[go: up one dir, main page]

WO1996037779A1 - A solid-phase method for assaying a hydrophobic-target binding substance - Google Patents

A solid-phase method for assaying a hydrophobic-target binding substance Download PDF

Info

Publication number
WO1996037779A1
WO1996037779A1 PCT/US1996/004589 US9604589W WO9637779A1 WO 1996037779 A1 WO1996037779 A1 WO 1996037779A1 US 9604589 W US9604589 W US 9604589W WO 9637779 A1 WO9637779 A1 WO 9637779A1
Authority
WO
WIPO (PCT)
Prior art keywords
hydrophobic
cholesterol
sterol
support material
phase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1996/004589
Other languages
French (fr)
Inventor
Shawn J. Green
Jacinta U. Aniagolu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Casi Pharmaceuticals Inc
Original Assignee
Entremed Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Entremed Inc filed Critical Entremed Inc
Priority to AU57118/96A priority Critical patent/AU5711896A/en
Publication of WO1996037779A1 publication Critical patent/WO1996037779A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Definitions

  • the present invention relates to a solid-phase method for assaying a hydrophobic-target binding substance, particularly to a solid-phase method for assaying a sterol binding substance.
  • a hydrophobic-target binding substance is a substance which binds to a hydrophobic-target substance (HTS).
  • HTS is a substance which tends to repel water and to leave the water phase.
  • HTSs include, but are not limited to, lipids such as steroids, steroids such as sterols, and sterols such as cholesterol and ergosterol.
  • Lipids are HTSs which are extracted from cells by nonpolar solvents. Lipids represent a heterogeneous collection of materials including, but not limited to, fatty acids, glycerides and glyceryl ethers, phospholipids, sphingolipids, alcohols and waxes, terpenes, steroids, glycolipids and vitamins.
  • Steroids are HTSs which are cyclic compounds, the basic nucleus of which consists of three 6-membered rings and one five membered ring, fused together to yield perhydrocyclopentanophenanthrene. Steroids represent a wide variety of compounds including, but not limited to, hormones, vitamins, cell membrane constituents, sterols and drugs. Sterols are HTSs which are steroids of 27 or more carbon atoms with an alcoholic hydroxyl group attached to position 3.
  • Sterols include, but are not limited to, cholesterol, ergosterol, cholecalciferol, cortisol, ergocalciferol, 5- dehydroepisterol, estriol, estradiol, lanosterol, and their analogs and derivatives.
  • ergosterol and 5-dehydroepisterol are major sterols found in many fungi, but not found in mammals.
  • ergosterol is antigenic (Berger et al. 1932 Z. Immunitat. 6: 16).
  • ergosterol binding substances could be useful as a vaccine against ergosterol synthesizing pathogenic organisms that infect mammals.
  • Methods used to measure sterol binding substances include the complement- fixation assay (Hahn et al. 1936 Z. Immunitat. 88: 16), the flocculation assay (Weil et al. 1937 Proc Soc. Exp. Biol. Med. 36:238), the complement- dependent liposome glucose release assay (Alving et al. 1977 /. Immunol. 1 18:342) and the conventional plastic microtiter plate enzyme-hnked immunosorbent assay (ELISA) (Alving et al. 1991 Crit. Rev. Immunol. 10:5).
  • the conventional ELISA involves contacting an inert solid-phase material, usually a plastic microtiter plate, with a solution containing a target substance which binds to the solid-phase material. This bound target substance is then contacted with an aqueous solution containing a target binding substance which reacts with the target substance. Unbound target binding substance is removed and the amount of reacted target binding substance is quantitated using any of a number of detection methods known to those skilled in the art. Hydrophobic substances, however, because of their low water solubility, absorb poorly to the plastic microtiter plates used in ELISAs.
  • Attempts to assay steroid binding activities in plastic microtiter plate ELISAs include covalently coupling the steroid to the plastic microtiter plate via a carrier molecule such as albumin (Tijssen et al. in Laboratory Techniques in Biochemistry and Molecular Biology. 15. R. Burdon and P.
  • Watanabe et al. (Infect. Immun. 1991 59:2200) used a plastic microtiter plate ELISA to measure antibodies to cholesterol. Using only a 1 :25 serum dilution and a cholesterol concentration range of 5.6 ⁇ g/ml to 90 ⁇ g/ml, they found no significant increase in cholesterol antibody titers. Because it is insensitive and because it requires a minimum of 22 hours to complete, this ELISA method is neither sensitive enough nor practical enough for rapid assessment of limited volume samples containing low concentrations of a cholesterol binding substance.
  • the present invention provides a simple, rapid, sensitive and reproducible solid-phase method for detecting a hydrophobic-target binding substance (HTBS) in a sample.
  • a hydrophobic-target substance HTS
  • the organic solvent, containing the dissolved HTS is applied to an inert solid-phase hydrophobic support material compatible with the organic solvent.
  • the organic solvent is removed such that the HTS is immobilized on and within the inert solid-phase hydrophobic support material.
  • the inert solid-phase hydrophobic support material is washed with an aqueous solution and is contacted with an aqueous sample containing a HTBS such that any HTBS in the aqueous sample binds to the HTg immobilized on and within the inert solid-phase hydrophobic support material. Binding of the HTBS to the HTS is detected. The amount of the HTBS bound to the HTS is measured and the amount of hydrophobic-binding substance in the sample is calculated.
  • the object of the present invention is to provide a solid-phase method for detecting a HTBS in an aqueous sample.
  • FIG. 1 Immunodetection of cholesterol binding substance in a PVDF membrane ELISA. Binding of pre- immune serum and of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol immobiUzed on and within PVDF membrane in a DOT-BLOT apparatus holder.
  • FIG. 2 Immunodetection of cholesterol binding substance in a nitrocellulose microtiter plate ELISA. Binding of pre-immune serum and of immune serum, at dilutions of approximately 1 :800 to 1 : 100,000, to cholesterol immobiUzed on and within nitrocellulose wells of a microtiter plate.
  • FIG 3. Immunodetection of cholesterol binding substance in a PVDF membrane ELISA. Binding of pre- immune mouse ascites fluid and of immune mouse ascites fluid, at dilutions of approximately 1 :800 to 1 :6,400, to cholesterol immobilized on and within PVDF membrane in a DOT-BLOT apparatus holder.
  • FIG. 4. Immunodetection of cholesterol binding substance in a PVDF microtiter plate ELISA. Binding of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol, at concentration of approximately 0 ⁇ g/well to 10 ⁇ g/well, immobiUzed on and within PVDF wells of a microtiter plate.
  • FIG. 5 Immunodetection of cholesterol binding substance in a plastic microtiter plate ELISA and in a PVDF microtiter plate ELISA using varying concentrations of cholesterol. Data presented is the mean ⁇ S.D. of triplicate samples.
  • FIG. 5A Binding of phosphate buffered saline (PBS) and of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol, at concentrations of approximately 0.01 ⁇ g/well to 25 ⁇ g/well, immobiUzed on polystyrene microtiter plate.
  • FIG. 5B Binding of PBS and of immune serum, at dilutions of approximately 1 :800 to
  • FIG. 5C Binding of pre-immune and of immune serum, at concentrations of approximately 1 :800 to 1 : 100,000, to cholesterol, at 5 ⁇ g/well, immobilized on and within PVDF wells of a microtiter plate and on polystyrene wells of a microtiter plate.
  • FIG. 6 Immunodetection of ergosterol binding substance in a PVDF membrane ELISA. Binding of preimmune serum and of immune serum, at 1 :800 to 1 :3,200, to ergosterol immobiUzed on and within PVDF wells of a microtiter plate.
  • FIG. 7 Detection of cholesterol transferred from a thin layer chromatography (TLC) plate to PVDF membrane.
  • FIG 7A Immunodetection of cholesterol transferred from a
  • FIG. 7B Detection of cholesterol transferred from a TLC plate to PVDF membrane using iodine vapor.
  • FIG. 8 Detection of cholesterol in very low density lipoprotein (VLDL) extracts transferred from a TLC plate to PVDF membrane using isopropanol. CE-cholesterol esters; TG-triglycerides; C-cholesterol; PL-phospholipids.
  • FIG. 8A Immunodetection of C in a VLDL extract transferred from a TLC plate to a PVDF membrane with 70% isopropanol (1 ) and with 100% isopropanol (2).
  • FIG. 8B Immunodetection of C in a VLDL extract transferred from a TLC plate to a PVDF membrane with 70% isopropanol (1 ) and with 100% isopropanol (2).
