WO1996030398A1 - Novel auto antigen - Google Patents

Abstract

This invention relates to polypeptides or peptides or analogues thereof, the amino acid sequences of which encode antigenic segments which immunologically react with multiple sclerosis induced antibodies. These polypeptides or peptides or analogues thereof are useful as diagnostic reagents for detecting the presence of antibodies or T cells from patients with multiple sclerosis and may also be useful as immunogens, in compositions and methods, to illicit anti-idiotypic antibodies against the abnormal autoimmune responses involved in the pathogenesis of multiple sclerosis.

Description

Novel Auto Antigen

This invention relates to polypeptides or peptides or analogues thereof, the amino acid sequences of which encode antigenic segments which immunologically react with multiple sclerosis induced antibodies. These polypeptides or peptides or analogues thereof are useful as diagnostic reagents for detecting the presence of antibodies or T cells from patients with multiple sclerosis and may also be useful as immunogens, in compositions and methods, to illicit anti-idiotypic antibodies against the abnormal autoimmune responses involved in the pathogenesis of multiple sclerosis.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a demyelinating disease of the nervous system which leads to damage of normal myelin. The disease is prevalent in temperate climates where between 50 and 100 per 100,000 people are afflicted with the symptoms of MS. Females are affected somewhat more often than males and most patients develop the first signs of the disease within 10 years of their 30th birthday.

Pathologically, multiple scattered areas of myelin breakdown called plaques, are found within the white matter in the central nervous system (CNS) of MS patients. Actual nerve fibres, the axons, remain intact but are unable to transmit impulses properly. The cause of the demyelination is currently unknown, but people with certain genetically determined immunological characteristics seem to be over-represented among MS populations.

Diagnosis of MS currently depends upon the demonstration of physical signs that imply the presence of plaques in multiple areas of the CNS of MS patients. When this cannot be determined clinically, electrical survey of the nervous system, using evoked potential testing, may be useful. Generally, the diagnosis of MS tends to be clinical; if laboratory support is not forthcoming, observation of the patients course over subsequent months or years may allow it to be made with confidence. There is now general agreement in the scientific community that abnormal autoimmune responses are involved in the pathogenesis of MS but despite intensive work over the last thirty years there is no consensus as to the importance of the various antigens which have been shown to react with antibodies or T cells from patients with MS. In other autoimmune diseases there has been considerable success in cloning new antigens through the use of antibodies from patients to probe cDNA libraries from patients with the disease. This has lead to the isolation of new antigens and in some cases the development of useful immunoassays which have been used in the early diagnosis of these autoimmune diseases. There are however few diagnostic tests for MS.

Summary Of The Invention

The present invention provides peptides and polypeptides or fragments thereof which are immunologically reactive with antibodies obtained from the serum and cerebrospinal fluid of patients with MS. Such peptides and polypeptides or fragments thereof may be employed in diagnostic assays for MS and in the preparation of anti-idiotypic antibodies which may be used in the treatment of the disease.

Thus, the present invention resides in a DNA sequence encoding at least a peptide that is immunologically reactive with MS induced antibodies wherein that DNA sequence is derived from the following nucleotide sequence:

TATCGGCGGAATCCCCGGTAATTCTCATAATCGCCCACGGGCTTACAT CCTCATTACTATTCTGCCTAGCAAACTCAAACTACGAACGCACTCACA GTCGCATCATAATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAAT AGC I I I I I GGATGACTTCTAGCAAGCCTCGCTAACCTCGCCTTACCCC

CCACTATTAACCTACTGGGAGAACTCTCTGTGCTAGTAACCACGTTCT CCTGATCAAATATCACTCTCCTACTTACAGGACTCAACATA

Sequence analyses were conducted on GenBank (releases 80 to 87, National Center for Biotechnology Information, National Library of Medicine, Bethesda, USA) to determine if this nucleotide sequence shared identity with any known or characterised nucleotide sequences. That analysis identified a greater than 98% identity between the above nucleotide sequence and a portion of a mitochondrial gene which encodes a protein, ND4, which is a part of the mitochondrial electron-transport chain.

The present invention also resides in an amino acid sequence which corresponds to the above nucleotide sequence, that sequence being:

He Leu Met He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg lie Met He Leu Ser Gin Gly Leu Gin Thr Leu Leu Pro Leu Met Ala Phe Trp Trp Leu

Leu Ala Ser Leu Ala Asn Leu Ala Leu Pro Pro Thr He Asn Leu Leu Gly Glu Leu Ser Val Leu Val Thr Thr Phe Ser Trp Ser Asn He Thr Leu Leu Leu Thr Gly Leu Asn Met

In the above amino acid sequences the three letter amino acid code is used wherein the letters mean:

Ala Alanine Met Methionine

Cys Cysteine Asn Asparagine

Asp Aspartic Acid Pro Proline

Glu Glutamic Acid Gin Glutamine

Phe Phenylalanine Arg Arginine

Gly Glycine Ser Serine

His Histidine Thr Threonine

He Isoleucine Val Valine

Lys Lysine Trp Tryptophan

Leu Leucine Tyr Tyrosine

Differences in codon usage during the synthesis of polypeptides and peptides between mitochondria and procaryotes (such as E. coli) or eucaryotes, have important implications with respect to the amino acid sequence recognised by MS induced antibodies. The differences between mitochondrial amino acid sequences and procaryote and eucaryotic amino acid sequences are as follows:-

Codon Organism Mitochondria Usual Meaning

ATG Common Tryptophan Termination

AGΔ Mammal Termination Arginine

G

ATA Mammal Methionine (initiation) Isoleucine

ATG Fruit Fly Methionine (initiation) Isoleucine

ATG Yeast Methionine (elongation) Isoleucine

CTA Yeast Threonine Leucine

AGA Fruit Fly Serine Arginine

The above amino acid sequence is written as it would be expressed in mitochondria. In procaryotic and eucaryotic cells the methionines at positions 3, 29 and 42 are replaced by isoleucine. Following the phenylalanines at position 44 and 45 the mitochondrial sequence has 2 tryptophan residues. In procaryotic and eucaryotic cells the codons for these mitochondrial tryptophans are recognised not as amino acids but as stop codons. Thus, the above nucleotide sequence when expressed in procaryotic or eucaryotic cells would produce a protein that is encoded as follows:

He Leu He He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He He He Leu Ser Gin Gly Leu Gin Thr Leu Leu Pro Leu He Ala Phe Trp Trp

The present invention encompasses the product of the above nucleotide sequence as expressed in mitochondria as well as the expression of such a sequence in procaryotic and eucaryotic cells. Polypeptide sequences described herein will be identified according to their origin of expression (ie mitochondrially expressed, procaryotically expressed etc). Where an amino acid sequence is not identified by an origin of expression, that sequence does not contain any amino acids which would be altered by an alternate origin of expression.

The invention further encompasses fragments of the above amino acid sequence(s) which fragments are immunologically reactive with MS induced antibodies. For example, MS IgG and IgA. Fragments within the scope of the invention include, but are not limited to:-

(1) He Leu Met He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He Met lie Leu Ser Gin Gly Leu Gin Thr Leu Leu Pro Leu He Ala Phe Trp Trp

(mitochondrial)

(2) Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg lie He He Leu Ser Gin Gly Leu Gin Thr Leu Leu Pro Leu He Ala Phe (bacterial)

(3) He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He He

(4) Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He Met He Leu Ser Gin Gly Leu (mitochondrial)

(5) Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg

It will be understood that the variation between genetic codes in mitochondria and procaryotes and eucaryotes is encompassed within the scope of the invention. Thus, a polypeptide or peptide fragment written as expressed in mitochondria also encompasses polypeptide or peptide fragments expressed by procaryotes or eucaryotes and vice versa.

Preferably the above sequences are of the general formulae X-Y-Z wherein: X and Z each represent individually of each other a hydrogen atom, or another amino acid, a protected amino acid, another sequence of the general formulae X-Y-Z, a peptide, a polypeptide, an amino group, a carboxyl group or an adjuvant; and Y represents one of the above amino acid sequences.

Such sequences may be glycosylated or non-glycosylated. At least one of X and Z may comprise at least one of the above sequences which may be the same or different from Y.

The invention also encompasses functionally equivalent analogues of the above polypeptide or peptide sequences, which have at least a immunological property in common with the immunological properties of the aforementioned sequences. Analogues as used herein refers generally to amino acid sequences which are functionally equivalent to the polypeptide or peptide sequences of the invention but which contain substitutions, deletions or additions made to those sequences.

The present invention encompasses oligo-peptides prepared from the amino acid sequences set out above or fragments, analogues thereof which include linear (continuous) epitopes, conformational epitopes or both which are immunologically reactive with MS antibodies.

The amino acid sequences of the invention are useful, alone or in combination, uncoupled or coupled to other molecules, in diagnostic methods for detecting MS, in treating MS, and in the production of polyclonal and monoclonal antibodies.

By employing one or more of the above cited peptides or polypeptide sequences in antibody production methodologies, described hereinafter, monoclonal or polyclonal antibodies may be produced which may find use in diagnostic assays or as immunogens for use in the treatment of MS.

