New York (1985)).
  • Polyclonal antibodies were preferably raised in rabbits, but other animals, such as sheep, pigs, mice, rats, goats, donkeys and the like, can also produce suitable antibodies. Antibody binding was tested by enzyme-linked immunosorbent assay. Cholesterol antigens, such as HDL, LDL, VLDL and IDL particles in 0.15 M saline, cholesterol in 95% ethanol (0.25 ⁇ g/well) and cholesterol esters in hexane (0.25 ⁇ g/well), were absorbed in polystyrene microtiter plate wells. The wells were then blocked with 10% (v/v) fetal calf serum or 1% (w/v) casein solutions.
  • Cholesterol antigens such as HDL, LDL, VLDL and IDL particles in 0.15 M saline, cholesterol in 95% ethanol (0.25 ⁇ g/well) and cholesterol esters in hexane
  • the antibody to be tested was incubated in the well and the well was washed with 0.05% (w/v) Tween 20 in 20 mM PBS, pH 7.0.
  • Enzyme labelled anti- antibody antibody such as goat anti-rabbit IgG conjugated to HRPO, was used to detect the presence of antibody bound to the cholesterol antigen absorbed in the well.
  • Rabbit antibody raised against the 6-(3-hydroxycholan-5-en-24- carbonylimino)hexanoic acid hapten coupled to BSA bound to cholesterol and cholesterol esters, but showed preferential binding to lipoprotein cholesterol particles in the order: HDL « VLDL > LDL > IDL.
  • Anti-cholesterol Antibody-HRPO conjugates were prepared as follows. Anti-cholesterol rabbit polyclonal antibody was purified by double precipitation with 33% saturated ammonium sulfate. The antibody (2.7 mg of IgG) was coupled to HRPO (1.44 mg) using the procedure described above for digitonin-HRPO conjugates (paragraph 6.b.).
  • the oxidized alkaline phosphatase was then mixed with the ethylenediamine derivatized digitonin ( 0.6 mL of a 1.67 mg/mL solution in 20 mM PBS, pH 7.4) prepared above (paragraph 6.b.) in 20 mM PBS, pH 7.4 in a digitonin:phosphatase weight ratio of 1 :5.
  • To the mixture was added 90 ⁇ L of 1 M bicarbonate buffer, pH 9.5 and the mixture was incubated at room temperature for 16 hours in the dark. Thirty microliters of sodium borohydride (5 mg/mL in 0.1 M bicarbonate buffer, pH 9.5) was added to the reaction and the reaction was incubated at 4°C for 4 hours.
  • the reaction was then dialyzed against 2 liters of a solution of 0.05 M TRIS, 0.1 M sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chloride and 0.1% (w/v) sodium azide, pH 8.0, at 4°C overnight.
  • a solution of 5% sodium deoxycholate in water (one-tenth the volume of the dialyzed material) and fatty-acid free BSA were added to make the BSA final concentration 5 mg/mL.
  • the digitonin-AP conjugate was sterile filtered through a 0.22 micro filter and stored at -20°C.
  • Tomatine-alkaline phosphatase conjugates were prepared in tomatine ⁇ hosphatase weight ratios of 1 :5 and 1 :10 according to the digitonin-AP conjugate procedure (paragraph 6.g.).
  • Amphotericin-alkaline phosphatase conjugates were prepared in amphotericin:phosphatase weight ratios of 1 :5 and 1 :10 according to the digitonin-AP conjugate procedure (paragraph 6.g.).
  • Antibody-alkaline phosphatase conjugates were prepared from rabbit anti-cholesterol IgG antibody (2.4 mg IgG) and AP (7.2 mg) according to the digitonin-AP conjugate procedure (paragraph 6.g.).
  • OPD o-Phenylenediamine
  • each conjugate (100 ⁇ L in each well) was titrated from 20 ⁇ g/mL in 1% alkali-treated casein in 50 mM TRIS, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chloride, 0.1% sodium azide, pH 8.0 (dilution buffer).