  • HTS hydrophobic target substance
  • HTBS hydrophobic-target binding substance
  • HTBSs include, but are not limited to, lipid binding substances such as steroid binding substances, steroid binding substances such as sterol binding substances, and sterol binding substances such as cholesterol binding substances and ergosterol binding substances.
  • the phrase "immobiUzed on and within” refers to insertion of a HTS on, between and among existing elements of an inert soUd-phase hydrophobic support material.
  • the present invention comprises a method for detecting a HTBS in an aqueous sample.
  • a HTS is dissolved in an organic solvent.
  • the organic solvent, containing the dissolved HTS is apphed to an inert soUd-phase hydrophobic support material compatible with the organic solvent.
  • the organic solvent is removed such that the HTS is immobilized on and within the inert solid-phase hydrophobic support material.
  • the inert solid-phase hydrophobic support material is washed with an aqueous solution and is contacted with an aqueous sample containing a HTBS such that any HTBS in the aqueous sample binds to the HTBS immobilized on and within the inert solid-phase hydrophobic support material. Binding of the HTBS to the HTS is detected.
  • the amount of the HTBS bound to the HTS is measured and the amount of hydrophobic-binding substance in the aqueous sample is calculated.
  • the present invention further comprises a method for detecting binding of a HTBS to a HTS in a mixture of hydrophobic substances.
  • the mixture of hydrophobic substances which includes the HTS, is separated into its individual hydrophobic substances.
  • the separated hydrophobic substances are transferred to an inert solid-phase hydrophobic support material in the presence of an organic solvent.
  • the organic solvent is removed such that the separated hydrophobic substances are immobiUzed on and within the inert solid-phase hydrophobic support material.
  • the present invention further comprises an assay kit for detecting a HTBS in an aqueous sample comprising an inert solid-phase hydrophobic support material having a HTS immobilized thereon and therein and further comprising a holder for the inert soUd-phase hydrophobic support material.
  • the hydrophobic-target substance (HTS) for use in the present invention can be any hydrophobic substance which is soluble in an organic solvent.
  • HTSs include, but are not Umited to, lipids, steroids, sterols and vitamins.
  • Preferred HTSs are sterols. More preferred HTSs are cholesterol, cholesterol derivatives and cholesterol analogs and ergosterol, ergosterol derivatives and ergosterol analogs.
  • the HTS can be obtained by methods well known to those skilled in the art.
  • the concentration of HTS for use in the present invention can range between about 0.0001 ⁇ g/ ⁇ l and about 50 ⁇ g/ ⁇ l.
  • the preferred range is between about 0.001 ⁇ g/ ⁇ l and about 10 ⁇ g/ ⁇ l.
  • the most preferred range is between about 0.01 ⁇ g/ ⁇ l and about 5 ⁇ g/ ⁇ l.
  • the HTS dissolved in organic solvent can be applied to the inert solid-phase hydrophobic support material by dipping, soaking, spotting, spraying, blotting or other convenient means.
  • Preferred methods include spotting, spraying and blotting. More preferred methods include spotting and blotting.
  • a preferred volume for spotting is between about 5 ⁇ l/well and 10 ⁇ l/well.
  • Determination of the amount of HTS to be used for each method of application is well within the knowledge of one skilled in the art.
  • a standard HTS-HTBS assay combination can be used to determine the amount of HTS to be appUed to the inert soUd-phase hydrophobic support material.
  • the organic solvent for use in the present invention can be any solvent which can solubiUze the HTS, and which is sufficiently miscible with water to be completely removed by subsequent thorough rinsing with an aqueous solution.
  • solvents include lower (C1 -C4) aliphatic alcohols including, but not limited to, methanol, ethanol, n- propanol, isopropanol, n-butanol, isobutanol, secondary-butyl alcohol, and tertiary-butyl alcohol; halogenated hydrocarbons including, but not Umited to, chloroform, ethylene chloride, carbon tetrachloride, trichloroethane methylene chloride, monochlorobenzene and perchloroethylene; lower aliphatic ketones including, but not Umited to, acetone, methyl ethyl ketone; dioxan, dimethyl formamide; dimethyl sulfoxide; acetonitrile; lower (
  • the inert solid-phase hydrophobic support material for use in the present invention includes materials having high hydrophobic properties, materials having low hydrophilic properties, and composites of materials having both high hydrophobic and low hydrophilic properties.
  • Materials having high hydrophobic properties include, but are not limited to, polyethylenes, polypropylenes, polyacetals, epoxy resins, polycarbonates, fluorine-contained resins, polyvinyls, polyvinylidines, polyvinylidene chlorides and polyvinyUdine fluorides.
  • Materials having low hydrophihc properties include, but are not limited to, nitrocellulose, nylon and cellulose.
  • Preferred materials are polyvinyUdine fluorides and nitrocellulose. More preferred materials are polyvinyUdine difluoride and nitrocellulose.
  • the inert solid-phase hydrophobic support material for use in the present invention can be in the form a membrane, a bead, or any other solid-phase hydrophobic support form known to those skilled in the art.
  • a preferred form is a membrane.
  • the inert solid-phase hydrophobic support material can be placed into a holder, including but not Umited to, a membrane sheet holder, a dot- blot apparatus, a microtiter plate, a column, and a filter.
  • Preferred holders include a membrane sheet holder, a dot-blot apparatus and a microtiter plate.
  • the blocking buffers for use in the present invention to prevent non-specific binding can be any suitable blocking buffer including, but not limited to, fetal calf serum, gelatin, low fat milk, and Tween-20 at various dilutions in an aqueous solution.
  • the washing solution for use in the present invention can be any suitable aqueous buffer including, but not limited to, phosphate buffered saline (PBS), tris(hydroxymethyl)amino methane (TRIS) and N-2- hydroxyethylpeperazine-N'-2-ethanesulfonic acid (HEPES).
  • PBS phosphate buffered saline
  • TMS tris(hydroxymethyl)amino methane
  • HEPES N-2- hydroxyethylpeperazine-N'-2-ethanesulfonic acid
  • the hydrophobic-target binding substance (HTBS) for use in the present invention is a substance which binds specifically to a HTS.
  • HTBSs include, but are not limited to, antibodies, receptors, antibiotics, and dyes.
  • Antibodies may be either monoclonal or polyclonal.
  • Preferred hydrophobic-target-binding substances are antibodies. More preferred hydrophobic-target-binding substances are antibodies to sterols. Most preferred HTBSs are antibodies to cholesterol and antibodies to ergosterol.
  • Any convenient indicator method can be used to detect binding of a HTBS to a HTS. Such methods include, but are not limited to, the use of enzymes, enzyme cofactors, enzyme effectors, chromogenic substances, fluorogenic substances, chemiluminescent substances, bioluminescent substances, and labeled antibodies.
  • Preferred indicator methods are the peroxidase-labeled antibody method and the alkaUne phosphatase-labeled antibody method.
  • NC nitrocellulose membrane
  • PVDF PolyvinyUdene difluoride membrane
  • PVDF membranes unexpectedly retain cholesterol at the lowest concentration appUed (10 ng/5 ⁇ l), the lower detection limit of iodine vapors. NC membranes also unexpectedly retain cholesterol, although less efficiently (>100 ng/5 ⁇ l), than PVDF membranes.
  • Lipids are aliquoted into pyrogen-free rotary evaporator flasks and are rotary evaporated to remove solvents. Lipid ratios are: dimyristoyl phosphatidylcholine (DMPC): dimyristoyl phosphatidyl-glycerol (DMPG): cholesterol, 9: 1 :7.5 (mol:mol:mol) (Avanti Polar Lipids,
  • Pre-immune serum is collected from 6 to 8 week old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME) by tail bleeds.
  • 100 ⁇ l of Uposomes containing about 71 mol % cholesterol, prepared as in Example 2 are injected intraperitoneally into BALB/c mice. After 2 weeks, the mice are boosted once with another 100 ⁇ l of cholesterol Uposomes and are bled 2 weeks later to collect immune serum containing cholesterol binding substance.
  • HYDROPHOBIC MEMBRANE ELISA A HTS, dissolved in an organic solvent is applied to an inert solid-phase hydrophobic support material.
  • the support material is dried at room temperature and in the dark to prevent spontaneous oxidation.
  • the inert solid- phase hydrophobic support material is rehydrated in blocking buffer.
