Classifications

• • 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
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

• the present invention is in the field of antibodies, particularly anti-cholesterol antibodies and more specifically relates to the diagnosis and treatment of atherosclerosis and diseases related to cholesterol.
• Arteriosclerosis a generic term for thickening and hardening of the arterial wall, is responsible for the majority of deaths in the United States and most westernized societies.
• One type of arteriosclerosis is atherosclerosis, the disorder of the larger arteries that underlies most coronary artery disease, aortic aneurysm, and arterial disease of the lower extremities, and also plays a major role in cerebrovascular disease.
• Atherosclerosis is by far the leading cause of death in the United States, both above and below age 65 in both sexes. E.L. Bierman, "Atherosclerosis and Other Forms of Arteriosclerosis," Ch. 208, p. 1 106 in Harrison's Principles of Internal Medicine. 13th edition, eds. KJ. Isselbacher, et al. (McGraw-Hill, Inc. NY 1994).
• Atherosclerosis and Cholesterol Atherosclerosis is characterized by infiltration of cholesterol and appearance of foam cells in lesions of the arterial wall. This is followed by a complex sequence of changes involving platelets, macrophages, smooth muscle cells, and growth factors that produces proliferative lesions. These distort the vessels and make them rigid. In individuals with elevated plasma cholesterol levels, there is an increased incidence of atherosclerosis and its complications. W.F. Ganong, Review of Medical Physiology, 17th edition, p. 281 (Appleton & Lange Norwalk, CT 1995).
• Cholesterol is absorbed from the intestine and incorporated into the chylomicrons formed in the mucosa. After the chylomicrons discharge their triglyceride in adipose tissue, the chylomicron remnants bring cholesterol to the liver. The liver and other tissues also synthesize cholesterol. Some of the cholesterol in the liver is excreted in the bile, both in the free form and as bile acids. Some of the biliary cholesterol is reabsorbed from the intestine.
• the transport system for endogenously produced cholesterol is made up of very low density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low- density lipoproteins (LDL), and high-density lipoproteins (HDL).
• VLDL very low density lipoproteins
• IDL intermediate-density lipoproteins
• LDL low- density lipoproteins
• HDL high-density lipoproteins
• the protein constituents of these proteins are called apoproteins.
• the major apoproteins are called apo E, apo C, and apo B.
• Apo B has two forms, apo B-48, characteristic of the exogenous lipid transport system, and apo B- 100, characteristic of the endogenous system.
• VLDL are formed in the liver and transport triglycerides formed from fatty acids and carbohydrates in the liver to extrahepatic tissues. After their triglyceride is largely removed by lipoprotein lipase, they become IDL. The IDL give up phospholipids and, through the action of lecithin- cholesterol acyltransferase, pick up cholesteryl esters formed from cholesterol in the HDL. Some IDL are taken up by the liver. The remaining IDL then lose more triglyceride and protein, and become LDL. During this conversion, they lose apo E, but apo B-l 00 remains. LDL provide cholesterol to the tissues.
• LDL are taken up by receptor-mediated endocytosis in coated pits.
• the receptors recognize the apo B-l 00 component of the LDL. They also bind apo E but do not bind apo B-48.
• LDL are also taken up by a lower affinity system in the macrophages and some other cells. When overloaded by a high plasma level of HDL, the macrophages become full of cholesteryl esters and make up the foam cells that appear early in atherosclerotic lesions.
• the LDL receptor on macrophages and related cells is called the scavenger receptor. W.F. Ganong, pp. 277-279; M. J. Malloy, et al, "Agents Used in Hyperlipidemia," ch. 34, p. 522, in Basic and Clinical Pharmacology, ed. B.G. Katzung (Appleton & Lange Norwalk, CT 1995).
• Cholesterol leaving cells is absorbed in HDL, lipoproteins that are synthesized in the liver and the intestine. Some of the HDL contain apo E and bind to LDL receptors on other cells, thus transporting cholesterol from one cell to another. W.F. Ganong, pp. 277-279.
• Antibodies that selectively and differentially bind to lipoproteins are described. Methods of use of anticholesterol antibodies are described for diagnosis and treatment of atherosclerosis. In one embodiment of the invention, anti-cholesterol antibodies are used to assay samples drawn from atherosclerotic lesions for the amount and relative ratio of lipoprotein molecules. In another embodiment of the invention, the anti-cholesterol antibodies are used as fusiogens to promote fusion of VLDL, IDL and LDL molecules and liposomes, to promote clearance of cholesterol from the body.