Nucleotide sequences of the present invention may be employed in a process for the production of a peptide or polypeptide of the invention or a fragment or analogue thereof comprising the steps of: culturing a host organism contain an expression vector containing a gene which encodes a polypeptide or peptide of the invention or a fragment, or analogue thereof under suitable conditions to permit expression of that peptide, polypeptide fragment, or analogue; and recovering the expressed peptide, polypeptide, fragment, or analogue.

Detailed Description Of The Invention

One of the major developments in autoimmunity in recent years has been the frequent occurrence of enzymes that act as autoantigens, for example thyroid peroxidase in thyroid disease and pyruvate dehydrogenase in primary biliary cirrhosis. Notably several of the autoantibodies which have been identified from these diseases, actually bind to the active site and inhibit their target enzyme.

During attempts to clone novel auto-antigens in Guillain-Barre Syndrome, pooled MS sera was used as one of the disease controls. Preliminary results from these experiments indicated that the MS sera were reacting with the cloned auto-antigens. From a λgtl 1 cDNA library that contained a wide range of central and peripheral myelin protein sequences, there was isolated two clones which reacted with MS sera. Those clones were subsequently sequenced and were found to share substantial identity to part of the human mitochondrial gene. In particular the sequences obtained were part of the mitochondrial encoded protein, ND4, which is one of forty one components of Complex I (NADH dehydrogenase or NADH:ubiquinone oxidoreductase). ND4 is one of the key components of the respiratory chain and is encoded by mitochondrial DNA.

Complex I is one of the components of the electron-transport chain which is involved in electron transport across mitochondrial membranes. Electrons from the reduced coenzyme NADH are passed to co-enzyme Q. The electrons then pass through a series of further complexes to oxygen. By coupling the transfer of electrons with a directional pumping of protons, some of the free energy released in the transport process is stored as an electrochemical potential that is in turn used to drive ATP synthesis in mitochondria.

Complex I is also the region where mutations arise in the most common form of Leber's Hereditary Optic Neuropathy (LHON). In its early stages LHON has been mistaken for MS but can easily be differentially diagnosed. The most common form of LHON occurs where a histidine replaces an arginine at position 340 in the ND4 amino acid sequence.

Due to differences in codon usage between mitochondria and procaryotes it was possible to know within 45 amino acids in ND4 where the immunologically reactive MS epitope was located. The amino acid sequence CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg was selected as a putative epitope based on amino acid sequence analysis from results of an epitope prediction program. Peptides were synthesised and conjugated to diphtheria toxoid to produce a strong immunogen. The immunogen generated a strong antipeptide antibody response in rabbits. Pooled MS IgG which has been used in the isolation of the ND4 clone reacted with the synthetic peptide attached to the diphtheria toxoid after thorough absorption of any antidiphtheria antibody activity. Pooled MS IgG also reacted to a synthetic peptide which contained 8 additional amino acids to the C terminal end of CysLeuAlaAsnSerAsnTyrGluArg ThrHisSerArg. That sequence being LeuAlaAsnSerAsnTyrGluArgThrHisSerArg HeMetlleLeuSerGlnGlyLeu.

Preliminary experiments with MS IgG and IgA from saliva MS patients have shown that the above mentioned peptides are highly immunoreactive with MS antibodies. Subsequent experiments have shown that IgG from patients with MS can inhibit NADH: ubiquinone reductase even at a dilution equivalent to 1μl of serum in 1 ml of enzyme assay.

DNA sequences described herein which encode peptides and polypeptides which are specific for MS antibodies are conspicuously valuable for the information which they provide concerning the mode of action of MS. The DNA sequences are also valuable as products useful in effecting the large scale microbial synthesis of antigens related to MS by a variety of recombinant and synthetic techniques. Put another way, DNA sequences provided by the invention are useful in generating new and useful DNA vectors, new and useful transformed and transfected microbial procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture). Peptides and polypeptides of the present invention embrace analogues and homologues. One may readily design and manufacture genes encoding for microbial expression of polypeptides having primary conformations which differ from those sequences specified herein in terms of identity or location of one or more residues (eg by substitutions, additions and deletions). Alternatively, modifications of cDNA and mitochondrial genes may be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogues of the specified peptides. Such analogues should possess at least one of the immunological properties of the sequences specified herein but may differ in others.

Amino acids residues that are functionally equivalent and that can be substituted in the same position for other amino acids to produce analogues without substantially effecting the conformational arrangement of an antibody are known to the art. For example, exchange of the positively charged amino acids arginine and lysine is considered to be a conservative substitution. Similarly substitution of the hydrophobic amino acids valine, leucine, isoleucine and methionine, the hydroxy amino acids threonine and serine and the acidic amino acids glutamic acid and aspartic acid are considered to be conserved.

Preparation of Antibodies Against MS Specific Epitopes

Immunogenic peptides or polypeptides as described above may be used to produce either monoclonal or polyclonal antibodies.

If polyclonal antibodies are required, a selected mammal (eg mouse, rat, sheep, monkey) is immunised with an immunogenic polypeptide bearing an MS specific epitope. For example the diphtheria toxoid immunogen discussed herein.

Serum from the immunised animal is then collected and treated according to known procedures (Linthicum etal 1981) which will depend on the ultimate use to which the polyclonal antibody will be employed. If serum containing polyclonal antibodies to the MS specific peptides and polypeptides contain contamination antibodies (ie. generated against other antigens) the antibody sera can be purified by for example immunoaffinity chromatography.

Alternatively polyclonal antibodies can be isolated from a patient suffering from MS. A full discussion of how such antibodies may be isolated is discussed below.

Monoclonal antibodies generated against the MS related peptides and polypeptides can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known (Campbell, 1984). Immortal antibody-producing cell lines can be created by cell fusion or by direct transformation of B lymphocytes with oncogenic DNA or by transfection with Epstein Barr Virus.

Antibodies, either monoclonal or polyclonal, which are directed against MS related peptides or polypeptides are useful in diagnosis. Further monoclonal antibodies, may be used to raise anti-idiotypic antibodies (ie which carry an internal-image of the antigen of the MS specific peptides or polypeptides). Such antibodies may be used in the treatment of MS (Thornton and Griggs, 1994). Techniques for raising anti-idiotypic antibodies are known in the art see, for example Dreesman etal (1985) (Poskitt etal, 1991) and (Linthicum and Farid, 1988).

Immunoassays And Diagnostic Kits

Peptides and polypeptides which react immunologically with serum containing MS immunoglobulins and the immunoglobulins raised against the MS specific epitopes are useful in immunoassays to detect the presence of MS immunoglobulins in biological samples, including cerebrospinal fluid, blood and saliva.

Design of diagnostic assays is subject to a great deal of variation. Such detection kits include, but are not limited to homogeneous and heterologous binding immunoassays, such as enzyme linked immunoabsorbant assays (ELISA), radioimmunoassays (RIA), Western Blot analysis, and enzyme inhibition assays.

Peptides and polypeptides of the invention may be labelled or unlabelled depending on the type of assay used. Labels which may be coupled to the peptides are those known in the art and include but are not limited to enzymes, radionucleotides, fluourogenic and chromogenic substrates, cof actors and, biotin-avidin, colloidal gold and magnetic particles.

The peptides and polypeptides can also be coupled to other peptides or polypeptides, solids supports and carrier polypeptides by any means known in the art. Such solid supports include for example polystyrene or polyvinyl microtitre plates, glass tubes, or glass beads and chromatographic supports such as paper, cellulose and cellulose derivates and silica. Carrier polypeptides include for example bovine serum albumen (BSA) and Keyhole Limpet hemocyanin (KLH).

Techniques especially useful for large scale clinical screening of patients sera, cerebrospinal fluid or saliva include ELISA and agglutination assays. Such techniques are preferred for their speed, and their ability to test numerous samples simultaneously and ease of automation.

Protocols upon which ELISA assays may be based include for example competition assays, direct reaction assays and sandwich type assays. In ELISA assays samples including for example body fluids and tissue samples may be added to a peptide coated wells in for example a microtitre tray where an immunological complex forms if MS antibodies are present in the sample. A signal generating means may be added to detect complex formation. A detectable signal is produced if MS specific antibodies are present in the sample.

Peptides or polypeptides of the invention are conveniently bound to the inside of microtitre wells. Peptides may be directly bound by hydrophobic interaction with the microtitre wells or attached covalently to a carrier polypeptide by means known in the art. The resulting conjugant being used to coat the wells.

Another example of an assay which may be used to detect the presence of MS antibodies is an agglutination assay. Such assays utilise latex support (eg beads) to which are bound at least one of the MS specific peptides. The coated latex beads are mixed with a small volume of patient sera and examined for agglutination. If MS specific antibodies are present in the patient's serum agglutination (clumping) of the latex beads will be observed. While this assay is not as specific as an ELISA, latex assays are quick and easy to perform and would be suitable for medical practitioners or naturopaths as initial screening.

The ELISA is a far more sensitive and quantifiable assay than the latex agglutination assay. Preliminary screening studies using MS specific peptides and polypeptides have shown that MS antibodies are found in low levels in patient's sera. Thus it is expected that the ELISA test would be at least a method of choice for the detection of MS antibodies.