  • TRIS 100 mM sodium chloride
  • 1 mM magnesium chloride 1 mM magnesium chloride
  • 0.1 mM zinc chloride 0.1% sodium azide
  • pH 8.0 dilution buffer
  • the plates were incubated at 37°C for one hour and washed eight times with TRIS-Tween.
  • p-Nitrophenolphosphate (100 ⁇ L of 2 mg/mL in dilution buffer) was added to each well. After incubation at room temperature for 16 minutes, the color reaction was stopped with 100 ⁇ L of 1 N sodium hydroxide.
  • the LDL specific monoclonal antibody 4B5.6 was diluted in 20 mM PBS, pH 7.4, to a final concentration of 5 ⁇ g/mL.
  • One-hundred microliters of the solution was added to each well of Maxisorb Nunc Immuno plates and incubated at room temperature with gentle shaking for two hours.
  • the plates were washed five times with PBS-Tween and then blocked with 200 ⁇ L of 5% BSA in 20 mM PBS by incubation at 37°C for one hour.
  • the plates were stored at 4°C with plastic sealers. Before use, the plates were washed five times with PBS- Tween for HRPO conjugates and TRIS-Tween for AP conjugates.
  • LDL-cholesterol standards 100 ⁇ LJwell in duplicate were incubated in the 4B5.6 plates (paragraph 6.k.) at 37°C for one hour. After washing the plates five times with PBS- Tween, 100 ⁇ L of digitonin-HRPO conjugate at 0.4 ⁇ g/mL in 1% casein in PBS or at 1.25 ⁇ g/mL in 1% casein in PBS was added to each well and incubated at 37°C for one hour.
  • DIG-HRPO digitonin-HRPO conjugate based assay
  • DIG-AP digitonin-AP conjugate based assay
  • TOM-HRPO tomatine- HRPO conjugate based assay
  • TOM-AP tomatine-AP conjugate based assay.
  • Standard curves were also prepared with the digitonin- AP and tomatine-AP conjugates using the same procedure except that the TRIS-Tween wash, the dilution buffer and p- nitrophenolphosphate substrate (100 ⁇ L of 2 mg/mL in dilution buffer) were used as in paragraph 6.j. A 16 minute substrate incubation was used and the reaction was stopped with 100 ⁇ L of 1 N sodium hydroxide. The absorbances were read at 405 nm.
  • Plasma samples in ethylenediaminetetraacetic acid (EDTA) were collected from normal individuals and patients. The samples were frozen at -20°C until used. Thawed samples were not used after two days storage at 4°C. The samples were diluted 600-fold in 1% casein in PBS and assayed for
  • the column was washed with binding buffer until the absorbance at 280 nm is ⁇ 0.02.
  • the bound IgG antibody was then eluted sequentially with 100 mM citrate buffer, pH 6 (for IgGi), pH 5 (for lgG2a) and pH 4 (for lgG2b).
  • Ten milliliters of each elution buffer was used for each milliliter of protein A in the column.
  • the column was regenerated by washing with 100 mM citrate buffer, (pH 3) until the absorbance at 280 nM is ⁇ 0.02 and then re- equilibrated with the binding buffer.
  • Each column was used at least five times without any loss of binding affinity.
  • Plasma samples from normal fasting subjects were collected into ethylenediaminetetraacetic acid (EDTA) and the red blood cells were removed by centrifugation. The plasma samples were then analyzed for Lp(a) using TERUMO ELISA kit. Plasma samples containing less than 1 mg/dL Lp(a)-cholesterol were selected for the purification of VLDL, IDL, LDL and HDL. Lipoprotein subtractions were prepared in a Beckman Ultracentrifuge with a SW 40 Ti rotor by successive ultracentrifugation at 4°C (Havel et al. (1955) J. Clin. Invest. 24:1345-1355).
  • VLDL was collected at a density of about d 1.0006 g/mL; IDL was collected at a density range of about d 1.006-1.019 g/mL; LDL was collected at a density range of about d 1.019-1.050 g/mL; and HDL was collected at a density range of about d 1.080-1.225 g/mL. All fractions were isolated by a tube-slicing technique. The lipoprotein fractions were dialyzed exhaustively against 0.15 M sodium chloride containing 0.1% EDTA and 0.1% sodium azide, pH 7.4 at 4°C.
