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WO2002006346A1 - Dosage utile pour identifier la nephropathie induite par la $g(a)2uglobuline - Google Patents

Dosage utile pour identifier la nephropathie induite par la $g(a)2uglobuline Download PDF

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Publication number
WO2002006346A1
WO2002006346A1 PCT/US2001/022906 US0122906W WO0206346A1 WO 2002006346 A1 WO2002006346 A1 WO 2002006346A1 US 0122906 W US0122906 W US 0122906W WO 0206346 A1 WO0206346 A1 WO 0206346A1
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Prior art keywords
globulin
antibody
assay
reservoir
immunochemical assay
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PCT/US2001/022906
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English (en)
Inventor
Alfred F. Fuciarelli
Jeffrey A. Dill
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Battelle Memorial Institute
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Priority to AU2001280651A priority Critical patent/AU2001280651A1/en
Publication of WO2002006346A1 publication Critical patent/WO2002006346A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to an immunochemical assay kit and associated methods for identification and assessment of ⁇ 2u-globulin-mediated nephropathy in rodent toxicity/carcinogenicity studies.
  • CIGA Induce 2u-Globulin Accumulation
  • Kidney disease arises as a result of the formation of hyaline droplets in the kidney proximal tubule epithelial cells. These droplets are the result of an excessive accumulation of the protein ⁇ 2u-globulin. Once present, these droplets induce a sequence of pathological events, which ultimately manifest as renal tubule neoplasia.
  • the protein ⁇ 2u-globulin is found in high concentrations in the urine of male rats, and is not present in the urine of other species.
  • ⁇ 2u-Globulin-induced renal nephropathy results from a conformational change in ⁇ 2u-globulin upon binding CIGA compounds.
  • any increase in cuu-globulin is observed by purifying protein from kidney, serum, and urine samples and performing SDS- polyacrylamide gel electrophoresis to detect a protein band that of the appropriate molecular size.
  • Prepared ⁇ 2u-globulin antiserum is used to confirm the presence of ⁇ 2u-globulin through Western blotting techniques.
  • U.S. Patent No. 5,139,932 to Cederholm et al. discloses an immunoassay for diagnosing IgA nephropathy, which includes the steps of: (i) preparing a substrate capable of binding fibronectin or IgA; (ii) contacting the substrate with a sample to bind any f ⁇ bronectin-IgA-complex present in the sample to the substrate; (iii) and determining the presence of the bound complex by using the reaction between the exposed part of the bound complex and an antibody which corresponds to the complex.
  • a kit version of this assay is also provided.
  • U.S. Patent Nos. 5,534,431 and 5,654,158 to McDonald disclose an immunoassay for determining the presence of nephropathy-related immunoglobulin-like protein (NRIg) in a sample of body fluid, which includes the steps of: (i) simultaneously contacting a test sample of body fluid with a first and second antibody which are specific for NRIg at different antigenic sites (the first antibody is detectably labeled and soluble in the body fluid, and the second antibody is bound to a solid carrier which is insoluble in the body fluid under conditions which allow the formation of an insoluble complex of the first antibody the NRIg, and the second antibody); (ii) separating the insoluble complex from the sample of body fluid and unreacted labeled first antibody; (iii) measuring the amount of labeled antibody bound to the insoluble complex or the amount of unreacted labeled antibody; and (iv) relating the amount of labeled antibody bound to the insoluble complex or the amount of unreacted labeled antibody to control samples and determining the
  • the prior art discussed above does not disclose an immunological assay kit with associated methods for utilizing ⁇ 2u- globulin as an indicator of 2u-globulin nephropathy in kidney proximal epithelial cells.
  • the prior art directed toward detecting renal toxicity either utilizes a different technical approach (i.e., the use of NRIg as a protein marker), or offers a far less sensitive measure of detection (i.e., SDS- polyacrylamide gel electrophoresis) as opposed to the immunological format of the present invention.
  • the present invention utilizes the enzyme-linked immunosorbent assay (ELISA) format of immunochemical assays, which results in a highly sensitive, highly specific, quantitative assay.
  • Immunochemical assays are dependent upon antibodies (polyclonal and/or monoclonal) with appropriate specificities for detection of the molecule of interest (i.e., distinct antigenic determinants on ⁇ 2u-globulin).
  • the present invention is very useful for detection and assessment of ⁇ 2u-globulin-mediated nephropathy in toxicity and carcinogenicity studies, as well as for evaluating the carcinogenic potential of certain toxic chemicals in general when rats are used as the test subjects.
  • a preferred embodiment of this invention is a competitive indirect ELISA kit for detection of ⁇ 2u-globulin in rat kidneys.
  • the preferred version of the kit includes: (1) mouse anti- ⁇ 2u-globulin monoclonal antibody for use as a primary antibody for specific binding to ⁇ 2u-globulin; (2) alkaline phosphatase- conjugated goat anti-mouse IgG for use as a secondary antibody for binding to mouse anti- ⁇ 2u-globulin for quantitation (3) 4-methylumbelliferyl phosphate (MUP) which is converted to a fluorogenic product (4-methylumbelliferone) following enzymatic cleavage by alkaline phosphatase conjugated to the secondary antibody for signal amplification; (4) a 96-well flat bottom, clear, methacrylate plate for reacting the ⁇ 2u-globulin with the mouse anti- ⁇ 2u- globulin monoclonal antibody; (5) a 96-well U-bottom, black pigmented, st
  • a preferred method for detecting accumulation of ⁇ 2u-globulin in tissue samples by means of an ELISA includes (1) purifying ⁇ 2u-globulin for use in preparation of calibration standards; (2) exposing male rats to a chemical known to induce ct2u-globulin nephropathy to generate control samples; (3) preparing necessary buffers and reagents; (4) preparing control samples and unknown samples by diluting the samples in the buffers; (5) performing a total protein assay to determine protein concentrations of control samples; (6) performing a reagent dilution assay for determining optimal concentrations of reagents for subsequent reactions; (7) running a competitive inhibition ELISA on the control samples and unknown samples; and (8) calculating results.