A more specific assay for detecting the presence of MS utilises an enzyme inhibition assay. In this assay, NADH: ubiquinone reductase activity is measured in the presence of sample material suspected of containing MS antibodies. In the presence of MS antibodies NADH: ubiquinone reductase enzymic activity is substantially reduced indicating the presence of MS in a patient. A detailed discussion of this assay is presented below.

At first sight it is difficult to see how the antibody to Complex I could be involved in the pathogenesis of MS, as it would have to get inside the oligodendrocyte and then inside the mitochondria. However, there is no need for this to occur because in the plasma membrane of human cells there is a quinone dependent enzyme Plasma Membrane Oxidoreductase (PMOR)(Lawen et al, 1994). This enzyme appears to act as a fall-back mechanism to enable cells to survive in the presence of damaged mitochondria, as it uses both NADH and quinones as substrates. Antibodies which react with its quinone binding site are likely to inactivate this enzyme. Because the enzyme is located in the plasma membrane the MS antibodies are able to easily interact with it. An accumulation of somatic mutations in oligodendrocytes in MS or damage to oligodendrocytes by a virus or viroid will cause an upregulation of PMOR in the plasma membrane of some but not all oligodendrocytes. Thus an enzyme inhibition assay which measures the activity of PMOR may serve as an alternative measure for the presence of MS antibodies and hence the presence of this disease.

As a result of this invention it is possible to devise a simple and highly specific assay kit which will depend upon the concept of detecting the MS antibody by inhibition of Complex I or inhibition of PMOR. The value of such assays will depend upon the prevalence of the MS antibody and its fluctuation with diseased state. An alternative to the detection of IgG in blood samples is the detection of IgA in saliva. An enzyme inhibition assay as described above when used in combination with an ELISA or as a separate diagnostic testing means would form a very simple and easily applied test for MS. Another possibility for a diagnostic aid is the ability to monitor the response to T cells in the MS patient to the synthetic peptide which reacts with the antibody.

The invention also provides a method of treating a patient suffering from MS wherein that treatment involves administration of an effective amount of suitable quinones to MS patients to restore the function of the damaged oligodendrocytes.

Alternatively the invention provides a method of treating a patient suffering from MS wherein that treatment involves administration of a humanised anti-idiotypic monoclonal antibody which will negate the effects of MS antibodies.

The present invention will now be described by reference to the following non- limiting figures and examples. It will be understood that all of the parameters prescribed in the examples are given as indicative only, and that parameters outside these limits may also provide useful results. In the Figures:

Figure 1 shows the electrophoretic pattern of products of amplification by PCR of the DNA clones isolated with MS IgG. λgtl 1 primers were used for the amplification. The marker DNA in lane 1 was a mixture of puc 19 DNA/MpA II and λ DNA Eco Rl/Hind III. Lanes 3 and 4 represent the products from clones M62 and M63 respectively.

Figure 2 shows the consensus sequence from analysis of nucleotides in clones M62 and M63 which were found to express peptides and polypeptides specific for multiple sclerosis antibodies. This sequence was found to be 98% identical to part of the human mitochondrial gene (HUMMTCG in GenBank release number 80) from nucleotide 11699 to 11960.

Figure 3 shows that the consensus nucleotide sequence in M62/63 is over 95% identical to six depositions of human mitochondrial DNA in GenBank

(release number 87). It is also homologous to 10 depositions of mitochondrial DNA from other primates.

Figure 4 shows amino acid sequences predicted from the nucleotide sequences. Figure 4a shows the predicted amino acid sequence for the protein expressed in clone M62 which reacted with antibodies from patients with multiple sclerosis based upon codon usage in E. coli. Figure 4b shows the predicted amino acid sequence for human mitochondrial ND4 protein from amino acids 315 to 367. The amino acid sequences presented in figures 4a and 4b are represented in single letter amino acid coding form, in accordance with the following table:

A Alanine M Methionine

C Cysteine N Asparagine

D Aspartic Acid P Proline

E Glutamic Acid Q Glutamine

F Phenylalanine R Arginine

G Glycine S Serine

H Histidine T Threonine

I Isoleucine V Valine

K Lysine w Tryptophan

L Leucine Y Tyrosine Figure 5 shows an epitope prediction analysis of the sequence in Figure 4a and the peptide sequences synthesised. Figure 5a represents the antigenic index analysis by MacVector 3.5. Figure 5b represents the conjugate prepared for immunisation of a rabbit and as an antigen (diphtheria toxoid -S-MS immunoreactive peptide) for testing MS IgG and

IgA. Figure 5c represents the peptide prepared for use as an antigen in peptide ELISA.

Figure 6 shows the reactivity of rabbit sera to diphtheria toxoid and diphtheria toxoid -S-MS immunoreactive peptide. The diphtheria toxoid was used to coat the wells 1 -5 and the conjugate was used in well 6. After

30 min incubation the supernatant from well 1 was moved to well 2, incubated for 30 min before the supernatant was moved to well 3. This process was repeated until the supernatant reached well 6.

Figure 7 shows the reactivity of rabbit sera to the 20 amino acid peptide presented in Figure 5c.

Figure 8 shows the reactivity of human MS IgG to diphtheria toxoid and diphtheria toxoid -S-MS immunoreactive peptide. Diphtheria toxoid was placed in wells 1 -3 and the peptide conjugate in well 4. Sequential transfer of the supernatant was done as described for Figure 6.

Figure 9 shows the reactivity of IgA in human saliva to the diphtheria toxoid

-S-MS immunoreactive peptide, after subtracting residual activity to diphtheria toxoid. Sample numbers 1 to 12 were normal saliva, 13 to 31 were from people with MS.

Figure 10 shows the reactivity of human IgG to the 20 amino acid sequence in Figure 5c. IgG from individual MS patients (1 to 10) was reacted at an equivalent of 1/200 dilution of original sera with the 20 amino acid peptide. Pooled normal human IgG is shown as 0.

Figure 11 shows the activity of Complex I from chicken brain submitochondrial particles (SMP) with additions of human IgG. The SMP were 10μl,(2 mg total protein/ml) were incubated for 90 min with the 10μl IgG (0.4 mg/ml) before the addition of NADH and decylubiquinone in K phosphate 20 mM buffer to 1ml total volume with final concentrations of substrates of 0.15 mM and 0.1 mM respectively.

Figure 12 shows the effect of time of incubation on inhibition of Complex I by MS IgG conditions as per Figure 11 except for time of incubation. Inhibition of enzyme activity was determined between 2 and 10 minutes.

Figure 13 shows inhibition of Complex I by increasing concentrations of MS IgG conditions as per Figure 11 except for the concentration of IgG.

Figure 14 shows the reactivity of rabbit serum to the diphtheria toxoid -S-

MS immunoreactive peptide with GST-M62 recombinant protein from clones 5, 9 and 19. Rabbit serum taken before immunisation was tested with clone 19.

Figure 15 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 3 patients with MS who were bled at various intervals. The numbers show the weeks between investigations.

Figure 16 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 54 patients with MS and 12 other people.

Figure 17 shows the reactivity of IgG in human sera to the diphtheria toxoid-S-MS immunoreactive peptide after subtracting residual activity to diphtheria toxoid from 8 patients with Leber's Hereditary Optic Neuropathy and 4 carriers of the LHON mutation (Mr/VD4*LHON11778A).

Examples

Sources of sera and saliva samples Initially normal and MS sera were obtained from collections used by Dr R.D. Cook, Murdoch University. Saliva samples were obtained from people with multiple sclerosis in Perth and from people with no apparent disease.

Source of λgtl 1 human foetal spinal cord (HFSC) library

A λgt11 library was obtained from Dr C. Campagnoni, Mental Retardation Research Center, University of California, Los Angeles. It was made from poly A⁺RNA from spinal cords at 14-16 weeks gestation. The cDNAs were cloned into λgtl 1 using Eco R1 linkers. The base was 1.5 x 10⁹ pfu per ml.

Immunoglobulin G (IgG) purification

Purification of immunoglobulin G from human or rabbit sera was performed using affinity column chromatography. 350 mg of commercially prepared protein A- Sepharose CL-4b (Pharmacia LKB, Upsala, Sweden) was swollen in 70 ml phosphate buffered saline (PBS) pH 7.4 and degassed with a vacuum pump for 15 min at room temperature. A Pasteur pipette was adapted as a column by fitting a piece of nylon mesh at the bottom. The protein A-Sepharose in PBS was packed in the column to a height of 10 cm and 2ml of the sample was loaded manually on the top of the column and left until the whole sample passed and then recirculated twice to produce the maximum binding. The column was connected to a peristaltic pump, UV detector (280nm) and recorder (Bio-Rad, model 1326). The column was washed by pumping with PBS until the detector had a stable reading. The bound IgG was washed off with elution buffer (0.1 M acetic acid in 0.15 M NaCI, pH 2.8), neutralised by adding 100μl of 1M Tris buffe H 8.9) per each ml eluted and dialysed (Spectraopor membrane MW cut off 6-8000) against 11 PBS(pH 7.4) overnight at 4°C (with stirring and 3 changes). The total yield of IgG was estimated by reading the optical density (OD) at 280nm and calculating as follows before storing at -20°C at a concentration of approximately 0.4 mg/ml for the MS samples.