  • Lipoprotein (a) was isolated from plasma samples with Lp(a)-cholesterol concentrations more than 15 mg/dL on a lysine-Sepharose column (Fless and Scanu, Arteriosclerosis (1987) 7:505A). The purity of each lipoprotein fraction was evaluated by electrophoresis under non-denaturing polyaccrylamide gradient gel electrophoresis (Lefevre et al. (1987) J. Lipid Res. 2& 1495- 1509). Gradient slab gels, 2-16% and 4-30% and electrophoresis apparatus GE-24 (Pharmacia LKB) were used in the analysis. The lipoprotein fractions containing no cross-contamination were used in the studies.
  • FIG. 3 A Maxisorb Nunc Immuno plate was coated with 100 ⁇ L of different lipoproteins by incubation at 37°C for 1/2 hour. After blocking the non ⁇ specific sites with 200 ⁇ L of 10% FBS in PBS at 37°C for one hour, and washing five times with PBS-Tween 20, 100 ⁇ L of B06-peroxidase conjugate (0.6 ⁇ g/mL diluted in 3% FBS in PBS) was added to each well. The plate was incubated at 37°C for 1/2 hour, washed eight times with PBS-Tween 20. One hundred microliters of OPD substrate solution was added to each well. After incubation at room temperature for five minutes, the color reaction was stopped with 100 ⁇ L of 1 N H2SO4. The plate was then read at 490 nm on a microplate reader.
  • CNBr-activated Sepharose 4B (Pharmacia LKB) was suspended in about 15 mL of 1 mM HCI. The gel was then transferred to a coarse-porosity sintered-glass funnel and washed with about 200 mL of 1 mM HCI. The gel was then washed with 25 mL of 0.1 M carbonate buffer in 0.5 M sodium chloride, pH 8.3 (coupling buffer). A gentle vacuum was applied to remove the buffer. The moist gel cake was. then transferred to a glass tube with a screw-capped stopper. Monoclonal antibody (10 mg, concentration 0.5 to 1 mg/mL), which was dialyzed against the coupling buffer at 4°C, was then added to the gel.
  • the mixture was then mixed gently end- over-end using an infiltration wheel at 4°C for 20 hours. The supernatant was checked by measuring the absorbance at 280 nm for the unbound antibody. For all the monoclonal antibodies used here, more than 95% were bound to the gel.
  • the gel was then transferred to a coarse-porosity sintered- glass funnel, washed with 50 mL of the coupling buffer and 25 mL of 0.1 M TRIS-HCI buffer, pH 8.0 (blocking buffer). The gel was then transferred to a glass tube, and mixed with 10 mL of the blocking buffer at room temperature for two hours. The antibody-immobilized gel was then washed with three cycles of alternating pH.
  • Each cycle consisted of a wash with acetate buffer (0.1 M, pH 4) containing sodium chloride (0.5 M) followed by a wash with TRIS buffer (0.1 M, pH 8) containing sodium chloride (0.5 M)
  • the final wash was done with 100 mL of TRIS-HCI buffer (0.05 M pH 7.4) containing sodium chloride (0.15 M) and sodium azide (0.01%) (storage buffer).
  • the gel was stored as a 25% suspension (14 mL) in the storage buffer at 4°C. Assuming 100% of the monoclonal antibody bound to the gel, 200 ⁇ L of gel suspension contains 143 ⁇ g of the monoclonal antibody.
  • hydrazide gel (Pierce Chemicals, Carbolink hydrazide, 50% suspension) were washed with 50 mL of 0.1 M phosphate buffer (pH 7.0) in a coarse-porosity sintered-glass funnel. After a gentle vacuum to remove the buffer, the moist gel was transferred to a glass tube with a screw-capped stopper. Five milligrams of monoclonal antibody (concentration, 2 mg/mL), which was dialyzed against 0.1 M phosphate buffer (pH 7.0) at 4°C, was oxidized with 10.5 mg sodium m-periodate at room temperature for 1/2 hour.
