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WO1994023302A1 - Dosage immunologique de lipoproteines humaines a faible densite modifiees par oxydation presentes dans le plasma - Google Patents

Dosage immunologique de lipoproteines humaines a faible densite modifiees par oxydation presentes dans le plasma Download PDF

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WO1994023302A1
WO1994023302A1 PCT/AU1994/000171 AU9400171W WO9423302A1 WO 1994023302 A1 WO1994023302 A1 WO 1994023302A1 AU 9400171 W AU9400171 W AU 9400171W WO 9423302 A1 WO9423302 A1 WO 9423302A1
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antibody
ldl
complex
labelled
plasma
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Michael Lionel Selley
Neville Gordon Ardlie
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Australian National University
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Australian National University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates generally to the immunodetection of oxidatively-modified low density lipoproteins (LDL), antibodies thereto or an immune complex thereof, in biological fluid. More particularly, the present invention relates to an enzyme-linked immunosorbent assay (ELISA) technique for the analysis of oxidatively-modified LDL, antibodies thereto or an immune complex thereof in mammalian plasma, and even more particularly, human plasma.
  • ELISA enzyme-linked immunosorbent assay
  • the present invention is also directed to a kit to facilitate the ELISA technique.
  • the present invention is particularly useful in the assessment of coronary heart disease risk.
  • Plasma low density lipoprotein (LDL) can undergo free radical induced oxidation either catalyzed by divalent ions such as Cu 2+ (Steinbrecher el aL I Proc. Natl. Acad.
  • oxidized LDL The functional changes of oxidized LDL include recognition by the scavenger receptor on macrophages (Steinbrecher el aL II supra; Parthasarathy el aL Proc. Natl. Acad. Sci. U.S.A. 84 507-540, 1987), cytotoxicity to human vascular smooth muscle cells and endothelial cells (Hessler el aL Atherosclerosis 32:213-229, 1979), chemotaxis for human monocytes (Quinn et aL I Proc. Natl. Acad. Sci. U.S.A.
  • Oxidized LDL attracts monocytes into the subendothelial space where the monocytes are converted to macrophages which then take up the oxidized LDL generating the foam cells characteristic of the early fatty streak
  • the mobility of the macrophages is inhibited, trapping them in lesion sites.
  • the oxidized LDL is also toxic to endothelial cells and could be responsible for endothelial injury.
  • Oxidatively modified LDL subtraction has been isolated from human plasma in amounts ranging from 5% to 10% of the total LDL (Arogaro el aL Arteriosclerosis 8 79-87, 1983). Increased blood concentrations of cholesterol are a well-established risk factor for coronary heart disease (Castelli el aL Circulation 67 730-734, 1983). Oxidatively modified LDL also exists in an immune complex with antibodies. Free antibodies to oxidatively modified LDL also exist.
  • oxidatively modified LDL is important in the pathogenesis of atherosclerosis then the measurement of oxidized LDL, antibodies thereto or an immune complex thereof in plasma may be a more reliable indicator of atherosclerotic risk than the measurement of cholesterol or LDL alone.
  • the present invention contemplates a method for detecting oxidatively-modified low density lipoprotein (LDL), or LDL in an immune complex with antibodies thereto, in biological fluid which method comprises contacting the fluid to be tested with an antibody specific to oxidized LDL for a time and under conditions sufficient for an LDL-antibody complex to form and then detecting said complex by a detection means.
  • LDL low density lipoprotein
  • the present invention is directed to a method for detecting oxidatively-modified LDL, or oxidized LDL in an immune complex with antibodies thereto, in human plasma which method comprises contacting the plasma to be tested with a monoclonal antibody specific to oxidised LDL, for a time and under conditions sufficient for an LDL-antibody complex to form and then detecting said complex by a detecting means.
  • Another aspect of the present invention relates to a method for detecting antibodies interactive with oxidized LDL or antibodies in an immune complex with oxidized LDL in biological fluid, such as human plasma, which method comprises contacting the fluid to be tested with oxidized LDL for a time and under conditions sufficient to form an antibody-LDL complex and then detecting said complex by a detection means.