Total IgG(mg) = (OD/1.4) x Volume(ml) The column was then washed with 10 bed volumes of PBS(pH7.4), regenerated by the same volume of 0.1 M Tris HCI, 0.5M NaCI, pH 8.5 followed by the 0.1 M Na acetate, 0.5M NaCI, pH 5.5. Finally the column was equilibrated with 10 bed volume PBS containing 0.01% Na azide and stored at 4°C.

Pre-absorption of the IgG solutions

All IgG solutions were preabsorbed to remove the anti-E. coli and anti-beta- galactosidase activity before use in screening of the λgtl 1 HFSC cDNA library.

A non-recombinant λgt11 phage was isolated from HFSC cDNA library using white/blue colour selection. Ten thousand phage from the library were plated on one 150mm Petri dish according to the procedure in the following section except that the top layer of agar was supplemented with 5-bromo-4-chloro-3-indolyl-β-D- galactoside (X-gal) 40mg/ml final concentration. One of the blue plaques was picked up after an overnight incubation at 37°C. The plaque was added to 300ml SM (Promega Protocols and Application Guide, Second Edition 1991) buffer containing one drop of chloroform, vortexed for 30 sec and incubated at least for 2 h at 4°C. The phage solution was replated three times in small plates until there were 100% blue plaques. One of non-recombinant blue plaques was added to 300ml of SM buffer plus 1 drop of chloroform before incubation for 2 h at 4°C. The phage solution (top layer) was added to 100ml of late phase culture of E. coli Y1090 and 900ml of LB media (Sambrook et al. 1989) was added to the culture and incubated for 2 h at 37°C with 250 rpm shaking.

The non-recombinant λgt11 phage was induced by raising the temperature to 45°C for 15 min. Isopropyl thiogalactoside (IPTG) was added to 10 mM final concentration and incubation continued for a further 2 h at 37°C with 250 rpm shaking. The bacteria was harvested by centrifugation at 500g for 15 min, resuspended in 50ml of PBS and then lysed by three freeze-thaw cycles using liquid nitrogen and 37°C water bath respectively. The bacterial lysate solution was then sonicated 3x30 sec bursts at maximum power to reduce the viscosity and stored at -20°C until used. A 137mm nitrocellulose disc (Hybond-c extra, Amersham, England) was soaked in the bacterial lysate solution for 30 min before incubation for 1 h in blocking solution (TBS-T containing 5% BSA). The filter was washed with TBST twice for 1 min, soaked in 50ml IgG solution (diluted 1 :10 with PBS) and incubated overnight at 4°C with slow shaking. This procedure was repeated 3 or 4 times to make sure that all anti-bacteria and anti-beta-galactosidase activity was absorbed. The IgG solution was tested with a dot blot procedure to make sure that there was no reactivity with the E. coli lysate proteins. In some cases when there was still some background activity the IgG solution was mixed with the equal volume of bacterial lysate, incubated overnight at 4°C with rotation and then centrifuged at 10O.OOOrpm, 15 m at 4°C. The supernatant was stored in 1 ml Ependorph tubes at -20°C until required.

Parallel lift method for immunoscreening

The bacterial parallel culturing method (Sambrook et al. 1989) was adapted for screening phage preparation production of duplicate filters carrying the expressed recombinant protein. An overnight culture of E.coli Y1090 was made in 10ml LB

(containing 0.2% maltose final concentration) and centrifuged at 3000g for 10 min at 4°C. The bacterial pellet was resuspended in minimal media. Bacteriophage and bacteria were mixed and incubated at 37°C for 20 min for uptake. The mixture was added to 5ml of top layer agar, prewarmed to 60°C, and poured onto an LB plate. Both bottom and top layer agar as well as LB media were supplemented with MgS0₄ to 10mM final concentration. Several combinations of phage were plated, at varying densities, to determine the optimum dilution. Lifts were taken from these phage plates using supported nitrocellulose filters pre- soaked in 10mM IPTG, and air dried in a laminar flow on sterile Whatman 3MM paper.

Parallel lifts were obtained by incubating the phage plates at 37°C for 4-6 h until the plaques became visible. One filter was placed on the first plate, marked for later orientation and transferred to a second plate after 1 min. The second plate had only a bacterial lawn, prepared at the same time as the phage was plated. The second duplicate filter was then placed on the first plate and marked. Both plates were incubated at 37°C for at least 4 h to overnight. The treatment of both filters was the same from this stage onwards.

Enhanced chemiluminescence (ECL) detection

Amersham's ECL Western blotting kit (Amersham Life Science, England), a sensitive non-radioactive method for detection of immobilised antigens, was adapted for screening the expressed proteins from a cDNA library. The filters from above were washed with TBS-T and immersed in blocking solution (5% BSA in TBS-T) for 1 h at room temperature with low speed shaking to block the non- specific binding sites. Washing the filters with TBS-T was similar for all stages of this procedure; two washes of 1 min, one of 15 min and a further two of 5 min at room temperature with slow shaking, except for the last washing which had two extra 5 min washings. The filters were washed before and after incubation in IgG solution and the secondary antibody (anti-human Ig, horseradish peroxidase linked F[Ab]2, Amersham, England). Incubation in each antibody was 1 h at room temperature with low speed shaking. Finally, the filters were immersed in ECL detection reagents for 1 min and exposed to the radiography film for 15 min according the manufacturer's procedure.

Isolation of positive clones

The positive clones were isolated from the HFS cDNA library by probing with a pool of IgG prepared from the individual sera. About 2 million recombinant phages from HFSC cDNA library were plated out on 150mm Petri dishes (40,000 in each) as described above. Expressed proteins were transferred to nitrocellulose discs as described above. Positive plaques which produced signals in duplicate filters were picked after alignment with the original plate by stabbing a sterile Pasteur pipette through both the top overlay and underlying agar. The plaque was transferred to a 1.5 ml Ependorph tube containing 300μl of SM buffer, vortexed for 30 sec after adding one drop of chloroform and incubated at 4°C overnight. Positive clones were rescreened three times in small plates (90mm, about 100 plaques each) until all plaques produced positive signals in duplicate filters.

Isolation of clones

A total of 6 clones which reacted with the pooled MS IgG, but did not reacted with pooled normal IgG, survived tertiary screening. These clones had inserts ranging from approximately 500 to 4,000bp. Fig 1 shows the results of PCR on some of these clones.

Amplification, titration and storage of phage

All positive recombinant phages isolated as described above were amplified using E.coli LE392 and stored for future work. An aliquot of each isolated phage preparation containing approximately 50,000 phage was transformed into E.coli LE392 cells (300ml, OD^o = 0.5) and plated out on 150mm agar plate. The plates were left at 37°C overnight before adding 12ml of SM buffer and then incubated overnight but at 4°C to allow the phage to diffuse into the buffer. The SM buffer was collected and centrifuged (10,000g, 10 min) to remove the agar and bacterial debris. Each phage solution was titrated by making 10^"3, 10^"5, 10^"7 and 10^"9 dilutions in SM buffer. 10 and 100μl of each phage dilution was added to 100μl of LE392 cells and incubated at 37°C for 20 min for transformation. The infected cells were then mixed with 4ml top layer agar (prewarmed to 60°C and supplemented with 10mM MgS0₄) and plated out on agar in 90mm Petri dishes. The plates were incubated at 37°C overnight upside down and then the plaques were counted to determine phage titres. Each phage solution was transferred to an Ependorph tube and stored at 4°C with 2% (v/v) chloroform or at -70°C, for long term storage, after adding 7% (v/v) DMSO and snap freezing in liquid nitrogen.

Large scale preparation of phage

A single colony of E.coli LE392 was inoculated in 100ml NZCYM media (Sambrook et al. 1989) in a 500ml flask and incubated overnight at 37°C with vigorous agitation (300 cycles/min in a rotary shaker). Four aliquot were withdrawn, each containing 10¹⁰ cells (1 ODeoo = 8 x 10 cell/ml), harvested (400g, 10 min) and added to 5 x 10⁷ pfu of phage, separately, after resuspending in 3ml SM buffer. The mixture was incubated at 37°C for 20 min before adding to 500ml of prewarmed (to 37°C) NZCYM in 21 flasks. The samples were incubated at 37°C for 8-12 h with vigorous shaking (300 cycles/min) until the lysis was completed. In cases where the lysis was not completed promptly an additional 500ml NZYCM was added to the culture and incubation was continued for 2-3 h more. Finally 10ml of chloroform was added to each flask and incubated for 10 min at 37°C with shaking.