  • the oxidized antibody (volume 2.5 mL) was then loaded on a Sephadex G-25 M PD-10 column (Pharmacia) which was pre-equilibrated with 0.1 M phosphate buffer (pH 7.0).
  • the oxidized antibody was eluted with 3 mL of 0.1 M phosphate buffer (pH 7.0) and mixed with the hydrazide gel end-over-end using an infiltration wheel at room temperature for seven hours. The supernatant was checked for the unbound antibody.
  • the amount of antibody bound to the gel ranged between 80- 85%.
  • the antibody-immobilized gel was filtered through a coarse-porosity sintered-glass funnel, washed with about 100 mL of 1 M sodium chloride and finally, with 0.05 M TRIS-HCI (pH 7.4) containing 0.15 M sodium chloride and 0.01% sodium azide (storage buffer).
  • the immobilized gel was stored as a 25% suspension in the storage buffer (12 mL) at 4°C.
  • the reaction mixture was cooled to room temperature and loaded onto a Sephadex G-25M PD-10 column (Pharmacia) which was pre-equilibrated with the reaction buffer.
  • the reduced antibody was then eluted with 3 mL of the reaction buffer and mixed with the sulfolink gel end-over-end using an infiltration wheel at room temperature for 15 minutes.
  • the reaction mixture was allowed to stand at room temperature for an additional hour.
  • the supernatant was checked for the unbound antibody.
  • the amount of antibody bound to the gel ranged between 75-80%.
  • the gel was filtered through a coarse-porosity sintered-glass funnel, washed with 50 mL of the reaction buffer and again transferred to a glass tube.
  • Dry Reagents Methods and formulation are described here to produce sensitive, rapid and stable assay reagents for the quantitation of cholesterol in a fluid phase. Two separate reagents were prepared and were mixed together at the time of the assay.
  • the first reagent formula was comprised of 1.62 g of 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt (Aldrich, Milwaukee, Wisconsin) (DCHBS) and 0.428 g of horseradish peroxidase (Amano International, specific activity 82.3 EZ/mg) (HRPO) dissolved in 20.4 mL 0.05 M of 3-(N- Morpholino)-2-hydroxypropanesulfonic acid sodium salt (Sigma) (MOPSO) at pH 7.
  • the peroxidase activity of each frit was 14.7 EZ.
  • the second reagent formula was comprised of 0.0113 g gCl2.6H2 ⁇ , 0.0276 g anhydrous CaCl2, 0.51 g lactose, 0.51 g dextran (Pharmachem, Mol. Wt.
  • Each of the second reagent frits weighed 11.86 mg and were comprised of BSA (0.85 g), dextran (0.425 g), lactose (0.425 g), glycerol (0.255 g), CaCI 2 (0.0.23 g), MgCI 2 (0.0094 g), AAP (0.156 g), CEH (0.124 g), CO (0.072 g) and MOPSO (0.525 g).
  • the enzyme activities of the second reagent frits were: CEH, 1.04 EZ; CO, 0.445 EZ. Both reagent frits have been found to be stable at room temperature for at least 16 months in terms of their assay performances, in terms of correlation and slope with known cholesterol standards.
  • reaction buffer which is also the extraction buffer contained the following materials: 0.05 M MOPSO (pH 7) (Sigma), 1% IgePal CO-530 (GAF), 0.2% Triton® X-100 (Bio-Rad) and 0.3% cholic acid (Sigma).
  • the buffer was sterile filtered and was stored at room temperature. The buffer is stable for at least six months.
  • Cholesterol assay reagent frits (one each of #1 and #2 from Example 7(a)) were added to each tube. The suspensions were mixed on a TOMY mixer for about eight minutes, centrifuged for one minute and the absorbances of the supernatant solutions were read on a DU7400 Spectrophotometer at 515 nm. The concentrations of the gel-bound cholesterol were determined from a cholesterol standard curve. The standard curve was prepared with purified LDL samples having concentrations of 0, 3, 6, 12, 24 and 48 mg/dL following the assay protocol described above (shown in Figure 9).