  • the detecting means comprises an antibody labelled with a reporter molecule capable of giving a detectable signal.
  • Yet another aspect of the present invention relates to a kit useful in the analysis of oxidatively-modified LDL, antibodies thereto or an immune complex thereof in human plasma.
  • the present invention also contemplates a method for assessing coronary heart disease risk in a human comprising quantitating the level of oxidatively-modified LDL, or oxidized LDL in an immune complex with antibodies thereto in plasma from said human by contacting said plasma with a monoclonal antibody specific to oxidized LDL for a time and under conditions sufficient to form an LDL-antibody complex and subjecting said complex to a quantifying detecting means and comparing the level so detected to a predetermined amount.
  • Another aspect of the present invention relates to a method for assessing coronary heart disease risk in a human comprising quantitating the level of antibodies specific for oxidatively-modified LDL or antibodies in an immune complex with oxidized LDL in plasma from said human by contacting said plasma with oxidized LDL for a time and under conditions sufficient to form an antibody-LDL complex and subjecting said complex to a quantifying detecting means and comparing the level so determined to a predetermined amount.
  • oxidatively-modified LDL as used in the present specification has the same meaning as “oxidized LDL”. Both terms are used interchangeably.
  • the present invention is directed to a method of detecting oxidatively-modified LDL, antibodies thereto or an immune complex thereof in biological fluid which comprises contacting the fluid to be tested with an antibody specific to oxidized LDL, or with oxidized LDL capable of binding to an antibody thereto for a time and under conditions sufficient for an LDL-antibody complex to form and then subjecting said complex to a detecting means.
  • oxidized LDL is intended to encompass oxidized LDL, antibodies thereto and/or an immune complex between said oxidized LDL and said antibodies.
  • the latter complex is also referred to as a circulating immune complex.
  • detecting is meant to include both quantitative and qualitative detection of oxidized LDL or antibodies thereto.
  • Detecting means includes, in the case of detecting oxidized LDL, a first antibody, specific or otherwise interactive to oxidized LDL and labelled with a reporter molecule capable of giving a detectable signal or a second antibody specific or otherwise interactive to said first antibody, where said second antibody is labelled with the reporter molecule.
  • the detecting means includes an anti-mammalian antibody capable of binding to the antibody to be detected, said anti-mammalian antibody labelled with a reporter molecule.
  • 'Biological fluid is used in its broadest sense and includes in.
  • oxidatively-modified LDL refers to any LDL having undergone free radical induced oxidation either catalyzed by divalent ions such as Cu 2+ , amongst others, or by incubation with cultured cells such as endothelial cells, vascular smooth muscle, monocytes, neutrophils or macrophages.
  • reference herein to oxidatively- modified LDL also includes reference to circulating populations of LDL subtractions such as from patients with cardiovascular disease.
  • Oxidatively-modified LDL have properties as hereinbefore described. Although for the purposes of exemplification Cu 2+ is described herein as being used to induce oxidation of LDL, this should not be construed as a limitation to a particular mode of oxidation. In the following disclosure, reference to oxidized LDL is also meant to encompass antibodies thereto and immune complexes thereof.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization of animals with oxidatively-modified LDL, and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the oxidatively-modified LDL, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of d e potential heterogeneity of the product.
  • the use of monoclonal antibodies in the present immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the produce.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, "Basic Facts About Hybridomas", in Compendium of Immunology, Bol. II, L. Schwartz (Ed.), 1981; Kohler and Milstein, I Nature. 256_:496-497, 1975; II European Journal of Immunology, &511-519, 1976).
  • the choice of animal is dependent on the availability of appropriate immortal lines capable of fusing with lymphocytes thereof.
  • Mouse and rat have been the animals of choice in hybridoma technology and are preferably used. Humans can also be utilized as sources for sensitized lymphocytes if appropriate immortalized human (or nonhuman) cell lines are available.