  • an immunochemical assay kit which may be made commercially available for evaluating c ⁇ u-globulin-induced nephropathy in rodent toxicity/carcinogenicity studies involving a variety of suspect chemicals.
  • hyaline droplet nephropathy The correlation of hyaline droplet formation, accumulation of 2u-globulin within the droplet, and increased cell replication at the same specific location in the proximal tubule of the kidneys indicates that chronic exposure to CIGAs will cause ⁇ 2u-globulin-mediated droplet formation and persistent cell proliferation as a result of unrepaired mutations resulting from enhanced DNA replication. This persistent cell proliferation may result in the formation of malignant tumors in the kidneys of the test rats.
  • the present invention provides an immunochemical assay and kit for detection of excessive accumulation of ⁇ 2u-globulin in rat tissues and fluids.
  • ⁇ 2u-Globulin is produced in the liver, transported through the bloodstream (serum) to the kidneys, and excreted in urine.
  • the detection of excessive ⁇ 2u-globulin accumulation may include analysis of liver or kidney tissue, serum, urine or combinations thereof.
  • the present invention is useful in toxicity/carcinogenicity studies for identifying organic compounds with significant CIGA potential.
  • the present invention may be used as an early indicator of potential pathological changes the renal function of rats chronically exposed to weaker CIGA compounds.
  • tissues and fluids include any tissue or fluid containing detectable amounts of oau-globulin.
  • Such fluids typically include kidney, liver, serum, and plasma (i.e., serum that has not had consumable clotting factors removed).
  • Kidney and urine samples are preferred for use in the present invention. Kidneys from rats may are only obtainable following terminal sacrifice of test animals; therefore, additional animals must be added to an ongoing toxicity/carcinogenicity study. Otherwise, ⁇ 2u-globulin analysis can only be conducted at the end of the study.
  • ⁇ 2u-globulin in urine samples is non-invasive and can be conducted repeatedly on the same test animals at frequent intervals during the study without the loss of animals from the cohort receiving chronic administration of the test chemical.
  • the present invention requires the production of polyclonal and/or monoclonal antibodies to ⁇ 2u- globulin.
  • ⁇ 2u-Globulin is an 18.7 kD protein with unique antigenic determinants, which are useful in inducing the production of polyclonal or monoclonal antibodies.
  • the antibodies produced by immunizing suitable host animals with purified ⁇ 2u-globulin bind specifically, and with very high affinity, to the antigenic determinants expressed in ⁇ 2u-globulin.
  • Polyclonal antibodies result from the production of antibodies to all recognizable sites on a substance injected into a host animal.
  • the "polyclonal antibody response" produces a broad range of antibodies of differing affinities and specificities for a particular immunogenic substance.
  • Multiple antibody- producing cells from multiple sites in the host's body produce antibodies to that part of the immunogenic substance for which they are activated; each antibody-producing a clone making only one type of antibody to only one antigenic epitope on the foreign substance.
  • Serum containing polyclonal antibodies may be harvested from the immunized animal for the duration of the animal's lifetime.
  • Monoclonal antibodies are produced by recovering specific immune lymphocytes from the immunized host animal and fusing these cells with myeloma tumor cells derived from the same animal species to form giant somatic cell hybrids.
  • Hybrid cell lines have the capacity to grow rapidly and indefinitely in tissue culture due to the characteristics of the myeloma parent cell, and they secrete large amounts of the antibody specified by the genes of the normal antibody-secreting lymphocyte parent. After appropriate selection and cloning, the hybridomas are propagated in tissue culture or in a genetically identical or immunocompromised animal for an indefinite period of time to continuously produce primary antibody.
  • radioactive substrates such as tritium, carbon-14, phosphorus-32, and iodine- 125
  • fluorescence substrates such as fluorescein, rhodamine, and phycoerythrin
  • luminescent substrates or enzyme markers such as horseradish peroxidase, alkaline phosphatase, and ⁇ -glactosidase.
  • enzyme markers require the addition of a substrate, which is converted to a product, which enhances signal amplification.
  • preferred substrates may include 4-nitrophenyl phosphate (colorimetric), 4-methylumbelliferyl phosphate (fluorogenic), 3-[2'- (spiroadamantane)-4-methyl-4-3"-phosphoryloxyphenyl-l-2-dioxetane, disodium salt] (luminescence), and avidin-biotin.
  • Immunochemical assays utilizing enzymes are typically classified as either competitive heterogeneous or competitive homogeneous enzyme immunoassays.
  • the competitive heterogeneous enzyme immunoassays which include the ELISA, are based on the separation of antigen-antibody complexes formed following incubation of antigen and antibodies from free antigen and antibody.
  • Competitive homogeneous enzyme immunoassays in which the activity of an enzyme-ligand conjugate is reduced when the ligand is complexed to an antibody, do not involve a separation step prior to measurement.
  • the usefulness of homogeneous enzyme immunoassays tends to be limited to the assay of low molecular weight haptens, whereas the heterogeneous enzyme assay is generally applicable to the measurement of almost any antigen.
  • the present invention provides an immunochemical assay for measurement of ⁇ 2u-globulin.
  • the preferred embodiment of this invention is an enzyme-linked immunosorbent assay (ELISA) comprising two major components.
  • the first component is the immunological reaction, which occurs between an antigen and an antibody.
  • An enzyme-labeled antibody is required, and may be either the primary antibody with specificity to the antigenic determinants in the target molecule (i.e., oau-globulin), or a secondary antibody with antigenic determinants directed against the primary antibody.
  • the second component of the ELISA requires the attachment or immobilization of a "capturing agent" (i.e., antigen or antibody) to a solid support made of an inert material such as glass, synthetic polymers, synthetic resins, cellulose, or various suitable metals.