  • ICMT solutions (Example 7(b)) were added to each tube to a final volume of 750 ⁇ L.
  • Cholesterol assay reagent frits (one each of #1 and #2 from Example 7(a)) were added to each tube.
  • the suspensions were mixed on a TOMY mixer for about eight minutes, centrifuged for one minute and the absorbances of the supernatant solutions were read on a DU7400 Spectrophotometer at 515 nm.
  • the concentrations of LDL-cholesterol in the plasma samples were determined by multiplying the concentration obtained from the standard curve shown in Figure 9 by 10. The results are shown in Tables 8 and 9, and Figures 10-13 and 16-17.
  • Plasma samples (7 mL each) were transferred to ultraclear tubes (Beckman, 14 x 95 mm) and then overlayered with 6 mL of d 1.006 g/mL KBr Solution. The samples were centrifuged on a SW40Ti rotor at 40,000 rpm at 4°C for 20 hours. The upper VLDL layers were recovered by a tube-slicing technique. LDL and HDL were recovered in the bottom fraction of each tube. Adequate recovery was verified by comparing the sum of cholesterol in each of the fractions to the total cholesterol of the sample.
  • the cholesterol concentrations of the upper VLDL and lower d >1.006 g/mL (intranet cholesterol) were determined with VISION cholesterol assays (Abbott Laboratories, Abbott Park, Illinois). Assays for HDL- cholesterol concentrations were performed with unfractioned plasma samples using VISION HDL-cholesterol assay (Abbott Laboratories). LDL-cholesterol concentrations were calculated as the difference between in intranet cholesterol and HDL-cholesterol. VLDL-cholesterol concentrations were calculated as the difference between total plasma cholesterol and intranet cholesterol. The results are shown in Table 7.
  • the monoclonal antibody 4B5-immobilized Sepharose 4B gel prepared as described in example 4 of this invention, was used in a dry format.
  • the following is a typical example that was used to demonstrate the proof of principle.
  • 400 uL of 4B5-Sepharose 4B gel suspension which contained 100 uL of gel and 280 ug antibody was added and then blocked with 1 mL of 5% alkali-treated casein in PBS at room temperature for one hour. The supernatant was aspirated off and the wet gel was lyophilized at 25 micron vacuum overnight.
  • This protocol was developed for the following reasons: 1) to demonstrate that 4B5-Sepharose 4B gel specifically and completely captured the LDL particles from plasma samples used in the direct immunocapture assay; 2) to develop an indirect LDL-cholesterol assay which could be useful in commercial instruments, such as Abbott Vision.
  • the assay format involves the specific capture of LDL particles on 4B5- Sepharose 4B gel and then assay the unbound supernatant containing lipoproteins other than LDL, namely VLDL, IDL, HDL and Lp(a). LDL-cholesterol is then calculated by subtract? f .g from the total cholesterol:
  • the present invention is further directed to a method for the direct measurement of Lp(a)-cholesterol in plasma, preferably using sandwich immunoassay methodology.
  • a specific binding agent preferably an antibody and more preferably a monoclonal antibody, specific for Lp(a) is used to capture Lp(a) particles in a plasma sample.
  • the cholesterol associated with the Lp(a) particles is then measured as described earlier herein.
  • the captured Lp(a) particles are separated from the remainder of the sample . prior to the cholesterol measurement.
  • the Lp(a) specific binding agent is preferably coupled to a solid phase as described earlier herein.
  • mice Female BALB/c mice were immunized four times in 2-3 week intervals with 50 ⁇ g of apo(a) protein which was emulsified with Ribi adjuvant (Ribi Immunochem Research, Inc., Hamilton, MT). Four days after the last boosing, the mice were sacrificed and the immune spleen cells were fused with myeloma cells SP2/0 according to the procedure reported by Getter, et al. (1977) Somatic Cell Genet. 2:231. After two to three weeks of hybrid cell growth in microtiter plate wells, tissue culture spent media were collected from hybrid growing wells and tested for Lp(a) binding monoclonal antibodies.
