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WO2003069309A2 - Dosage immunocapteur et fonctionnel pour complexe i mitochondrial - Google Patents

Dosage immunocapteur et fonctionnel pour complexe i mitochondrial Download PDF

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WO2003069309A2
WO2003069309A2 PCT/US2003/004567 US0304567W WO03069309A2 WO 2003069309 A2 WO2003069309 A2 WO 2003069309A2 US 0304567 W US0304567 W US 0304567W WO 03069309 A2 WO03069309 A2 WO 03069309A2
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complex
sample
kda
subunits
patient
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WO2003069309A3 (fr
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Michael F. Marusich
Roderick A. Capaldi
Devin Oglesbee
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University of Oregon
Oregon State
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • 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
    • 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

Definitions

  • This invention relates to immunoassays, in particular, to immunoassays for determimng disorders of mitochondrial energy metabolism and diseases associated with late onset mitochondrial disorders.
  • Oxidative phosphorylation Oxidative phosphorylation
  • Oxidative phosphorylation Isolated enzymatic deficiency of the first OXPHOS complex, NADH:ubiquinone oxidoreductase (EC 1.6.99.3) or Complex I, is one of the most frequent causes of mitochondrial respiratory chain disorders (2).
  • Complex I is the first multiprotein complex of the OXPHOS system (3) and participates in the formation of a proton gradient across the inner mitochondrial membrane coupled to transfer of electrons from NADH to ubiquinone. This proton gradient provides part of the proton-motive force used for ATP production.
  • Two other sites in the cytochrome chain also couple electron transfer to ATP production in the same way so that for every pair of electrons from NADH that are oxidized by O 2 3 ATPs are produced. Alterations in Complex I reduce or eliminate energy production in mitochondria and so are pathogenic.
  • Complex I is the largest of the respiratory chain complexes, made up of seven different subunits encoded on mitochondrial DNA (mtDNA; ND1-6 and ND4L) and 38 or more different subunits encoded by nuclear genes (4, 5): Together, these subunits form a complex with an estimated molecular mass of about 900,000 daltons (3). Mutations in both the mitochondrial and nuclear encoded genes are known to cause Complex I deficiencies (6). However, in addition to the structural genes, there may be additional genes encoding proteins required for the assembly of a functional Complex I. So-called “assembly factors" involved in assembly of Complex IV and the ATP synthase have already been reported(7-9). For example, mutations in SU F1, an assembly factor required for full assembly of Complex IV, has been shown to cause cytochrome c oxidase deficiency in many of the reported cases of Leigh's disease (7, 10, and 11).
  • the present invention overcomes these and other problems in the art by providing methods by providing immunoassays useful for detecting deficiencies in Complex I and various diseases associated with such deficiencies.
  • the invention provides methods for determining the amount of Complex I in a biological sample of a mammalian patient by contacting isolated antibodies that specifically immunoprecipitate Complex I with a sample comprising solubilized Complex I so that the antibodies bind to Complex I present in the sample to form an antibody/Complex I complex. Remaining sample contents are separated from the antibody/Complex I complex and the amount of Complex I in the antibody/Complex I complex in the sample is determined.
  • the invention provides methods for detecting Complex I deficiency in a patient by contacting monoclonal antibodies specific for each of a plurality of Complex I subunits with a patient sample so that the antibodies immunocapture Complex I subunits present in the sample, wherein the antibodies are each tagged with a detectable label.
  • the amount of each Complex I subunit immunocaptured by a respective Complex I subunit antibody is then determined and the amount of each of the Complex I subunits is compared with an amount thereof present in a corresponding normal sample, wherein a decrease in the amount of any of the Complex I subunits in the patient sample as compared to the normal sample indicates the presence of a Complex I deficiency in the patient.
  • the invention provides kits for assaying Complex I activity in a sample that contain a plurality of separate solid supports, each coated with a monoclonal antibody specific for a Complex I subunit selected from 30 kDa, 20 kDa, 15 kDa, and 8 kDa subunits of normal Complex I.
  • 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-Complex I monoclonal antibodies in the presence and absence of the agent; and Complex I 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 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 Complex I subunits are contacted with a patient sample comprising solubilized Complex I subunits so that the antibodies bind to Complex I subunits present in the sample to form an antibody/Complex I subunit complex.