• Figure 1 is a graph of the induction of antibodies to cholesterol with 71 mol % cholesterol liposomes.
• BALB/c mice were inoculated twice (2 weeks apart) with liposomes containing 71 mol % cholesterol.
• serum assessed for anticholesterol-binding activity using the PVDF-ELISA Absorbance values on the ordinate are the are the mean of triplicate determinations, with the standard deviation indicated. The ordinate gives the reciprocal of the serum dilution. Background absorbance amounted to 0.200. (squares): immune serum; (diamonds): pre-immune serum.
• Figure 2 is a bar graph of the effect of epitope density of liposomal cholesterol on induction of antibodies to cholesterol.
• BALB/c mice were inoculated twice (2 weeks apart) with liposomes containing various amounts of cholesterol. Serum was collected one week after the boost and anticholesterol activity assessed with the PVDF-ELISA as described below.
• Antibody titers (ordinate) are the mean of triplicate determinations. Pre-immune titers were less than 800. The abscissa presents the mole percentage of liposomal cholesterol.
• Figure 3 is a bar graph of the determination of anti-phospholipid and anti-lipid A antibodies in immune serum.
• PVDF-ELISA wells were coated with cholesterol (5 micrograms), DMPC (7.5 micrograms), or DMPG (7.5 micrograms) (abscissa).
• Polystyrene-ELISA wells were coated with lipid A (0.5 micrograms).
• Immune serum (obtained as for Figure 1) was assessed for binding activity to the individual liposomal constituents as described below. Titers were calculated from triplicate determinations (abscissa). Titers were calculated from triplicate determinations. Pre-immune titers were less than 800.
• Figure 4 is a graph of the binding of the anticholesterol monoclonal antibody 2C5-6 to cholesterol.
• the binding of the affinity chromatography-purified, murine IgM anticholesterol monoclonal antibody 2C5-6 was assessed using the PVDF-ELISA.
• Absorbance values +/- SD are the mean of triplicate determinations, (squares): anticholesterol IgM; (circle): nonspecific murine IgM.
• the absorbance at 405 nm is presented on the ordinate, the nanograms per milliliter of monoclonal antibody is presented on the abscissa.
• Figure 5 are three graphs of anti-cholesterol antibody binding to intact human VLDL/IDL, LDL and HDL, in ELISA wells coated with various amounts of lipoprotein protein. As indicated, varying amounts of HDL, LDL and VLDL/IDL were used to coat the wells of polystyrene-ELISA plates. After removal of unbound lipoprotein, dilutions of immune serum were assessed for binding activity. Absorbance values are the mean of triplicate determinations. Absorbance values of pre-immune serum did not exceed 0.250. On the abscissa is plotted the reciprocal of serum dilution. On the ordinate is measured the absorbance at 405 nm.
• Figure 6 is a graph of anti-cholesterol antibody binding to intact human VLDL/IDL, LDL and HDL, in ELISA wells coated with corresponding amounts of unesterified cholesterol.
• Polystyrene-ELISA plates were coated with 50 microliters of a solution containing either 0.05 micrograms VLDL/IDL, 0.15 micrograms LDL, or 220 micrograms HDL (based on protein). After removal of unbound lipoprotein, binding of the monoclonal antibody 2C5-6 was assessed. After performing the ELISA, the amounts of total and unesterified cholesterol adsorbed to the wells was determined for each individual lipoprotein. The obtained values are indicated in the Figure. On the abscissa is plotted the reciprocal of the serum dilution, on the ordinate is plotted the absorbance at 405 nm.
• Figures 7 A and B are graphs of the binding of anti-cholesterol antibodies to total lipid extracts from human lipoprotein.
• Figure 7 A. is a graph of the total lipid extracted from either VLDL/IDL, LDL, or HDL was bound to the PVDF membrane of ELISA plate wells (10 micrograms of total cholesterol per well).
• Figure 7B is a graph of the matched amounts of the intact lipoproteins (10 micrograms total cholesterol per well) were used to coat the wells of PVDF-ELISA plates. Both the lipid extracts (A) and the intact lipoproteins (B) were incubated with the indicated dilutions of immune and pre-immune serum and further processed as described in the Methods section. Absorbance values are the mean plus or minus the standard deviation of triplicate determinations. On the abscissa is plotted the absorbance values at 405 nm, on the ordinate is plotted the reciprocal of the dilution.
• Figure 8 is a picture of an anti-cholesterol immunoblot of TLC-separated lipid extracts from human lipoproteins.