  • the screening procedure was carried out by first incubating the tissue culture spent media on a Lp(a) or Apo(a) coated microtiter plate. Then, after removing the media and washing the wells, horseradish peroxidase labeled goat anti- mouse antibody was incubated in the wells. The wells were again washed and o-phenylenediamine was added to each well for signal development.
  • the microtiter plate was read at 492 nm using a microtiter plate reader. The presence and/or amount of signal development indicated the presence and/or concentration of anti-Lp(a) antibody produced in the tissue culture spent media.
  • the Lp(a) specific monoclonal antibodies 4D2, 1 E1 and 4F2 prepared by the above method were purified on a Protein A-Sepharose 4B column (commercially available from Pharmacia) using 100 mM citrate buffer, pH 6.0, to elute the antibodies from the column.
  • the antibodies did not show any cross-reactivity with LDL, VLDL, IDL and HDL. Also, these antibodies did not show any significant inhibition of binding to Lp(a) coated microtiter plates with human plasminogen up to 1 mg/mL.
  • Lp(a) concentrations in fresh plasma samples were measured using a commercial ELISA test for Lp(a) (TERUMO Medical Corp., Elkton, MD). Plasma samples with high Lp(a) concentrations were ultracentrifuged for 20 hours at 40,000 rpm at a density of 1.080 g/mL. The upper lipoprotein fraction containing Lp(a), LDL, VLDL and IDL was dialyzed and then the Lp(a) was affinity purified on a Lp(a) specific monoclonal antibody (4F2) Sepharose 4B column using standard procedures well known in the art.
  • 4F2 monoclonal antibody
  • the purity of the Lp(a) obtained from the column was determined by polyacrylamide gel electrophoresis under denatured conditions, by SDS-PAGE electrophoresis under reducing conditions and by Western Blot.
  • the protein content of the Lp(a) obtained form the column was measured by Lowry assay and the cholesterol concentration was measured using the Abbott Vision® Cholesterol Assay (commercially available from Abbott Laboratories, Abbott Park, IL).
  • Lp(a) standards having a protein concentration within the range of about 0.3 mg/mL and about 0.6 mg/mL were prepared from the purified Lp(a) by dilution in 20 mM phosphate buffered saline at pH 7.4 or 1% alkali-treated casein in 20 mM phosphate buffered saline at pH 7.4.
  • Calibrators having Lp(a)-cholesterol concentrations of 0, 2.4, 4.85, 9.7, 19.5, 38.9, 77.8, 155.6 and 311 ⁇ g/mL were prepared by dilution of the Lp(a) standards in 20 mM phosphate buffered saline at pH 7.4 or 1% alkali-treated casein in 20 mM phosphate buffered saline at pH 7.4.
  • the Lp(a) standards and calibrators were stored at 4°C.
  • HRPO horseradish peroxidase
  • HRPO-digitonin conjugate was sterile filtered through a 0.22 micro filter (Coaster Labs) and stored at -20°C.
  • the wells were washed five times with 0.05% w/v Tween 20 in 20 mM PBS, at pH 7.4 (PBS-Tween) and then blocked with 200 ⁇ L of 5% w/v BSA in 20 mM PBS, at pH 7.4, by incubation at 37°C for one hour.
  • the HRPO-digitonin conjugate solution was serially diluted in the wells with a solution of 5 ⁇ g/mL of alkali-treated casein in 20 mM PBS, at pH 7.4 (100 ⁇ L total volume in each well). After incubation at 37°C for one hour, the wells were washed eight times with PBS-Tween.
  • the monoclonal antibody 1E1 was diluted in 20 mM PBS, at pH 7.4, to a concentration of 5 ⁇ g/mL.
  • One hundred microliters of the 1 E1 solution was added to the wells of Maxisob Nunc Immuno plates and the plates were incubated at room temperature on a rotator at 100 rpm for two hours.
  • the plates were washed five times with PBS-Tween solutions and then blocked with 200 ⁇ L of 5% w/v BSA in 20 mM PBS, at pH 7.4, by incubation at 37°C for one hour.
