WO2003104421A2 - Dosage immunocapteur et fonctionnel pour atpase f1/f0 mitochondriale - Google Patents

Dosage immunocapteur et fonctionnel pour atpase f1/f0 mitochondriale Download PDF

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WO2003104421A2
WO2003104421A2 PCT/US2003/018114 US0318114W WO03104421A2 WO 2003104421 A2 WO2003104421 A2 WO 2003104421A2 US 0318114 W US0318114 W US 0318114W WO 03104421 A2 WO03104421 A2 WO 03104421A2
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atpase
sample
activity
patient
subunits
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PCT/US2003/018114
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WO2003104421A3 (fr
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Roderick A. Capaldi
Michael F. Marusich
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The State Of Oregon, Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon
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Priority to AU2003245425A priority Critical patent/AU2003245425A1/en
Publication of WO2003104421A2 publication Critical patent/WO2003104421A2/fr
Publication of WO2003104421A3 publication Critical patent/WO2003104421A3/fr
Priority to US10/997,819 priority patent/US20050153381A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • This invention relates to immunoassays, in particular, to immunoassays for determining disorders of mitochondrial energy metabolism and diseases associated with late onset mitochondrial disorders.
  • Oxidative phosphorylation Oxidative phosphorylation
  • a typical adult human utilizes approximately 50 kg of ATP per day under normal activity levels, requiring roughly a lOOOx turnover of the 50 g of ATP/ADP present in the body.
  • the Fl/FO-type ATPase also known as ATPsynthase and Complex V of OXPHOS, produces the great majority of this ATP in mitochondria through the process of oxidative phosphorylation.
  • Fl/FO ATPase is composed of two parts, the F 0 , which is an integral membrane complex that functions as a proton pore, and F 1; which can catalyze ATP hydrolysis.
  • F 0 is an integral membrane complex that functions as a proton pore
  • F 1 which can catalyze ATP hydrolysis.
  • the F 0 complex is a proton channel and can let the protons run down the concentration gradient, releasing free energy.
  • the close association of Ft with Fo causes the released free energy to be used to reverse the hydrolysis of ATP, with the net synthesis of ATP from ADP and Pi.
  • the proton channel can be blocked by the classic inhibitor of the enzyme, oligomycin.
  • Fl/FO ATPase functions as a small molecular motor in which catalytic site events in the Fi part are coupled to proton translocation in the F 0 part by rotation of a mobile domain or "crankshaft" within the protein complex.
  • JF ⁇ an inhibitor protein
  • JF ⁇ Its binding to Fl/FO ATPase depends on pH. Below neutrality, JF ⁇ is dimeric and forms a stable complex with the enzyme. At higher pH values, for example 8.0, or 8.5, JF ⁇ forms tetramers and is inactive. Tetramer formation masks the inhibitory region, preventing binding of IF i to ATP synthase.
  • the mammalian form of this enzyme has been extensively studied, using beef heart and rat liver as a source. It is complex of 16 different subunits with ⁇ 3 ⁇ 3 ⁇ and ⁇ comprising the Fi part and a, b, c, d, e,f g, A6L, OSCP and coupling factor 6 providing the F 0 and stator (Capaldi R.A., and Aggeler, R. (2002) Trends Biochem. Sci. 27, 154-160 and Ko, Y. H., et al. (2000) J. Biol. Chem. 275, 32931-32939). Also associated in the complex at physiological pH, is an intrinsic inhibitor protein JFi (Ko, Y. H., et al. (2000) J. Biol. Chem. 275, 32931-32939 and Karrash, S., and Walker, J. E. (1999) J. Mol Viol. 290, 379-384).
  • the invention is based on the seminal discovery of a monoclonal antibody against human F ⁇ that can be used to isolate active human mitochondrial Fl/FO ATPase in a one step immunological assay.
  • the immunoprecipitated Fl/FO ATPase contains a full complement of subunits that have been identified with specific antibodies against five of the subunits combined with analysis using mass spectroscopy studies.
  • the invention provides methods for determining the amount of Fl/FO ATPase of the mitochondrial respiratory chain (Fl/FO ATPase) in a biological sample of a mammalian patient by contacting an isolated antibody that specifically immunoprecipitates Fl/FO ATPase with a sample comprising solubilized Fl/FO ATPase so that the antibody binds to Fl/FO ATPase present in the sample to form an antibody/Fl/FO ATPase complex. Remaining sample contents are separated from the antibody/Fl/FO ATPase complex and the amount of Fl/FO ATPase in the antibody/Fl/FO ATPase complex in the sample is determined.
  • the invention provides methods for detecting Fl/FO ATPase deficiency in a patient by contacting a monoclonal antibody specific for each of a plurality of Fl/FO ATPase subunits with a patient sample so that the antibodies immunocapture Fl/FO ATPase subunits present in the sample, wherein the antibodies are each tagged with a detectable label.
  • the amount of each Fl/FO ATPase subunit immunocaptured by a respective Fl/FO ATPase subunit antibody is detected; and the amount of each of the Fl/FO ATPase subunits is compared with an amount thereof present in a corresponding normal sample.
  • a decrease in the amount of any of the Fl/FO ATPase subunits in the patient sample as compared to the normal sample indicates the presence of a Fl/FO ATPase deficiency in the patient.
  • the invention provides methods for detecting Fl/FO ATPase deficiency in a patient by contacting monoclonal antibodies specific for each of a plurality of Fl/FO ATPase subunits with a patient sample so that the antibodies immunocapture Fl/FO ATPase subunits present in the sample, wherein the antibodies are each tagged with a detectable label.
  • the amount of each Fl/FO ATPase subunit immunocaptured by a respective Fl/FO ATPase subunit antibody is then determined and the amount of each of the Fl/FO ATPase subunits is compared with an amount thereof present in a corresponding normal sample, wherein a decrease in the amount of any of the Fl/FO ATPase subunits in the patient sample as compared to the normal sample indicates the presence of a Fl/FO ATPase deficiency in the patient.
  • kits for assaying Fl/FO ATPase activity in a sample that contain a plurality of separate solid supports, each coated with a monoclonal antibody specific for a Fl/FO ATPase Fi unit or a subunit of Fl/FO ATPase subunit selected from CN- ⁇ , CN- ⁇ , CV-d, CV-OSCP, CV-IFi subunits of normal Fl/FO ATPase.
  • the invention provides screening methods for identifying an agent that causes a mitochondrial disorder.
  • agent screening method samples containing cells are contacted with one or more anti-Fl/FO ATPase monoclonal antibodies in the presence and absence of the agent so as to allow interaction between the agent, Fl/FO ATPase in the cells, and the antibodies; and Fl/FO ATPase activity is determined separately in each sample, wherein a lower level of activity in the sample in the presence of the agent indicates that the agent than in the absence of the agent causes a mitochondrial disorder.