• Total lipid extracts of VLDL/IDL, LDL, and HDL (10 micrograms total cholesterol) were separated by TLC and lipids were then transferred to PVDF membranes. Membranes were probed with anticholesterol immunoreactive serum and stained with peroxidase- conjugated anti-IgM antibody as described below.
• Figure 8 A TLC plate stained after the transfer with ferric chloride spray.
• Figure 8 B Immunoblot (mirror image).
• C cholesterol
• CE cholesterol esters
• TG triglycerides
• PL phospholipids.
• Figures 9 A, B, and C are three graphs of the effect of temperature on anticholesterol antibody binding to cholesterol, cholesterol-rich liposomes, and VLDL/IDL. Cholesterol (panel A), 71 mol % cholesterol liposomes
• VLDL/IDL Figure 10A
• LDL Figure 1 OB
• HDL Figure IOC
• Figures 11 A and B are photomicrographs of lipoproteins and liposomes after incubation with anticholesterol antibodies.
• Figure 12 is a graph of showing the detection of antibodies to cholesterol in the serum of BALB/c mice inoculated with liposome containing 71 % cholesterol and 2 micrograms monophosphoryl lipid A, using polyvinylnitrocellulose composite on which cholesterol was adsorbed. On the abscissa is plotted cholesterol
• Figure 13 is a graph of cholesterol detection using various dilutions of immune sera added to wells containing cholesterol immobilized on polyvinyl nitrocellulose. On the abscissa is plotted cholesterol (micrograms/well), and on the ordinate is plotted absorbance at 405 nm.
• Novel antibodies that selectively and differentially bind to cholesterol and to the different classes of lipoproteins are provided by the present invention.
• a method of determining the amount VLDL, IDL and LDL, in a biological sample comprising the steps of: (a) contacting a biological sample with an anti ⁇ cholesterol antibody;
• the biological sample may be derived from any suitable biological source, such as blood, serum, tissue and the like, in one embodiment of the method it is desirable that the biological sample is derived from an atherosclerotic lesion.
• the antibody is produced by the hybridoma cell line deposited at the ATCC under Designation Number ATCC 8995.
• the ATCC 8995- derived anti-cholesterol antibodies are used to assay samples drawn from atherosclerotic lesions for the amount and relative ratio of VLDL, IDL, LDL, and HDL molecules.
• the anti- cholesterol antibodies are used as fusiogens to promote fusion of VLDL, LDL molecules and liposomes, to promote clearance of cholesterol from the body.
• the antibodies of the present invention can be labeled with paramagnetic ions, such as Eu or Gd, or a stable free radical for in vivo immunodetection. Additionally, the antibodies of the present invention can be labeled with ! Hn for use in computer assisted tomography. Still further, labels useful for in vitro assays, such as radioactive isotopes, color generating reagents or light emitting reagents, may be conjugated or otherwise associated with the antibodies of the present invention.
• paramagnetic ions such as Eu or Gd
• ! Hn for use in computer assisted tomography
• labels useful for in vitro assays such as radioactive isotopes, color generating reagents or light emitting reagents, may be conjugated or otherwise associated with the antibodies of the present invention.
• Labeled antibodies of the present invention may be administered to a patient to localize cholesterol and lipoprotein areas by non-invasive imaging techniques.
• Labeled antibodies of the invention may be contacted with biological samples in vitro assays systems to determine the presence and amount of cholesterol and the relative ration of various lipoproteins.
• cholesterol as used herein regarding the binding specificity of the antibodies of the invention, includes structural analogs of cholesterol that are bound by the antibodies.
• the invention may be further understood with reference to the following non-limiting examples. All publications cited herein are incorporated by reference.
• Example 1 Induc tion of anti - cholesterol antibodies in mice after inoculation with cholesterol-rich liposomes.
• DMPC dimyristoyl-sn-glycero-3-phosphocholine
• DMPC l ,2-dimyristoyl-snglycero-3-phos ⁇ hoglycerol
• cholesterol 5-cholesten-3-beta-ol
• Cholesteryl beta-epoxide (cholestan-5-beta,6-beta-epoxy-3- beta-ol); 7-dehydrocholesterol (5,7-cholestadien-3-beta-ol); epicholesterol (5-cholesten-3-alpha-ol); 7-ketocholesterol (5-cholesten-3-beta-ol-7-one); 4-beta-hydroxycholesterol (5-cholesten-3-beta-4-beta-diol); 7-beta-hydroxycholesterol (5-cholesten-3-beta-7-beta-diol); and lanosterol (8,24,(5- alpha)cholestadien-4,4, 14-alpha-trimethyl-3-beta-ol), were obtained from Steraloids, Inc. (Wilton, NH). Cholestene
• Cholesterol liposomes Cholesterol-rich multilamellar vesicles (MLV; 71 mol % cholesterol) used for induction of antibodies to cholesterol were composed of DMPC, DMPG, and cholesterol in a molar ratio of 9: 1 :25 and contained the adjuvant monophosphoryl lipid A at 25 micrograms per mole phospholipid.