  • the animal of choice may be injected with from about 20ug to about 500 ug of the oxidatively-modified LDL and preferrably 50 ⁇ g to about 200 ⁇ g.
  • the injecting material is emulsified in Freund's complete adjuvant. Boosting injections may also be required.
  • Lymphocytes can be obtained by removing the spleen or lymph nodes of sensitized animals in a sterile fashion and carrying out fusion. Alternatively, lymphocytes can be stimulated or immunized in vitro, as described, for example, in Reading, J. Immunol. Meth. 53:261-291, 1982.
  • a number of cell lines suitable for fusion have been developed, and the choice of any particular line for hybridization protocols is directed by any one of a number of criteria such as speed, uniformity of growth characteristics, deficiency of its metabolism for a component of the growth medium and potential for good fusion frequency.
  • Intraspecies hybrids work better than interspecies fusions.
  • Several cell lines are available, including mutants selected for the loss of ability to secrete myeloma immunoglobulin. Included among these is the mouse myeloma line NSI (Kohler et aL III Eur. J. Immunol. 6:292-295, 1987).
  • Another useful myeloma is P3-XAg8.653 (Kearney et at J. Immunol. 12& 1548, 1982)
  • the present invention extends to subclones and other myeloma derived from P3- XAg8.653.
  • Cell fusion can be induced either by virus, such as Epstein-Barr or Sendai virus, or polyethylene glycol.
  • Polyethylene glycol (PEG) is the most efficacious agent for the fusion of mammalian somatic cells. PEG itself may be toxic for cells, and various concentrations should be tested for effects on viability before attempting fusion.
  • the molecular weight range of PEG may be varied from 1000 to 6000. It gives best results when diluted to from about 20% to about 70% (w/w) in saline or serum-free medium. Exposure to PEG at 37 °C for about 30 seconds is preferred in the present case, utilizing murine cells. Extremes of temperature (i.e., about 45 °C) are avoided, and preincubation of each component of the fusion system at 37 °C prior to fusion gives optimum results.
  • the successfully fused cells can be separated from the myeloma line by any technique known by the art.
  • the most common and preferred method is to choose a malignant line which is Hypoanthine Guanine Phosphoribosyl Transferase (HGPRT) deficient, which will not grow in an aminopterin-containing medium used to allow only growth of hybrids and which is generally composed of hypoanthine lxlO "4 M, aminopterin lxlO "5 M, and thymidine 3xlO "5 M, commonly known as the HAT-containing culture medium immediately after the fusion 24 hours later.
  • the feeding schedules usually entail maintenance in HAT medium for two weeks and then feeding with either regular culture medium or hypoanthine, thymidine-containing medium.
  • the growing colonies are then tested for d e presence of antibodies that recognise the antigenic preparation.
  • Detection of hybridoma antibodies can be performed using an assay where the antigen is bound to a solid support and allowed to react to hybridoma supernatants containing putative antibodies.
  • the presence of antibodies maybe detected by "sandwich” techniques using a variety of indicators. Most of the common methods are sufficiently sensitive for use in the range of antibody concentrations secreted during hybrid growth.
  • Cloning of hybrids can be carried out after 21-23 days of cell growth in selected medium. Cloning can be performed by cell limiting dilution in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilution, cell suspensions are diluted serially to yield a statistical probability of having only one cell per well. For the agarose technique, hybrids are seeded in a semisolid upper layer, over a lower layer containing feeder cells. The colonies from the upper layer may be picked up and eventually transferred to wells.
  • Antibody-secreting hybrids can be grown in various tissue culture flasks, yielding supernatants with variable concentrations of antibodies. In order to obtain higher concentrations, hybrids may be transferred into animals to obtain inflammatory ascites. Antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection. The ascites contain a higher concentration of antibodies but include both monoclonals and immunoglobulins from the inflammatory ascites. Antibody purification may then be achieved by, for example, affinity chromatography.