  • the plates can be stored at 4°C with plastic sealers at least for ten days prior to use.
  • Plasma samples were diluted 201-fold with 1% w/v alkali-treated casein in 20 mM PBS, at pH 7.4. One hundred microliters of the diluted samples were added to each well of the 1E1 plates and the plates were incubated at 37°C for one hour. After washing the plates five times with PBS-Tween, 100 ⁇ L of HRPO-digitonin conjugate (2 ⁇ g/mL in 1% w/v alkali-treated casein in 20 mM PBS at pH 7.4) were added to each well. The plates were incubated at 37°C for one hour and then washed ten times with PBS-Tween.
  • Lp(a)-cholesterol standards were prepared from Lp(a) standard solutions as described in Example 15. Calibrators having Lp(a)-cholesterol concentrations of 0, 2.4, 4.85, 9.7, 19.5, 38.9, 77.8, 155.6 and 311 ⁇ g/mL were assayed by the method described in Example 17. The concentrations were multiplied by 201 to generate the standard curve because the plasma samples were diluted 201 -fold prior to performing the assay. A plot of Lp(a)-cholesterol concentration versus absorbance was prepared from the resulting data. Figure 23 is illustrative of such a plot. The Lp(a)-cholesterol concentration in unknown samples can be determined from the calibration curve. Generally the calibrators and the plasma samples were assayed on the same plate to minimize the effect of variations in the reagents, materials and conditions. The number and concent* tion of calibrators can be readily altered depending on th>, desired accuracy of the results.
  • lipid profiles of 64 plasma samples from individuals without known cardiac problems (“N”) and patients with mixed hyperlipidemia (“MHL”), hypocholesterolemia (“HC”) and mild hypocholesterolemia (“MHC”) were determined by the methods described earlier herein and the results are shown in Table 11.
  • Total cholesterol, HDL-cholesterol and triglyceride concentations were measured using an Abbott Vision® instrument and reagents (commercially available from Abbott Laboratories, Abbott Park, IL).
  • the TERUMO ELISA method tended to produce erroneous results for the cardiac patient samples, especially those with high concentrations of Lp(a) (>40 mg/dL).
  • MOLECULE TYPE peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

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Abstract

La présente invention se rapporte à un procédé permettant de mesurer directement des concentrations de cholésterol associées à des lipoprotéines spécifiques dans un échantillon de plasma. Le procédé consiste à capturer de manière spécifique des particules de lipoprotéines intactes contenant du cholestérol dans un échantillon de plasma au moyen d'un agent de liaison de lipoprotéine spécifique. Le niveau de cholestérol de lipoprotéines spécifiques présent dans l'échantillon est ensuite mesuré par détection de la quantité de complexes agent de liaison/lipoprotéines qui ont été formés au cours de la réaction. L'on peut détecter le cholestérol contenu dans ces complexes en faisant réagir les complexes avec des agents de liaison de cholestérol spécifiques et marqués, et en mesurant le niveau d'agent de marquage qui y est lié, ou en libérant le cholestérol dans les complexes et en mesurant le niveau de cholestérol libéré. L'agent de liaison de lipoprotéines spécifiques peut être lié à un support solide. Ce procédé de dosage peut également comprendre une étape supplémentaire qui consiste à détecter la présence de cholestérol lié au support solide.