  • the Complex I subunits are separated from the remaining sample contents and the presence of post-translational modification in the one or more separated Complex I subunit is detected, wherein the presence of a post-translational modification indicates the patient is suspected of having a late onset mitochondrial disorder.
  • Fig. 1 is a bar graph showing isolated Complex I deficiency in patient fibroblast cell lines as revealed by Western blots using the invention monoclonal antibodies.
  • Western blot signals were quantitated, and the levels of the Complex 139-kDa subunit (CI-39), Complex ⁇ 70-kDa subunit (CII-70), Complex El core 2 subunit (CHI-Core 2), Complex IV subunit IV (CIV-IV), and Complex V subunit (CV-alpha) in normal and RhoO MRC5 fibroblasts and the 11 patient cell lines in relation to the control skin fibroblasts ( were plotted. All subunits were set to 100% for the control fibroblasts. All lanes were standardized using porin as the control for equal loading.
  • Fig. 2 is a bar graph showing variations in Complex I assembly as identified by Western blot. Western blot signals were quantitated, and the levels were plotted of the indicated Complex I subunits in normal and RhoO MRC5 fibroblasts and the 11 patient cell lines in relation to control skin fibroblasts. All subunits were set to 100% for the control fibroblasts. All lanes were standardized using porin as the control for equal loading.
  • Figs. 3 A-F are series of graphs showing the relationship between loss of Complex I subunits and residual Complex I enzymatic activity.
  • the level of each of the indicated subunits as detected by Western blot was plotted in relation to the residual Complex I enzymatic activity.
  • the dashed line represents what would be expected if there were a perfect correlation between loss of subunit and loss of activity.
  • Figs.4A-C are a series of graphs showing the results of Western blot analysis of three different patient cell lines and a control MRC5 cell line after sucrose gradient centrifugation. Shown are gradients of patient 1 (0), patient 7 (D), patient 11 (x), and control MRC5 fibroblasts (o). In each case the darkest intensity band for each antibody and each sample was set to 100%.
  • Fig. 4A shows the distributions of the Va subunit of Complex TV; Fig. 4B shows the 39-kDa subunit of Complex I; and Fig. 4C shows a 20-kDa subunit of Complex I.
  • Fig. 5 is a chart showing the results of separation of 10 ⁇ g Immunopurified human heart Complex I separated by 10-20 % acrylamide SDS-PAGE. Lanes were stained with (1) Coomassie brilliant blue or (2) a mass spectrometry compatible silver nitrate staining procedure. Table MALDI-TOF and LC/MS/MS was used to detect tryptic peptides from complex I subunits. Complex I proteins (bold) are represented where possible by their gene names (HUGO prefix NDUFx) and the theoretical mass of the mature human polypeptide. Minor contaminating proteins are shown in parentheses. l Neuronal protein, Genbank 13938442, 2 17.2 kDa protein related to the 13 kDa Differentiation Association Protein.
  • 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 Complex I deficiencies of all types. For example, it appears that different assembly profiles occur when different Complex I subunits are mutated.
  • the invention provides methods for determining the amount of Complex I in a biological sample of a mammalian patient.
  • the invention assay comprises contacting isolated antibodies that immunoprecipitate Complex I with a sample comprising solubilized Complex I so that the antibodies bind to Complex I present in the sample to form an antibody/Complex I complex, (i.e., under suitable conditions and for a time suitable to form the antibody/Complex I complex). Remaining sample, for example unbound sample contents, is then separated from the antibody/Complex I complex; and the amount of antibody/Complex I 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 Complex I 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 Complex I in the sample.
  • the term "activity" or "functional activity” as applied to Complex I means all aspects of natural Complex I activity, including, but not limited to Complex I enzymatic activity in oxidative phosphorylation.
  • antibodies that are known to bind specifically to a particular Complex I 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-39 kDa, -30 kDa, -20 kDa, -15 kDa, and -8 kDa subunit antibodies can be used to determine the amount of the respective subunits in the patient sample.
  • a mixture of monoclonal antibodies containing anti-30 kDa, -20 kDa, -15 kDa, and -8 kDa Complex I 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 (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 Complex I peptides that assemble into the Complex I or from a representative group of such normal patients).
  • a comparable sample obtained from a normal patient 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 Complex I peptides that assemble into the Complex I 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).
  • Complex I-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 Complex I can be isolated from any mammalian cell, including human cells, with cells having high energy requirements having the largest supply of mitochondrial Complex I.