• LUV multilamellar vesicles
• liposomes having lower amounts of cholesterol or completely lacking lipid A were occasionally also prepared.
• the lipids were aliquoted from stock solutions in chloroform into a pyrogen-free, round-bottom flask, and the solvent was removed by rotary evaporation. The lipids were further dried under high vacuum for 2 hours.
• the dry film was hydrated by adding sterile deionized water to achieve a 50 mM total phospholipid and vortexing until all the lipid was resuspended. The material was subsequently incubated for 2 hours at room temperature and occasionally vortexed. Aliquots of the liposomal preparations were lyophilized in vaccine vials and prior to use reconstituted in PBS to 10 mM with respect to total phospholipids.
• MLV prepared without lipid A were sonicated to obtain small unilamellar vesicles (SUV).
• One to two ml of an MLV suspension (diluted in PBS to 5 mM phospholipid) was transferred to a capped, glass culture-tube.
• the lipid was sonicated under a nitrogen atmosphere to opalescence (four 15 minute periods with intermittent vortexing) in a bath-type sonicator (Laboratory Supplies, Inc., Hicksville, NY) at 30 °C.
• the SW were used on the day of preparation.
• mice For induction of antibodies to cholesterol, groups of 5 male inbred mice (C3H/HeJ, C3H/HeOuJ, C57BL/6J, C57BL/LDLr-/-, C3H.MRLFaslpr, DBA/2J, CBA/CaJ, CBA/CaHN-xid/J, A/J, BXSB/MpJYaa, NZB/B 1NJ, HRS/J, BALB/cByJ, and BALB/cByJ-nu strain; Jackson Lab, Bar Harbor, ME) were immunized intraperitoneally with 100 microliters of the cholesterol-rich MLV (2.5 micromoles of cholesterol). After 2 weeks, the mice were boosted and bled 1 to 2 weeks later from the tail vein to collect immune serum. Pre-immune sera were collected just before the primary inoculation.
• the hybridoma cell line 2C5-6 (ATCC 8995), secreting a monoclonal IgM antibody that reacts with cholesterol (Swartz, G. M., et al , 1988, Proc. Natl Acad. Sci. USA 85: 1902. ), was a gift from Dr. C. R. Alving, Walter Reed
• IgM was purified by affinity chromatography on a mannan-binding protein column (Pierce, Rockford, IL).
• Lipoprotein extracts and pure, lipid standards were separated on TLC silica gel plates and detected according to the procedure of Aniagolu, S. J. Green, et al, 1995, J. Immunol Methods 182: 85.).
• the separated lipids were transferred to hydrophobic polyvinylidenefluoride membranes (PVDF), incubated with antibodies and stained as described (Aniagolu, J., et al , 1995, /. Immunol Methods 182: 85.).
• Serum samples were serially diluted in blocking buffer, and 100 microliters was added to duplicate wells. After 1 hour incubation with slow agitation, wells were washed four times with the blocking buffer, followed by a 1 hour incubation with a 1/1500 dilution of peroxidase conjugated anti-mouse IgG (H+L chain; BioRad, Richmond, CA) or IgM (Kirkegaard & Perry, Gaithersburg, MD).
• PVDF-ELISA was occasionally also used for the determination of anti-phospholipid and anti-steroid activity.
• VLDL/IDL, LDL, HDL, or liposome dilution in PBS containing the indicated amounts of protein or lipid.
• PVDF plates were occasionally also used to assess binding to either lipoproteins or liposomes.
• the wells were washed and further processed as described for the cholesterol ELISA. In some lipoprotein ELISA experiments, the wells washed extensively after determination of the absorption, to remove residual chromophore. Next, the cholesterol was extracted from individual wells by three washes with 100 ml isopropanol.
• the combined extracts of 12 wells were pooled and dried in a Speed Vac concentrator and total cholesterol was assayed after resuspension in 50 microliters isopropanol. Free cholesterol was calculated from the free over total cholesterol ratios measured for each of the lipoprotein stock solutions used in the ELISA.