DNA purification from phage

Solid NaCI was added to each large scale phage culture to a final concentration of 1 M, dissolved and left in ice for 1 h to dissociate the phage particles from bacterial particles. The clear phage solutions were poured, after removing the bacterial debris by centrifugation at 11 ,000g for 10 min at 4°C. The bacteriophage particles were precipitated by adding solid polyethylene glycol (PEG 8000) to a final concentration of 10% w/v, incubated in ice for 1 h and centrifuged at 11 ,000g for 10 min at 4°C. Each phage pellet was resuspended in 8ml SM buffer and then extracted with an equal volume of chloroform vortexed for 30 sec and centrifugation at 3000g for 15 min at 4°C. Solid CsCI was added to the top aqueous phase (0.5g / ml supernatant) and dissolved by gentle mixing. A CsCI step gradient was prepared by layering 1.45, 1.50 and 1.70 g/ml (CsCI dissolved in SM buffer) respectively and finally overlayed by CsCI bacteriophage solution and centrifuged at 22,000 rpm for 2 h at 4°C in a Beckman SW41 or SW28 rotor.

Polymerase chain reaction

PCR technique was employed to amplify the inserted DNA from all isolated positive clones using the materials purchased from Biotech International, Perth Western Australia. Lambda gt11 forward and reverse primers (20ng each) were mixed with dNTP (1ml of 2mM), MgCI2 (1ml of 25mM), 0.7 unit of Tth plus DNA polymerase and 10 x PCR reaction buffer (Biotech International, Western Australia) in a 10ml total volume. As DNA template, 40ng of phage DNA, 0.5μl of phage solution or a touch a fresh plaque by tip of a pipette and left in PCR solution for 1 min was used. To prevent the evaporation of solution in high temperature 1 drop of paraffin oil (Biotech International, Western Australia) was added to the top of the solution and spun in a microcentrifuge for 5 sec. The amplification proceeded for 25 cycles in a DNA thermal cycler (Perkin Elemer Cetus, Norwalk, USA). Each cycle involved a 94°C denaturing step for 1 min followed by 1 min annealing temperature at 62°C and then 2 min polymerization step at 72°C. Denaturing time for the first cycle was 5 min to destroy the phage protein coat and DNA super coil. Polymerisation time for the last cycle was 10 min to get full length of amplified products. Each PCR products (5ml) was run on 1% agarose gel for identification of the amplified DNA.

Purification of DNA for sequencing

In cases where the amplified DNA has produced a sharp band, the PCR product was extracted from low melt agarose gel and used for sequencing. Low melt agarose (Bio-RAD, California) was dissolved in 0.5 x TBE buffer (1% w/v final concentration) by boiling. The gel was stained by immersing in ethidium bromide (EtBr) solution (1 Omg/ml in TBE buffer) for 2 min. The DNA samples were loaded after mixing with 6 x gel loading buffer. The electrophoresis was then conducted in 0.5 x TBE buffer in a horizontal apparatus (Bio-RAD) at 60 V until the blue dye passed 2/3 of the gel length. The DNA band was monitored under the UV light. The desired DNA bands were excised from agarose gel using a clean, sterile razor blade and spun down in a calibrated microcentrifuge for 30 sec to estimate its volume. The gel was melted by incubation for 10 min at 70°C after adding the ^Λh volume of TE buffer and 1/10 volume of 1 M NaCI solution. The mixture was cooled to room temperature before adding an equal volume of phenol vortex for 30 sec and spun at 4000g for 10 min. The upper aqueous phase was transferred to another tube and re-extracted once with phenol/chloroform and once with chloroform. The sample was incubated at -80°C for 30 min after adding Na acetate solution (3 M, pH 5) to a final concentration of 0.3 M and 2 volume of cold 100% ethanol. DNA was then precipitated with centrifugation (14000 rpm at 4°C in a microcentrifuge), washed with 70% ethanol, dried in a vacuum centrifuge for 2 min and resuspended in sterile distilled water or TE buffer. The samples were stored at -20°C until used.

DNA sequencing

Sequencing reactions were preformed by the dideoxynucleotide chain termination method using a PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems Foster City, USA). To 1mg of each DNA template (purified PCR product) was added 3.2 pmol of λgtl 1 forward (or reverse) primer, 9.5μl of terminator premixture and sterile distilled water to 20μl final volume. The mixture was overlayed with one drop of mineral oil to avoid evaporation before the thermal cycling. A total of 25 cycles were employed in a Perkin-Elemer Cetus thermocycler (Model 480). Each cycle included denaturing at 96°C for 30 sec, annealing at 50°C for 15 sec and polymerization at 60°C for 4 min. The cycling program had a rapid thermal ramp to the desired temperature before each section and final soaking at 4°C. The mixture was briefly centrifuged before adding 80μl of water and then purified using the phenol/chloroform extraction method. The mineral oil was dissolved in 10Oμl chloroform and removed with a pipette. The terminators were extracted twice with adding 100μl of phenol:H₂0:chloroform (68:18:14) reagent was added, vortexed and centrifuged. The extracted products were precipitated by adding 15μl of 2 M Na acetate, pH 4.5 and 300μl of 100% ethanol before incubation at -80°C for 20 mh and then centrifuged (14,000 m at 4°C). The pellet was washed with 70% ethanol and dried in a vacuum centrifuge for 2 min. The samples were applied on 6% acrylamide gel and the sequences were analysed using the Applied Biosystem sequencing system, model 373A.

Sequence comparisons were done with BLAST and FASTA using ANGIS (Sydney University).

Sequencing of the clones

Three of the clones M62, M63 and M64 had a very high degree of similarity over the first 300 nucleotides. A concensus sequence was determined from that in M62 and M63 (Fig 2). The consensus nucleotide sequence for clone M62 was compared to all sequences in the GeneBank database. It was 99% identical over 260 nucleotides to a known human sequence HUMMTCG (Fig 2). It is clear from this comparison that clone M62 contained an insert which had originated from part of the human mitochondrial DNA sequence (Fig 3). This is believed to be the first autoantibody discovered to any protein encoded by mitochondrial DNA.

Amino acid sequence in M62

The nucleotide sequence identified in the Fig 2 codes for a protein known as ND4. ND4 is one of the 41 components which comprise the enzyme known as Complex I (NADH:ubiquinone reductase). ND4 has a total of 459 amino acids. Because of differences in codon usage between mitochondria and E. coli in the synthesis of proteins there are important implications with respect to the amino acid sequence recognised by the MS IgG. The predicted amino acid sequence which has been synthesized by E. coli in the λgtl 1 library is shown in Fig 4. The main differences between the mitochondrial amino acid sequence and the cloned sequence are at positions 3, 29 and 42 where the methionine is replaced by an isoleucine in the E. coli sequence. Following the phenyalanine at position 44 and 45 the mitochondrial sequence has 2 tryptophan residues. However in E. coli the codons for these mitochondrial tryptophans are recognised not as amino acids but as stop codons. Thus it would appear that the antibody had recognised quite a short sequence comprising 44 amino acids.

Epitope prediction

The MacVector 3.5 (IBI, New Haven, USA) program was used to predict likely epitopes in the amino acid sequence. The region which had a positive antigenic index was used in the design of peptides chosen for synthesis (Figure 5a).

Epitope analysis of the cloned sequence

Although epitope analysis programs have only about a 50% chance of predicting epitopes in a protein it was considered worthwhile examining the cloned sequence for potential epitopes. Fig 5 shows a peak between amino acids 18 and 24 which suggests that this region could be an epitope. Two peptides were synthesized. The first peptide CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg was linked to diphtheria toxoid as a carrier through a maleimido-thiol bond on its C-terminal cysteine. This peptide was used an immunogen to generate antibody in a rabbit and as an antigen in ELISA assays.

The second peptide had the sequence LeuAlaAsnSerAsnTyrGluArgThrHisSer ArglleMetlleLeuSerGlnGlyLeu was used as an antigen in ELISA assays.

Peptide synthesis and immunisation of rabbit

Two peptides were synthesized by Chiron Mimotopes, Melbourne. A 13 amino acid sequence, CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg, was conjugated through its N-terminal cysteine via an maleimido-thiol bond to diphtheria toxoid for use as an immunogen and as an antigen (Figure 5b). The peptide was 84% pure and conjugated in ratio of 1.2mg peptide to 14mg of toxoid. Antibodies were generated in a rabbit by immunising with 250μg of the conjugate in Freund's complete adjuvant, distributed over 4 sites, followed by a booster after 3 weeks with the same conjugate (150μg) in Freund's incomplete adjuvant.

A second peptide of 20 amino acids, LeuAlaAsnSerAsnTyrGluArgThrHisSer ArglleMetlleLeuSerGlnGlyLeu, was synthesized for use in peptide ELISA assays (Figure 5c). The peptide was 39% pure by mass spectrographic analysis.

EUSA assay for antisera or purified IgG

Enzyme linked immunosorbant assay (ELISA) technique Linthicum et al 1981 was used to screen the activity of antibody against the synthetic peptides or conjugated proteins. Assays were carried out in 96-well micro-titer plates (NUNC ). A Trtertech multi-channel pipette (Flow Laboratories ) was used to dispense all the buffers or solutions into the wells. The volume of the used solutions was 10Oμl per well, except for the blocking buffer which was 300μl. Washing of the wells was carried out with phosphate buffered saline (PBS, 0.01 M Na phosphate in 0.15 M NaCI, pH 7.2) containing 0.1% Tween 20. The washing step that was repeated four times. The excess buffer was removed from the wells after the final washing by vigorously "slapping" the plate, well down, on a benchtop covered with paper towels. Incubation was preformed with gentle shaking on a rotary shaker at room temperature.