  • 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.
  • tissue samples that can be utilized to obtain cells for use in the invention methods include saliva, mucosal cells and semen, for example.
  • the assay can be performed utilizing Complex I 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 studies contained in the Examples herein show that when any one Complex I subunit is mutated, the levels of assembled Complex I, and hence of Complex I activity, are reduced.
  • the invention provides methods for detecting Complex I deficiency in a patient by contacting monoclonal antibodies specific for each of a plurality of Complex I subunits with a patient sample so that the antibodies immunocapture Complex I subunits present in the sample, wherein the antibodies are each tagged with a detectable label.
  • the antibodies specific for different subunits of Complex I 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 fluoresence of different colors, and gold or other metallic labels can be used.
  • the amount of each Complex I subunit immunocaptured by a respective Complex I 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 Complex I subunits in the patient sample as compared to the amount in a normal sample indicates the presence of a Complex I deficiency in the patient.
  • the invention assay can be used to detect a decrease in Complex I 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-Complex I or anti- Complex I subunits or the invention methods can be repeated to independently assess the amount of each of the plurality Complex I subunits assayed in the sample by assaying the Complex I 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 Complex I assembly factor.
  • 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 Complex I of the mitochondrial respiratory chain and immunoprecipitating Complex I, wherein Complex I retains functional activity. It should be understood that the invention monoclonal antibodies may be able to immunoprecipitate complex I in the absence of all 45 subunits being present, although antibodies that precipitate the entire 45 subunit complex are preferred.
  • the invention also provides isolated monoclonal antibodies that specifically bind to subunits of Complex I, for example the 39 kDa, 30 kDa, 20 kDa, 18 kDa, 15 kDa, and 8 kDa subunits of Complex I. In particular, anti-15 kDa subunit and anti- 20 kDa subunit monoclonal antibodies can be used to immunoprecipitate the fully assembled complex of complex I.
  • Hybridoma cell lines producing monoclonal antibodies useful in the invention methods for immunocapture of Complex I are commercially available by hybridoma name as used to identify the monoclonal antibodies Table I and Table II below from Molecular Probes (Eugene, OR) or from the Monoclonal Antibody Facility at the University of Oregon (Eugene, OR).
  • b OE overexpression of the human form of the protein in E. coli
  • WB Western blot with biochemically and immunopurified bovine Complex I and human mitochondria.
  • c ND not determined.
  • CM+ antibody not purified, but antibody-containing conditioned medium works well
  • epitope refers to an antigenic determinant on an antigen, such as a Complex I or a Complex I subunit, to which the paratope of an antibody, such as an antibody that binds to a Complex I or a Complex I 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 Complex I, or a Complex I 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.
  • carrier protein e.g., a carrier protein
  • 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.
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or
  • 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, Pqroduction of Polyclonal Antisera, in hnmunochemical 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-Complex I 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. USA, 86:3833 (1989), which is hereby incorporated in its entirety by reference.
  • 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.
  • 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 VL chains. This association may be noncovalent, as described in Inbar et al, Proc. Nat'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 V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V domains connected by an oligonucleotide.
  • 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 Enzymology, 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 Enzymology, Vol. 2, page 106 (1991).
  • the levels of the 20- and 18-kDa subunits were higher than the levels of functional complex as measured by enzymatic activity, whereas the levels of the 15- and 8-kDa subunits were lower.
  • the RhoO cells tested seem to behave differently, because the 15- and 8-kDa subunits are present in higher amounts relative to the 20- and 18- kDa subunits.
  • patient 11 is the most likely to involve a mutation in an assembly factor for Complex I.
  • the levels of subunits are low, and these subunits are not in a fully assembled complex based on the sucrose gradient data.
  • the comparison of subunit profiles based on the results of the invention methods allows patients to be sorted, as in genetic complementation studies, so that with wider screening of patients, a group of possible Complex I assembly factor mutants can be collected for chromosomal analysis and gene identification, as was done for the SURFl mutations of cytochrome c oxidase.
  • the invention antibodies and methods also are useful for adding to the understanding of genotype-phenotype relationships of mutations already specified genetically.
  • Patient 7 was shown to carry a mutation, T423M, in subunit NDUFVl, the flavin-containing subunit of Complex I.