• the lipid A ELISA was performed as described by Freudenburg, et al , 1988, Infection 7: 322. , using polystyrene immulon-2 plates. The wells were coated with 0.5 micrograms lipid A in 50 microliters ethanol and after drying, the plates were further processed as described for the cholesterol ELISA.
• antibody titers are defined as the reciprocal of the highest serum dilution having an absorbance of at least two standard deviations above the background level.
• the background was determined by replacing the antiserum or purified primary (monoclonal) antibody with blocking buffer.
• Lipoprotein or liposome dilution (10 microliters) containing the indicated amounts of protein or lipid were admixed with 10 microliters of a purified 1 mg/ml solution of anti-cholesterol monoclonal IgM, diluted in 300 ul saline (sterile, tissue culture grade, Sigma Chem. Co., St. Louis, MO), and incubated for 30 minutes in polypropylene tubes at 4°C.
• the lipoproteins were also incubated with 10 ug of control, non-specific IgM. Subsequently, the samples were analyzed by FACSort (Becton-Dickinson, San Jose, CA) flow cytometery.
• Total cholesterol and free, unesterified cholesterol in lipoproteins and lipid extracts were determined using enzymatic colorimetric methods purchased as kits from Wako Chemicals USA, Inc. (Richmond, VA).
• the specificity of the antibodies was to unesterified cholesterol and structurally similar sterols containing a 3-beta-hydroxyl group (i.e. , ergosterol).
• anti-cholesterol binding activity was significantly diminished if the 3-beta-hydroxyl was altered by epimerization (i.e. , epicholesterol), by substitution with hydrogen (i. e. , cholestene), by oxidation to a keto-group (i. e. , cholestenone), or by esterification (i. e. , cholesteryl sulfate or cholesteryl oleate).
• Changes in the structure of the ⁇ -ring degree of saturation, i.e., dihydrocholesterol or
• Figure 6 shows that, even with a slight excess of free-HDL-cholesterol bound to the wells, antibody titers for binding of the anti-cholesterol monoclonal antibody to both VLDL/IDL and LDL were in excess of 20,000, whereas negligible interaction was observed with HDL (titer ⁇ 800). Similar results were obtained when anti-cholesterol immune serum was tested under these conditions (not shown).
• VLDL and LDL presumably resulted in aggregation of the lipoproteins, as indicated by the change in size distribution.
• large (up to 5 micrometers diameter) spherical, droplet-like structures were found in suspensions of VLDL/IDL and LDL incubated with the anti-cholesterol monoclonal IgM antibody (Figure IIA).
• Control IgM, or an irrelevant antiphosphatidylinositolphosphate monoclonal antibody had no effect (data not shown). No visible structures could be detected in the suspensions containing HDL and anti ⁇ cholesterol antibodies ( Figure IIA).
• the vesicles were found to have increased in size, the majority having dimensions larger than 1 micrometer (not shown).
• the SUV products did not disintegrate at higher temperatures, but even fused into larger, more complex structures upon warming (not shown).
• the large vesicular structures could also be formed from the SUV at room temperature or at 37 °C.
• anti-cholesterol antibodies (1 ) recognize the 3-beta-hydroxyl group of sterols, (2) bind to the lower density lipoproteins, i. e. , VLDL/IDL and LDL, but not HDL, and (3) induce a fusion-like reaction upon binding to these lipoproteins.
• Only anti-cholesterol antibodies of the IgM subtype could be detected in the sera of mice boosted with lipid A containing, cholesterol-rich liposomes.
• naturally occurring antibodies to cholesterol observed in humans were reported to be of both the IgM and IgG subtype. Swartz, G. M., et al , 1988, Proc. Natl. Acad. Sci.
• antibody-lipoprotein binding at 37 °C may result in the release of antibody-lipoprotein fragments from the ELISA plate. Therefore, it is possible that the ELISA at 37 °C underestimates the actual interaction of anti- cholesterol antibodies with the fixed lipoprotein.
• EXAMPLE 2 Binding of murine monoclonal and polyclonal IgM antibodies at 37 degrees Celsius to lipid particles isolated from advanced human athromas, but not to LDL derived from serum.
• Tissue and Serum Samples Human aortic tissue was obtained from either autopsy of individuals within 24 hours of death or from surgery samples during aortic aneurysm repair and frozen (at -70°C) immediately. In order to obtain grossly normal tissue, abdominal aortic samples from pediatric cases and thoracic or aortic arch material from patients with no to minimal disease was used. Samples were grouped into three categories: as grossly normal or no lesion, minimal presence of plaques or fatty streaks, or severe disease or ulcerating lesions. Table 2 summarizes the source of tissue assessed for anti-cholesterol IgM antibody.