  • HTS cholesterol
  • hydrophobic support material is rehydrated in blocking buffer, 4% or 10% heat inactivated (56°C for 30 min) fetal calf serum (FCS) deficient in cholesterol binding substance and negUgible ( ⁇ 1 ng) in cholesterol content (Biofluids, Inc. Rockville, MD) in phosphate buffered saUne, pH 7.2-pH 7.4 (PBS), for 1 hour at room temperature.
  • FCS fetal calf serum
  • Serum or ascites fluid samples are diluted in blocking buffer, and are contacted with the hydrophobic support material. After 1 hr incubation with slow agitation at room temperature, the hydrophobic support material is washed 3-4-times with blocking buffer. 3.
  • HTBS e.g. anticholesterol antibody
  • a 1/1500 dilution of peroxidase conjugated goat anti-mouse IgG (H+L), IgM or IgGl, IgG2a in blocking buffer is appUed to the hydrophobic support material.
  • the hydrophobic support material is washed 3-4- times with PBS, and are incubated with a soluble peroxidase substrate, 2,2'- azino-di[3-ethyl-bezthiazoline sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 1-45 min until adequate color is developed.
  • ABTS peroxidase substrate system 2,2'- azino-di[3-ethyl-bezthiazoline sulfonate
  • PVDF membrane (8 X 10 cm) is sprayed with 2-3 ml of an ethanol.xhloroform (1:1) solution containing 5 mg/ml of purified cholesterol. The membrane is dried at room temperature and in the dark to prevent spontaneous oxidation. For use, the membrane is rehydrated in blocking buffer and placed into a 96 well DOT-BLOT apparatus holder (BioRad Laboratories, Hercules, CA).
  • the cholesterol containing PVDF membrane in the DOT-BLOT apparatus holder is rehydrated in blocking buffer (4% FCS) for 1 hour at room temperature.
  • IgG2a in blocking buffer is added to each well. After 1 hour of incubation with slow agitation, the wells are washed 3-4- times with PBS, and are incubated with 100 ⁇ l of a soluble peroxidase substrate, 2,2'-azino-di[3- ethyl-bezthiazohne sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 15-20 min until adequate color is developed. Ninety ⁇ l of the reactant is then transferred to a round bottom 96- well clear plastic microtiter plate well and absorption is measured for each sample at 405 nm using a Microtiter
  • pre-immune serum shows no cholesterol binding activity and immune serum, at dilutions between 1 :800 and 1 : 12,800, shows dilution dependent cholesterol binding activity in this PVDF membrane ELISA.
  • Duplicate wells show comparable color intensity demonstrating that immune serum reproducibly and uniformly recognizes cholesterol bound to PVDF and indicating that PVDF immobilizes cholesterol in an orientation which allows for cholesterol binding substance recognition of the sterol.
  • nitrocellulose well microtiter plate BA85 nitrocellulose well microtiter plate.
  • the plates are stored in the dark at 4° C for two weeks.
  • the wells are rehydrated with blocking buffer and the standard assay protocol of Example 4 is followed using 100 ⁇ l aUquots of pre-immune serum and 100 ⁇ l aUquots of immune serum diluted from 1 :1,000 to 1:100,000.
  • 100 ⁇ l of a 1/1500 dilution of peroxidase conjugated goat anti-mouse IgG (H+L), IgM or IgGi, IgG2a i blocking buffer is added to each well.
  • the wells are washed 3- 4 times with PBS, and are incubated with 100 ⁇ l of a soluble peroxidase substrate, 2,2'-azino-di[3-ethyl-bezthiazoline sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 10-20 min until adequate color is developed.
  • ABTS peroxidase substrate system 2,2'-azino-di[3-ethyl-bezthiazoline sulfonate
  • FIG. 2 shows the unexpected result that cholesterol binding substance is measurable in immune serum at a dilution of approximately 1 :75,000, whereas cholesterol binding substance is not detectable in pre-immune serum in this nitrocellulose well microtiter plate ELISA.
  • FIG. 3 shows the unexpected result that cholesterol binding substance is detectable at dilutions of 1 :6,400 in immune ascites fluid, whereas cholesterol binding substance is not detectable in pre-immune ascites fluid in this
  • microtiter plate The plate is stored in the dark overnight at room temperature. For use, the plate is rehydrated in blocking buffer and the standard assay protocol of Example 4 is followed using 100 ⁇ l of immune serum diluted 1 :800, 1 : 1 ,600, 1 :3,200 and 1 :6,400 and
  • FIG. 4 shows cholesterol binding substance is detectable at 1 ⁇ g cholesterol and that approximately 5 ⁇ g cholesterol is optimum for detecting cholesterol binding substance in this PVDF membrane microtiter plate ELISA.
  • FIG. 5A shows the polystyrene microtiter plate
  • FIG. 5B shows that the PVDF microtiter plate ELISA of the present invention enables a dose response effect and shows consistent binding with increasing concentrations of cholesterol.
  • FIG. 5C shows that, using 5 ⁇ g/well of cholesterol, the dose response effect of the PVDF microtiter plate ELISA of the present invention is 4-6 fold more sensitive than that of the conventional polystyrene microtiter plate ELISA.
  • PVDF membrane microtiter plate ELISA of the present invention provides more reproducible results and unexpectedly enables detection and quantitation of significantly lower amounts of cholesterol binding substance than the conventional plastic microtiter plate ELISA.
  • dose response effect of the PVDF membrane microtiter plate ELISA of the present invention is 4-6 fold more sensitive than the conventional plastic microtiter plate ELISA.
  • FIG. 6 shows the unexpected result that ergosterol binding substance is measurable in immune serum at dilutions >1 :3,200 and that there is at least a two-fold difference in absorbance at 405 nm for immune serum relative to pre- immune serum.
  • Cholesterol, transferred to PVDF paper, is detected using the standard assay protocol of Example 4 with 1 :1,000 dilutions of pre-immune and of immune mouse serum. The color reaction is stopped by rinsing the membrane with distilled water.
  • FIG. 7 A shows that after 15 minutes of blotting, 200 ng of cholesterol applied to the TLC plate are detectable on PVDF membrane with immune serum and that after 15 minutes of blotting 500 ng of cholesterol apphed to the TLC plate are not detectable on PVDF membrane with preimmune serum.
  • FIG. 7B shows the TLC plate after 15 minutes of blotting and after exposure to iodine vapor. Blotting for 15 minutes results in almost complete transfer of 200 ng of cholesterol. Blotting for 3 to 5 minutes results in partial transfer of 200 ng of cholesterol (data not shown). Blotting for > 30 minutes results in excessive diffusion of the 200 ng of transferred cholesterol (data not shown). This demonstrates that cholesterol transfer depends on blotting time.
  • VLDL very low density lipoproteins
  • the extracted VLDL are separated on the TLC plate in a 80 40 2 hexane:ether:glacial acetic acid (v:v:v:) solvent and the TLC plates are air dried.
  • the air-dried TLC plates are overlaid with a PVDF membrane, pre-wetted by spraying with isopropanol to facilitate passive transfer of lipids from the silica gel. Due to the difference in hydrophobicity of the various lipids, mixtures of isopropanol/water (v/v) of 50%, 70% or 100% (v/v) isopropanol are evaluated.
  • the PVDF membrane is covered with a sheet of cellophane and with a flat weight (200 g) for 15 minutes to 30 minutes. After the transfer the PVDF membrane is separated from the siUca plate and air dried.
  • Cholesterol transfer to PVDF is detected using the standard assay protocol of Example 4 with 1 : 1 ,000 dilutions of pre-immune and of immune mouse serum. The color reaction is stopped by rinsing the membrane with distilled water. Cholesterol transfer to PVDF is further confirmed using filipin complex (Smejkel et al. 1994 Biotech. 16:68). Lipid transfer is confirmed by exposing the PVDF membrane to iodine vapor.
  • FIG. 8A1 shows, using cholesterol binding substance in immune serum to visuaUze cholesterol, that cholesterol transfers from the TLC plate to the PVDF membrane in the presence of 70% isopropanol in 15 minutes.
  • FIG. 8A2 shows, using iodine vapor to visualize lipids, that cholesterol and phosphoUpids, but not triglycerides and cholesterol esters, transfer from the TLC plate to PVDF membrane in the presence of 70% isopropanol.
  • FIG. 8B 1 shows, using cholesterol binding substance in immune serum to visualize cholesterol, that cholesterol transfers from the TLC plate to the PVDF membrane in the presence of 100% isopropanol.
  • FIG. 8B2 shows, using iodine vapor to visualize lipids, that cholesterol triglycerides and cholesterol esters, but not phosphoUpids, transfer from the TLC plate to PVDF membrane in the presence of 100% isopropanol.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