  • the subunit profile and, more definitively, the sucrose gradient experiments described in Examples show that the -25% loss of activity of Complex I is due to altered catalytic function and not a failure to assemble the complex. This result is different than the results obtained in other patients studied in the Examples herein, such as patients 5, 6, and 8, in whom the levels of all of the subunits probed were significantly decreased, suggesting a more profound defect.
  • the invention provides methods for screening for an agent that causes mitochondrial disorders by contacting samples containing cells with an anti-Complex I antibody in the presence and the absence of the agent and determining the complex I 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 Complex I is assayed.
  • the invention method can be used as a simple screen for when to remove patients from drugs that affect Complex I 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 complex I activity in the cells of the patient.
  • the method can be repeated at suitably spaced intervals, with decreased Complex I activity over time indicating increased damage.
  • suitably 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 Complex I 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 HIV 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 Complex I subunit is involved.
  • late onset mitochondrial disorders include, for example, late onset diabetes (NIDDM), Huntington's, Parkinson's and Alzheimer's diseases, ALS (amyotrophic lateral sclerosis), Schizophrenia, and the like.
  • NIDDM late onset diabetes
  • ALS amyotrophic lateral sclerosis
  • Schizophrenia and the like.
  • post translational modifications to Complex I subunits are thought to be caused by free radical damage and result in one or more Complex I subunit having a molecular weight different than the molecular weight of the corresponding normal Complex I 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 Complex I subunits with a patient sample comprising solubilized Complex I subunits so that the antibodies bind to Complex I subunits present in the sample to form an antibody/Complex I subunit complex; b) separating the Complex I subunits from the remaining sample contents; and c) detecting the presence of aberrant post-translational modification in the one or more separated Complex I 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 -). 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 modification.
  • matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has previously been used to characterize peptides containing 3-nitrotyrosine (Sarver A, et al. JAm SocMass Spectrom 2001 Apr 12:439-48).
  • MALDI matrix-assisted laser desorption/ionization
  • mass spectrometry is used to detect post-translational modifications of various Complex I subunits by comparing the molecular weight of particular Complex I subunits obtained from the patient with those of a corresponding normal Complex I subunit. A difference in molecular weight between the two indicates that the patient's Complex I activity is impaired and that the patient should be more thoroughly screened for aberrant post translational modification of Complex I subunits, suggesting late onset diseases.
  • Complex I 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 Complex I subunits involves an immunoassay wherein the Complex I subunits obtained from a patient sample are separated (see description of Fig.
  • nitrotyrosine antibody 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 Complex I subunit indicates that the patient's Complex I activity is impaired and that the patient should be more thoroughly screened for late onset diseases, such as disclosed herein.
  • the invention provides a kit for determining complex I activity in a cell.
  • the kit comprises one or more anti-Complex I antibody.
  • the invention kit may 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/Complex I complex when the antibody is contacted with a Complex I containing sample.
  • the invention kit may contain any of the monoclonal antibodies contained in Tables II and III.
  • 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 Complex I using the contents of the invention kit.
  • the assays described herein are based on the specificity of the anti-Complex I monoclonal antibodies as well as upon antigen function. Any general biochemical activity of Complex I will suffice as a marker of Complex I activity function as the antibody/antigen capture of functional Complex I provides specificity to the assay.
  • the assay can also detect all categories of defects in Complex I 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 Complex I 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 Complex I.
  • 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, ELIS A (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, ELIS A (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 RJJPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 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 RJJPA buffer (1% NP-40 or Triton X-100,
  • 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 125 1) 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 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.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELIS As known in the art. For further discussion regarding ELIS As 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. [0060] 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.
  • labeled antigen e.g., 3 H or 125 1
  • 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.
  • a screening protocol for characterizing the invention monoclonal antibodies for antibody specificity is set forth in the Examples herein.
  • Immunopurified bovine heart Complex I was generated by solubilizing bovine heart mitochondria in 1% N-dodecyl- ⁇ -D-maltoside (LM; Calbiochem, S_D_, CA), centrifuging twice (10,000 x g, 12 min) to remove insoluble material, passing the supernatant over an immunoaffinity column generated as described previously (16) using the 15-kDa Complex I mAb 17G3D9E12 created as described below, washing with phosphate-buffered saline (PBS) containing 0.05% LM, and eluting with 100 mM glycine, pH2.5.
  • PBS phosphate-buffered saline
  • MRC5 fibroblasts were obtained from the American Type Culture Collection (ATCC), and MRC5-Rho0 fibroblasts were derived from theMRC5 fibroblasts by culturing the cells in permissive medium supplemented with 50 ng/ml ethidium bromide as described previously (17).