  • the invention provides methods for screening patients to identify patients suspected of having a late onset mitochondrial disorder.
  • isolated antibodies that immunoprecipitate Fl/FO ATPase subunits are contacted with a patient sample comprising solubilized Fl/FO ATPase subunits so that the antibodies bind to Fl/FO ATPase subunits present in the sample to form an antibody/Fl/FO ATPase subunit complex.
  • the invention provides methods for screening patients for a condition associated with a Fl/FO ATPase function.
  • an isolated antibody that immunoprecipitates active Fl/FO ATPase is contacted with a patient sample comprising solubilized Fl/FO ATPase so that the antibody binds to Fl/FO ATPase present in the sample to form an antibody/Fl/FO ATPase complex.
  • the Fl/FO ATPase is obtained from the remaining sample contents; and the Fl/FO ATPase is contacted with oligomycin under conditions suitable to allow interaction. Then a decrease in production of inorganic phosphate in the presence of the oligomycin is detected as compared to in absence of the oligomycin, wherein the decrease in ATP hydrolysis activity indicates function of the Fl/FO ATPase and absence of the decrease indicates a defect in Fl/FO ATPase function of the patient.
  • the invention provides methods for identifying a modulator of JF ⁇ inhibition of Fl/FO ATPase activity by contacting a sample containing Fl/FO ATPase in the presence of JFi and a test compound under conditions suitable to allow interaction at a pH in the range from about 6.0 to about 7.0; and determining a change in ATP hydrolysis activity caused by the presence of the test compound as compared to in the absence of the test compound. A change indicates the test compound modulates JFi inhibition of Fl/FO ATPase in the sample.
  • FIG. 1 is a graph showing human Fl/FO ATPase immunocaptured by the anti- Fl/FO ATPase mAb 12F4AD8AF8 is active as an ATPase and is sensitive to oligomycin inhibition.
  • Human heart mitochondria were solubilized, captured and ATPase activity measured all at pH 7.5 All measurements were done in triplicate and the data points show the specific signal (ATPase activity) captured by the anti-Fl/FO ATPase mAb minus the background signal captured by a null antibody. Activities were measured in the absence (•) and presence of 1.5 ⁇ g/ml oligomycin (D), respectively.
  • Figures 2A and 2B are graphs showing ATP hydrolysis activity of immunocaptured mitochondrial Fl/FO ATPase from human heart at low and high pH.
  • Figure 2A shows activity of human heart Fl/FO ATPase when solubilized, immunocaptured and assayed at pH 6.5.
  • Figure 2B shows human heart Fl/FO ATPase activity when solubilized, immunocaptured and assayed at pH 7.5.
  • Added IFi remains bound to Fl/FO ATPase during activity measurements carried out at pH 6.5, but can be subsequently stripped (strip JF ) from the enzyme by brief treatment at pH 8 Bars show the average with standard deviations, of samples tested in triplicate.
  • Figures 3A and 3B are graphs showing immunocapture purifies human fibroblast mitochondrial Fl/FO ATPase from the bulk of total cellular ATPase enzymatic activities.
  • Figure 3A shows detergent solubilized cells assayed directly in solution for ATPase activity.
  • Figure 3B shows equivalent samples where the Fl/FO ATPase was immunocaptured on micro well plates before ATPase activity measurements. Activities were measured in duplicate in the absence (O) and presence of 1.5 ⁇ g/ml oligomycin ( ⁇ ), respectively.
  • Figure 4 is a graph showing that immunocapture of Complex V can reveal defects in fibroblast mitochondrial Fl/FO ATPase MF1/F0 ATPase).
  • MF1/F0 ATPase was immunocaptured from human heart, normal fibroblasts and fibroblasts from patient GW28 and assayed in the presence and absence of 1.5 ⁇ g/ml oligomycin. Bars show the average with standard deviations, of samples tested in triplicate at 20 ⁇ g mitochondrial protein per well.
  • FIG. 5 is a graph showing that GM28 fibroblast mitochondria obtained with a patient with Lucas's disease contain sufficient endogenous IFi to almost completely inhibit GM28 mitochondrial Fl/FO ATPase and are indistinguishable from control fibroblasts in this regard.
  • Mitochondrial solubilization as well as Fl/FO ATPase immunocapture and ATPase activity measurements were all done at pH 6.5. Under these conditions, immunocapture Fl/FO ATPase has negligible ATPase activity, due to the association of JFi with the F ⁇ F 0 .
  • Recombinant IFi was added at 5 ⁇ g/ml. Bars show the average with standard deviations, of samples tested in triplicate at 20 ⁇ g mitochondrial protein per well. DETAILED DESCRIPTION OF THE INVENTION
  • Mitochondrial F 1/FO ATPase couples the synthesis of ATP from ADP and inorganic phosphate to a flux of protons down the electrochemical gradient from the intermembrane space to matrix in mitochondria.
  • the enzyme is composed of a membrane-embedded complex, F 0 , which forms a proton channel to which is attached a large water-soluble complex, Ft-ATPase, exposed to the intermembrane space and which has the subunit composition ⁇ 3, ⁇ 3, ⁇ , ⁇ , ⁇ .
  • the classic inhibitor of the enzyme, oligomycin can block the proton channel.
  • Fl/FO ATPase functions as a small molecular motor in which catalytic site events in the F ⁇ part are coupled to proton translocation in the Fo part by rotation of a mobile domain or "crankshaft" within the protein complex.
  • the invention provides a micro scale method for the immunocapture and functional detection of active mitochondrial Fl/FO ATPase from solubilized human mitochondria using a specific anti-human F t monoclonal antibody in an immunocapture format.
  • the assay is suitable for high-throughput screening of samples containing mitochondria, for example obtained from human heart, human brain, human cultured fibroblast or bovine heart.
  • the assay is specific because the captured ATPase activity is oligomycin-sensitive, a characteristic of the mitochondrial Fl/FO ATPase. Irrelevant antibodies fail to capture detectable activity.
  • the assay is also quantitative and can be used to measure the amount of solubilized mitochondrial Fl/FO ATPase in samples relative to a reference control containing a known amount of Fl/FO ATPase, for example.
  • the invention assay can be used to detect disorders in production and or utilization of Fl/FO ATPase in patient samples.
  • the invention assays are also sensitive, requiring as little as 10 nanograms of mitochondrial protein per test, and have a wide dynamic range of at least 1000-fold. For example, when human heart mitochondria are used as a target, the assay is quantitative over a range from 10 nanograms to 10 micrograms of mitochondrial protein per sample. Thus the invention methods are suitable for use in high-throughput screening assay formats.