For coating the wells with protein the plates were left exposed to the air at 37°C overnight to allow the solution (1 OOμl of 5 μg / ml protein in water) to evaporated to dryness. For coating with short peptides the wells were pre-coated with glutaraldehyde (0.2% v/v) for 45 min at room temperature. The wells were washed twice before dispensing the peptide solution (2μg/100μl PBS) into wells and incubating overnight at 4°C. The plate was washed once and blocked by adding the 200μl of a solution containing 3% w v BSA in PBS or 1 % BSA plus 0.1 M glycine in PBS. The plates were incubated for 2 h at room temperature to block any remaining unblocked attachment sites on the wells.

The purified IgG was added after washing the plates and incubated 30 min to 2 h at room temperature. To find the optimum reactivity between antigen and antibody, different dilution of IgG and antisera prepared in blocking solution were tested. Each dilution was in duplicate or triplicate. Only blocking solution was added to some of the wells as a blank. The wells were washed before adding the secondary enzyme linked antibody and incubated 60 min at room temperature. The secondary antibody which was conjugated to horseradish peroxidase (same as above) was used in 1 :1000 dilution in blocking solution. The wells were washed and then finally washed with PBS without Tween before the substrate was added.

Colour development of the plates was preformed by adding 100ml of substrate solution of 0.4 M ABTS (2,2-azinobis 3-ethylbenthiazoline sulfonic acid diammonium salt, Sigma, Sydney (1 OOμi) and an initiator of 1.5 mM H₂0₂ (10μl) in 9.9ml of 0.05M citrate buffer (pH 4.0). The plate was incubated on a rotary shaker at room temperature for 15 to 45 min until enough colour was produced. Finally, the absorbance was read in a dual wavelength at 405 nm against a reference wavelength of 495 nm in a Bio-Rad ELISA plate reader (Bio-Rad 3550-UV microplate reader).

Detection of antibody to MS Immunoreactive Peptides by ELISA

Plates coated with the diphtheria toxoid peptide were used to detect antibody produced in the rabbit immunized with the diphtheria toxoid peptide. Fig 6 shows the reactivity of the rabbit antibody with the peptide after absorption of the antibody activity to the diphtheria toxoid carrier. The rabbit antipeptide antibody was also used to compare the coating of the ELISA plates with the 20 amino acid peptide to ensure that the wells were evenly coated with the peptide (Fig 7).

Pooled MS IgG but not normal IgG clearly reacted with the peptide after removal of antibody activity to diphtheria toxoid (Fig 8). Preliminary experiments show that 40% of a group of 14 MS patients had IgA in their saliva which reacted more strongly with the peptide conjugated with diphtheria toxoid than 12 saliva samples from normal people (Fig 9). Samples from individual people with MS were tested for reactivity to the 20 amino acid peptide (Fig 10). It can be seen that there was quite a variation in the reactivity of individual MS IgG but there was no reactivity with the pooled normal human IgG.

Enzyme inhibition assay for MS antibody

The reaction of the antibody with the peptide antigens is particular interesting as these peptides are contained within the proposed enzyme active site for NADH:ubiquinone reductase (Complex I). In October 1994 Delgi Esposti et al. published a study of the effect of mutation in this enzyme on its activity. Patients with Leber's Hereditary Optic Neuropathy (LHON) and in particular those with the /VfTΛ/D4*LHON11778A have an arginine at 340 replaced by a histidine. This arginine is the arginine at the end of our 13-amino acid epitope. Delgi Esposti et al. showed that the binding to Complex I of quinone substrates and the well known inhibitor of respiration rotenone were defective in patients with this mutation. This enzyme is extremely important in the provision of energy throughout the body as it is associated with one-third of the ATP produced by mitochondria. Since the MS IgG was binding with a peptide within this proposed active site it was likely to affect the activity of Complex I.

Preparation of mitochondrial Complex I

Mitochondria were purified from heart and brain tissue using the procedure of Beckman et al. (1993). For example three brains from chickens (4 weeks old) were placed in ice just after slaughtering All subsequent procedures were carried out at 4°C. Fat and connective tissues were trimmed before cutting into the approximately 5mm cubes. Chilled homogenization buffer (100mM Tris-HCI, pH 7.4, 250mM sucrose, 10mM EDTA) was added to the samples (3-4 g weight each) in a ratio of 1 :5 (w/v) tissue to buffer. The mixtures were homogenized using a Polytron homogenizer equipped with basic unit K and hand unit PM10S for 1 min at 600 rpm. Nuclei and cellular debris were removed by centrifugation at 18,500 g for 10 min at 4°C. The supernatant was transferred to a fresh tube and centrifuged at 10,000 g for 10 min at 4°C to sediment the mitochondria.

Sub mitochondrial particles (SMP) were prepared from mitochondrial pellets according the procedure of Estornell (1993). The pellets were resuspended in a solution 0.15 M in KCI, 0.01 M in Tris-HCI pH 7.5 and 5 mM in EDTA to a final concentration of 30 mg/ml total protein. The pH was adjusted to 7.5 with 1 M KOH and subjected to sonic treatment for 1 min in an ultrasonic, XL-series sonicator, Mixonix at sonicator. The temperature was maintained below 5°C using an ice bucket with NaCI powder added to the top of the ice during the sonication. The pH of the sonicated suspension was adjusted to 7.5 with 1 M/KOH. The mixture was centrifuged at 50,000 rpm a Beckman ultracentrifuge model L8-M (50TI rotor) for 90 min at 4°C to precipitate the SMP. The SMP pellets, containing the Complex I, were resuspended in cold Tris-HCI buffer (25 mM, pH 8.0 ) supplemented with histidine and sucrose to the final concentration of 0.5 mM and 0.33 mM respectively (TSH buffer). The concentration of the samples were adjusted to 0.2 mg/ml of total protein using the cold TSH buffer and stored at -20°C until used. The NADH-ubiquinone reductase (Complex I) activity of each SMP sample was measured at 340 nm and 30°C according the procedure Hatefi (1978) using the Beckman spectrophotometer model DU-50 equipped with thermostable water circulation system. To each 1-ml quartz cuvettes were added 20μl of K phosphate buffer (1 M, pH 8.0), 20μl of Na azide (0.1 M), 100μl of decylubiquinone (Sigma, Sydney), 10μl of 15 mM NADH (Boehringer Mannheim, Germany) and water to a final volume of 1 ml. Decylubiquinone was dissolved in ethanol and then added to distilled water to make 1 mM, 10% ethanol final concentration. The cuvettes were placed in the spectrophotometer and the absorbance was measured for 1 min duration time adding 10μl of enzyme solution (SMP fraction) for total 30 min. To check the enzyme inhibition, samples were mixed with the IgG solution and incubated at room temperature for 90 min before adding to the cuvette. The rate was calculated between 0 and 10 min and the % inhibition calculated.

Inhibition of Complex I by MS IgG

Fig 11 shows the effect of pooled normal IgG and pooled MS IgG on the activity of Complex I in chicken brain submitochondrial particles. Chicken was chosen because of the close similarity between the amino acid sequence in the peptide of Figure 5b from human and chicken ND4. Similar results were obtained with horse heart SMP. Horse heart SMP was chosen because of their higher activity and stability (Degli Esposti, Personal Communication).

The incubation time was studied for optimising the time required for interaction of the antibody with Complex I. From the data in Fig 12, 90 min was chosen as the time for incubation of the IgG with the enzyme prior to the addition of the substrate.

The influence of dilution of the MS IgG on the inhibition shown in Fig 13. Close to maximum inhibition of enzyme activity was found at dilution of approximately 1 μl of serum equivalents per ml of the assay mixture. Production of recombinant antigen

The PCR product from M62 was cloned into Sma 1 site of the PGEX-1N expression vector to produce the recombinant protein. The Sma 1 site is located in glutathione S-transferase (GST) gene that can be activated to express the protein by induction with isopropyl thiogalactoside (IPTG) (Amrad, Australia). The method used was adopted from Lorens (1991) and Liu and Schwartz (1992).

The 3' A overhang PCR products were purified using the GENECLEAN II kit (Bresatec Ltd., South Australia), filled in by Klenow enzyme and phosphorylated with T4 polymerase kinase.

The Klenow /kinase reaction was set up by adding the dNTPs (0.2 mM final concentration), 4 units of kinase and 5 units of Klenow to KK buffer. The 10xKK buffer was Tris-HCI (300 mM, pH 7.8 ) containing MgCI2 (100 mM), DDT (100 mM) and 5 mM ATP 300 mM. The purified PCR products (500 ng to 1 mg) was added to the mixture and the volume was adjusted to 25 μl by adding the sterile distilled water. The tube was then incubated at 25°C for 2 h to complete the reaction. The blunt ended product was purified by the GENECLEAN kit.