PCT/US1993/002011 1992-03-06 1993-03-04 Dosage par immunocapture pour quantification directe de niveaux de cholesterol de lipoproteines specifiques WO1993018067A1 (fr)

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WO1996000903A1 (fr) * 1994-06-30 1996-01-11 Oklahoma Medical Research Foundation Anticorps diriges contre les lipoproteines et les apolipoproteines, et leurs procedes d'utilisation
US5753517A (en) * 1996-03-29 1998-05-19 University Of British Columbia Quantitative immunochromatographic assays
EP0904538A1 (fr) * 1995-12-05 1999-03-31 EntreMed, Inc. Methode de diagnostic et de traitement de l'atherosclerose a l'aide d'anticoprs anti-cholesterol
WO1999036785A1 (fr) * 1998-01-16 1999-07-22 Abbott Laboratories Dosage immunologique utilise pour detecter des lipoproteines a tres basse densite et anticorps appropries
WO1999036784A1 (fr) * 1998-01-20 1999-07-22 Abbott Laboratories Anticorps specifiques au kringle 5 de l'apolipoprotein a et procedes d'utilisation a cet effet
US6165798A (en) * 1996-10-10 2000-12-26 University Of British Columbia Optical quantification of analytes in membranes
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
US6808889B2 (en) 1999-03-16 2004-10-26 Serex, Inc. Method and device for detection of Apo A, Apo B and the ratio thereof in saliva
EP1918300A2 (fr) * 2001-04-05 2008-05-07 Forskarpatent I Syd Ab Thérapie d'immunisation à base de peptide pour le traitement d'athérosclérose
US7785589B2 (en) 2001-04-05 2010-08-31 Forskarpatent I Syd Antibodies against a peptide epitope of apolipoprotein B
US8119590B2 (en) 2001-09-28 2012-02-21 Cedars-Sinai Medical Center Prevention and treatment of restenosis by local administration of drug
US8926958B2 (en) 2004-04-06 2015-01-06 Cedars-Sinai Medical Center Prevention and treatment of vascular disease with recombinant adeno-associated virus vectors encoding apolipoprotein A-I and apolipoprotein A-I milano
US9205139B2 (en) 2010-11-12 2015-12-08 Cardiovax, Llc Immunomodulatory methods and systems for treatment and/or prevention of aneurysms
US9205141B2 (en) 2010-11-12 2015-12-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of hypertension
EP3425406A1 (fr) * 2014-10-16 2019-01-09 Sysmex Corporation Procédé de mesure de la capacité de lipoprotéine à accepter le cholestérol et trousse de réactifs
WO2022076459A1 (fr) * 2020-10-05 2022-04-14 The Regents Of The University Of California Procédé à haut débit pour la quantification de lp(a)-cholestérol

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NATURE, Volume 323, issued 23 October 1986, T.J. KNOTT et al., "Complete Protein Sequence and Identification of Structural Domains of Human Apolipoprotein B", pages 734-738. *

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* Cited by examiner, † Cited by third party
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US7098036B2 (en) 1994-06-30 2006-08-29 Okalahoma Medical Research Foundation Antibodies to lipoproteins and apolipoproteins and methods of use thereof
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US5753517A (en) * 1996-03-29 1998-05-19 University Of British Columbia Quantitative immunochromatographic assays
US6165798A (en) * 1996-10-10 2000-12-26 University Of British Columbia Optical quantification of analytes in membranes
WO1999036785A1 (fr) * 1998-01-16 1999-07-22 Abbott Laboratories Dosage immunologique utilise pour detecter des lipoproteines a tres basse densite et anticorps appropries
WO1999036784A1 (fr) * 1998-01-20 1999-07-22 Abbott Laboratories Anticorps specifiques au kringle 5 de l'apolipoprotein a et procedes d'utilisation a cet effet
US6210906B1 (en) 1998-01-20 2001-04-03 Abbott Laboratories Specific antibodies to kringle 5 of apo(a) and methods of use therefor
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
US6808889B2 (en) 1999-03-16 2004-10-26 Serex, Inc. Method and device for detection of Apo A, Apo B and the ratio thereof in saliva
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US9205139B2 (en) 2010-11-12 2015-12-08 Cardiovax, Llc Immunomodulatory methods and systems for treatment and/or prevention of aneurysms
US9205141B2 (en) 2010-11-12 2015-12-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of hypertension
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US10436806B2 (en) 2014-10-16 2019-10-08 Sysmex Corporation Method of measuring lipoprotein's capacity to accept cholesterol
WO2022076459A1 (fr) * 2020-10-05 2022-04-14 The Regents Of The University Of California Procédé à haut débit pour la quantification de lp(a)-cholestérol

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