  • the invention Fl/FO ATPase functional immunocapture assay is suitable for use as a diagnostic assay to detect any type of activity-affecting defect of mitochondrial Fl/FO ATPase in humans, such as catalytic defects, the presence or absence of target subunit antigen, defects in assembly of the enzyme complex, and the like.
  • the invention provides a Fl/FO ATPase functional immunocapture assay for determining interactions between human mitochondrial Fl/FO ATPase and the inhibitor protein (IFi), which can be added or removed from the captured enzyme in a dose-dependent, and pH-sensitive fashion that mimics normal interactions between Fl/FO ATPase and IFi.
  • IFi inhibitor protein
  • ATP hydrolysis activity of mitochondrial Fl/FO ATPase solubilized, immunocaptured and assayed at pH in the range from about 6.0 to about neutrality is relatively low, but the enzyme could be greatly activated (>10-fold) by conditions that strip the JF from the protein (i.e., 30 min exposure to pH above 7.0, for example 8.0, 8.2 or about 8.5.
  • Recombinant human IFi can also be added or removed from the captured enzyme in a dose-dependent, and pH-sensitive fashion that mimics normal interactions between Fl/FO ATPase and IFi.
  • a method is also provided by the invention for obtaining solubilized mitochondrial Fl/FO ATPase that is fully saturated with IFi for use in such assays in a patient-specific manner. Therefore, the functionality of endogenous IFi or endogenous Fl/FO ATPase can be determined or monitored within small samples, e.g. nanosamples. Such an assay is valuable as a research tool for studying the interactions between human mitochondrial Fl/FO ATPase and its inhibitor.
  • the invention provides methods for screening to detect agents, such as small molecules, drugs, or proteins that modify the inhibitor activity of IFi for human mitochondrial Fl/FO ATPase, for example by binding to IFi so as to prevent its inhibitor activity.
  • agents such as small molecules, drugs, or proteins that modify the inhibitor activity of IFi for human mitochondrial Fl/FO ATPase, for example by binding to IFi so as to prevent its inhibitor activity.
  • Such small molecules, drugs, or proteins are desirable therapeutic agents that could be used to regulate ATPase activity of Fl/FO ATPase and thereby the energy balance and efficiency of energy utilization of cells and tissues.
  • modulators have utility in treatment of disorders of energy production or utilization.
  • Invention screening assay comprises contacting a sample containing Fl/FO ATPase in the presence of IFi and a test compound and determining the degree to which the test compound modifies the inhibitor activity of IFi in the sample, wherein a decrease of IFi inhibitor activity indicates the test compound inhibits IFi- Compounds that increase IFi inhibitor activity may also be useful in treating disorders of energy production or utilization by inhibiting ATPase activity.
  • the invention screening assay can also be used to determine the degree to which a test compound increases IFi inhibitor activity.
  • the invention assays can be used to determine the specific activity (units of activity per mg enzyme protein) of Compound V.
  • a distinction can be made between defects in enzyme turnover rates and defects in production of sufficient amounts of enzyme.
  • the invention provides isolated monoclonal antibodies characterized as specifically binding to mitochondrial Fl/FO ATPase and immunoprecipitating the entire 16 subunit complex, wherein the complex retains functional activity. It should be understood that the monoclonal antibody may be able to immunoprecipitate the functional complex in the absence of all 16 subunits being present, although 16 is preferred. Specifically, a monoclonal antibody wherein the antibody has the specificity and avidity of mAb MM#l-12F4AD8A:f8 is included. An additional mAb, MM#I-8EI2, captures human and bovine Fl/FO ATPase while retaining enzyme activity.
  • hybridoma cell line that produces a monoclonal antibody having the specificity of a monoclonal antibody as described herein.
  • the invention also provides a kit for determining Fl/FO ATPase activity in a cell comprising an antibody of the invention.
  • the kit may contain a detectable label such as a fluorescent label or an enzymatic label.
  • the assay described herein is based on the specificity of the monoclonal antibodies as well as antigen function. Any general biochemical activity of antigen will suffice as a marker of antigen function as the antibody/antigen capture provides specificity to the assay.
  • Fl/FO ATPase Alterations in Fl/FO ATPase reduce or eliminate energy production in mitochondria and so are pathogenic.
  • the literature shows that Fl/FO ATPase is affected by a variety of environmental toxins including pesticides, impurities in narcotic drugs, and damage to organelles caused by drugs used to treat other diseases, among others. In effect these inhibit Fl/FO ATPase activity, for example, enzymatic activity, to varying degrees.
  • Mutations of Fl/FO ATPase in patients (genetically derived) can also affect activity and produce the same sequellae as the toxins. Such mutations first affect and then destroy (by apoptosis) those cells with the highest need for ATP.
  • These cells include selected brain cells such as those of the substancia nigra cells, whose impairment results in Parkinson's disease; frontal cortex cells, whose impairment is implicated in Alzheimer's disease or dementia, pancreatic cells, which are involved in insulin secretion; cardiocyte cells, whose destruction leads to cardiomyopathy, and the like.
  • the present invention provides evidence of the utility of antibody analysis in the characterization of Fl/FO ATPase deficiencies of all types.
  • the invention provides methods for determining the amount of Fl/FO ATPase in a biological sample of a mammalian patient.
  • the invention assay comprises contacting isolated antibodies that immunoprecipitate Fl/FO ATPase with a sample comprising solubilized Fl/FO ATPase so that the antibodies bind to Fl/FO ATPase present in the sample to form an antibody/Fl/FO ATPase complex, (i.e., under suitable conditions and for a time suitable to form the antibody/Fl/FO ATPase complex).
  • Remaining sample for example unbound sample contents, is then separated from the antibody/Fl/FO ATPase complex; and the amount of antibody/Fl/FO ATPase complex in the sample is detected.
  • any suitable immunoassay format known in the art and as described herein can be used to detect and quantify the amount of antibody that binds to an antigen of interest. If the activity of the Fl/FO ATPase in the sample is also known, for example the enzymatic activity, the results of the invention method can be used to calculate the specific activity of the Fl/FO ATPase in the sample.
  • the term "activity" or "functional activity” as applied to Fl/FO ATPase means all aspects of natural Fl/FO ATPase activity at physiological pH, including, but not limited to Fl/FO ATPase enzymatic activity in oxidative phosphorylation.
  • antibodies that are known to bind specifically to a particular Fl/FO ATPase subunit can be used to determine the amount of the respective subunit being produced by the patient whose sample is being diagnosed.
  • any combination of the invention antibodies, wherein each different antibody binds specifically to a different one of the subunits in the sample can also be used in practice of the invention methods.