An improved solid-phase method for assaying a HTBS comprising immobilizing a hydrophobic-target substance dissolved in an organic solvent on an inert solid-phase hydrophobic support material compatible with the organic solvent, contacting the hydrophobic-target substance with an aqueous sample containing a HTBS, detecting binding of the HTBS to the hydrophobic-target substance and determining the amount of the HTBS in the sample.

Description

A SOLID-PHASE METHOD FOR ASSAYING A HYDROPHOBIC-TARGET BINDING SUBSTANCE
Technical Field
The present invention relates to a solid-phase method for assaying a hydrophobic-target binding substance, particularly to a solid-phase method for assaying a sterol binding substance.
Background of the Invention
A hydrophobic-target binding substance (HTBS) is a substance which binds to a hydrophobic-target substance (HTS). A HTS is a substance which tends to repel water and to leave the water phase. HTSs include, but are not limited to, lipids such as steroids, steroids such as sterols, and sterols such as cholesterol and ergosterol.
Lipids are HTSs which are extracted from cells by nonpolar solvents. Lipids represent a heterogeneous collection of materials including, but not limited to, fatty acids, glycerides and glyceryl ethers, phospholipids, sphingolipids, alcohols and waxes, terpenes, steroids, glycolipids and vitamins.
Steroids are HTSs which are cyclic compounds, the basic nucleus of which consists of three 6-membered rings and one five membered ring, fused together to yield perhydrocyclopentanophenanthrene. Steroids represent a wide variety of compounds including, but not limited to, hormones, vitamins, cell membrane constituents, sterols and drugs. Sterols are HTSs which are steroids of 27 or more carbon atoms with an alcoholic hydroxyl group attached to position 3. Sterols include, but are not limited to, cholesterol, ergosterol, cholecalciferol, cortisol, ergocalciferol, 5- dehydroepisterol, estriol, estradiol, lanosterol, and their analogs and derivatives.
Cholesterol is a HTS which is a major sterol in animal tissues and is a precursor of bile acids and of steroid hormones. Although viewed as an unconventional immunogen, cholesterol is antigenic (Sato et al. 1976 Biomed. 24:385). Both polyclonal and monoclonal antibodies to cholesterol have been induced in mammals (Alving et al. 1991 Crit. Rev. Immun. 10:441) and naturally-occurring antibodies to cholesterol have been reported in animals and humans (Swartz et al. 1988 Proc Natl. Acad. Sci. 85:1902). However, studies of humoral responses to cholesterol and to other sterols have been hindered by the lack of a simple, sensitive and reproducible assay for sterol binding substances.
Such studies could provide information of significant clinical importance. For example, high levels of serum cholesterol are associated with vascular diseases, such as atherosclerosis. Current strategies for reducing atherosclerotic risk include reduction in dietary cholesterol, interference with cholesterol metabolism and interference with intestinal absorption of cholesterol. Preliminary experiments demonstrate that immunization with cholesterol-ester conjugated to serum albumin reduces the development of dietary-induced atherosclerosis in rabbits (Bailey et al. 1967 Nature 201 :407) and that induction of antibodies to low- density lipoproteins (LDL), the principal carriers of cholesterol in serum, also reduces the development of dietary- induced atherosclerosis in rabbits (Gero et al. 1961 Lancet 1 : 1 1 19). However, to exploit the use of anti-cholesterol antibodies in reducing atherosclerotic risk it is necessary to have a simple, sensitive and reproducible assay to detect and quantify cholesterol binding substances in serum. Such an assay has not been available.
Further, many pathogenic organisms that infect mammals synthesize sterols that are not synthesized by mammals. For example, ergosterol and 5-dehydroepisterol are major sterols found in many fungi, but not found in mammals. Although viewed as an unconventional immunogen, ergosterol is antigenic (Berger et al. 1932 Z. Immunitat. 6: 16). As ergosterol is not found in mammals and as ergosterol is antigenic, ergosterol binding substances could be useful as a vaccine against ergosterol synthesizing pathogenic organisms that infect mammals. To examine the feasibility of inducing a protective immune response to pathogen-specific sterols, it is necessary to have a simple, sensitive and reproducible assay for ergosterol binding substances. Such an assay has not been available.
The lack of a simple, sensitive and reproducible assay for sterol binding substances which are water soluble is due to the hydrophobicity of sterols which are relatively water insoluble. Because of the water insolubility of sterols, water soluble sterol binding substances cannot be assayed easily or accurately by conventional means.
Methods used to measure sterol binding substances include the complement- fixation assay (Hahn et al. 1936 Z. Immunitat. 88: 16), the flocculation assay (Weil et al. 1937 Proc Soc. Exp. Biol. Med. 36:238), the complement- dependent liposome glucose release assay (Alving et al. 1977 /. Immunol. 1 18:342) and the conventional plastic microtiter plate enzyme-hnked immunosorbent assay (ELISA) (Alving et al. 1991 Crit. Rev. Immunol. 10:5). Because of the inherent difficulties in measuring the binding of a water soluble HTBS to a relatively water insoluble HTS, these assays have, thus far, proven impractical or unreliable. The complement fixation assay is riddled with technical problems, including the anti-complement effects of serum which make conclusive immunological judgments difficult. The flocculation assay requires subjective analysis of experimental results. The complement-dependent, liposome glucose release assay is tedious, insensitive and expensive. Moreover, as glucose can be induced non-specifically, only a subpopulation of antigen reactive antibodies can be measured and only a small number of samples can be assayed at one time.
The conventional ELISA involves contacting an inert solid-phase material, usually a plastic microtiter plate, with a solution containing a target substance which binds to the solid-phase material. This bound target substance is then contacted with an aqueous solution containing a target binding substance which reacts with the target substance. Unbound target binding substance is removed and the amount of reacted target binding substance is quantitated using any of a number of detection methods known to those skilled in the art. Hydrophobic substances, however, because of their low water solubility, absorb poorly to the plastic microtiter plates used in ELISAs.
Attempts to assay steroid binding activities in plastic microtiter plate ELISAs include covalently coupling the steroid to the plastic microtiter plate via a carrier molecule such as albumin (Tijssen et al. in Laboratory Techniques in Biochemistry and Molecular Biology. 15. R. Burdon and P.
Van Knippenberg eds, Elsevier, Amsterdam, 1985) and modifying the steroid molecule covalently to bind it to the plastic microtiter plate (Sondergard-Andersen et al 1990 J. Immunol. Meth. 131 :99). These methods are tedious, time consuming and expensive. Moreover, they do not provide reproducible results.
Attempts to assay sterol binding substances in plastic microtiter plate ELISAs using unmodified sterols as HTSs results in high noise-to-signal ratios, low sensitivity, unacceptable background levels and wide variations in observed values. These problems result mainly from the non- uniform binding of the hydrophobic sterol to the plastic wells of the microtiter plates. That is, when the organic solvent in which the sterol is dissolved evaporates, the sterol precipitates out of solution resulting in nonuniform distribution of the sterol, clustering of sterol crystals, and nonuniform removal of the sterol from the surface of the plastic wells during washings of the microtiter plate. This makes sterol plastic microtiter plate ELISAs insensitive and unreliable.
For example, Watanabe et al. (Infect. Immun. 1991 59:2200) used a plastic microtiter plate ELISA to measure antibodies to cholesterol. Using only a 1 :25 serum dilution and a cholesterol concentration range of 5.6 μg/ml to 90 μg/ml, they found no significant increase in cholesterol antibody titers. Because it is insensitive and because it requires a minimum of 22 hours to complete, this ELISA method is neither sensitive enough nor practical enough for rapid assessment of limited volume samples containing low concentrations of a cholesterol binding substance.
Therefore, there is a need for a simple, rapid, sensitive and reproducible method for assessing, both quaUtatively and quantitatively, a HTBS which interacts with a HTS.
Summary of the Invention
The present invention provides a simple, rapid, sensitive and reproducible solid-phase method for detecting a hydrophobic-target binding substance (HTBS) in a sample. In this solid-phase method, a hydrophobic-target substance (HTS) is dissolved in an organic solvent. The organic solvent, containing the dissolved HTS is applied to an inert solid-phase hydrophobic support material compatible with the organic solvent. The organic solvent is removed such that the HTS is immobilized on and within the inert solid-phase hydrophobic support material. The inert solid-phase hydrophobic support material is washed with an aqueous solution and is contacted with an aqueous sample containing a HTBS such that any HTBS in the aqueous sample binds to the HTg immobilized on and within the inert solid-phase hydrophobic support material. Binding of the HTBS to the HTS is detected. The amount of the HTBS bound to the HTS is measured and the amount of hydrophobic-binding substance in the sample is calculated. The object of the present invention is to provide a solid-phase method for detecting a HTBS in an aqueous sample.
It is another object of the present invention to provide a sohd-phase method for quantitating the amount of a HTBS in an aqueous sample.
It is another object of the present invention to provide a sohd-phase method for assaying a HTBS in an aqueous solution that solves previously encountered problems related to the aqueous insolubiUty of the hydrophobic-target substance.
It is another object of the present invention to provide a method to immobihze a HTS on and within an inert solid-phase hydrophobic support material such that the HTS does not precipitate in the absence of an organic solvent. It is another object of the present invention to provide a method for assaying a HTBS wherein the HTS is immobilized on and within an inert solid-phase hydrophobic support material and remains accessible for reaction with the HTBS. It is another object of the present invention to provide a sohd-phase method for assaying a HTBS that is rapid.
It is another object of the present invention to provide a solid-phase method for assaying a HTBS that is sensitive.
It is another object of the present invention to provide a solid-phase method for assaying a HTBS that is reproducible. It is another object of the present invention to provide a solid-phase method for assay of a HTBS that is qualitative.
It is another object of the present invention to provide a solid-phase method for assaying a HTBS that is quantitative.