The vector was digested by Sma 1 and dephosphorylated by calf intestine phosphatase before using in the ligation. Dephosphorylation of the 5' end of the cut vector was carried out to prevent re-ligation of the linear plasmid. Approximately 2 μg of PGEX-1N plasmid (AMRAD, Australia) was added to a mixture of buffer A and 10 units of Sma 1 restriction enzyme. The reaction was carried out by incubation at 25°C for 2 h after adjusting the volume to 50 ml by adding the sterile distilled water. To check efficiency of restriction, 2 μl of the sample was run on a 0.6% agarose gel along with the undigested plasmid samples (roughly the same concentration). The sample was stored on ice until the restriction was confirmed before purification with the GENECLEAN procedure.

The purified digested DNA (approximately 1 μg in 34 μl volume) was dephosphorylated using 2 units of calf intestine phosphatase (CIP) enzyme. The CIP buffer was added before incubation at 37°C for 30 min. A further 2 μl of enzyme was added to the mixture and the incubation was continued for 30 min more. The reaction was terminated by the addition of 8 μl TNE buffer, 2 μl of 20% SDS and 30 μl of sterile distilled water. The product was stored at -20°C after purification with GENECLEAN and dissolved in sterile distilled water to a final concentration of 40 ng per μl.

The blunt ended PCR product was ligated into the cut and dephosphorylated PGEX-1N plasmid using the T4 DNA ligase (Progen, New South Wales, Australia). Three different reaction mixtures were set up by mixing the insert and vector in a molar ratio of 1 :1 , 1 :3 and 3:1. Two additional mixtures were set up, one without insert to test the vector self-ligation and another one without insert and vector as negative control. The T4 DNA ligase (2 units) was added to each tube as well as 10xligation buffer and sterile distilled water to 25 μl final volume. The mixtures were incubated overnight (12 to 16 h) at 22°C in a thermocycler.

The host strain E.coli JM83 was made competent by calcium chloride method (Sambrook et al. 1989). The competent cells made by this method were used directly or preserved at -70°C for future work. A single colony was picked from a fresh grown plate and transferred into 100 ml of LB broth medium in a 11 flask.

The culture was then incubated at 37°C with maximum vigorous shaking for about

3 h until the ODgoo of the sample achieved to 0.5. The grown culture was transferred to sterile ice-cold polypropylene centrifuge tube before 10 min incubation in ice. The cells were recovered by centrifugation at 4000 rpm for 10 min in a Sorvall GS3 rotor.

The supernatant was totally removed and the pellet was resuspended in 10 ml of ice-cold 0.1 M CaCI₂ before storing on ice for 10 min. The cells were recovered by centrifugation at 4,000 rpm for 10 min at 4°C. in a Sorvall centrifuge. The cells were resuspended in 4 ml of ice-cold 0.1 M CaCI₂ (2ml for each 50 ml of original culture). These competent cells have been directly used for transformation or frozen at -70°C until used.

To prepare frozen stocks of competent cells, 140 ml of dimethyl sulfoxide (DMSO) was mixed with 4 ml of resuspended cells and stored in ice for 15 min. An additional 140 ml of DMSO was added to the mixture before dispensing aliquots (50 ml each) into chilled, sterile microfuge tubes. The tubes were stored at -70°C after snap-freezing in liquid nitrogen for several months. The chimeric plasmid was transformed into competent E. coli cells using the method of (Sambrook et al. 1989). A bach of frozen competent cells was incubated in ice for 10 min after being gently thawed by holding the tube in the palm of the hand. Seven transformation tubes were been set up by mixing the DNA and competent cells according the following table:

Tube 1 2 3 4 5 6 7

PGEX uncut - X - - - - -

PGEX cut - - X - - - -

PGEX cut + dephosphorylation - - - X x x X

M62 (insert) - - - - x X X Footnote to table:

The ratio of vector to insert in tube 5 was 1 :1 ; 6, 1 :3; 7, 3:1.

The mixtures were incubated in ice for 10 min before induction by incubating 45 sec at 42°C. The samples were transferred to an ice bath and allowed to chill for 1 to 2 min. Preheated SOC media (Sambrook et al. 1989) 800 ml, 37°C was added to each tube and incubated at 37°C with 220 rpm shaking for 45 min.

The transformed competent cells (200 ml per 90mm plate) were spread over the surface of the agar plate containing 20 mM MgS04. The plates were left at room temperature for 15 min to absorb the liquid before incubating overnight at 37°C in invert position.

Screening of transformants

To screen for inserts the transformants were check by colony hybridization, PCR and Southern blot analysis with 32 _r P labelled M62 DNA Production and testing of recombinant M62

Colony hybridisation method (as described above) was used to confirm the success of cloning the isolate. The bacterial colonies were transferred onto nitrocellulose filers and probed with ³²P labelled DNA from M62. About 50% of the total bacterial colonies showed the signal in duplicate filters. The recombinant positive colonies (about 10) were picked and used for PRC testing and protein production. PCR amplification of these bacterial colonies (bacterial colony as DNA template and λgt11 primers), showed the correct size of insert. A non-recombinant colony (containing PGEX-1 N) was used in all of these experiments as a negative control.

The 20 recombinant bacterial colonies (plus one non-recombinant) were grown in LB media where the GST fusion protein was induced by adding IPTG. The bacterial cultures were sonicated before electrophoresis with 12.5% acrylamide followed by Coomassie blue staining. In the non-recombinant bacterial colonies and 9 of the recombinants a band of GST protein at 26Kd was visible. As expected the predicted (Fig 4) amino acid sequence from ND4 (5Kd) together with the GST (26Kd) would give rise to a 31 Kd band, three clones producing the 31 Kd band were chosen for production of recombinant.

The bacterial lysate mixture from this clone was diluted 1 :10 with PBS (pH 7.4) and used for testing with the ELISA method. Each well of a microlitre plate was coated with 100 μl of the bacterial lysate. Bacterial lysate from non-recombinant clone was used as negative control. Probing the plate with the anti-ND4 antibody from the rabbit showed a significant increase for recombinant clones compared to the non-recombinant clones. Pre-immune sera from the rabbit had a low activity against the recombinant protein compared to the immune sera (Fig 14).

Presence of antibody to diphtheria toxoid-S-MS immunoreactive peptide.

Human sera were examined for antibody to the diphtheria toxoid-S-MS immunoreactive peptide with an ELISA assay modified from that described in Fig 8. A microtitre plate was coated with diphtheria toxoid (5.8μg in 100μl PBS) and the peptide CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg conjugated to diphtheria toxoid (Fig 5b, 6.3mg, containing 0.5mg of the peptide, in 100μl PBS) so that there were five wells with the former to one well of the latter. Binding to plastic was blocked with 300ml 5% BSA in PBS in all wells. Serum (100μl, 1/100 in PBS) was added to the first well and incubated for 1 h before moving the sample to the second well and so on with 1 h incubation in each before it was moved into the sixth well which contained the peptide conjugated to diphtheria toxoid. This procedure removes non-specific binding of IgG to diphtheria toxoid, BSA and plastic. After 1h plates were washed with 0.1% Tween in PBS, then the bound human IgG was determined with alkaline phosphatase conjugated to goat antihuman IgG (Sigma, Sydney) and the optical density measured at 405nm. The presence of antibody to the peptide was determined by the difference in optical density between well six and well five for each sample which was analysed in triplicate.

Sequential samples of sera from three MS patients were obtained from Dr W.W. Tourtellotte, Los Angeles. The level of antibody to the diphtheria toxoid-S-MS immunoreactive peptide fluctuated in two of the patients (Fig 15).

Figure 16 shows the detection of antibody to the diphtheria toxoid-S-MS immunoreactive peptide to MS and other sera. Samples of MS sera were obtained from Professor D.A.S. Compston, Cambridge 44; Professor C.A.A. Bernard, Melbourne 5; Dr W. Carroll, Perth 2; Dr R. Edis, Perth 1 ; and Dr W.W. Tourtellotte 2. Other sera were from 9 people with no obvious disease (2 from Professor D.A.S. Compston, 4 from Professor C.A.A. Bernard, and 3 from Dr R.M. Chalmers, London), 2 with Guillain-Barre syndrome from Dr Pollard, Sydney, and 1 with breast cancer from Murdoch University. Antibody to the peptide was detected in 11/54 samples of MS sera but in none of the Other" samples. This result shows that a subgroup of MS patients can be detected using an ELISA assay to the peptide. Because the peptide contains an arginine which is mutated to histidine in the commonest form of Leber^*s Hereditary Optic Neuropathy (MTND4*LHON11778A) and because it has been speculated that patients with the LHON mutation do not develop clinical disease until an autoimmune reaction occurs, 11 sera were obtained from Dr R.M. Chalmers, London; and 1 sample was from Dr R.D. Simmons, Canberra. Antibody was detected in 7/8 patients with clinical LHON but not in 3 carriers of the MTΛ/D4*LHON11778A mutation who had no disease nor in 1 carrier who had MS rather than LHON (Fig 17).