  • a combination or mixture of anti- CV- ⁇ , CV- ⁇ , CV-d, CN-OSCP, CN-IFi subunit antibodies can be used to determine the amount of the respective subunits in the patient sample.
  • a mixture of monoclonal antibodies containing, for example, anti- CN- ⁇ , CN- ⁇ , CV-d, CN-OSCP, CN-IFi Fl/FO ATPase subunit antibodies can be used for this purpose.
  • monoclonal antibodies are preferred.
  • Such assays can also be used to determine whether a particular subunit is produced in low quantity as compared with what would be expected in a comparable sample obtained from a normal patient or a normal sample (i.e., obtained from a single patient that has been screened to eliminate the possibility of genetic defects in nucleotide sequences known to produce the various Fl/FO ATPase peptides that assemble into the Fl/FO ATPase or from a representative group of such normal patients).
  • "corresponding samples” would be mitochondria isolated from a patient fibroblast cell line and mitochondria isolated from a control skin fibroblast cell line (i.e. isolated from skin fibroblasts of a normal individual).
  • Fl/FO ATPase- containing samples for use in the invention methods can be obtained from whole cell extracts of the patient or from mitochondria isolated from such cells.
  • fibroblast cells are particularly convenient as a source of patient samples for diagnostic assays, it should be understood that Fl/FO ATPase could be isolated from any mammalian cell, including human cells, with cells having high-energy requirements having the largest supply of mitochondrial Fl/FO ATPase.
  • cells that can be used in the invention methods include neural cells, cardiomyocytes, pancreatic islet cells, hematopoietic cells, liver cells, kidney cells, T cells, B cells and other cell types.
  • the assay can be performed utilizing Fl/FO ATPase or mitochondria that have been immunopurified from patient cells or experimental cells by any method known in the art, such as the methods described in the Examples herein.
  • the invention provides methods for detecting Fl/FO ATPase deficiency in a patient by contacting monoclonal antibodies specific for each of a plurality of Fl/FO ATPase subunits with a patient sample so that the antibodies immunocapture Fl/FO ATPase subunits present in the sample, wherein the antibodies are each tagged with a detectable label.
  • the antibodies specific for different subunits of Fl/FO ATPase can be tagged with labels that are readily distinguishable from one another.
  • fluorescent labels can be selected to emit at different wavelengths or to produce fluorescence of different colors, and gold or other metallic labels can be used.
  • the amount of each Fl/FO ATPase subunit immunocaptured by a respective Fl/FO ATPase subunit antibody is then determined and compared with the amount thereof determined to be present in a corresponding normal sample, wherein a decrease in the amount of any of the Fl/FO ATPase subunits in the patient sample as compared to the amount in a normal sample indicates the presence of a Fl/FO ATPase deficiency in the patient.
  • the invention assay can be used to detect a decrease in Fl/FO ATPase enzymatic activity in the cells of the patient whose sample is tested.
  • the invention methods can be conducted using a single one of the anti-Fl/FO ATPase or antiFl/FO ATPase subunits or the invention methods can be repeated to independently assess the amount of each of the plurality Fl/FO ATPase subunits assayed in the sample by assaying the Fl/FO ATPase subunits one at a time, or by assaying a portions of or subsets of the subunits together until all of the subunits of interest have been assayed. If the proportionate amounts of the various subunits tested vary greatly from what would be expected in a corresponding normal sample, the patient can be identified as a good candidate for having a defect in a Fl/FO ATPase assembly factor.
  • the invention immunological tests for Fl/FO ATPase activity can be used in a high throughput format using any technique known in the art, such as FASC screening as is described below in greater detail.
  • Detectable labels suitable for binding to antibodies used in the invention methods include radiolabels linked to the antibodies using various chemical linking groups or bifunctional peptide linkers.
  • a terminal hydroxyl can be esterified with inorganic acids, e.g., 32 P phosphate, or 14 C organic acids, or else esterified to provide linking groups to the label.
  • Enzymes of interest as detectable labels will primarily be hydrolases, particularly esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and so forth.
  • Chemiluminescers include luciferin, and 2, 3- dihydrophthalazinediones (e.g., luminol), and the like.
  • the invention methods use monoclonal antibodies characterized as specifically binding to Fl/FO ATPase of the mitochondrial respiratory chain and immunoprecipitating Fl/FO ATPase, wherein Fl/FO ATPase retains functional activity. It should be understood that the invention monoclonal antibodies may be able to immunoprecipitate Fl/FO ATPase in the absence of all 16 subunits being present, although antibodies that precipitate the entire 16 subunit complex are preferred.
  • the invention also provides isolated monoclonal antibodies that specifically bind to subunits of Fl/FO ATPase, for example the CN- ⁇ , CN- ⁇ , CV-d, CN-OSCP, CN-IFi subunits of Fl/FO ATPase.
  • the anti-IFi monoclonal antibodies can be used to immunoprecipitate the fully assembled complex of Fl/FO ATPase with full activity.
  • Hybridoma cell lines producing monoclonal antibodies useful in the invention methods for immunocapture of Fl/FO ATPase are commercially available by hybridoma name as used to identify the monoclonal antibodies Table I below from Molecular Probes (Eugene, OR) or from the Monoclonal Antibody Facility at the University of Oregon (Eugene, OR).
  • epitope refers to an antigenic determinant on an antigen, such as a Fl/FO ATPase or a Fl/FO ATPase subunit, to which the paratope of an antibody, such as an antibody that binds to a Fl/FO ATPase or a Fl/FO ATPase subunit.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • Additional antibodies that bind to fully assembled Fl/FO ATPase, or a Fl/FO ATPase subunit can be prepared by those of skill in the art using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis, which can be conjugated to a carrier protein, if desired.
  • Such commonly used carriers, which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference).
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first monoclonal antibody.
  • Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies.
  • the preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992), which are hereby incorporated by reference.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
  • Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
  • Large-scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
  • Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., osyngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane tetramethylpentadecane prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
  • an anti-Fl/FO ATPase antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci.
  • Antibodies for use in the invention methods also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, Methods: A Companion to Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al, Ann. Rev. Immunol. 12:433 (1994), which are hereby incorporated by reference.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from Stratagene Cloning Systems (La Jolla, CA).
  • antibodies for use in the invention methods may be derived from a human monoclonal antibody.
  • Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7:13 (1994); Lonberg et al, Nature, 368:856 (1994); and Taylor et al, Int. Immunol., 6:579 (1994), which are hereby incorporated by reference.
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • Fv fragments comprise an association of V H and NL chains. This association may be noncovalent, as described in Inbar et al, Proc. ⁇ at'l Acad. Sci. USA, 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra.