  • a "capturing agent” i.e., antigen or antibody
  • a solid support made of an inert material such as glass, synthetic polymers, synthetic resins, cellulose, or various suitable metals.
  • This attachment is accomplished by covalent or non-covalent linkage, adsorption, or other process.
  • the inert material to which the capturing agent is attached has an extensive, continuous form, such as a membrane or sheet, which is either flat or molded into convenient shapes such as multiwell plates
  • the inert material is in the form of discrete particles or beads.
  • an immunochemical assay kit for assessing ⁇ 2u-globulin-mediated nephropathy in carcinogenicity and toxicity studies includes: (1) mouse anti-oau-globulin monoclonal antibody for use as a primary antibody for binding to ⁇ 2u-globulin; (2) alkaline phosphatase-conjugated goat anti-mouse IgG for use as a secondary antibody for binding to mouse anti- ⁇ 2u-globulin (3) 4- methylumbelliferyl phosphate (MUP) for creating a fluorimetric change w en contacted with the alkaline phosphatase-conjugated goat anti-mouse IgG; (4) a 96-well flat bottom, clear, methacrylate plate for reacting the ⁇ 2u-globulin with the mouse anti- ⁇ 2u-globulin monoclonal antibody; (5) a 96-well U- bottom, black pigmented,
  • MUP 4- methylumbelliferyl phosphate
  • ⁇ 2u-Globulin may be purified from the urine of male rats and characterized as described in Mao et al., Analysis of ⁇ u-globulin in Rat Urine and Kidneys by Liquid Chromatography-Electrospray Ionization Mass Spectrometry, Chemical Research in Toxicology, 11:953-961 (1998). Additionally, isolation, purification, and characterization of ⁇ 2u-globulin may be accomplished by several types of separation techniques including: SDS gel electrophoresis; Western blotting, immunochemical assays; or liquid chromatography-electrospray ionization mass spectrometry. Preferred methods for isolation, purification, and characterization are discussed below.
  • Urine is collected over dry ice from mature (about 10 to 12 weeks of age) male rats (Sprague-Dawley, Fischer 344/N or equivalent) and kept frozen at about minus 20°C prior to isolation of ⁇ 2u-globulin.
  • Low molecular weight proteins are isolated using a combination of molecular weight cut-off filters and high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • 90 mL of rat urine is concentrated to about 9.0 mL using Centriprep-10 centrifugal concentrators (Amicon, Inc., Beverly, VA), which also eliminates low molecular weight ( ⁇ 10,000 daltons) materials. Aliquots (typically 500 ⁇ l) of the concentrated urine are then subjected to HPLC.
  • ⁇ 2u-globulin is purified from about 4.0 mL of male rat urine.
  • Identity confirmation and estimation of purity of the isolated ⁇ 2u-globulin fraction is accomplished using one or more of these methods: (i) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) to assess the apparent molecular mass of the material, (ii) gel electrophoresis in combination with antibodies directed against oau-globulin (i.e., Western blotting) for specific identification of ⁇ 2u-globulin in the gel banding pattern, or (iii) liquid chromatography-electrospray ionization mass spectrometry to assess the molecular mass of the protein. Preferred versions of methods are described in greater detail below.
  • SDS-PAGE is accomplished using 18% gels run on a SE 250 Mighty SmallTM Vertical Slab Minigel Unit (Hoefer Scientific Instruments, San Francisco, CA) using methods described in Protein Electrophoresis Applications Guide (Hoefer Scientific, 1994). Gels (7 x 8 cm, 1 mm thick) are self-cast using a SE 245 Gel Caster (Hoefer Scientific Instruments, San Francisco, CA).
  • Urine samples are lyophilized to dryness using a SpeedVacTM (Savant Instruments, Inc., Farmingdale, NY) and diluted in 10 mM Tris-HCI (pH 8.0), 1 mM EDTA, 5% ⁇ - mercaptoethanol, 2.5% (w/v) SDS and heated to 95°C for five minutes. After cooling to room temperature 5 ⁇ l samples is applied to the gels and run at constant current at 30 mA using a BioRad 3000/300 Power Supply (Bio-Rad Laboratories, Hercules, CA) at ambient temperature.
  • the gels are stained with a solution consisting of 0.025% Coomassie Brilliant Blue R 250, 40% methanol and 7% acetic acid and destained with a sequential treatment of methanol :acetic acid (40%:7%) and acetic acid: methanol (5%:7%) solutions.
  • a prestained low molecular weight range (43, 29, 18.4, 14.3, 6.2, and 3 kDa) calibration mixture (Life Technologies, Gaithersburg, MD) is run on each gel and used to determine the molecular weight range of the proteins.
  • Immunoblotting of purified ⁇ 2u-globulin fractions with anti- ⁇ 2u-globulin monoclonal antibodies can be conducted to confirm identity.
  • Purified samples from the ⁇ 2u-globulin fractions and commercially available low range molecular weight markers (Life Technologies, Gaithersburg, MD) concurrently are resolved by SDS-PAGE as previously described.
  • the gel is divided and one portion of the gel is stained with Coomassie Blue as previously described, and proteins from the second portion of the gel are electrophoretically transferred to sheets of nitrocellulose (Bio-Rad Laboratories, Hercules, CA) using a semi dry blotter (Integrated Separation Systems, Natick, MA).
  • TBS- Tween Tris-buffered saline containing 0.1% Tween-20
  • TBS-Tween Tris-buffered saline containing 0.1% Tween-20
  • Tris-HCI pH 7.4
  • 137 mM NaCI 0.1% Tween-20
  • the sheets are then washed with TBS- Tween (once for 15 minutes and then three times at 5 minutes each) and incubated with a solution of the mouse anti- ⁇ 2u-globulin monoclonal antibodies (1:5000 dilution with 2% bovine serum albumin (BSA) in TBS- Tween) for 60 minutes at 37°C.