  • oxidatively-modified LDL contemplated herein in a biological fluid, such as in human plasma can be detected utilizing antibodies prepared as above, eidier monoclonal or polyclonal, in virtually any type of immunoassay.
  • antibodies specific to oxidized LDL can be detected, using, for example, anti-human antibodies.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043 4,424,279 and 4,018,653. This, of course, includes both single-site and two-site, or "sandwich", assays of the non-competitive types, as well as in the traditional competitive binding assays.
  • a sandwich assay technique A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by d e present invention.
  • an unlabelled antibody is immobilized in a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • oxidatively-modified LDL is referred to as the antigen.
  • a second antibody labelled with a reporter molecule capable of producing a detectable single is then added and incubated, allowing time sufficient for the formation of a tertiary complex of antibody-antigen-labelled antibody (e.g.
  • any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to die bound antibody. These techniques are well known to those skilled in the art, and tihen possible of minor variations will be readily apparent.
  • a two component assay is used comprising the antigen to be detected and a labelled antibody.
  • a first antibody having interactivity for oxidatively-modified LDL as contemplated by the present invention is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplats, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalendy binding or physically adsorbing the molecule to the insoluble carrier. Following binding, the polymer-antibody complex is washed in preparation for the test sample.
  • An aliquot of the sample to be tested is dien added to the solid phase complex and incubated at 25 °C for a period of time sufficient to allow binding of any subunit present in the antibody.
  • the incubation period will vary but will generally be in die range of about 2-40 minutes.
  • d e antibody subunit solid phase is washed and dried and incubated witii a second antibody specific for a portion of the antigen or first antibody.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of die second antibody.
  • reporter molecule is meant a molecule which, by its chemical nature, provides an analytically identifiable or detectable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes).
  • an enzyme is conjugated to die second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to die skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, streptavidin peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used witii the specific enzymes are generally chosen for the production, upon hydrolysis by die corresponding enzyme, of a detectable colour change.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine, 5-aminosalicyclic acid, or tolidine are commonly used.
  • fluorogenic substrates which yield a fluorescent product ratiier than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to die first antibody-antigen complex, allowed to bind, and tiien the excess reagent is washed away.
  • a solution containing die appropriate substrate is then added to die tertiary complex of antibody-antigen-antibody.
  • the substrate will react with the enzyme linked to die second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectophotometricly, to give an indication of die amount of hapten which was present in the sample.
  • fluorescent compounds such as fluorescein and rhodamine, may be chemically coupled to antibodies witi out altering tiieir binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination witii light of a particular wavelength, the fluorochrome-labelled antibody absorbs die light energy, inducing a state of excitability in the molecule, followed by emission of die light at a characteristic colour visually detectable witii a light microscope.
  • the fluorescent labelled antibody is allowed to bind to die first antibody-antigen complex.
  • the fluorescence observed indicates die presence of the antigen of interest
  • immunofluorescene and ELISA techniques are botii very well established in die art and are particularly preferred for d e present metiiod.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit d e required purpose. It will also be apparent that the foregoing can be used to detect direcdy or indirecdy (i.e., via antibodies or immune complexes) the LDL of this invention.
  • antibodies to oxidized LDL are readily detectable in biological fluid, e.g. human plasma, by contacting die fluid with oxidized LDL in order to form an immune complex between oxidized LDL and its antibodies.
  • biological fluid e.g. human plasma
  • oxidized LDL e.g. human plasma
  • Such an immune complex can be detected using labelled anti-mammalian antibodies capable of binding to die antibody in the immune complex.
  • the present invention is also directed to a kit for the rapid and convenient assay of oxidatively-modified LDL, antibodies tiiereto or immune complexes thereof in mammalian body fluids, such as human plasma.
  • the kit is compartmentalized to receive a first container adapted to contain an antibody to oxidatively-modified LDL, and a second container adapted to contain a second antibody to said first antibody, said second antibody being labelled witii a reporter molecule capable of giving a detectable signal as hereinbefore described. If the reporter molecule is an enzyme, then a third container adapted to contain a substrate for said enzyme is provided.