  • the F v fragments comprise N H and N L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by Whitlow et al, Methods: A Companion to Methods in ⁇ nzymology, Vol. 2, page 97 (1991); Bird et al, Science, 242:423 (1988); Ladner et al, U.S. patent No. 4,946,778; Pack et al, Bio/Technology, 11:1271 (1993); and Sandhu, supra.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al, Methods: A Companion to Methods in ⁇ nzymology, Vol. 2, page 106 (1991).
  • the invention provides methods for screening for an agent that causes mitochondrial disorders by contacting samples containing cells with an anti-Fl/FO ATPase antibody in the presence and the absence of the agent and determining the Fl/FO ATPase activity in the samples, wherein a lower level of activity in the sample in the presence of the agent indicates that the agent causes a mitochondrial disorder.
  • the agent can be a toxin, such as an environmental toxin.
  • the agent can be an experimental drug whose possible deleterious effect on Fl/FO ATPase is assayed.
  • the invention method can be used as a simple screen for when to remove patients from drugs that affect Fl/FO ATPase functioning, e.g., in HIV or statin therapy, screening for novel drugs for prophylaxis, as well as for the treatment of early- onset and late-onset mitochondrial disorders, or for screening for potential side effects of drugs being designed for other human conditions.
  • the invention methods can also be used to assess toxin damage to Fl/FO ATPase activity in the cells of the patient.
  • the method can be repeated at suitably spaced intervals, with decreased Fl/FO ATPase activity over time indicating increased damage.
  • suitable spaced intervals will vary according to the type of toxin, or drug or the type of disorder whose progress is being monitored as well as according to the general health of the patient.
  • a suitably spaced interval may range from one day to 1 year, or 10 days to six months, or 30 days to 3 months, depending upon the disorder being monitored, the magnitude of the toxicity suspected, the circumstances of the patient's exposure to the toxin or drug, frequency of exposure or administration, and the like.
  • Alteration of Fl/FO ATPase functioning due to reduced synthesis and/or alteration of mtDNA can also be detected and/or monitored (e.g., for onset or stage of the disorder) using the invention methods.
  • .Thus such neurodegenerative diseases as Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, Downs Syndrome, Schizophrenia, late-onset type II diabetes (NIDDM) and the aging process itself can be predicted, diagnosed or monitored using the invention methods.
  • conditions such as reperfusion injury, therapy for HIN infection with nucleoside reverse transcriptase inhibitors, such as AZT and DDC, and the myopathy that is an occasional side effect of statin use to treat hypercholesterolemia can all be predicted, diagnosed or monitored using the invention methods.
  • nucleoside reverse transcriptase inhibitors such as AZT and DDC
  • myopathy that is an occasional side effect of statin use to treat hypercholesterolemia can all be predicted, diagnosed or monitored using the invention methods.
  • the invention assays can be used in a method for diagnosing late onset mitochondrial disorders in which post translational modifications of one or Fl/FO ATPase subunit is involved.
  • late onset mitochondrial disorders include, for example, late onset diabetes ( ⁇ IDDM), Huntington's, Parkinson's and Alzheimer's diseases, ALS (amyotrophic lateral sclerosis), Schizophrenia, and the like.
  • ⁇ IDDM late onset diabetes
  • ALS amyotrophic lateral sclerosis
  • Schizophrenia and the like.
  • Such post translational modifications to Fl/FO ATPase subunits are thought to be caused by free radical damage and result in one or more Fl/FO ATPase subunit having a molecular weight different than the molecular weight of the corresponding normal Fl/FO ATPase subunit attributable to post translation modification.
  • the invention provides methods for screening patients to identify patients suspected of having a late onset disease, said method comprising a)contacting isolated antibodies that immunoprecipitate Fl/FO ATPase subunits with a patient sample comprising solubilized Fl/FO ATPase subunits so that the antibodies bind to Fl/FO ATPase subunits present in the sample to form an antibody/Fl/FO ATPase subunit complex; b) separating the Fl/FO ATPase subunits from the remaining sample contents; and c) detecting the presence of aberrant post- translational modification in the one or more separated Fl/FO ATPase subunit, wherein the presence of the post-translational modification indicates the patient is suspected of having the late onset disease.
  • Late onset disease as the term is used herein means such diseases as late onset diabetes (Diabetes Type I), Huntington's, Parkinson's and Alzheimer's diseases, ALS (amyotrophic lateral sclerosis), Schizophrenia and the like, wherein the patient is free of the disease in early life, but develops the disease during puberty or thereafter, sometimes as late as age 70 or 80.
  • Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite (ONO 2 -).
  • reactive oxygen species including those containing nitrogen such as peroxynitrite (ONO 2 -).
  • Peroxynitrite has been shown in vitro to target tyrosine residues in proteins through free radical addition to produce 3-nitrotyrosine.
  • Mass spectral patterns associated with 3-nitrotyrosine containing peptides allow identification of peptides containing this post-translational modification.
  • matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has previously been used to characterize peptides containing 3-nitrotyrosine (Sarver A, et al. J Am Soc Mass Spectrom 2001 Apr 12:439-48 .
  • a unique series of ions were found for these peptides in addition to the mass shift of +45 Da corresponding to the addition of the nitro group.
  • mass spectrometry is used to detect post- translational modifications of various Fl/FO ATPase subunits by comparing the molecular weight of particular Fl/FO ATPase subunits obtained from the patient with those of a corresponding normal Fl/FO ATPase subunit. A difference in molecular weight between the two indicates that the patient's Fl/FO ATPase activity is impaired and that the patient should be more thoroughly screened for aberrant post translational modification of Fl/FO ATPase subunits, suggesting late onset diseases.
  • Fl/FO ATPase subunits obtained from a patient sample as described herein can be separated by Western blotting with antiphosphotyrosine or antinitrotyrosine antibodies followed by mass spectrometry, for example using LC/MS/MS.
  • Yet another method for determining the presence of post-translational modifications of Fl/FO ATPase subunits involves an immunoassay wherein the Fl/FO ATPase subunits obtained from a patient sample are separated and are contacted with an antibody that binds specifically to nitrotyrosine, such as the anti-nitrotyrosine antibody, rabbit IgG fraction commercially available from Molecular Probes (Eugene, OR, Cat # A- 21285) Binding of the anti-nitrotyrosine antibody to a Fl/FO ATPase subunit indicates that the patient's Fl/FO ATPase activity is impaired and that the patient should be more thoroughly screened for late onset diseases, such as disclosed herein.
  • an antibody that binds specifically to nitrotyrosine such as the anti-nitrotyrosine antibody, rabbit IgG fraction commercially available from Molecular Probes (Eugene, OR, Cat # A- 21285)
  • the invention provides a kit for determining Fl FO ATPase activity in a cell.
  • the kit comprises one or more anti-Fl/FO ATPase antibody.