  • BSA bovine serum albumin
  • the sheets are then washed with TBS- Tween (once for 15 minutes and then three times at 5 minutes each) and incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG (heavy + light chain) antibody (Pierce Chemical Co., Rockford, IL or equivalent) with 2% BSA in TBS-Tween (1:5000 dilution) for 60 minutes at 37°C.
  • the sheets are washed with TBS-Tween (once for 15 minutes and then three times at 5 minutes each) and placed into a protective cover.
  • Atto- PhosTM substrate reagent JBL Scientific, Huntington, England
  • Detection of fluorescence activity is performed on a Vistra Fluorescence Fluorlmager SI (or equivalent) interfaced to a Dell Dimension XPS P90 computer with Image QuaNTTM software (Molecular Dynamics, Inc. Austin, TX).
  • 90 ⁇ l of HPLC-purified ⁇ 2u-globulin in 1 mM Tris-HCI (pH 7.4) are desalted thrice with 1.0 mL of deionized water using sterile ultra spin cellulose triacetate 10,000 amu molecular weight cutoff filters (Alltech Assoc. Inc. Deerfield, IL).
  • the filters are centrifuged using a Microcentrifuge (Alltech Assoc, Inc., Deerfield, IL, or equivalent) operating at 2,000 g. Material retained on the filter is resuspended in 100 ⁇ L of methanol: water (1:1) containing 1% acetic acid yielding a final concentration of 900 ⁇ g/mL.
  • the spectra are acquired with a Finnigan MAT TSQ 7000 (Finnigan MAT, San Jose, CA or equivalent) triple quadrupole mass spectrometer equipped with a Finnigan atmospheric pressure electrospray ionization (ESI) source.
  • the sample is infused into the source by a syringe pump (Model 22, Harvard Apparatus, South Natick, MA) at a flow rate of approximately 2 ⁇ L/minutes.
  • the electrospray voltage is set at 3.9 kV, the heated capillary is operated at 200°C and nitrogen is used as a sheath gas at 55 psi.
  • the quadrupole manifold and metal ESI ion inlet capillary are heated to 70°C and 200°C.
  • the mass spectrometer is initially tuned and calibrated using a solution of myoglobin (25 ⁇ g/mL).
  • the quadrupoles are scanned over the mass range from 1000 to 2500 amu with a scan time of 1.9 seconds per scan.
  • Bioworks software (Finnigan MAT, San Jose, CA) is used for deconvolution of the charge envelop.
  • the molecular mass of o&j-globulin is 18,730 daltons.
  • Mouse (or other suitable host species) anti- ⁇ 2u-globulin monoclonal antibody (or polyclonal antiserum) is also required in the preferred embodiment of the present invention.
  • Mouse anti- ⁇ 2u-globulin monoclonal antibody is prepared by methods known by those skilled in the art using ⁇ 2u- globulin extracted and purified from rat urine (see, for example, methods described in Cellular and Molecular Immunology, Abbas et al., 1991; Monoclonal Antibodies: Principals and Practice, Goding, 1996; Purification Tools for Monoclonal Antibodies, Gagnon, 1996.
  • the alkaline phosphatase- conjugated goat anti-mouse IgG is a commercially available product.
  • a competitive indirect ELISA initially requires optimization of experimental conditions using a reagent dilution assay.
  • the reagent dilution assay performed whenever a new preparation of antibodies or antigen is used, is conducted to determine: (i) the optimal dilution of antigen for coating wells of microtiter plates; and (ii) the dilution at which an ELISA reading of 50% maximum value occurs for optimal interactions in subsequent competition experiments.
  • Measurement of ⁇ 2u-globulin is then accomplished by a competitive inhibition experiment wherein reactions are prepared that involve a competition for antibodies between oau-globulin adsorbed to wells of microtiter plates and free ⁇ 2u-globulin in solution.
  • the assay is calibrated by comparison of the ELISA reading obtained upon measurement of a sample to that obtained upon measurement of known concentrations of ⁇ 2u-globulin in a standard curve.
  • the following is a description of a preferred method for conducting the required reagent dilution assay in accordance with the present invention.
  • a typical 96-well microtiter plate consists of eight stacked, horizontal rows of wells (labeled A-H) each row consisting of 12 individual wells organized into vertical columns.
  • the wells in rows B, C, D, E, F, and G of U-bottom shaped MicroFluor 96 well microtiter plates are equilibrated overnight at 4°C with 100 ⁇ L aliquots of purified ⁇ 2u-globulin prepared at target concentrations of 100, 250, 500, 750, 1000, and 2000 ng/mL in phosphate buffered saline (PBS, 0.14 M NaCI, 1.0 M KH 2 P0 4 , 8 mM Na 2 HP0 4 , 3 mM KCI, pH 7.4).
  • Negative controls are established in rows A and H which are filled with 100 ⁇ L aliquots of PBS, and PBS/0.05% Tween containing
  • the plates are washed by alternately filling and emptying each well five times with PBS-Tween.
  • the washing step is accomplished manually using a plastic squirt bottle containing PBS-Tween or an automated microtiter plate washer (i.e., Dynatech Ultrawash Plus or equivalent).
  • An automated microtiter plate washer i.e., Dynatech Ultrawash Plus or equivalent.
  • Two hundred microliter aliquots of PBS-Tween containing 1% bovine serum albumin (BSA) are added to each well of the plate and allowed to equilibrate for 2 hours at 37°C in an effort to reduce non-specific binding of primary and secondary antibodies in subsequent stages of the experiment.
  • BSA bovine serum albumin
  • Serial dilutions of the primary antibody e.g., mouse anti-c ⁇ u-globulin monoclonal antibody diluted from 1:2000 to 1:10 8
  • the primary antibody e.g., mouse anti-c ⁇ u-globulin monoclonal antibody diluted from 1:2000 to 1:10 8
  • 100 ⁇ L of each diluted solution is added to the wells of columns 1 through 12 of the microtiter plate after washing the wells five times with PBS-Tween.