  • the first container may contain oxidized LDL and the second container contains anti-mammalian (eg.
  • a sample to be tested for oxidatively-modified LDL, antibodies tiiereto or an immune complex thereof is contacted to the contents of the first container for a time and under conditions for an LDL antibody complex to form. If such a complex does form, it is detected by die second antibodies of the second container which will bind to the secondary complex to form a tertiary complex and, since said second antibodies are labelled with a reporter molecule, when subjected to a detecting means, the tertiary complex is detected.
  • die “containers” described above may include a microtitre tray or other similar device or may include a paper material. Accordingly, the present invention provides a rapid and convenient assay for oxidatively-modified LDL, antibodies thereto or immune complexes thereof in biological fluid, such as human plasma. This is particularly important in the assessment of coronary heart disease risk. It is also useful in monitoring therapeutic procedures for treating atherosclerosis.
  • Figure 1 is a graphical representation of the elution profile of LDL by ion exchange chromatography, •-• 280nm; o-o 254nm.
  • Figure 2 is a graphical representation of the fraction spectrum of LDL isolated by ultracentrifugal techniques; Region A: lOO ⁇ g/ml; Region B: 400 ⁇ g/ml; Region C: 205 ⁇ g/ml.
  • a high density salt solution NaCl and KBr, containing EDTA
  • the preparation is washed and dialysed at 4°C for 24 hours against modified Tyrode's buffer (NaCl 140mM, KC12.68mM, NaHC0 3 11.9mM, NaH 2 P0 4 ,H 2 O 0.32mM, pH7.4) containing EDTA (0.27m).
  • modified Tyrode's buffer NaCl 140mM, KC12.68mM, NaHC0 3 11.9mM, NaH 2 P0 4 ,H 2 O 0.32mM, pH7.4
  • EDTA EDTA
  • the protein content is determined by the method of Lowry (Lowry el aL J. Biol. Chem. 122:265-275, 1951). 1.2 Lipoprotein Oxidation.
  • Lipoprotein are oxidized by incubation LDL (200 microgram protein/mL) with 5 microgram CuS0 4 in modified Tyrode's buffer without EDTA for 20 hours at 37 °C (Steinbrecher el aL II supra). Oxidation of lipoprotein preparations is assessed as tiiiobarbituric acid reacting substances (Lowry et aL supra).
  • Venous blood is drawn from a normolipidemic female subject receiving 500 microgram bd of the antioxidant drug probucol and native LDL isolated from the plasma by density gradient ultracentrifugation as described above.
  • the LDL is chromatographed on a 2.5 x 22 cm column of DEAE Sepharose CL6b (Pharmacia) equilibrated with 0.04% (w/v) NH 4 HCO 3 , pH7.4, containing EDTA (0.1 mg/mL) and GSH (0.1 mg/mL).
  • the LDL is eluted with a linear gradient from 0 to 0.8M NaCl prepared with a GM-1 gradient mixer (Pharmacia) at a flow rate of 12 mL/hr. Each 2 mL fraction is monitored at a wavelength of 280 nm for protein and 254 nm for conjugated dienes formed by lipid peroxidation (Pryor el aL Methods Enzymol, 105; 293-299, 1984).
  • the elution pattern of LDL on the ion exchange column is shown in Figure 1.
  • the elution of LDL determined by optical density at 254 nm, shows a peak at an NaCl concentration of 0.46M, overlapping the main protein peak, with a larger peak at an NaCl concentration of 0.62M overlapping the smaller protein peak.
  • Blood was collected from patients undergoing coronary angiography with or without angiographically-proven coronary artery disease. Blood was collected into lmg/ml EDTA, which was subsequentiy present throughout the isolation of the LDL subtraction.
  • LDL was isolated from the plasma by isopyonic ultracentriguation followed by density gradient ultracentriguation as described below.
  • Plasma density was adjusted by 1.090 with solid KBr, overlaid with density solution of 1.090, and spun at 60000rpm, 4.7 hours at 4 °C in a Beckman 60Ti fixed angle rotor.