  • the invention kit may additionally contain a detectable label, such as a fluorescent label or an enzymatic label, with which the antibody can be tagged for detection of formation of an antibody/Fl/FO ATPase complex when the antibody is contacted with a Fl/FO ATPase containing sample.
  • the invention kit may contain any of the monoclonal antibodies contained in Table I.
  • the antibodies can be bound to a solid support, such as a 96 well micro-titer plate or beads.
  • the invention kit may further comprise instructions for performing immunoassay of a sample containing Fl/FO ATPase using the contents of the invention kit.
  • the assays described herein are based on the specificity of the anti-Fl/FO ATPase monoclonal antibodies as well as upon antigen function. Any general biochemical activity of Fl/FO ATPase will suffice as a marker of Fl/FO ATPase activity function as the antibody/antigen capture of functional Fl/FO ATPase provides specificity to the assay.
  • the assay can also detect all categories of defects in Fl/FO ATPase multisubunit enzyme complexes, for example the presence/absence of one or more target subunits; misassembly of the enzyme complex, catalytic defects, and the like.
  • the assay described herein can be quantitative. If combined with quantitation of captured antigen protein, the assay can be used to determine the specific activity of Fl/FO ATPase enzyme. Using the methods of the invention one can then distinguish alterations in enzyme turnover rates from alterations in enzyme amounts.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or immunoprecipitation of Fl/FO ATPase.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • Various immunoassay screening techniques can be utilized using the invention monoclonal antibodies, and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., a bead or micro-titer plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999).
  • immunoassays that can be used in practice of the invention methods include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation as
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 MNaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4°C, adding protein A and or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4°C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxy
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
  • ELIS comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 1) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3 H or 125 1) in the presence of increasing amounts of an unlabeled second antibody.
  • Normal human fibroblasts (MRC5, a diploid strain derived from fetal human lung) were obtained from the American Type Culture collection and used between PD 30 and 45.
  • p°-MRC5 cells (lacking mtDNA) were derived by culturing normal MRC5 cells for 12-14 population doublings in ethidium bromide (50 ng/ml).
  • Patient-derived GM00028 fibroblasts were obtained from the HIGMS Human Genetic Mutant Cell Repository at the Coriell Institute for Medical Research and used at early passage levels.
  • cultured cells were grown to confluence, dissociated by trypsinization, the trypsin inactivated with fetal calf serum, the cells washed twice with PBS, cells counted, stored frozen as cell pellets and then used as described below.
  • Mitochondria were solubilized at 5 mg/ml and whole cells were solubilized at 2 x 10 7 cells/ml. Samples were mixed well and then kept on ice for 30 min with occasional agitation. Insoluble material was then removed by centrifugation at 16,000 x 6 for 20 minutes at 4° C. [0083] Immunocapture. Assays were run in 96-well plates (Falcon Probind) using 100 ⁇ l/well.
  • the wells were prepared for immunocapture by first adsorbing goat-anti-mouse IgG-Fe (IgGi+IgG 2a +lgG 2b +lgG 3 ) at 5 ⁇ g/ml in TBS (50 mM Tris pH 7.5, 150 mM NaCl) with 0.02% azide overnight at 4°C, washing 3 times with TBS alone, incubating for 1 hr at room temperature with the anti-ATPase capture mAb 12F4AD8AF8 at 5 ⁇ g/ml in TBS with 2.5% BSA, and then washing 4 times with TBS.
  • TBS 50 mM Tris pH 7.5, 150 mM NaCl
  • a non-specific mouse monoclonal antibody was used as a null capture mAb.
  • Wells were then loaded with solubilized test samples diluted in TBS with 2.5% BSA, incubated for 2 hr at room temperature and then washed 4 times with TBS.
  • ATPase activity ATPase reaction buffer (25 mM HEPES pH 7.5 or 10 mM MOPS pH 6.5, with 25 mM KC1, 5 mM MgCl 2 , 5 mM KCN and 2 mM ATP) was added at 100 ⁇ l/well and incubated for 3 hr at 37° C. The reaction was stopped and free Pi generated by ATP hydrolysis was detected essentially as described by J. E. Walker et al. ((1995) Methods Enzymol. 260, 163-190) by addition of 0.5% ammonium molybendate in 0.7 M H 2 SO 4 (42 ⁇ l/well) followed by 10% ascorbic acid (4 ⁇ l/well). After incubation for 30 min at room temperature to allow color development, absorbance was measured at 690 nm.
  • IFi was dissociated from previously immunocaptured IFi/FiFo complexes by exposure to IFi stripping buffer (30 mM Tris-sulfate pH 8, 250 mM KC1, 2 mM EDTA and 75 mM sucrose) for 30 min at 37° C (Van Raaij, M. J., et al. ((1996) Biochemistry 35, 15618-15625). The wells were then washed with either a pH 7.5 wash buffer (TBS) or a pH 6.5 wash buffer (10 mM MOPS pH 6.5, 150 mM NaCl and 1 mM MgCl 2 ), and ATPase activity measured at either pH 7.5 or pH 6.5 as described above.
  • IFi stripping buffer (30 mM Tris-sulfate pH 8, 250 mM KC1, 2 mM EDTA and 75 mM sucrose) for 30 min at 37° C (Van Raaij, M. J., et al. ((1996) Biochemistry
  • JFi Addition conditions Recombinant human IFi was obtained by PCR-based amplification of the appropriate gene from a human heart cDNA library (Clontech), cloning into pET15b (Novagen), transformation of E. coli and overexpression and purification of the His-tagged protein on a Ni-column by standard protocols (Novagen). Isolated protein was diluted in pH 6.5 wash buffer (as described above) containing 1 mM ATP and incubated with previously immunocaptured F ⁇ F 0 . The wells were then washed with pH 6.5 wash buffer and ATPase activity measured at pH 6.5 as described above.
  • Proteins were separated by electrophoresis at 100 V for 2 hr at room temperature, after which the gels were soaked with gentle mixing for 30 min in cold CAPS transfer buffer (10% methanol in 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid, pH 11). The proteins were then electrophoretically transferred (2 hr at 100 mA) to 0.45 ⁇ PVDF membranes (Millipore) in ice-cold 3-(Cyclohexylamino)-l-propanesulfonic acid (CAPS) transfer buffer.