  • 100 ⁇ L of an alkaline phosphatase conjugated goat anti-mouse IgG (heavy and light chain) secondary antibody (1:1000 dilution in PBS-Tween containing 1% BSA) is added to all wells.
  • the secondary antibody conjugate is allowed to equilibrate at 37°C for 2 hour and then the wells of the plate are washed five times with PBS-Tween and twice with 50 mM 2-amino-2-methyl-l,3-propanediol containing 0.01% BSA.
  • One hundred microliters of 0.1 mM 4-methylumbelliferyl phosphate (MUP) prepared in 50 mM 2-amino-2-methyl-l,3-propanediol containing 0.01% BSA is added to the wells and incubated for 30 minutes at room temperature.
  • the fluorescence intensity is measured using a microplate reader (such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA) using a 360 nm/460 nm excitation/emission filter pair.
  • the minimum concentration of ⁇ 2u-globulin required to coat the wells of the microtiter plates is 750 ng/mL.
  • Lower concentrations of ⁇ 2u-globulin in the coating solution lead to a decrease in the maximum relative fluorescence intensity observed at various dilutions of antibody. Therefore, the preferred methods require a concentration of 2000 ng/mL to coat the wells of microtiter plates for subsequent ELISA measurements. This concentration is suitable over a wide range of antibody dilutions.
  • the preferred range of the analytical method is based on analysis of the available scientific literature concerning accumulation of ⁇ 2u-globulin in kidney samples following exposure to chemical agents such as unleaded gasoline, 2,2,4-trimethylpentane, 1,4 dichlorocbenzene, isophorone, decalin and o imonene (Swenberg et al., Toxicol. Appl. Pharmacol., 97: 35-46, 1989; Borghoff et al., Toxicol. Appl. Pharmacol., 107: 228-238, 1991; Flamm and Lehman-McKeeman, Regul. Toxicol.
  • the preferred method performance evaluation is designed to permit measurements of ⁇ 2u-globulin ranging from about 0.005 to 1.0 ng/ng total soluble protein in kidney homogenates.
  • the present invention uses a calibration curve spiked with soluble female rat kidney homogenate when study samples are analyzed, and measures sufficient blank female kidney homogenate samples to ensure that ⁇ 2u-globulin concentrations in the matrix are below the detection limits of the ELISA.
  • Spiked Tissue Standards Blank female rat kidney homogenates are used as a matrix in calibration standards and QC samples.
  • kidneys from female rats are removed and homogenized in 4X (w/v) ice-cold 67 mM sodium/potassium phosphate buffer (pH 7.2) in a 50-mL polypropylene centrifuge tube using an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH).
  • the homogenate is centrifuged in a Beckman T6-J table top refrigerated centrifuge at a setting of 10 for 15 minutes at 4°C. Following centrifugation, the supernatant is removed and stored at -70°C.
  • kidney homogenate Prior to ELISA, the kidney homogenate is freshly diluted 1:50 with 67 mM sodium/potassium phosphate buffer, and total protein content is determined using Pierce BCA reagent (Pierce Chemical Co., Rockford, IL) with bovine serum albumin (BSA) as the standard. Aliquots containing 500 ng of female rat kidney homogenate are then dispensed into each of several clean, 5.0-mL polypropylene culture tubes. These samples are then spiked with 1.0, 2.5, 5.0, 10, 25, 50, 100, 250, 500, 1000, and 2500 ng of ⁇ 2u-globulin and the volume of each standard solution brought to 1.000 mL with PBS-Tween. Every other calibration standard is prepared from an alternate, independently prepared stock solution of ⁇ 2u-globulin.
  • Positive Tissue Controls In a preferred method for generating positive control tissue samples, male Sprague-Dawley rats (Taconic Farms, Germantown, PA) 10 to 12 weeks of age are gavaged (force-fed) with either corn oil (control group), 100, or 200 mg/kg of decalin in corn oil for four consecutive days at a volume of 5 mL/kg. On day five, rats are anesthetized with carbon dioxide, exsanginated, and their kidneys immediately excised. The dissected kidneys are weighed and then immediately quick-frozen in liquid nitrogen. The kidneys are stored frozen at -70°C until homogenized.
  • Homogenized is conducted in 2X (weight to volume) ice-cold 67 mM sodium/potassium phosphate buffer (pH 7.2) in a 50-mL polypropylene centrifuge tube using an Ultra-Turrax (Tekmar Co, Cincinnati, OH) homogenizer. Between samples, the homogenizer probe is rinsed with 3 changes each of water, ethanol, and buffer prior to the next sample. The homogenates are centrifuged in a Beckman T6-J table top refrigerated centrifuge at a setting of 10 for 15 minutes at 4°C. Following centrifugation, the supernatant is removed and stored at minus 70°C.
  • kidney homogenates Prior to the ELISA, kidney homogenates are freshly diluted 1:50 with 67 mM sodium/potassium phosphate buffer and total protein content is determined using Pierce BCA reagents (Pierce Chemical Co., Rockford, IL) with bovine serum albumin (BSA) as the standard.
  • ELISA is performed on kidney homogenates freshly diluted to 2.5 ⁇ g/mL total protein in PBS-Tween. The concentration of ⁇ 2u-globulin observed in kidney homogenates from male rats administered corn oil mixtures containing up to 200 mg/kg of decalin over the course of four consecutive days increases linearly as a function of decalin exposure.
  • Kidney homogenates from male rats receiving only corn oil are found to have 0.0056 ng ⁇ 2u-globulin /ng soluble kidney tissue protein.
  • level of ⁇ 2u-globulin increased linearly with doses of up to 200 mg/kg, but that oral administration of 400 mg/kg decalin resulted in a significantly lower concentration of ⁇ 2u-globulin (0.030 ng/ng protein) than observed at a dose of 200 mg/kg.