  • the clearly visable and distinct LDL band was removed and the following gradient produced in Beckman polyallomer ultracentrifuge tubes: pl.102 (4.5mL), LDL at pl.090 (9.0mL), pl.060 (3.0mL), pl.056 (3.0mL), pl.045 (3.0mL, P1.034 (6mL), pl.024 (6mL), pl.019 (3.0mL) and pl.006 (2.25mL).
  • the gradient was spun in a Beckman SW28 rotor at 28000rpm, at 10 °C for 39 hours.
  • mice 6-8 weeks old are injected with 100 microgram of oxidized LDL in complete Freund's ajuvant (50 microgram subscutaneously and 50 microgram intraperitoneally) 4 weeks apart.
  • a booster injection of 150 microgram of oxidized LDL in phosphate buffered saline (PBS), pH7.0 is given intraperitoneally.
  • PBS phosphate buffered saline
  • a single fusion of spleen cells to the mouse myeloma cell line NSI (Kohler el aL III supra) is performed using polyethylene glycol (Hnatowich el aL J. Nucl. Med. 28:1294-1302, 1987).
  • hybridomas are screened for antibodies directed against oxidized LDL using an EISA as described in Example 3.1. Positive hybridomas are obtained and cloned. Using this method, hybridoma 4C12 was positive and produced 100 positive colonies on recloning. These clones produce two classes of antibody, IgG2b and IgM as determined by isotyping (CSL Misotype Kit). The hybridomas were further cloned to produce a monoclonal IgG2b designated Mab 216. The clones were then expanded and Mab 216 separated by affinity chromatography on protein A-Sepharose (Pharmacia). EXAMPLE 3 3.1 Enzyme-linked Immunosorbent Assay.
  • the hybridomas are screened using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Ninety-six well microtiter plates (Flow Laboratories) are coated with 2mg/ml of oxidatively-modified LDL at 4°C overnight. The plates are then washed four times with PBS containing 0.05% (v/v) Tween 20 and then incubated for 4hr with 200 L of PBS containing 1% (w/v) bovine serum albumin (BSA). The plates are then incubated overnight at 4°C with varying concentrations of the diluted antibody preparation.
  • PBSA bovine serum albumin
  • a 50% (w/v) ammonium suphate cut of the culture supernatant is dialysed against NaHC0 3 ,pH8.5, for 36 hours and the protein concentration adjusted to 1 mg/mL.
  • the solution is gently stirred on ice and the succinamide ester of biotin (sulfosuccinimidyl 6- (biotinamido) hexanoate, NHS-LC-biotin, Pierce Chemicals) in saline added to a final concentration of 20 microgram of NHS-LC-biotin, Pierce Chemicals) in saline added to a final concentration of 20 microgram of NHS-LC-biotin per mg of protein (Kohler el aL III supra).
  • the reaction is allowed to proceed for 2 hrs and the product is separated from free biotin by dialysis against 2 x 2L of PBS for 24 hrs..
  • Microtiter wells are incubated overnight at 4°C with Mab 216 (100 L of culture supernatant), blocked with PBS containing 1% (w/v) BSA for 1 hr at 37 °C and washed with PBS containing 0.05% (v/v) Tween 2.0. .All subsequent washes are done in the same way.
  • Biotinylated Mab 216 100 ⁇ g/mL prepared as described in 3.2 is added to each well (100/mL) and incubated for 1 hr at 37 °C. The plates are washed and 100 L of streptavidin peroxidase conjugate (BRL) (0.24 ⁇ g/mL) is then added to each well and incubated for 60 min.
  • BBL streptavidin peroxidase conjugate
  • the plates are washed and 100 L of ⁇ -phenylenediamine (Sigma Chemicals) dissolved in 25mM sodium citrate/50mM sodium phosphate, pH6, containing 0.4 L/mL H 2 0 2 is added. The reaction is terminated after 30 min with 50 L of 4M sulphuric acid and the absorbence determined at 492 nm (See 3.1).