It is another object of the present invention to provide a sohd-phase method for assaying a HTBS wherein the HTS does not have to be altered to enable binding on and within the inert sohd-phase support material. It is another object of the present invention to provide a sohd-phase method for assaying a HTBS wherein neither couphng of the HTS to another molecule nor chemical modification of the HTS is necessary for immobiUzation of the HTS on and within the inert sohd-phase support material. It is another object of the present invention to detect lower concentrations of HTBSs than possible in currently available sohd-phase methods for detecting HTBSs
It is another object of the present invention to provide a solid-phase method for assaying a steroid binding substance including, but not limited to, steroid hormone binding substances, steroid vitamin binding substances, animal steroid specific binding substances, plant steroid specific binding substances, pathogen steroid specific binding substances, steroid drug binding substances, and steroid dye binding substances. It is another object of the present invention to provide a solid-phase method for assaying a sterol binding substance.
It is another object of the present invention to provide a solid-phase method for assaying a cholesterol binding substance.
It is another object of the present invention to provide a solid-phase method for assaying an ergosterol binding substance. It is another object of the present invention to provide a solid-phase method for assaying a sterol binding substances that requires small volumes of reagents, is simple to perform, can be completed in a short period of time, and requires no special expertise. It is another object of the present invention to provide a simple, rapid, sensitive and reproducible method to assess the efficacy of a sterol vaccine and to evaluate changes in the immunologic status of a host.
It is another object of the present invention to provide a method for efficient transfer of a heterogeneous mixture of HTSs from a thin layer chromatography plate to a inert soUd-phase hydrophobic support material.
It is another object of the present invention to provide a method for detecting a HTBS using a HTS transferred from a thin layer chromatography plate to an inert soUd-phase hydrophobic support material.
It is another object of the present invention to use cholesterol transferred from a thin layer chromatography plate to a inert solid-phase hydrophobic support material for detecting a cholesterol binding substance in an aqueous sample.
It is another object of the present invention to using ergosterol transferred from a thin layer chromatography plate to a inert solid-phase hydrophobic support material for detecting an ergosterol binding substance in an aqueous sample. Others object, features and advantages of the present invention will become apparent upon reading the following detailed description of the preferred embodiment of the invention when taken in conjunction with the drawings and the appended claims.
Brief Description of the Drawings
FIG. 1. Immunodetection of cholesterol binding substance in a PVDF membrane ELISA. Binding of pre- immune serum and of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol immobiUzed on and within PVDF membrane in a DOT-BLOT apparatus holder.
FIG. 2. Immunodetection of cholesterol binding substance in a nitrocellulose microtiter plate ELISA. Binding of pre-immune serum and of immune serum, at dilutions of approximately 1 :800 to 1 : 100,000, to cholesterol immobiUzed on and within nitrocellulose wells of a microtiter plate.
FIG 3. Immunodetection of cholesterol binding substance in a PVDF membrane ELISA. Binding of pre- immune mouse ascites fluid and of immune mouse ascites fluid, at dilutions of approximately 1 :800 to 1 :6,400, to cholesterol immobilized on and within PVDF membrane in a DOT-BLOT apparatus holder. FIG. 4. Immunodetection of cholesterol binding substance in a PVDF microtiter plate ELISA. Binding of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol, at concentration of approximately 0 μg/well to 10 μg/well, immobiUzed on and within PVDF wells of a microtiter plate.
FIG. 5. Immunodetection of cholesterol binding substance in a plastic microtiter plate ELISA and in a PVDF microtiter plate ELISA using varying concentrations of cholesterol. Data presented is the mean ± S.D. of triplicate samples. FIG. 5A. Binding of phosphate buffered saline (PBS) and of immune serum, at dilutions of approximately 1 :800 to 1 : 12,800, to cholesterol, at concentrations of approximately 0.01 μg/well to 25 μg/well, immobiUzed on polystyrene microtiter plate. FIG. 5B. Binding of PBS and of immune serum, at dilutions of approximately 1 :800 to
1 :12,800, to cholesterol, at concentrations of approximately 0.01 μg/well to 25 μg/well, bound on and within PVDF wells of a microtiter plate. FIG. 5C. Binding of pre-immune and of immune serum, at concentrations of approximately 1 :800 to 1 : 100,000, to cholesterol, at 5 μg/well, immobilized on and within PVDF wells of a microtiter plate and on polystyrene wells of a microtiter plate.
FIG. 6. Immunodetection of ergosterol binding substance in a PVDF membrane ELISA. Binding of preimmune serum and of immune serum, at 1 :800 to 1 :3,200, to ergosterol immobiUzed on and within PVDF wells of a microtiter plate.
FIG. 7. Detection of cholesterol transferred from a thin layer chromatography (TLC) plate to PVDF membrane. FIG 7A. Immunodetection of cholesterol transferred from a
TLC plate to PVDF membrane using immune serum and pre- immune serum. FIG. 7B. Detection of cholesterol transferred from a TLC plate to PVDF membrane using iodine vapor.
FIG. 8. Detection of cholesterol in very low density lipoprotein (VLDL) extracts transferred from a TLC plate to PVDF membrane using isopropanol. CE-cholesterol esters; TG-triglycerides; C-cholesterol; PL-phospholipids. FIG. 8A. Immunodetection of C in a VLDL extract transferred from a TLC plate to a PVDF membrane with 70% isopropanol (1 ) and with 100% isopropanol (2). FIG. 8B.
Iodine vapor detection of CE, TG and C in a VLDL extract transferred from a TLC plate to a PVDF membrane with 70% isopropanol (1) and with 100% isopropanol. Detailed Description of the Invention
As used herein, the phrase "hydrophobic target substance (HTS)" refers to a substance that tends to repel water and to leave the water phase. HTSs include, but are not limited to, Upids such as steroids, steroids such as sterols, and sterols such as cholesterol and ergosterol.
As used herein, the phrase "hydrophobic-target binding substance (HTBS)" refers to a substance that tends to interact specifically with a HTS. HTBSs include, but are not limited to, lipid binding substances such as steroid binding substances, steroid binding substances such as sterol binding substances, and sterol binding substances such as cholesterol binding substances and ergosterol binding substances.
As used herein, the phrase "immobiUzed on and within" refers to insertion of a HTS on, between and among existing elements of an inert soUd-phase hydrophobic support material.
As used herein, the word "pre-immune" refers to samples containing no HTBS. As used herein, the word "immune" refers to samples containing a HTBS.
The present invention comprises a method for detecting a HTBS in an aqueous sample. In this soUd-phase method, a HTS is dissolved in an organic solvent. The organic solvent, containing the dissolved HTS, is apphed to an inert soUd-phase hydrophobic support material compatible with the organic solvent. The organic solvent is removed such that the HTS is immobilized on and within the inert solid-phase hydrophobic support material. The inert solid-phase hydrophobic support material is washed with an aqueous solution and is contacted with an aqueous sample containing a HTBS such that any HTBS in the aqueous sample binds to the HTBS immobilized on and within the inert solid-phase hydrophobic support material. Binding of the HTBS to the HTS is detected. The amount of the HTBS bound to the HTS is measured and the amount of hydrophobic-binding substance in the aqueous sample is calculated.
The present invention further comprises a method for detecting binding of a HTBS to a HTS in a mixture of hydrophobic substances. The mixture of hydrophobic substances, which includes the HTS, is separated into its individual hydrophobic substances. The separated hydrophobic substances are transferred to an inert solid-phase hydrophobic support material in the presence of an organic solvent. The organic solvent is removed such that the separated hydrophobic substances are immobiUzed on and within the inert solid-phase hydrophobic support material. The inert solid-phase hydrophobic support material is washed with an aqueous solution and is contacted with an aqueous sample containing a HTBS such that any HTBS in the aqueous sample binds to the any HTS among the hydrophobic substances immobiUzed on and within the inert solid-phase hydrophobic support material. Binding of the HTBS to the HTS is detected. Because of the hydrophobicity of the inert solid- phase hydrophobic support material, when the organic solvent in which the HTS is dissolved evaporates, the HTS is immobiUzed on and within the inert soUd-phase hydrophobic support material and does not precipitate. Moreover, the HTS remains immobilized, non-precipitated and accessible on the inert solid-phase hydrophobic support material for binding of the HTBS in the aqueous sample.
The present invention further comprises an assay kit for detecting a HTBS in an aqueous sample comprising an inert solid-phase hydrophobic support material having a HTS immobilized thereon and therein and further comprising a holder for the inert soUd-phase hydrophobic support material.
The hydrophobic-target substance (HTS) for use in the present invention can be any hydrophobic substance which is soluble in an organic solvent. HTSs include, but are not Umited to, lipids, steroids, sterols and vitamins. Preferred HTSs are sterols. More preferred HTSs are cholesterol, cholesterol derivatives and cholesterol analogs and ergosterol, ergosterol derivatives and ergosterol analogs. The HTS can be obtained by methods well known to those skilled in the art.
The concentration of HTS for use in the present invention can range between about 0.0001 μg/μl and about 50 μg/μl. The preferred range is between about 0.001 μg/μl and about 10 μg/μl. The most preferred range is between about 0.01 μg/μl and about 5 μg/μl. The HTS dissolved in organic solvent can be applied to the inert solid-phase hydrophobic support material by dipping, soaking, spotting, spraying, blotting or other convenient means. Preferred methods include spotting, spraying and blotting. More preferred methods include spotting and blotting. A preferred volume for spotting is between about 5 μl/well and 10 μl/well. Determination of the amount of HTS to be used for each method of application is well within the knowledge of one skilled in the art. For example, a standard HTS-HTBS assay combination can be used to determine the amount of HTS to be appUed to the inert soUd-phase hydrophobic support material.