  • ELISA Competitive Inhibition Assay A preferred method for measuring ⁇ 2u-globulin in standards, QC samples, and unknown samples (all analyses are conducted at least in triplicate) during the competitive inhibition experiment initially involves coating the wells of a CytoFluor flat-bottomed 96 well microtiter plate (Plate 1: Biosearch Instruments, Millipore Corp., Bedford, MA or equivalent) with 200 ⁇ L of PBS-Tween overnight at 4°C.
  • Plate 1 is then used for incubation of free antigen with primary antibody prior to addition of this solution to a second microtiter plate which, in the preferred method is a black U-bottom shaped MicroFluor 96 well microtiter plate (Plate 2: Dynatech Laboratories, Inc., Chantilly, VA or equivalent) that is previously incubated overnight at 4°C with 2000 ng ⁇ 2u-globulin per mL of PBS.
  • the optimal concentration of oc2u- globulin is determined from the reagent dilution assay, and several wells on each microtiter plate should be incubated with PBS alone to serve as negative control wells for assessing non-specific binding of reagents.
  • Plate 1 is washed three times with PBS-Tween and 60 ⁇ L aliquots of matrix-spiked kidney homogenate standards, positive and negative control samples and blanks, and either solvent standards or samples containing unknown amounts of ⁇ 2u-globulin, are added to designated wells of the microtiter plates. To these samples, 60 ⁇ L of the primary antibody prepared in PBS-Tween is added at a dilution which yields 50% maximum ELISA readings.
  • Calibration curves are performed in triplicate on each ELISA plate using a wide range of concentrations of ⁇ 2u-globulin to ensure that relative fluorescence measurements plateau at either extreme. Samples spiked with 1000 and 2500 ng/mL of ⁇ 2u-globulin tend to almost completely inhibit fluorescence activity in competition experiments. Conversely, ⁇ 2u-globulin concentrations below 2.5 ng/mL are insufficient to produce a fluorescence value different from blank samples. Samples in the high and low ends of the calibration curve plateau, falling off the log-linear relationship, and are thus useful as one type of assay control.
  • More explicit negative controls include female kidney homogenates diluted to the exact concentration as are the spiked samples, wells that contain only primary antibody, male rat kidney homogenates diluted tenfold less than used for measurements, and wells not containing oau-globulin adsorbed to the microtiter plate.
  • the optimal dilution of stock primary antibody is made in PBS-Tween to achieve 50% of the maximum value.
  • Positive controls should be established in several wells containing only primary antibodies, PBS-Tween buffer spiked with a diluted solution of female kidney homogenate. Plate 1 (containing the primary antibody and antigen in solution) is incubated for 2 hours at 37°C. During this same time period, the wells of Plate 2 are incubated with 200 ⁇ L of PBS-Tween containing 1% BSA in an effort to reduce non-specific binding of primary and secondary antibodies during subsequent phases of the assay.
  • each well of plate 2 is washed five times with PBS-Tween.
  • the washing step is accomplished manually using a plastic squirt bottle containing PBS-Tween or an automated microtiter plate washer (i.e., Dynatech Ultrawash Plus or equivalent).
  • 100 ⁇ L from each well in Plate 1 is directly transferred to the corresponding wells in Plate 2.
  • the competition reaction initiated in Plate 2 is then allowed to proceed at 37°C for 2 hours after which the wells are washed five times with PBS-Tween.
  • MUP 4-methylumbelliferyl phosphate
  • 2-amino-2-methyl-l,3-propanediol containing 0.01 % BSA is added to the wells and incubated for 30 minutes at room temperature.
  • the fluorescence intensity is measured using a microplate reader (such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA) using a 360 nm/460 nm excitation/emission filter pair.
  • a characteristic feature of ELISA calibration data is the log-linear relationship of the instrument response as a function of inhibitor concentration.
  • the analyst may decide to modify the concentrations of ⁇ 2u-globulin in the calibration standards to achieve a set of conditions where all calibration standards are used for the ultimate generation of a standard curve.
  • the sensitivity obtained with ELISA is not only dependent on the type and design of the assay, the enzyme used and how its activity is measured, but also to a great extent on the affinity of the antibodies used in the assay.
  • the measurement of ⁇ 2u-globulin in rat kidney homogenates using an ELISA includes the following general steps (1) preparation of buffers and reagents; (2) preparation of samples; (3) completion of a total protein assay; (4) completion of a reagent dilution assay; and (5) completion of the ELISA competitive inhibition assay and calculation of results. These steps are discussed in greater detail below.
  • Buffers and Reagents First prepare the homogenization buffers as follows: (1) 0.106 M sodium phosphate dibasic buffer; (2) 2 M potassium phosphate monobasic buffer; and sodium/potassium phosphate buffer, pH 7. Next prepare the ELISA reagents as follows: (1) Phosphate Buffered Saline (PBS, Coating Buffer), pH 7.2; (2) PBS-Tween Washing Buffer (PBS-Tween), pH 7.2; (3) Blocking Reagent consisting of 1% BSA in PBS; (4) 4-Methylumbelliferyl phosphate (MUP) Dilution Buffer: 50 mM 2-amino-2-methyl-l,3-propanediol buffer containing 0.01% (w/v) BSA; (5) 2000 ng ⁇ 2u -globulin per mL of PBS Stock Antigen (for plate coating): purified stock ⁇ 2u-globulin; (6) secondary antibody solution: for each plate to be ass
  • Kidney Homogenization (1) thaw and weigh kidney samples; transfer samples to 50-mL polypropylene culture tubes; (2) homogenize kidney tissue in 2X weight/volume sodium/potassium phosphate buffer (pH 7.2) using an Ultra-Turrax homogenizer (Tekmar Co.); between samples, rinse the homogenizer in succession with water (changed after each sample), ethanol, and water; (3) pellet homogenized tissue in refrigerated centrifuge; (4) Following centrifugation, remove the supernatant and store the supernatant at -70°C; (5) make appropriate dilutions (e.g. 1:10, 1:50, 1:10,000, etc.).
  • Total Protein Assay Perform a total protein determination on an appropriate dilution of the kidney homogenates (i.e. 1 :50 dilution) using the BCA assay or a similar assay.