  • ⁇ -phenylenediamine Sigma Chemicals
  • the ELISA described in 3.1 above is used to examine fractions obtained from the separation of oxidatively-modified LDL isolated from human plasma (See Example 1.3). The fractions (100 ⁇ L) are added to microtiter wells containing adsorbed antibody Mab 216 and the ELISA carried out as described in 3.1. Maximum absorbence at 405 nm corresponds to the well containing the fraction representing oxidatively-modified LDL ( Figure 1).
  • the methodology of the present invention includes detection of epitopes lost or gained due to the oxidative event as determined by a panel of monoclonal antibodies to both native and oxidised LDL.
  • direct detection of oxidised LDL in plasma would be the most straight forward procedure, other procedures are also possible and are encompassed by the present invention.
  • Such other procedures include the detection of straight forward total LDL by ELISA and comparing quantitively differences with specific monoclonal antibodies which recognise epitopes sensitive to oxidative processes. This indirect measurement would be tested using procedures as described above.

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Abstract

La présente invention concerne généralement l'immunodétection de lipoprotéines à faible densité (LDL) modifiées par oxydation, des anticorps contre lesdites lipoprotéines ou un complexe immun desdites protéines, dans un fluide biologique. Plus particulièrement, la présente invention concerne une technique d'essai d'immuno-absorption enzymatique (ELISA) pour l'analyse de LDL modifiées par oxydation, des anticorps contre lesdites LDL ou un complexe immun desdites LDL dans du plasma de mammifère, et plus particulièrement dans du plasma humain. La présente invention concerne également une trousse destinée à faciliter la technique ELISA. Ladite invention est particulièrement utile dans l'évaluation du risque de coronaropathie.
PCT/AU1994/000171 1993-04-07 1994-04-07 Dosage immunologique de lipoproteines humaines a faible densite modifiees par oxydation presentes dans le plasma Ceased WO1994023302A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64202/94A AU6420294A (en) 1993-04-07 1994-04-07 Immunological assay of oxidatively modified human low density lipoproteins in plasma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPL8188 1993-04-07
AUPL818893 1993-04-07

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WO1994023302A1 true WO1994023302A1 (fr) 1994-10-13

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WO1998059248A1 (fr) * 1997-06-20 1998-12-30 Leuven Research & Development Vzw Dosages, anticorps et etalons de detection de lipoproteines de basse densite, oxydees et modifiees par mda
WO2000014548A1 (fr) * 1998-09-04 2000-03-16 Leuven Research & Development Vzw Detection et determination des stades d'une maladie des arteres coronaires
EP0904538A4 (fr) * 1995-12-05 2001-04-11 Entremed Inc Methode de diagnostic et de traitement de l'atherosclerose a l'aide d'anticoprs anti-cholesterol
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
US6716410B1 (en) * 1999-10-26 2004-04-06 The Regents Of The University Of California Reagents and methods for diagnosing, imaging and treating atherosclerotic disease
US6727102B1 (en) 1997-06-20 2004-04-27 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
EP1272666A4 (fr) * 2000-03-31 2004-09-22 Univ California Dosage fonctionnel de lipoprot ine haute densit
US6869568B2 (en) 2000-03-31 2005-03-22 The Regents Of The University Of California Functional assay of high-density lipoprotein
US7250304B2 (en) 2000-03-31 2007-07-31 The Regents Of The University Of California Functional assay of high-density lipoprotein