  • CAPS transfer buffer 10% methanol in 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid, pH 11
  • the proteins were then electrophoretically transferred (2 hr at 100 mA) to 0.45 ⁇ PVDF membranes (Millipore) in ice-cold 3-(Cyclohexylamino)-l-propanesulfonic acid (CAP
  • Mitochondrial protein-loaded membranes were blocked overnight in 5% non-fat dry milk in Phosphate-Buffered Saline (PBS) at 4° C then incubated overnight at 4° C in mixtures of subunit-specific monoclonal antibodies as desired (see Table ⁇ 1) in 5% milk/CMF-PBS with 0.02% azide, washed four times with PBS combining 0.05% TWEEN-20, incubated 2 hr at room temperature in 5% milk/PBS containing HW-GAM-lgG-Fe (Jackson ImmunoReseareh) at 0.08 ⁇ g/ml, washed as above, rinsed with PBS and the bound antibodies visualized with ECL-Plus (Amersham) on a STORM imaging system (Molecular Dynamics), reading blue fluorescence.
  • PBS Phosphate-Buffered Saline
  • M FiF 0 mitochondria from human heart were washed with 10 mM Tris, 250 mM sucrose, 0.5 mM EDTA, pH 7.5 and resuspended at 3 mg/ml in the presence or absence of 1 mM ADP in 20 mM Bis-Tris, 150 mM Sucrose, 0.5 mM EDTA, pH 6.5, supplemented with protease inhibitors leupeptin (2 ⁇ g/ml), pepstatin (2 ⁇ g/ml) and PMSF (2 mM).
  • the mitochondria were solubilized by addition of 1 volume of the same buffer without inhibitors, containing 0.45% n-dodecyl- ⁇ -D- maltoside and incubation for 20 minutes at 4° C.
  • the extract was centrifuged in a TLA100.2 at 65000 rpm for 30 min at 4° C.
  • Protein G Agarose beads with bound nonspecific antibody (NSA) were added at 40 ⁇ l/1 ml supernatant and shaken at room temperature for 1 hour. After centrifugation for one minute at 10000-x g, the supernatant was exposed a second time to 40 ⁇ l NSA beads, followed by immunoprecipitation with 40 ⁇ l 12F4AD8 beads for 2h at room temperature.
  • the beads were then washed 6 times with the same buffer, containing 0.05% n-dodecyl- ⁇ -D-maltoside and the ATP synthase eluted twice with 70 ⁇ l 0.1M glycine, 0.05% n-dodecyl- ⁇ -D-maltoside, pH 2.5. Na- phosphate, pH 8.0 was added to a final concentration of 0.1 M to adjust the pH to about 7.5.
  • Subunits were identified by the number of matched fragments (x), and the coverage of the full sequence of mature protein (y%): subunit ⁇ (32,55%), subunit ⁇ (28,50%), subunit ⁇ (12,34%), subunit b (18,57%), OSCP (13,71%), subunit d (12,56%), subunit a (5,8%), subunit ⁇ (2,16%), subunit f 34
  • Protein concentrations were determined with BCA (Pierce). Polyacrylamide gels were stained with Coomassie Brilliant Blue (Serva) or for higher sensitivity with Sypro-Ruby (Molecular Probes Inc., Eugene, OR) as described in Marusich, M. F. ((1988) J. Immunol. Methods 114, 155-159).
  • Monoclonal antibodies (mAbs) used for Western blotting were produced in the Monoclonal Antibody Facility at the University of Oregon using standard protocols for hybridoma development and antibody purification and are specific for the following subunits: CV- ⁇ (7H10BD4F9), CN- ⁇ (3D5AB1), CN- (7F9BG1), CN-OSCP (4C11C10D12), CN-IFi (5E2D7), CI-39 (20C11B11B11), CII-30 (21A11AE7), CHI-core 2 (13G12AF12BB11), and CIN-II (12C4F12).
  • mice were immunized with purified bovine Fl/FO ATPase, and the resulting immune splenocytes used to construct mouse-mouse hybridomas. MAbs were then screened for ability to bind immobilized Fl/FO ATPase in ELISA assays and the positives were re-screened to identify mAbs that could capture active enzyme.
  • Human mitochondrial Fl/FO ATPase can be isolated by immunoprecipitation as a complex of 16 different subunits. When immobilized on beads or 96 well plates, anti-Fl/FO ATPase "capture" mAb (12F4AD8AF8) immunocaptured Fl/FO ATPase from detergent-solubilized extracts of human tissues or cell culture material as described above.
  • Gel pieces were destained by two washes in 0.2 ml 50% acetonitrile in 0.1 M ⁇ H- ⁇ HCO 3 for 45 min at 37° C. After dehydration with 0.1 ml acetonitrile the gel pieces were dried in a Speed- Vac, 100 ⁇ l of 0.1 M DTT in 0.1 M NH 4 HCO 3 was added for one hour at 56° C followed by one wash with water and addition of 0.1 M iodoacetamide for 30 min in the dark. The gel pieces were then washed twice with 0.1 ml NE- 4 HCO 3 for 15 min and once with 50% acetonitrile in 0.1 M NH- ⁇ HCO 3 .
  • gel pieces were dried in a 3 Speed- Vac and 20 ⁇ l freshly prepared modified trypsin at 25 ⁇ g/ml (Promega) in 10% acetonitrile and 50 mM HiHCOs was added. After 10 min, the gel pieces were overlaid with 50 ⁇ l buffer and incubated at 37° C over night. The digestion was terminated by adding 2 ⁇ l trifluoroacetic acid. After addition of 0.1 ml water, the supernatant was placed in a siliconized pre-washed eppendorff tube. The gel pieces were extracted twice for 30 min with 100 ⁇ l 50% acetonitrile and 0.2% trifluoroacetic acid with occasional shaking.
  • the supernatants were pooled and the samples dried in a SPEED-VAC and re-dissolved with 20 ⁇ l 5% acetonitrile, 0.1% acetic acid for MALDI-TOF analysis with a Voyager DE- subunit-specific mAbs and mass spectrometry using both MALDI-TOF and LC/MS/MS procedures as described above.
  • the ⁇ , ⁇ , ⁇ , b, OSCP, d, a, ⁇ and/subunits were each detected as a single band.
  • Subunits g, F6, e, A6L and ⁇ were observed in more than one closely spaced band, based on the detection of fragments by mass spectrometry.
  • subunit c Also present near the bottom of the gel was what was interpreted to be subunit c. This subunit could not be identified by the mass spectrometry analysis because it generated too few tryptic digest fragments of appropriate molecular weights. However, subunit c must be present as the enzyme was active and inhibitor (oligomycin) sensitive as isolated. In support of this conclusion, the presence of subunit c was recently confirmed in human M Fl/FO ATPase labeled with 14 C-DCCD, solubilized in the same detergent as used here, but affinity purified with a Sepharose-EAH column (Garcia, J. J., et al. (2000) J. Biol. Chem. 275, 11075-11081). Finally, immunoprecipitated Fl/FO ATPase also was shown to contain JFi when solubilization and capture conditions allow Fl/FO ATPase/IFi interactions.