The organic solvent for use in the present invention can be any solvent which can solubiUze the HTS, and which is sufficiently miscible with water to be completely removed by subsequent thorough rinsing with an aqueous solution. Such solvents include lower (C1 -C4) aliphatic alcohols including, but not limited to, methanol, ethanol, n- propanol, isopropanol, n-butanol, isobutanol, secondary-butyl alcohol, and tertiary-butyl alcohol; halogenated hydrocarbons including, but not Umited to, chloroform, ethylene chloride, carbon tetrachloride, trichloroethane methylene chloride, monochlorobenzene and perchloroethylene; lower aliphatic ketones including, but not Umited to, acetone, methyl ethyl ketone; dioxan, dimethyl formamide; dimethyl sulfoxide; acetonitrile; lower (C1 -C4) glycols including, but not Umited to, ethylene glycol and propylene glycol; and, lower (C3-C6) aUphatic ethers including, but not Umited to, 2-methoxyethanol and 2-ethoxyethanol. Mixtures of solvents can also be used. Preferred solvents are lower aliphatic alcohols, chloroform, ether, acetone and isopropanol. Most preferred solvents are ethanol, chloroform and isopropanol.
The inert solid-phase hydrophobic support material for use in the present invention includes materials having high hydrophobic properties, materials having low hydrophilic properties, and composites of materials having both high hydrophobic and low hydrophilic properties. Materials having high hydrophobic properties include, but are not limited to, polyethylenes, polypropylenes, polyacetals, epoxy resins, polycarbonates, fluorine-contained resins, polyvinyls, polyvinylidines, polyvinylidene chlorides and polyvinyUdine fluorides. Materials having low hydrophihc properties include, but are not limited to, nitrocellulose, nylon and cellulose. Preferred materials are polyvinyUdine fluorides and nitrocellulose. More preferred materials are polyvinyUdine difluoride and nitrocellulose.
The inert solid-phase hydrophobic support material for use in the present invention can be in the form a membrane, a bead, or any other solid-phase hydrophobic support form known to those skilled in the art. A preferred form is a membrane. In addition, the inert solid-phase hydrophobic support material can be placed into a holder, including but not Umited to, a membrane sheet holder, a dot- blot apparatus, a microtiter plate, a column, and a filter. Preferred holders include a membrane sheet holder, a dot-blot apparatus and a microtiter plate.
The blocking buffers for use in the present invention to prevent non-specific binding can be any suitable blocking buffer including, but not limited to, fetal calf serum, gelatin, low fat milk, and Tween-20 at various dilutions in an aqueous solution. The washing solution for use in the present invention can be any suitable aqueous buffer including, but not limited to, phosphate buffered saline (PBS), tris(hydroxymethyl)amino methane (TRIS) and N-2- hydroxyethylpeperazine-N'-2-ethanesulfonic acid (HEPES).
Such aqueous buffers and their appropriate pHs are well known to those skilled in the art.
The hydrophobic-target binding substance (HTBS) for use in the present invention is a substance which binds specifically to a HTS. Examples of HTBSs include, but are not limited to, antibodies, receptors, antibiotics, and dyes. Antibodies may be either monoclonal or polyclonal. Preferred hydrophobic-target-binding substances are antibodies. More preferred hydrophobic-target-binding substances are antibodies to sterols. Most preferred HTBSs are antibodies to cholesterol and antibodies to ergosterol.
Any convenient indicator method can be used to detect binding of a HTBS to a HTS. Such methods include, but are not limited to, the use of enzymes, enzyme cofactors, enzyme effectors, chromogenic substances, fluorogenic substances, chemiluminescent substances, bioluminescent substances, and labeled antibodies. Preferred indicator methods are the peroxidase-labeled antibody method and the alkaUne phosphatase-labeled antibody method. The invention also relates to an assay kit for detecting a hydrophobic-target binding substance in an aqueous sample comprises an inert solid-phase hydrophobic support material having a hydrophobic-target substance immobilized thereon and therein and may optionally further comprise a holder for the inert soUd-phase hydrophobic support material.
The invention further relates to a composition comprising, an inert soUd-phase hydrophobic support material having a hydrophobic-target substance immobilized thereon and therein. In a preferred embodiment the solid-phase hydrophobic support material is selected from the group consisting of polyvinylidene difluoride and hydrophobic nitrocellulose.
The invention will be illustrated by the following examples, which are in no way intended to Umit the scope of the present invention.
Example 1
BINDING OF PURIFIED CHOLESTEROL TO INERT SOLID-PHASE HYDROPHOBIC SUPPORT MATERIALS
To determine whether an inert solid-phase hydrophobic support material will immobiUze cholesterol in a non-precipitated and accessible manner for binding of the HTBS in the aqueous sample, purified cholesterol (Aldrich Chemical Company, Inc., Milwaukee, WI) is dissolved in
100% ethanol to a final concentration of between approximately 1 μg to 2 μg per μl ethanol. Serial dilutions of between about 10 ng to 500 ng cholesterol in 5 μl ethanol are spotted onto the following commercially available inert soUd- phase hydrophobic support materials:
1. "Biorad-Zeta" amine derivatized nylon membrane (ADN)
(BioRad Laboratories, Hercules, CA)
2. "Nytran" nylon membrane (NY) (Schleicher & Schuell, Keene, NH)
3. "BioRad Pure" nitrocellulose membrane (NC) (BioRad
Laboratories, Hercules, CA)
4. "BA85" nitrocellulose membrane (NC) (Schleicher &
Schuell, Keene, NH) 5. "Westran" polvinyUdene difluoride membrane (PVDF)
(Schleicher & Scheull, Keene, NH) 6. "Immobilon-P" polvinyUdene difluoride membrane (PVDF) (Millipore Corporation, Bedford, MA). The spots are air dried and washed repeatedly with phosphate buffered saUne, pH 7.2 -pH 7.4, (PBS). To detect cholesterol immobiUzed on the membranes, the membranes are exposed to iodine vapors, sprayed with ferric chloride, or are stained with fiUpin-complex, an antibiotic which binds to cholesterol and to other sterols, and washed with PBS. ADN and NY membranes retain no cholesterol even at the highest concentration applied (500 ng/5 μl). PVDF membranes unexpectedly retain cholesterol at the lowest concentration appUed (10 ng/5 μl), the lower detection limit of iodine vapors. NC membranes also unexpectedly retain cholesterol, although less efficiently (>100 ng/5 μl), than PVDF membranes.
Example 2 PREPARATION OF CHOLESTEROL LIPOSOMES
Lipids are aliquoted into pyrogen-free rotary evaporator flasks and are rotary evaporated to remove solvents. Lipid ratios are: dimyristoyl phosphatidylcholine (DMPC): dimyristoyl phosphatidyl-glycerol (DMPG): cholesterol, 9: 1 :7.5 (mol:mol:mol) (Avanti Polar Lipids,
Alabaster, AL) and 25 μg monophosphoryl Upid A per μmol phosphoUpid. Trace solvent is removed by vacuum desiccation for 2 hrs. Dry lipid is hydrated by adding sterile, pyrogen- free deionized water to achieve a 50 mM total phosphoUpid concentration. The sample is vortexed vigorously until all the lipid is resuspended and is incubated for 2 hrs at room temperature. The sample is transferred to vaccine vials and lyophilized overnight. The lyophiUzed lipid is reconstituted in PBS to a final phosphoUpid concentration of 10 mM (Bartlett, 1959 J. Biol. Chem. 234:466). Between about 50% and about
71% of the lipid membrane of these cholesterol Uposomes is cholesterol (Swartz et al. 1988 Proc Natl Acad Sci. 85: 1902). Example 3
INDUCTION OF CHOLESTEROL BINDING SUBSTANCE
Pre-immune serum is collected from 6 to 8 week old male BALB/c mice (Jackson Laboratories, Bar Harbor, ME) by tail bleeds. To induce the generation of cholesterol binding substance in mice, 100 μl of Uposomes containing about 71 mol % cholesterol, prepared as in Example 2, are injected intraperitoneally into BALB/c mice. After 2 weeks, the mice are boosted once with another 100 μl of cholesterol Uposomes and are bled 2 weeks later to collect immune serum containing cholesterol binding substance.
Example 4
STANDARD ASSAY PROTOCOL FOR -MMUNODETECTION OF HYDROPHOBIC-TARGET BINDING SUBSTANCE USING A
HYDROPHOBIC MEMBRANE ELISA A HTS, dissolved in an organic solvent is applied to an inert solid-phase hydrophobic support material. The support material is dried at room temperature and in the dark to prevent spontaneous oxidation. For use, the inert solid- phase hydrophobic support material is rehydrated in blocking buffer.
The following standard assay protocol is followed:
1. The HTS (cholesterol) containing hydrophobic support material is rehydrated in blocking buffer, 4% or 10% heat inactivated (56°C for 30 min) fetal calf serum (FCS) deficient in cholesterol binding substance and negUgible (<1 ng) in cholesterol content (Biofluids, Inc. Rockville, MD) in phosphate buffered saUne, pH 7.2-pH 7.4 (PBS), for 1 hour at room temperature.
2. Serum or ascites fluid samples are diluted in blocking buffer, and are contacted with the hydrophobic support material. After 1 hr incubation with slow agitation at room temperature, the hydrophobic support material is washed 3-4-times with blocking buffer. 3. To detect the binding of HTBS (e.g. anticholesterol antibody) in the serum or ascites fluid, a 1/1500 dilution of peroxidase conjugated goat anti-mouse IgG (H+L), IgM or IgGl, IgG2a in blocking buffer is appUed to the hydrophobic support material. After 1 hour of incubation with slow agitation, the hydrophobic support material is washed 3-4- times with PBS, and are incubated with a soluble peroxidase substrate, 2,2'- azino-di[3-ethyl-bezthiazoline sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 1-45 min until adequate color is developed.
4. Absorption is measured for each sample at 405 nm using a Microtiter Plate Reader (BioRad Laboratories). The concentration and amount of cholesterol binding substance in each serum sample are quantitated by calculations well known to those skilled in the art.
Example 5 IMMUNODETECTION OF CHOLESTEROL BINDING SUBSTANCE
IN SERUM USING A PVDF MEMBRANE ELISA
PVDF membrane (8 X 10 cm) is sprayed with 2-3 ml of an ethanol.xhloroform (1:1) solution containing 5 mg/ml of purified cholesterol. The membrane is dried at room temperature and in the dark to prevent spontaneous oxidation. For use, the membrane is rehydrated in blocking buffer and placed into a 96 well DOT-BLOT apparatus holder (BioRad Laboratories, Hercules, CA).
The standard assay protocol of Example 4 is followed:
1. The cholesterol containing PVDF membrane in the DOT-BLOT apparatus holder is rehydrated in blocking buffer (4% FCS) for 1 hour at room temperature.
2. Serum samples are diluted serially in blocking buffer, and 100 μl of each dilution is added to dupUcate wells. After 1 hr incubation with slow agitation at room temperature, the wells are washed 4-times with blocking buffer.
3. To detect the binding of cholesterol binding substance in the serum, 100 μl of a 1/1500 dilution of peroxidase conjugated goat anti-mouse IgG (H+L), IgM or IgGl ,
IgG2a in blocking buffer is added to each well. After 1 hour of incubation with slow agitation, the wells are washed 3-4- times with PBS, and are incubated with 100 μl of a soluble peroxidase substrate, 2,2'-azino-di[3- ethyl-bezthiazohne sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 15-20 min until adequate color is developed. Ninety μl of the reactant is then transferred to a round bottom 96- well clear plastic microtiter plate well and absorption is measured for each sample at 405 nm using a Microtiter
Plate Reader (BioRad Laboratories).