  • Patient fibroblasts were obtained from skin biopsies of young children in whom an isolated Complex I deficiency has been confirmed in muscle tissue as well as in cultured fibroblasts, using the slightly modified method of Fischer et al. (18). The phenotypes and genotypes of the patients included in this study have been extensively described by Loeffen et al. (2). Control fibroblasts were obtained from postcircumcision tissue from a child in the same age range in whom biochemical enzyme analyses revealed normal results.
  • the monoclonal antibodies used in this study were developed at the University of Oregon (Eugene, OR) by immunizing mice with purified bovine Complex I as described previously (22).
  • the antigen used to generate monoclonal antibodies was beef heart Complex I purified according to Hatefi (13).
  • Virgin female BALB-c mice, 6-8 weeks old were given a primary intraperitoneal immunization of an emulsion consisting of one part aqueous antigen solution and three parts complete Freund's adjuvant.
  • Subsequent boosts were given at 3-4 week intervals, and were also delivered intraperitoneally, but consisted of antigen emulsified in incomplete Freund's adjuvant (IF A).
  • the final boosts were given 3 and 4 days before the splenocytes were harvested, and were delivered intraperitoneally, in either IFA or saline.
  • Splenocytes were then mixed with the appropriate number of myeloma cells and immediately processed for cell fusion.
  • the remaining splenocytes were collected by centrifugation, resuspended at a concentration of 4.5 x 10 7 cells/ml in freezing medium (one part DMSO, nine parts 20F-HgDMEM) and 1.5 ml aliquots sealed in cryotubes.
  • the cryotubes were then placed in a Styrofoam box, frozen at -80 °C for one day, and then transferred to a liquid nitrogen freezer.
  • Cell fusions were conducted as known in the art except that macrophage feeder cells were replaced in cell culture media by P388D1 cells (ATCC) producing plasmacytoma-growth factor.
  • the newly generated monoclonal antibodies were sequentially screened for 1) binding to purified bovine Complex I adsorbed to polystyrene; 2) binding specifically to a single subunit in denaturing Western blots of bovine Complex I; 3) binding to a single subunit in denaturing Western blots of the flavoprotein, iron-sulfur protein, or hydrophobic protein subtractions of bovine Complex I; 4) binding to a single subunit in denaturing Western blots of immunopurified bovine Complex I; 5) binding to a single subunit in denaturing Western blots of human mitochondria; and 6) reactivity and mitochondrial localization in immunohistochemistry of human mitochondria. Immunohistochemistry was carried out as described previously (17).
  • the monoclonal antibody concentrations used in these studies were: anti- Complex 1-39 kDa, anti-Complex 1-15 kDa, anti-Complex 1-8 kDa, anti-Complex IV Va, and anti-Complex V- ⁇ at 2.0 ⁇ g/ml, anti-Complex 11-70 kDa at 0.15 ⁇ g/ml (17), anti- Complex Ill-core Complex at 0.3 ⁇ g/ml, anti-Complex II at 3.0 ⁇ g/ml, anti-Complex TV at 0.5 ⁇ g/ml (23), and anti-Complex 1-20 kDa and anti-Complex 1-25 kDa as twice-diluted hybridoma cell culture supernatants.
  • the commercially obtained anti-porin antibody (Calbiochem) used in the assay as the control for equal loading of Western blots was diluted 1:120,000.
  • Screening involved an assay for binding to native Complex I, Western blotting, and/or immunohistochemistry.
  • Biochemically purified and immunopurified bovine heart Complex I, biochemically purified iron-sulfur protein, flavoprotein, and hydrophobic protein subfractions of bovine Complex I and human fibroblast cell lines from controls and patients were used in the screening.
  • Unequivocal identification of two of the antibodies was also made by Western blotting after overexpressingthe candidate human subunit antigens subunits (NDUFA9 and NDUFS3) in E. coli as described above.
  • Fibroblasts were cultured from 11 patients in whom an isolated enzymatic Complex I deficiency had been confirmed in muscle tissue as well as cultured fibroblasts. In seven of the patients, the pathogenic mutation was identified genetically, and for four patients the genetic defect was not identified. The residual Complex I activities of the 11 patient fibroblast cell lines ranged from 35 to 85%. Specifics of the results for each patient are provided in Table III below.