  • Reagent Dilution Assay The reagent dilution assay is used to determine optimal concentrations of reagents for subsequent competitive inhibition assays. The overall strategy is to optimize the concentrations of both the antigen and antibody in the reagent dilution phase. Use these results to select appropriate concentrations of antigen and antibody for competitive inhibition experiments. Follow this procedure for each new lot of primary antibody and whenever optimal reagent concentrations are in question.
  • Coating Solution Preparation Prepare 4.0 mL of solutions containing ⁇ 2u-globulin at concentrations of 40, 100, 200, 300, 400, 800 ng/mL for coating ELISA plate wells (100 ⁇ L of appropriate solution per well) using a 2000 ng ⁇ 2u-globulin per mL of PBS stock antigen solution.
  • Primary Antibody Solution Preparation-.
  • the current version of the anti- ⁇ 2u-globulin monoclonal antibody is diluted with PBS-Tween to final dilutions ranging from 1:2.5 x 10 3 to 1:1.0 x 10 8 .
  • Day 1 (1) Add 100 ⁇ L of ⁇ 2u-globulin (4 ng to 80 ng per well) to designated wells of a black pigmented styrene U-Bottom shaped 96-well microtiter plate. Include negative control wells containing PBS-Tween (no ⁇ 2u-globulin). Let this plate sit overnight at 4°C in a covered plastic container to prevent evaporation. This step allows sufficient time for the antigen to bind non-specifically to the plastic wells. Day 2: (2) wash each well with PBS-Tween (A Dynex Ultrawash Plus plate washer, or equivalent may be used if available).
  • PBS-Tween A Dynex Ultrawash Plus plate washer, or equivalent may be used if available.
  • the wash cycle includes three repeats of the following sequence: wells filled with about 300 ⁇ L of PBS-Tween, soaked for 10 seconds and emptied under vacuum; (3) in the blocking step, add 200 ⁇ L of PBS-Tween containing 1% BSA to each well of the plate and incubate for 90 minutes at 37 ⁇ 2°C. The wells in the plate are now adequately blocked with 1% BSA in an effort to reduce non-specific binding of primary and secondary antibodies; (4) repeat washing steps described in (2); (5) add 100 ⁇ l of each preparation of the serial dilution of primary antibody (range of l:2.5xl0 3 to 1:1.0 x 10 8 ) in PBS-Tween to the microtiter plate.
  • An enzyme, alkaline phosphatase, is coupled to the secondary antibody that is directed toward mouse IgG.
  • this incubation step provides the secondary antibody with sufficient time to bind to the primary antibody; (8) Repeat washing steps described in (2); (9) Wash wells two times with the MUP dilution buffer, draining plate after each wash; (10) add 100 ⁇ L of 0.1 mM MUP solution to each well.
  • MUP is converted to umbelliferyl phosphate (UP) by alkaline phosphatase; (11) read the initial fluorescence at excitation 360 nm and emission 460 nm using a microplate reader (such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA), then hold the plate at room temperature and read fluorescence at about 15 minutes, about 30 minutes and beyond if level of fluorescence continues to change significantly.
  • a microplate reader such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA
  • Female Rat Kidney Matrix Dilute a homogenate prepared from an untreated female rat kidney to an appropriate dilution (i.e., range of 1:10 to 1:10,000 depending on the dilution of kidney tissue require for analysis of a set of experimental samples) with PBS-Tween for use as negative controls and as the blank matrix for use in preparation of calibration standards;
  • Standard Curve Preparation Prepare a 10 mL working solution with a final concentration of 5000 ng/mL oau-globulin by diluting the appropriate volume of purified ⁇ 2u-globulin stock to 10.0 mL with PBS-Tween.
  • Sample analysis is conducted on 96-well microtiter plates, with wells designated as negative controls in which no antigen will be adsorbed to the walls of the wells and wells designated as positive controls in which only antibody diluted in matrix is added.
  • Appropriate spiked matrix standards typically 2.5 to 2,500 ng/mL
  • containing an appropriate amount of female kidney homogenate are also analyzed in triplicate for calibration purposes.
  • the remaining wells on the plate are used for analysis of samples, typically in triplicate.
  • Day 1 (1) coat each well of a flat-bottomed clear methacrylate 96-well microtiter plate (Clear Plate) with 200 ⁇ L of 1% BSA/PBS-Tween. Let plate stand overnight at about 4°C in a covered plastic container to prevent evaporation; (2) add 100 ⁇ L of 2000 ng/mL ⁇ 2u-globulin coating solution to the appropriate wells of a black U-Bottom shaped pigmented styrene 96-well microtiter plate (Black Plate). Add 100 mL PBS to uncoated wells for use as negative controls. Let plate stand overnight at about 4°C in a covered plastic container to prevent evaporation.
  • Day 2 (3) thaw standards, primary antibody, matrix, and samples; (4) wash Clear Plate with PBS-Tween.
  • the wash cycle includes three repeats of the following sequence: wells filled with about 300 ⁇ L of PBS-Tween, soaked for 10 seconds and emptied under vacuum; (5) add 60 ⁇ L of appropriate dilutions of samples, standards, or kidney matrix into the clear plate. Kidney matrix is added to both positive (antigen coated) and negative (PBS coated) control wells (in reference to sample positions on Black Plates).
  • An ⁇ 2u- globulin calibration curve prepared at least at eight concentration levels between 2.5 to 2500 pg/mL will also be included; (6) on the Clear Plate, add 60 ⁇ L of an appropriate dilution of primary antibody (determined in Reagent Dilution Assay) prepared in PBS-Tween to each well; (7) incubate Clear Plate containing the antibody and antigen in solution for 90 minutes at 37 ⁇ 2°C. The Clear Plate holds up to 96 different reactions involving binding of the antibodies to "free" antigen in solution; (8) transfer ⁇ 2u-globulin from wells in Black Plate to labeled plastic trough to be saved for coating the next day's plate(s).