As shown in FIG. 1, pre-immune serum shows no cholesterol binding activity and immune serum, at dilutions between 1 :800 and 1 : 12,800, shows dilution dependent cholesterol binding activity in this PVDF membrane ELISA. Duplicate wells show comparable color intensity demonstrating that immune serum reproducibly and uniformly recognizes cholesterol bound to PVDF and indicating that PVDF immobilizes cholesterol in an orientation which allows for cholesterol binding substance recognition of the sterol.
Example 6
IMMUNODETECTION OF CHOLESTEROL BINDING SUBSTANCE IN SERUM USING A NITROCELLULOSE WELL MICROTTTΕR
PLATE ELISA
Five μl of absolute ethanol containing 5 μg of purified cholesterol is spotted directly into each well of an
BA85 nitrocellulose well microtiter plate. The plates are stored in the dark at 4° C for two weeks. For use, the wells are rehydrated with blocking buffer and the standard assay protocol of Example 4 is followed using 100 μl aUquots of pre-immune serum and 100 μl aUquots of immune serum diluted from 1 :1,000 to 1:100,000. To quantitate cholesterol binding substance in the serum, 100 μl of a 1/1500 dilution of peroxidase conjugated goat anti-mouse IgG (H+L), IgM or IgGi, IgG2a i blocking buffer is added to each well. After 1 hour of incubation with slow agitation, the wells are washed 3- 4 times with PBS, and are incubated with 100 μl of a soluble peroxidase substrate, 2,2'-azino-di[3-ethyl-bezthiazoline sulfonate (ABTS peroxidase substrate system) (Kirkegaard & Perry) and monitored for 10-20 min until adequate color is developed. Ninety μl of the reactant is then transferred to a round bottom 96-well clear plastic microtiter plate well and absorption is measured for each sample at 405 nm using a
Microtiter plate Reader (BioRad Laboratories). Duplicate wells show comparable color intensity.
FIG. 2 shows the unexpected result that cholesterol binding substance is measurable in immune serum at a dilution of approximately 1 :75,000, whereas cholesterol binding substance is not detectable in pre-immune serum in this nitrocellulose well microtiter plate ELISA.
Example 7 IMMUNODETECTION OF CHOLESTEROL BINDING SUBSTANCE
IN ASCITES FLUID USING A PVDF MEMBRANE ELISA
To detect cholesterol binding substance in mouse ascites fluid, 300 μg of purified cholesterol is dissolved in 1 ml of ethanol: chloroform ( 1 : 1) and is sprayed onto a PVDF membrane. The membrane is stored in the dark overnight at room temperature. For use, the membrane is rehydrated in blocking buffer and placed in a DOT-BLOT (BioRad Laboratories) apparatus holder. The standard assay protocol of Example 4 is followed using 100 μl aUquots of pre-immune ascites fluid and 100 μl aUquots of immune ascites fluid diluted from 1 : 1 ,000 to 1 :6,400. Duplicate wells show comparable color intensity.
FIG. 3 shows the unexpected result that cholesterol binding substance is detectable at dilutions of 1 :6,400 in immune ascites fluid, whereas cholesterol binding substance is not detectable in pre-immune ascites fluid in this
PVDF membrane ELISA.
Example 8 IMMUNODE -ΕCTION OF CHOLESTEROL BINDING SUBSTANCE
USING VARIOUS CONCENTRATIONS OF CHOLESTEROL IN A PVDF WELL MICROTTTER PLATE ELISA Ten μl aUquots of ethanol containing from 0 μg purified cholesterol to 10 μg purified cholesterol are spotted into each PVDF well of an Immobilon-P (Millipore
Corporation, Bedford, MA) microtiter plate. The plate is stored in the dark overnight at room temperature. For use, the plate is rehydrated in blocking buffer and the standard assay protocol of Example 4 is followed using 100 μl of immune serum diluted 1 :800, 1 : 1 ,600, 1 :3,200 and 1 :6,400 and
1 : 12,800. DupUcate wells show comparable color intensity. The concentration and amount of cholesterol-binding substance in each serum sample are quantitated by calculations well known to those skilled in the art. FIG. 4 shows cholesterol binding substance is detectable at 1 μg cholesterol and that approximately 5 μg cholesterol is optimum for detecting cholesterol binding substance in this PVDF membrane microtiter plate ELISA.
Example 9
COMPARISON OF A CONVENTIONAL POLYSTYRENE PLASTIC
MICROTITER PLATE ELISA WTTH THE NOVEL PVDF MEMBRANE
MICROTTTER PLATE ELISA OF THE PRESENT INVENTION
This example compares immunodetection of a HTBS in a conventional polystyrene plastic well microtiter plate ELISA and in the novel PVDF well microtiter plate ELISA of the present invention.
Purified cholesterol at concentrations of 0.01 to 25 μg/5 μl absolute ethanol are added to wells of a polystyrene microtiter plate and to wells of a PVDF microtiter plate. The plates are stored in the dark overnight, rehydrated with blocking buffer and the standard assay protocol of Example 4 is followed using 100 μl aUquots of immune serum diluted between 1 :800 and 1 : 12,800. FIG. 5A shows the polystyrene microtiter plate
ELISA does not enable a dose response effect and shows inconsistent binding with increasing concentrations of cholesterol.
FIG. 5B shows that the PVDF microtiter plate ELISA of the present invention enables a dose response effect and shows consistent binding with increasing concentrations of cholesterol.
FIG. 5C shows that, using 5 μg/well of cholesterol, the dose response effect of the PVDF microtiter plate ELISA of the present invention is 4-6 fold more sensitive than that of the conventional polystyrene microtiter plate ELISA.
A dose response effect is evident with the PVDF well microtiter plate ELISA, whereas inconsistent binding is found with the polystyrene microtiter plate ELISA at increasing concentrations of cholesterol. The high frequency of experimental variation observed with plastic microtiter plate ELISA likely results from non-uniform distribution of cholesterol on polystyrene surfaces. The PVDF membrane microtiter plate ELISA of the present invention provides more reproducible results and unexpectedly enables detection and quantitation of significantly lower amounts of cholesterol binding substance than the conventional plastic microtiter plate ELISA. Moreover, the dose response effect of the PVDF membrane microtiter plate ELISA of the present invention is 4-6 fold more sensitive than the conventional plastic microtiter plate ELISA.
Example 10 IMMUNODETECTION OF ERGOSTEROL BINDING SUBSTANCE IN
USING A PVDF WELL MICROTTTER PLATE ELISA
To detect ergosterol binding substance in immune serum containing ergosterol binding substance, 5 μg of purified ergosterol in 5 μl of absolute ethanol is spotted into each well of a PVDF membrane microtiter plate. The plate is dried and stored in the dark overnight. For use, the wells are rehydrated in blocking buffer and the standard assay protocol of Example 4 is followed using preimmune and immune serum. DupUcate wells show comparable color intensity. FIG. 6 shows the unexpected result that ergosterol binding substance is measurable in immune serum at dilutions >1 :3,200 and that there is at least a two-fold difference in absorbance at 405 nm for immune serum relative to pre- immune serum.
Example 11
LMMUNODETECπON OF CHOLESTEROL BINDING SUBSTANCE USING DILUTIONS OF CHOLESTEROL TRANSFERRED FROM A THIN LAYER CHROMATOGRAPHY PLATES TO PVDF MEMBRANE A TLC silica gel 60 plate (Sigma Chemical Co.,
St. Louis, MO) is pre-run with 100% methanol. Dilutions of cholesterol dissolved in chloroform are streaked on the TLC plate, run in a hexane-methanol solvent and air dried. The TLC plate is sprayed with 70% isopropanol, covered with a sheet of PVDF, a sheet of cellophane and pressed with a flat weight (200 g).
Cholesterol, transferred to PVDF paper, is detected using the standard assay protocol of Example 4 with 1 :1,000 dilutions of pre-immune and of immune mouse serum. The color reaction is stopped by rinsing the membrane with distilled water.
FIG. 7 A shows that after 15 minutes of blotting, 200 ng of cholesterol applied to the TLC plate are detectable on PVDF membrane with immune serum and that after 15 minutes of blotting 500 ng of cholesterol apphed to the TLC plate are not detectable on PVDF membrane with preimmune serum. This demonstrates the unexpected finding that relatively small amounts of cholesterol transferred from a TLC plate to a PVDF membrane are detectable by cholesterol binding substance in immune serum.
FIG. 7B shows the TLC plate after 15 minutes of blotting and after exposure to iodine vapor. Blotting for 15 minutes results in almost complete transfer of 200 ng of cholesterol. Blotting for 3 to 5 minutes results in partial transfer of 200 ng of cholesterol (data not shown). Blotting for > 30 minutes results in excessive diffusion of the 200 ng of transferred cholesterol (data not shown). This demonstrates that cholesterol transfer depends on blotting time.
Example 12
IMMUNODETECTION OF CHOLESTEROL IN LIPIDS SEPARATED
IN THIN LAYER CHROMATOGRAPHY PLATES AND
TRANSFERRED TO PVDF MEMBRANE Commercially purified very low density lipoproteins (VLDL) (Organon Teknika Corp.-CAPPEL Research Products, Durham, NC) are extracted, rotary evaporated, resuspended in 100% chloroform and stored at -20°C. TLC sihca gel 60 plates (Sigma Chemical Co . St.
Louis, MO) are pre-run with 100% methanol. The extracted VLDL are separated on the TLC plate in a 80 40 2 hexane:ether:glacial acetic acid (v:v:v:) solvent and the TLC plates are air dried. The air-dried TLC plates are overlaid with a PVDF membrane, pre-wetted by spraying with isopropanol to facilitate passive transfer of lipids from the silica gel. Due to the difference in hydrophobicity of the various lipids, mixtures of isopropanol/water (v/v) of 50%, 70% or 100% (v/v) isopropanol are evaluated. The PVDF membrane is covered with a sheet of cellophane and with a flat weight (200 g) for 15 minutes to 30 minutes. After the transfer the PVDF membrane is separated from the siUca plate and air dried.
Cholesterol transfer to PVDF is detected using the standard assay protocol of Example 4 with 1 : 1 ,000 dilutions of pre-immune and of immune mouse serum. The color reaction is stopped by rinsing the membrane with distilled water. Cholesterol transfer to PVDF is further confirmed using filipin complex (Smejkel et al. 1994 Biotech. 16:68). Lipid transfer is confirmed by exposing the PVDF membrane to iodine vapor.
With 50% isopropanol, there is no transfer of cholesterol esters and triglycerides and only minimal transfer of cholesterol and phosphoUpids. FIG. 8A1 shows, using cholesterol binding substance in immune serum to visuaUze cholesterol, that cholesterol transfers from the TLC plate to the PVDF membrane in the presence of 70% isopropanol in 15 minutes.
FIG. 8A2 shows, using iodine vapor to visualize lipids, that cholesterol and phosphoUpids, but not triglycerides and cholesterol esters, transfer from the TLC plate to PVDF membrane in the presence of 70% isopropanol.
FIG. 8B 1 shows, using cholesterol binding substance in immune serum to visualize cholesterol, that cholesterol transfers from the TLC plate to the PVDF membrane in the presence of 100% isopropanol.
FIG. 8B2 shows, using iodine vapor to visualize lipids, that cholesterol triglycerides and cholesterol esters, but not phosphoUpids, transfer from the TLC plate to PVDF membrane in the presence of 100% isopropanol. These data demonstrate that, using a soUd-phase hydrophobic support material with appropriate transfer buffers, one HTS, among a heterogeneous mixture of HTSs, can be evaluated using the hydrophobic membrane ELISA of the present invention.
It should be understood, of course, that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein, without departing from the spirit and the scope of the invention as set forth in the appended claims.