JP2019174432A (ja) * 2018-03-29 2019-10-10 株式会社明日香特殊検査研究所 粥状動脈硬化による心筋梗塞や脳梗塞のリスクが高いと判定する易酸化性ＶＬＤＬ（ＶＬＤＬ ｓｕｓｃｅｐｔｉｂｉｌｉｔｙ ｔｏ ｏｘｉｄａｔｉｏｎ）および易酸化性ＬＤＬ（ＬＤＬ ｓｕｓｃｅｐｔｉｂｉｌｉｔｙ ｔｏ ｏｘｉｄａｔｉｏｎ）の簡便な測定方法および測定装置
CN117192134A (zh) * 2023-09-14 2023-12-08 宁波美康盛德生物科技有限公司 一种氧化型低密度脂蛋白的检测试剂盒、检测方法

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Cited By (24)

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Publication number Priority date Publication date Assignee Title
EP0904538A4 (fr) * 1995-12-05 2001-04-11 Entremed Inc Methode de diagnostic et de traitement de l'atherosclerose a l'aide d'anticoprs anti-cholesterol
WO1998059248A1 (fr) * 1997-06-20 1998-12-30 Leuven Research & Development Vzw Dosages, anticorps et etalons de detection de lipoproteines de basse densite, oxydees et modifiees par mda
US7579159B2 (en) * 1997-06-20 2009-08-25 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US7390627B2 (en) 1997-06-20 2008-06-24 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US7378250B2 (en) 1997-06-20 2008-05-27 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US7229776B2 (en) 1997-06-20 2007-06-12 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US6727102B1 (en) 1997-06-20 2004-04-27 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US7229775B2 (en) 1997-06-20 2007-06-12 Leuven Research & Development Vzw Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
US7166469B2 (en) 1998-09-04 2007-01-23 Leuven Research & Development Vzw Detection and determination of the stages of coronary artery disease
EP1503213A3 (fr) * 1998-09-04 2005-07-13 Leuven Research & Development V.Z.W. Detection et determination des stades d'une maladie des arteres coronaires
US7604952B2 (en) 1998-09-04 2009-10-20 Leuven Research & Development Vzw Detection and determination of the stages of coronary artery disease
WO2000014548A1 (fr) * 1998-09-04 2000-03-16 Leuven Research & Development Vzw Detection et determination des stades d'une maladie des arteres coronaires
US6309888B1 (en) 1998-09-04 2001-10-30 Leuven Research & Development Vzw Detection and determination of the stages of coronary artery disease
US6762062B2 (en) 1999-01-27 2004-07-13 Matsushita Electric Industrial Co., Ltd. Method of determining cholesterol and sensor applicable to the same
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
US7556927B2 (en) 1999-10-26 2009-07-07 The Regents Of The University Of California Methods for diagnosing, imaging and treating atherosclerotic disease
US6716410B1 (en) * 1999-10-26 2004-04-06 The Regents Of The University Of California Reagents and methods for diagnosing, imaging and treating atherosclerotic disease
US7575873B2 (en) 1999-10-26 2009-08-18 The Regents Of The University Of California Reagents for diagnosing, imaging and treating atherosclerotic disease
EP1272666A4 (fr) * 2000-03-31 2004-09-22 Univ California Dosage fonctionnel de lipoprot ine haute densit
US7250304B2 (en) 2000-03-31 2007-07-31 The Regents Of The University Of California Functional assay of high-density lipoprotein
US6869568B2 (en) 2000-03-31 2005-03-22 The Regents Of The University Of California Functional assay of high-density lipoprotein
EP1650312A3 (fr) * 2000-03-31 2006-05-17 The Regents of the University of California Dosage fonctionnel de lipoprotéine haute densité
JP2019174432A (ja) * 2018-03-29 2019-10-10 株式会社明日香特殊検査研究所 粥状動脈硬化による心筋梗塞や脳梗塞のリスクが高いと判定する易酸化性ＶＬＤＬ（ＶＬＤＬ ｓｕｓｃｅｐｔｉｂｉｌｉｔｙ ｔｏ ｏｘｉｄａｔｉｏｎ）および易酸化性ＬＤＬ（ＬＤＬ ｓｕｓｃｅｐｔｉｂｉｌｉｔｙ ｔｏ ｏｘｉｄａｔｉｏｎ）の簡便な測定方法および測定装置
CN117192134A (zh) * 2023-09-14 2023-12-08 宁波美康盛德生物科技有限公司 一种氧化型低密度脂蛋白的检测试剂盒、检测方法

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