  • control and patient fibroblasts were prepared for immunoprecipitation of Complex I and Complex I subunits using the monoclonal antibodies described in Table II.
  • Control and patient fibroblasts were grown in Ml 99 (Life Technologies), 5 mg/liter TWEEN 20® medium with 10% fetal calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin.
  • Approximately 30 x 10 6 cells were harvested at 95% confluence after mild trypsinization (3-5 min) with 2-3 ml 0.25% trypsin solution/175- cm 2 (5 x 10 6 cells) cell culture.
  • Cells were resuspended in 50 ml 10% fetal calf serum- phosphate buffered saline (PBS). Cells were rinsed three times with 1% fetal calf serum- PBS as well as with PBS and finally frozen at 80 C. To obtain mitochondrial pellets, cells were solubilized in 5 ml homogenization buffer (1 mM EDTA, 0.25 M sucrose, 10 mM Tris, pH 7.4) containing protease inhibitors (0.5 ⁇ g/ml leupeptin, 0.5 ⁇ g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride).
  • PBS fetal calf serum- phosphate buffered saline
  • the mitochondrial pellets were washed twice (15 min, 10,000 x g) with 2 ml washing buffer (1 mM EDTA, 0.25 M sucrose, 10 mMTris/HCl, pH 7.5) including protease inhibitors (0.5 ⁇ g/ml leupeptin, 0.5 ⁇ g/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Finally, pellets were saved in 200 ⁇ l protease inhibitors/washing buffer and stored frozen at 80 °C. Protein amounts were estimated yAy ⁇ zo determination.
  • proteins were transferred electrophoretically (2 h, 0.10 A) to 0.45- ⁇ m polyvinyhdine difluoride membranes in transfer buffer (10% methanol in 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid, pH 11) on ice.
  • transfer buffer 10% methanol in 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid, pH 11
  • transfer buffer 10% methanol in 10 mM 3-[cyclohexylamino]-l-propanesulfonic acid, pH 11
  • CMF-PBS Dulbecco's phosphate-buffered saline
  • the blots were treated with primary anti-Complex I monoclonal antibodies (Table II) diluted in 5% milk CMF-PBS for 2 h.
  • the blots were incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse IgG + IgM (heavy and light chain) at 0.2 ⁇ g/ml (Jackson hnmunoResearch, WestGrove, PA) in CMF-PBS. Specific detection of the secondary antibody was obtained with ECL Plus® chemiluminescent Western blotting detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ) after rinsing the blots with CMF-PBS three times.
  • Mitochondria were isolated from each of the patient fibroblast cell lines, a control skin fibroblast cell line, and normal and RhoO MRC5 fibroblasts (a lung fibroblast cell line). Samples of each were examined by Western blotting with mixtures of antibodies, including ones specific to the 39-kDa subunit of Complex I, the70-kDa subunit of succinate dehydrogenase (Complex II), core II protein of Complex III, subunit II of cytochrome c oxidase (Complex TV), subunit IV of Complex IV, the ⁇ subunit of F ⁇ F 0 (Complex V), and porin (as a control for equal loading of lanes).
  • the mitochondrial samples of the 11 patient fibroblasts were examined for levels of six different subunits of Complex I (referred to by their apparent molecular weights as listed in Table II.
  • mAbs to the 30-, 20-, 15-, and 8-kDa subunits were used as an antibody mixture along with porin, and the amounts of the 39- and the 18-kDa subunits were quantitated relative to porin separately.
  • a bar graph of the levels of the six components of Complex I in the different samples is shown in Fig 2. A significant reduction in the levels of one or more components of the complex was seen in the patient samples, except for patient 7, who had a mutation inNDUFVl.
  • the patterns of subunit loss were similar in patients 3 and 4, each of which has a different mutation in NDUFS4. Similarly, the pattern of subunit loss was the same in patients 5 and 6, each with a different mutation in the same subumt, NDUFS2. Patients 9 and 10, both of which have unidentified mutations, show remarkable similarity in the pattern of subunit loss. This pattern most closely resembles that of patients 3 and 4.
  • RhoO cells where there is an absence of the mitochondrially encoded subunits of Complex I, a different pattern from any of the patient samples was observed. In this case, the levels of the 20- and 18-kDa subunits are as low or lower than those of the 15- and 8- kDa subunits. Subunit amounts were lowest in patient 11, identifying this as a likely candidate for a mutation in an assembly factor (see below).