  • ⁇ 2u-globulin solution may be used to coat up to 3 plates on subsequent days. Discard solution if this cannot be accomplished in that time frame. Wash Black Plate as in Step (4); (9) to the Black Plate, add 200 ⁇ L of PBS-Tween containing 1% BSA to each well and incubate for 90 minutes at 37 ⁇ 2°C.
  • the secondary antibody incubation step is performed by adding 100 ⁇ L of the secondary antibody preparation to each well and incubating the microtiter plate for 90 minutes at 37 ⁇ 2°C.
  • An enzyme, alkaline phosphatase is coupled to the secondary antibody that is directed toward mouse IgG. Therefore this incubation step enables secondary antibody to bind to the primary antibody;
  • Step (14) Repeat washing steps described in Step (4) after 90 minute incubation in Step (13) is complete; (14) Wash wells manually two times with the MUP buffer, draining plate after each wash; (15) Add 100 ⁇ L of 0.1 mM MUP buffer solution to each well of Black Plate. MUP is converted to umbelliferyl phosphate (UP) by alkaline phosphatase. Read the initial fluorescence at excitation 360 nm and emission 460 nm using a microplate reader (such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA), then hold the plate at room temperature and read fluorescence at about 15 minutes, about 30 minutes and 60 minutes.
  • a microplate reader such as the Cytofluor II Microplate Fluorescence Reader Biosearch Instruments, Millipore Corp., Bedford, MA
  • nmol ⁇ 2u-globulin /g kidney (ng ⁇ 2u-globulin / ⁇ g Soluble Protein) * (lxlO 6 ⁇ g Soluble Protein/1 g Soluble Protein) * (lg ⁇ 2u-globulin / lxlO 9 ng ⁇ 2u-gIobulin) * (g Soluble Protein / 100 mL kidney supernatant) * (1 mL kidney supernatant / 1 g kidney supernatant) * (2 g kidney supernatant / lg kidney tissue) * (mol ⁇ 2u-globulin /18,730 g ⁇ 2u-globulin) * (lxlO 9 nmol ⁇ 2u-globulin / 1 mol ⁇ 2u-globulin).

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Abstract

La présente invention concerne une trousse de dosage immunochimique et une méthodologie associée utilisées pour identifier la néphropathie induite par la α2u-globuline dans des études portant sur la toxicité/cancérogénicité chez les rongeurs. Cette trousse comprend : un premier anticorps de liaison à la α2u-globuline; un deuxième anticorps conjugué à une enzyme prévu pour se lier au premier anticorps; un substrat chimique destiné à créer un changement décelable lorsqu'il se trouve en contact avec le deuxième anticorps conjugué à une enzyme; un premier réservoir servant à mettre en contact la α2u-globuline avec le premier anticorps; un deuxième réservoir servant à mettre en contact le premier anticorps, le deuxième anticorps conjugué à une enzyme et le substrat; des échantillons test positifs et négatifs; et de la α2u-globuline purifiée. La présente invention concerne également des procédés de préparation de tampons et de réactifs; la préparation d'échantillons test et d'échantillons inconnus; la réalisation de dosages de protéines brutes et de dosages de dilutions de réactifs; l'exécution d'un dosage immunoenzymatique par compétition (ELISA) sur des échantillons test et des échantillons inconnus; et le calcul des résultats.
PCT/US2001/022906 2000-07-19 2001-07-19 Dosage utile pour identifier la nephropathie induite par la $g(a)2uglobuline WO2002006346A1 (fr)

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AU2001280651A AU2001280651A1 (en) 2000-07-19 2001-07-19 Assay for identifying theta2u-globulin-mediated nephropathy

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114018882A (zh) * 2021-10-27 2022-02-08 浙江理工大学 一种基于流式荧光技术的同步检测吗啡和冰毒的检测方法及试剂盒

Citations (4)

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Publication number Priority date Publication date Assignee Title
US5139932A (en) * 1987-05-08 1992-08-18 Biocarb Ab Method and a kit for the diagnosis of iga nephropathy
JPH05333025A (ja) * 1992-05-28 1993-12-17 Sumitomo Chem Co Ltd α２ｕ− グロブリン腎症の早期診断マーカー
JPH06118076A (ja) * 1992-10-08 1994-04-28 Sumitomo Chem Co Ltd 毒性試験方法
US5534431A (en) * 1994-02-28 1996-07-09 The Board Of Regents Of The University Of Nebraska Hybridomas and monoclonal antibodies specific for unique determinants of nephropathy-related immunoglobulin G and complexes thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5139932A (en) * 1987-05-08 1992-08-18 Biocarb Ab Method and a kit for the diagnosis of iga nephropathy
JPH05333025A (ja) * 1992-05-28 1993-12-17 Sumitomo Chem Co Ltd α２ｕ− グロブリン腎症の早期診断マーカー
JPH06118076A (ja) * 1992-10-08 1994-04-28 Sumitomo Chem Co Ltd 毒性試験方法
US5534431A (en) * 1994-02-28 1996-07-09 The Board Of Regents Of The University Of Nebraska Hybridomas and monoclonal antibodies specific for unique determinants of nephropathy-related immunoglobulin G and complexes thereof
US5654158A (en) * 1994-02-28 1997-08-05 Board Of Regents Of The University Of Nebraska Methods for detection of nephropathy-related immunoglobulin G using monoclonal antibodies specific for nephropathy-related immunoglobulin G

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Title
PATENT ABSTRACTS OF JAPAN vol. 018, no. 162 (P - 1712) 17 March 1994 (1994-03-17) *
PATENT ABSTRACTS OF JAPAN vol. 018, no. 399 (P - 1776) 26 July 1994 (1994-07-26) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114018882A (zh) * 2021-10-27 2022-02-08 浙江理工大学 一种基于流式荧光技术的同步检测吗啡和冰毒的检测方法及试剂盒

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