• tissue from thoraic aorta * tissue from thoraic aorta; all others tissue from aortic arch.
• the supernatant was passed through a 0.45 micron filter and was concentrated on a stirred cell concentrator with a 10,000 MW cut-off. After dialyzing overnight against 1.5 M NaCl (pH 7.4) containing 0.1 % EDTA, 0.05% glutathione and 0.02% NaN3, the extract was subjected to gel filtration chromatography (Bio-gel A- 50m, Richmond CA) using a 1.6 x 50 cm column as described. Next, The void volume fractions containing cholesterol were pooled and subjected to density gradient centrifugation.
• the gradient was constructed from bottom to top using 3 ml of 1.100 g/ml, 3 ml of void volume fraction (density adjusted to 1.061 g/ml), 3 ml of 1.019 g/ml, and 2.5 ml of 1.006 g/ml NaCl solution and centrifuged at 170,000 g for 22 hours at 4°C. Fractions containing liposomes of various densities were located visually and collected by aspiration from the top. Fractions were then dialyzed against PBS and 0.02% NaN3. ELISA for anti-cholesterol IgM binding activity to cholesterol and liposomes. To quantify antibodies specific for these antigens, PDVF microtiter plates (Immobilon P
• Millipore were either spotted with 10 microliters of a 1 mg/ml ethanol solution of cholesterol or cholesterol oleate and allowed to air dry. If coated with liposomes the plates were washed 4 times with 250 ul of PBS per well. Next, the plates were blocked with 10% heat inactivated fetal calf serum and then incubated with primary and secondary antibody as described (Aniagola J., et al., 1995, J. Immunol. Methods 182: 85). In all cases specific absorbance was calculated by subtracting a no-antigen or cholesterol oleate negative control from that of the test material.
• IgM levels PDVF plates were coated with anti-total human immunoglobulin. After blocking for non-specific binding, titration curves of serial dilutions of the aortic homogenates were then constructed using either affinity purified IgM (m) specific anti-sera as the secondary antibody (Kirkegard and Perry). Results of the curve were then compared with known commercial standards to quantify the amount of IgM. Anti-cholesterol IgM concentration in the aortic homogenates was calculated by subtracting the quantity of IgM bound to the negative control well from that bound to the cholesterol coated well. The same standard curve noted above was used for determining the quantity of both the cholesterol specific and non-specific IgG in this calculation. When possible these assays were performed in the same microtiter trays to control for plate to plate variation.
• Murine antibody The production of the murine anti ⁇ cholesterol anti-sera and the murine anti-cholesterol monoclonal antibody (2C5-6) are described in Swartz, G. M., et al, 1988. PNAS, 85: 1902. Isolation of LDL. Plasma was obtained from healthy fasted subjects. LDL was isolated by sequential ultracentifugation as described in Patsch, J. R., et al, 1986, in Methods in Enzymology, Vol. 129, J. Albers, et al, eds., Academic Press, New York, NY, p. 37.
• the murine monoclonal anti-cholesterol antibody selectively binds to both of the lesion-derived lipid particles at 37°C, whereas no reactive was observed with LDL under the same conditions.
• Example 3 An ELISA for detection of anti-sterol antibodies using polyvinylnitrocellulose and anti- sterol antibodies
• the ELISA plate uses a polyvinylnitrocellulose composite material that can resist organic solvent (i.e. , chloroform/ethanol) which are necessary for solubilizing sterols.
• organic solvent i.e. , chloroform/ethanol
• the initial coating of the sterol-antigen is done in the presence of a chloroform/ethanol solution. Due to the hydrophobic nature of the nitrocellulose composite, cholesterol and ergosterol tightly binds allowing the replacement of the organic solvent with aqueous buffer without causing the sterol to precipitate.
• the proposed ELISA using immobilized sterol on polyvinyl nitrocellulose, is superior to the traditional radioimmunoassays and bioassays because it does not require a radioligand; the ligand does not have to be chemically modified or cross-linked for binding; insolubility of the ligand is of no concern; it is equally as sensitive and reproducible as a radioimmunoassay; the assay is both quantitative and qualitative; it is a quicker and cheaper assay than the RIA and other traditional methods; it is direct and can be converted for use for any hydrophobic sterol or steroid; and it is not biologically hazardous.

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