  • Mitochondria from three cells lines i.e. from patient 7 with a mutation in NDUFVl, patient 1 with a mutation in NDUFS7, and patient 11, with an unknown mutation, were each dissolved in 1% LM and subjected to sucrose gradient centrifugation using a discontinuous gradient as follows.
  • Mitochondria (1 mg) from control MRC5 fibroblasts and three patient cell lines (patients 1, 7, and 11) were pelleted (10,000 * g, 10 min, 4 °C) and resuspended at a protein concentration of 5 mg/ml in 100 mM Tris/HCl, 1 mM EDTA, pH 7.5, 1 ⁇ g/ml pepstatin, 1 ⁇ g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% LM.
  • the mitochondria were incubated in this solution for 20 min on ice with stirring before any insoluble membranes were pelleted again by centrifugation (10,000 x g, 10 min, 4 °C).
  • the supernatant was layered on top of a discontinuous sucrose gradient composed of 250 ⁇ l of 35% sucrose, 500 ⁇ l of 30%) sucrose, 750 ⁇ l of 27.5% sucrose, 1 ml of 25% sucrose, 1 ml of 20% sucrose, and 1 ml of 15% sucrose. All sucrose solutions contained 100 mM Tris/HCl,pH 8.0, 0.05% LM, 1 ⁇ g/ml pepstatin,l ⁇ g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride. The gradient was then centrifuged overnight at 4 °C (128,000 x g, 16.5 h, SW 50.1). The sucrose gradient was fractionated from the bottom of the tube into 500 ⁇ l fractions, which were frozen at 80 °C. For Western blotting, 20 ⁇ l of each fraction was loaded per lane.
  • Fig. 4 shows the distribution in the gradient of the Va subunit of cytochrome c oxidase as well as the 39- and 20-kDa subunits of Complex I for the three patient cell lines and a control of MRC5 fibroblasts.
  • FP complex I flavoprotein
  • the artificial electron acceptor hexamineruthinium (III) chloride
  • This oxidant has the ability to accept electrons from NADH through the FP fraction of complex I. It is an important and useful indicator for general complex I function. Assay conditions are identical to those described above for ubiquinone-1. i one well of a 96-well plate, one microgram of immunopurified complex I or ten micrograms of isolated frozen-thawed mitochondrial membranes are added to phosphate buffer containing antimycin A, potassium cyanide, and hexamineruthinium (El) chloride.
  • the initial rate of NADH oxidation at 37 °C is followed by reading the absorbance of the sample at 340 nm with a spectrophotometer plate reader for 2-5 min after the addition of NADH to the mixture.
  • a reference sample without proteins is used to the record the background NADH oxidation activity of hexamineruthinium chloride (III). This reaction is not sensitive to the potent complex I inhibitor, rotenone.
  • a sensitive fluorescent assay for complex I function uses a non-fluorescent compound, resazurin, which is reduced by NADH and complex I to produce the fluorescent product, resorufin.
  • the assay conditions are identical to those described above for ubiquinone-1 and hexaminerathinium (111) chloride.
  • one microgram of immunopurified complex I or ten micrograms of isolated frozen-thawed mitochondrial membranes are added to phosphate buffer containing antimycin A, potassium cyanide, and resazurin. The initial rate of resazurin reduction at 37 °C is followed by reading the fluorescence (ex. 530 nm, em.

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Abstract

Des anomalies du complexe I sont l'une des causes les plus fréquentes de maladies et de troubles associés à des troubles de la chaîne respiratoire mitochondriale, notamment des maladies génétiquement associées, et des maladies neurodégénératives à début tardif. L'invention concerne des méthodes d'utilisation d'anticorps monoclonaux qui réagissent aux sous-unités du complexe I de 39, 30, 20, 18, 15, et 8-kDa de façon à aider à diagnostiquer les carences de complexe I. Ces procédés consistent à effectuer un criblage rapide permettant de détecter le début d'une variété de maladies ou la phase dans laquelle elle se trouve. L'invention concerne en outre des procédés de criblage d'agents, tels que des médicaments, pour déterminer leur effet sur l'activité du complexe I et des procédés de dépistage de patients dont l'activité du complexe I est altérée et de patients suspects d'avoir une maladie à début tardif associée à la modification post-translationnelle d'au moins une sous-unité du complexe I.
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