A collection of 50 sera was obtained from Associate Professor P.N. Hollingsworth , Perth. These sera were from people who had been examined for a variety of autoantibodies which are indicators of various autoimmune diseases. In preliminary experiments, the following results were obtained

No. of sera Autoantiαen ELISA result to diphtheria toxoid- classification S-MS immunoreactive DβDtide

- + ++ +++

5 DNA 2 3

5 Acetylcholine receptor 1 2 1 1 5 Islet cell 1 1 3

5 Nuclear antigen 1 2 2 5 Parietal cell 4 1

5 Rheumatoid factor 4 1

5 Smooth muscle 4 1

5 Thyroid microsomal 4 1

10 No autoantigens detected 9 1

50 Total 28 10 7 5

+++, ++, +, - were equivalent to delta OD's Of >0.1 , <0.1 , <0.06, <0.04, respectively

These results indicate that the antibody to the MS immunoreactive peptide was found in association with antibody to other autoantigens in patients being examined for other autoimmune diseases. This suggests that tissue damage in these patients has led to the exposure of mitochondrial antigens to the immune system or that the MS reactive peptide was also present within a protein in microorganism to which these patients had mounted an immune response. The presence of several different types of autoantibodies in patients with autoimmune disease is a well known phenomenon.

Molecular mimicry between epitopes in human proteins and proteins in microorganisms

Molecular mimicry between epitopes in human proteins and proteins in microorganisms may explain the induction of autoimmune disease. The ND4 peptide CysLeuAlaAsnSerAsnTyrGluArgThrHisSerArg was used to search GenPeptide with the BLOSUM50 matrix and FastA for similarities between the peptide and sequences in bacteria and viruses.

A match was made to a predicted epitope in a surface protein from Borrelia burgdorferi. This sequence is encoded, AlaAsnSerAsnTyrGlu, in a linear plasmid (Norris et al 1992 Infection and Immunity. 60, 4662-4672) which has the capability of easily moving to other species of spirochaetes. There have been strong arguments put forward that spirochaetes are involved in the pathogenesis of MS and in older papers structures resembling Borrelia sp were reported.

If the autoantibody is really directed against spirochaetes then the ELISA assay to the MS immunoreactive peptide could be useful in identifying a subgroup of MS patients who are infected with spirochaetes. Such patients could be treated with appropriate antibiotic therapy. There are reports that a proportion of randomly selected MS patients do improve with such a treatment.

If the autoantibody to ND4 is not just a marker for tissue damage in MS and LHON but is involved in the pathogenesis then it might be possible to generate an antiidiotypic antibody to block the autoantibody.

References Beckman etal. (1993) Purification of mitochondrial DNA with wizard™ minipreps DNA purification system. Promega Notes Magazine 43:10-13.

Campbell A.M. (1984) in: Laboratory techniques in biochemistry and molecular biology (Burdon R.H. and van Knippenberg P.H. ed) Elsevier, Amsterdam. Degli Esposti etal. (1994) Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy. FEBS Letters 352:375-379.

Dreesman etal. (1985) Estornell etal (1993) Assay conditions for the mitochondrial NADH oenzyme Q Oxidoreductase. FEBS Letters 332:127-131.

Hatefi (1978) Preparation and properties of NADH:Ubiquinone Oxidoreducase (Complex I), EC 1.6.5.3. Methods in Enzymology 53:5-21.

Lawen etal (1994) The university of bioenergetic disease: the role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase. Molec. Aspects Med. 15 Sup:s13-s27.

Linthicum etal (1981) Detection of antibodies to central nervous system antigens by solid phase radioimmunoassay. J Neurosci. Res. 6:567-578. Linthicum and Farid (1988) Anti-ldiotypes, receptors, and molecular mimicry. Springer- Verlag New York.

Poskit eta/ (1991) Internal image (Ab2β) anti-idiotype vaccines. Theoretical and practical aspects. Vaccine 9:792-796.

Sambrook etal (1989) Molecular cloning a laboratory manual. Cold Spring Harbour Laboratory Press New York.

Thornton and Griggs (1994) Plasma exchange and intravenous immunoglobulin treatment of neuromuscular disease. Annals of Neurology 35:260-267.

Seαuence Listings

SEQ ID N0.1 :

TATCGGCGGA ATCCCCGGTA ATTCTCATAA TCGCCCACG 40

GGCTTACATC CTCATTACTA TTCTGCCTAG CAAACTCAAA 80 CTACGAACGC ACTCACAGTC GCATCATAAT CCTCTCTCAA 120

GGACTTCAAA CTCTACTCCC ACTAATAGCT TTTTGGATGA 160

CTTCTAGCAA GCCTCGCTAA CCTCGCCTTA CCCCCCACTA 200

TTAACCTACT GGGAGAACTC TCTGTGCTAG TAACCACGTT 240

CTCCTGATCA AATATCACTC TCCTACTTAC AGGACTCAAC 280 ATA

SEQ ID N0:2

He Leu Met He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu 1 5 10 15

Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg lie Met He Leu Ser 20 25 30

Gin Gly Leu Gin Thr Leu Leu Pro Leu Met Ala Phe Trp Trp Leu

35 40 45

Leu Ala Ser Leu Ala Asn Leu Ala Leu Pro Pro Thr lie Asn Leu 50 55 60 Leu Gly Glu Leu Ser Val Leu Val Thr Thr Phe Ser Trp Ser Asn

65 70 75

He Thr Leu Leu Leu Thr Gly Leu Asn Met 80 85

SEQ ID NO:3

He Leu Met He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu

1 5 10 15

Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He Met He Leu Ser

20 25 30

Gin Gly Leu Gin Thr Leu Leu Pro Leu lie Ala Phe Trp Trp 35 40 45 SEQ ID NO:4

Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser Asn Tyr Glu Arg 1 5 10 15

Thr His Ser Arg He He He Leu Ser Gin Gly Leu Gin Thr Leu Leu 20 25 30

Pro Leu He Ala Phe 35

SEQ ID NO:5

He Ala His Gly Leu Thr Ser Ser Leu Leu Phe Cys Leu Ala Asn Ser 1 5 10 15

Asn Tyr Glu Arg Thr His Ser Arg He He 20 25

SEQ ID NO:β

Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg He Met He Leu 1 5 10 15

Ser Gin Gly Leu 20

SEQ ID NO:7

Cys Leu Ala Asn Ser Asn Tyr Glu Arg Thr His Ser Arg 1 5 10

SEQ ID NO:8

Ala Asn Ser Asn Tyr Glu 1 5

Claims

THE CLAIMS defining the invention are as follows:

1. A DNA sequence encoding at least a peptide that is immunologically reactive with MS induced antibodies wherein the polypeptide or peptide's DNA sequence is derived from SEQ ID NO:1.

2. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:2.

3. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:3.

4. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:4.

5. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:5.

6. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:6.

7. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:7.

8. A DNA sequence according to claim 1 wherein the DNA sequence encodes the polypeptide or peptide described as SEQ ID NO:8.

9. A polypeptide sequence that is immunologically reactive with MS induced antibodies wherein the polypeptide sequence comprises part or all of SEQ

ID NO:2.

10. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:3.

11. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:4.

12. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:5.

13. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:6.

14. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:7.

15. A polypeptide sequence according to claim 8 wherein the polypeptide sequence described as SEQ ID NO:8.

16. A polypeptide sequence according to anyone of claims 8 to 13 wherein the sequence is part of the general formulae X-Y-Z wherein: X and Z each represent individually of each other a hydrogen atom, or another amino acid, a protected amino acid, diphtheria toxoid-S-, another sequence of the general formulae X-Y-Z, another peptide sequence, another polypeptide sequence, an amino group, a carboxyl group or an adjuvant; and Y represents the selected polypeptide sequence.

17. A monoclonal antibody directed against any one of SEQ ID NO:2 to SEQ ID NO:8.

18. A polyclonal antibody directed against any one of SEQ ID NO:2 to SEQ ID NO:8.

19. An immunoassay for detecting mitochondrial damage comprising:

(a) incubating a sample suspected of containing antibodies directed against against any one of SEQ ID NO:2 to SEQ ID NO:8, with any one of SEQ ID NO:2 to SEQ ID NO:8 under conditions which allow the formation of an antigen-antibody complex; and

(b) detecting an antigen-antibody complex.

20. An immunoassay for detecting mitochondrial damage comprising:

(a) incubating a sample suspected of containing antibodies directed against against any one of SEQ ID NO:2 to SEQ ID NO:8, with a sequence defined by claim 14 under conditions which allow the formation of an antigen-antibody complex; and

(b) detecting an antigen-antibody complex.

21. An enzyme inhibition assay for detecting mitochondrial damage comprising: (a) preparing a sub-mitochondrial particle preparation containing complex 1 or a plasma membrane preparation containing oxidoreductase;

(b) adding quinone substrate and NADH substrate to the preparation prepared in step (a); and (c) measuring the decrease in NADH in the preparation

22. An immunoassay for detecting antibodies which react to both a spirochaete surface protein and a mitochondrial protein comprising:

(a) incubating a sample suspected of containing antibodies directed against against SEQ ID NO:8 with a sequence defined by SEQ ID

NO:8 under conditions which allow the formation of an antigen- antibody complex; and

(b) detecting an antigen-antibody complex.