  • Human Fl/FO ATPase captured by the mAb is active as an ATPase.
  • a micro scale 96-well format was used to facilitate measurements of ATPase activity of immunoprecipitated material.
  • Goat anti-mouse IgG specific for the Fe portion of mouse IgG was absorbed to 96 well plates and used to capture mouse anti-Fl/FO ATPase mAb in favorable orientations. The wells were then incubated with solubilized tissue or cell samples, washed, and any immunocaptured Fl/FO ATPase was detected by colorimetric ATP hydrolysis measurements.
  • this assay of ATPase activity of Fl/FO ATPase is sensitive, quantitative, and has a large dynamic range. Positive signals were obtained from as little as 10 ng/well of solubilized human heart mitochondrial protein, and the assay only saturated at 10 ⁇ g/well, i.e. a range of three orders of magnitude.
  • Figure 1 shows the results of activity measurement of the enzyme in the presence and absence of oligomycin. When isolated by the invention methods for one-step immunoprecipitation, ATPase activity of human Fl/FO ATPase could be inhibited more than 90% by oligomycin. This is a higher level of inhibition than reported for enzyme isolated by fractionation or column chromatography methods.
  • Antibody capture can be used to isolate Fl/FO ATPase from human fibroblast cell lines, including p° cells with partly assembled enzyme.
  • Sypro Ruby was used as stain rather than Coomassie Brilliant Blue and the greater sensitivity of Sypro Ruby revealed minor impurities readily, as was seen comparing the gel subunit profile of Fl/FO ATPase from human heart mitochondria in gels. Importantly, all of the subunits of the enzyme seen in human heart Fl/FO ATPase could also be resolved in the fibroblast immunoprecipitate.
  • GM0028 fibroblasts were included in this study because they were previously suggested to have a defect in IFi (Yamada, E.W., and Huzel, N. J. (1992) Biochim. Biophys. Ada 1139, 143-147).
  • GM00028 mitochondria also appeared to have the same levels of functional IFi as MRC5 and the Fl/FO ATPase was responsive to IFi inhibition. Enzyme solubilized, immunocaptured and analyzed at pH 6.5 was inhibited by its endogenous IFi, and ATP hydrolysis activity could be increased upon removal of this inhibitor protein by treatment with high pH after capture.
  • Fibroblast mitochondria (5 ⁇ g per lane) were analyzed by probing with a cocktail of mAbs (anti-IFi at 0.5 ⁇ g/ml; anti-Cl-39 at 2 ⁇ g/ml; anti-CII-30 at 5 ⁇ g/ml; anti-CIII-core 2 at 0.4 ⁇ g/ml; anti-CIV-II at 2 ⁇ g/ml; and anti-CV- ⁇ at 4 ⁇ g/ml), and visualizing with chemiluminescence using HRP-conjugated goat anti-mouse antibodies and ECL + .
  • mAbs anti-IFi at 0.5 ⁇ g/ml; anti-Cl-39 at 2 ⁇ g/ml; anti-CII-30 at 5 ⁇ g/ml; anti-CIII-core 2 at 0.4 ⁇ g/ml; anti-CIV-II at 2 ⁇ g/ml; and anti-CV- ⁇ at 4 ⁇ g/ml
  • GM28 fibroblast cells mitochondria contain normal levels of IFi as well as the OXPHOS complexes I, II, III, IV and V. It was noted that p° cells lack mtDNA-encoded subunit II of complex IV, indicating full depletion of mtDNA. Thus, the assay showed the expected absence of mtDNA-encoded subunits in MRC5-p° cells, but also revealed clearly that GM00028 mitochondria exhibited a normal OXPHOS subunit profile, indistinguishable from control cells MRC5. Moreover GM00028 cells contained normal levels of IFi, a result consistent with the activity measurements above.

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Abstract

Des anomalies de ATPase F1/F0 sont l'une des causes les plus fréquentes de maladies et de troubles en rapport avec des troubles de la chaîne respiratoire mitochondriale, notamment des maladies génétiquement associées et des maladies neurodégénératives à apparition tardive. L'invention concerne des méthodes d'utilisation d'un anticorps capteur anti-ATPase F1/F0 et des anticorps réagissant avec des sous-unités de ATPase F1/F0 qui facilitent le diagnostic d'anomalies de ATPase F1/F0 et qui peuvent s'utiliser pour un criblage rapide visant à déceler l'apparition ou à déterminer l'étape de développement de maladies diverses. De plus, l'invention porte sur des méthodes immunologiques de criblage d'agents, de médicaments par exemple, pour déterminer leur action sur l'activité de ATPase F1/F0 et sur des méthodes de dépistage propres à détecter des patients chez lesquels l'activité de ATPase F1/F0 est altérée et ceux dont on soupçonne qu'ils ont une maladie à apparition tardive associée à une modification post-translationnelle d'une ou de plusieurs sous-unités de ATPase F1/F0.
PCT/US2003/018114 2002-02-14 2003-06-06 Dosage immunocapteur et fonctionnel pour atpase f1/f0 mitochondriale WO2003104421A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003245425A AU2003245425A1 (en) 2002-06-06 2003-06-06 Immunocapture and functional assay for mitochondrial f1/f0 atpase
US10/997,819 US20050153381A1 (en) 2002-02-14 2004-11-24 Immunocapture of mitochondrial protein complexes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38708902P 2002-06-06 2002-06-06
US60/387,089 2002-06-06

Related Child Applications (1)

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US91725404A Continuation 2002-02-14 2004-08-11

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WO2003104421A2 true WO2003104421A2 (fr) 2003-12-18
WO2003104421A3 WO2003104421A3 (fr) 2004-07-08

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AU (1) AU2003245425A1 (fr)
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962646A (en) * 1997-03-17 1999-10-05 Incyte Pharmaceuticals, Inc. ATP synthase Fo subunit
US6020474A (en) * 1997-01-15 2000-02-01 Incyte Pharmaceuticals, Inc. ATP synthase subunits
US6376211B1 (en) * 1999-07-12 2002-04-23 Xoma Technology Ltd. Agents and methods for inhibiting F1/F0 ATPase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6020474A (en) * 1997-01-15 2000-02-01 Incyte Pharmaceuticals, Inc. ATP synthase subunits
US5962646A (en) * 1997-03-17 1999-10-05 Incyte Pharmaceuticals, Inc. ATP synthase Fo subunit
US6376211B1 (en) * 1999-07-12 2002-04-23 Xoma Technology Ltd. Agents and methods for inhibiting F1/F0 ATPase

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AU2003245425A1 (en) 2003-12-22
AU2003245425A8 (en) 2003-12-22
WO2003104421A3 (fr) 2004-07-08

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