Claims

CLAIMSWe claim:
1. A method for detecting a hydrophobic- target binding substance in an aqueous sample, comprising the steps of: a. dissolving a hydrophobic-target substance in an organic solvent to yield a hydrophobic-target substance- containing solvent mixture; b. applying the hydrophobic-target substance- containing solvent mixture to a solid-phase hydrophobic support material; c. removing the solvent such that the hydrophobic-target substance is immobiUzed on or within the soUd-phase hydrophobic support material; d. washing the solid-phase hydrophobic support material with an aqueous solution to remove any hydrophobic-target substance not immobiUzed on or within the soUd-phase hydrophobic support material; e. contacting the solid-phase hydrophobic support material with the aqueous sample such that hydrophobic-target binding substance in the aqueous sample binds to the hydrophobic-target substance immobilized on or within the solid-phase hydrophobic support material; and, f. detecting the hydrophobic-target binding substance bound to the hydrophobic-target substance.
2. The method of Claim 1 , wherein the amount of the hydrophobic-target binding substance bound to the hydrophobic-target substance is quantitated.
3. The method of Claim 1, wherein the inert solid-phase hydrophobic support material is selected from the group consisting of polyvinyUdene difluoride and hydrophobic nitrocellulose.
4. The method of Claim 1 , wherein the hydrophobic-target substance is a sterol.
5. The method of Claim 4, wherein the sterol is selected from the group consisting of cholesterol, cholesterol derivatives, cholesterol analogs, ergosterol, ergosterol derivatives, and ergosterol analogs.
6. The method of Claim 5, wherein the sterol is cholesterol.
7. The method of Claim 1 , wherein the hydrophobic-target binding substance is a sterol binding substance.
8. The method of Claim 7, wherein the sterol binding substance is selected from the group consisting of a cholesterol binding substance and an ergosterol binding substance.
9. The method of Claim 8, wherein the sterol binding substance is a cholesterol binding substance.
10. A method for detecting a sterol binding substance in an aqueous sample, comprising the steps of: a. dissolving a sterol in an organic solvent; b. applying the sterol-containing solvent to a solid-phase hydrophobic support material; c. removing the solvent such that the sterol is immobilized on or within the soUd-phase hydrophobic support material; d. washing the solid-phase hydrophobic support material with an aqueous solution to remove sterol not immobilized on or within the soUd-phase hydrophobic support material; e. contacting the solid-phase hydrophobic support material with the aqueous sample such that sterol binding substance in the aqueous sample binds to the sterol. f . detecting the sterol binding substance bound to the sterol.
1 1. The method of Claim 10, wherein the amount of the sterol binding substance bound to the sterol is quantitated.
12. The method of Claim 10, wherein solid- phase hydrophobic support material is selected from the group consisting of polyvinylidene difluoride and hydrophobic nitrocellulose.
13. The method of Claim 10, wherein the sterol is selected from the group consisting of cholesterol, cholesterol derivatives, cholesterol analogs, ergosterol, ergosterol derivatives, and ergosterol analogs.
14. The method of Claim 13, wherein the sterol is cholesterol.
15. The method of Claim 13, wherein the sterol is er ' cgov sterol.
16. An assay kit for detecting a hydrophobic- target binding substance in an aqueous sample compπsing an inert solid-phase hydrophobic support material having a hydrophobic-target substance immobiUzed thereon and therein.
17. The assay kit of Claim 16, wherein the soUd-phase hydrophobic support material is selected from the group consisting of polyvinyUdene difluoride and hydrophobic nitrocellulose.
18. The assay kit of Claim 16, wherein the hydrophobic-target substance is a sterol.
19. The assay kit of Claim 18, wherein the sterol is selected from the group consisting of cholesterol, cholesterol derivatives, cholesterol analogs, ergosterol, ergosterol derivatives, and ergosterol analogs.
20. The assay kit of Claim 19, wherein the sterol is cholesterol.
21. The assay kit Claim 19, wherein the sterol is ergosterol.
22. A composition comprising, an inert solid- phase hydrophobic support material having a hydrophobic- target substance immobiUzed thereon and therein.
23. The composition of Claim 22, wherein the soUd-phase hydrophobic support material is selected from the group consisting of polyvinyUdene difluoride and hydrophobic nitrocellulose.
PCT/US1996/004589 1995-04-05 1996-04-03 A solid-phase method for assaying a hydrophobic-target binding substance Ceased WO1996037779A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57118/96A AU5711896A (en) 1995-04-05 1996-04-03 A solid-phase method for assaying a hydrophobic-target bindi ng substance

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41702595A 1995-04-05 1995-04-05
US08/417,025 1995-04-05

Publications (1)

Publication Number Publication Date
WO1996037779A1 true WO1996037779A1 (en) 1996-11-28

Family

ID=23652276

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/004589 Ceased WO1996037779A1 (en) 1995-04-05 1996-04-03 A solid-phase method for assaying a hydrophobic-target binding substance

Country Status (2)

Country Link
AU (1) AU5711896A (en)
WO (1) WO1996037779A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079847A1 (en) * 2000-04-17 2001-10-25 Orgentec Diagnostika Gmbh Method for coating plastic solid phases
CN119438435A (en) * 2024-11-15 2025-02-14 华中农业大学 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4774174A (en) * 1981-01-23 1988-09-27 Baxter Travenol Laboratories, Inc. Solid phase system for ligand assay
US5116733A (en) * 1988-08-22 1992-05-26 Konica Corporation Method of assaying target substance
WO1994014454A1 (en) * 1992-12-29 1994-07-07 Entremed, Inc. Vaccines against sterols

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4774174A (en) * 1981-01-23 1988-09-27 Baxter Travenol Laboratories, Inc. Solid phase system for ligand assay
US5116733A (en) * 1988-08-22 1992-05-26 Konica Corporation Method of assaying target substance
WO1994014454A1 (en) * 1992-12-29 1994-07-07 Entremed, Inc. Vaccines against sterols

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ARTERIOSCLEROSIS AND THROMBOSIS, December 1994, Vol. 14, No. 12, MAGGI et al., "LDL Oxidation in Patients with Severe Carotid Atherosclerosis", pages 1892-1899. *
BIOCHEMICAL SOCIETY TRANSACTIONS, 1989, Vol. 17, ALVING et al., "Naturally Occurring Autoantibodies to Cholesterol in Humans", pages 637-639. *
BIOTECHNIQUES, May/June 1986, Vol. 4, No. 3, PLUSKAL et al., "Immobilon PVDF Transfer Membrane: A New Membrane Substrate for Western Blotting of Proteins", pages 272-283. *
IMMUNOLOGY, 1991, Vol. 10, No. 5, ALVING et al., "Antibodies to Cholesterol, Cholesterol Conjugates and Liposones: Implication for Atherosclerosis and Autoimmunity", pages 441-453. *
MILLIPORE DIRECT CATALOG, Blotting, Transfer, Assay Section, 1992, pages 199-203. *
PROC. NATL. ACAD. SCI. U.S.A., March 1988, Vol. 85, SWARTZ et al., "Antibodies to Cholesterol", pages 1902-1906. *
THE JOURNAL OF IMMUNOLOGY, 01 November 1989, Vol. 143, No. 9, WASSEF et al., "Anaphylactoid Reactions Mediated by Autoantibodies to Cholesterol in Miniature Pigs", pages 2990-2995. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079847A1 (en) * 2000-04-17 2001-10-25 Orgentec Diagnostika Gmbh Method for coating plastic solid phases
CN119438435A (en) * 2024-11-15 2025-02-14 华中农业大学 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition

Also Published As

Publication number Publication date
AU5711896A (en) 1996-12-11

Similar Documents

Publication Publication Date Title
US5780319A (en) Immunoassays to detect antiphospholipid antibodies
US5776487A (en) Liposome reagents for immunoassays
US4483921A (en) Immunoassay with antigen or antibody labeled liposomes sequestering enzyme
JP2727127B2 (en) Nucleophilic polysubstituted aryl acridinium esters, their production method and assays using the same
JPH0312705B2 (en)
Bora et al. Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures
JPS5994069A (en) Special coupling analysis using analysis object-citricine conjugate body
US5187066A (en) Methods for detecting amphiphilic antigens
IE912625A1 (en) Biologically active reagent, analytical element and methods¹for use of the reagent
Pauillac et al. Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins
JPH0355790B2 (en)
JP3715348B2 (en) Analytical elements, compositions and methods using modified apopoperoxidase
Unson et al. An immunoassay for the detection and quantitative determination of mycothiol
WO1996037779A1 (en) A solid-phase method for assaying a hydrophobic-target binding substance
Rupprecht et al. Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs
JP3808527B2 (en) Specific binding methods and test kits
IE911599A1 (en) Use of heme-containing proteins as stabilizers for¹enzyme-labeled immunoreactants
Aniagolu et al. Analysis of anticholesterol antibodies using hydrophobic membranes
JP2001281250A (en) Method for detecting low molecular weight compounds using antigen-antibody reaction
JP4272518B2 (en) Ligand for β2-glycoprotein I and uses thereof
JPH07248329A (en) Detecting method of tubercular infectivity inspection membrane and tubercular infectivity related antibody therewith
JPH01295165A (en) Immunoassay
US20100291705A1 (en) Antigen exposing micelle and unordered aggregate
RU2412443C1 (en) Method for immunoenzymometric detection of t-2 mycotoxin in liquid samples
RU2038600C1 (en) Membrane for separating components of immunologic reaction

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase