Butyrylcholinesterase as target/marker for insulin resistance

Type 2 diabetes is a disease of fast growing worldwide importance and can be described as a failure of the pancreatic beta-cell (beta-cell failure) to compensate, with enhanced insulin secretion of the beta-cells, for peripheral insulin resistance.

Insulin resistance can be considered the first step in the development of Type 2

Diabetes and develops years before diabetes is diagnosed. During this first stage, patients remain normoglycaemic and compensate for reduced insulin responsiveness of muscle and liver by an enhanced secretion of insulin. At later stages in the development of Type 2 Diabetes, beta cell function decreases, leading to impaired glucose tolerance and, finally, diabetes. Early intervention by either weight loss, exercise, or pharmaceutical treatment, was shown to delay or even prevent the development of diabetes in patients with impaired glucose tolerance (Diabetes Prevention Program Research Group, N. Engl. J. Med. 346 (2002) 393-403). Therefore, an early diagnosis of insulin resistance would allow early intervention by anti-diabetic treatment or other measures that would prevent progression of the disease. To date, the only reliable possibility to detect insulin resistance is by the euglycemic-hyperinsulinemic clamp (EHC). HOMA modeling is often used for assessing insulin resistance but is not an accepted diagnostic method (Wallace et al., Diabetes Care 27(2004) 1487ff.). Due to them being time consuming and labor intensive, these methods do not lend themselves to broad patient screening programs. A molecular marker for insulin resistance would therefore be extremely useful for the detection of this condition.

Most currently used Type 2 Diabetes treatments do not directly address Insulin resistance. Safety concerns exist for those that do primarily act at the level of peripheral glucose uptake (e.g. insulin sensitizers). Therefore, it would also be useful to identify additional, better targets for treatment and markers for detection of Insulin Resistance or efficacy that are more sensitive or more reliable than the markers commonly used, such as the EHC or HOMA method.

Furthermore, it would be an advantage to identify markers that can be detected in plasma.

The aim of the present invention is to identify and provide a novel target to screen for compounds that prevent, attenuate, or inhibit Insulin Resistance, and for a marker that allows for monitoring and/or diagnosis of Insulin Resistance at an earlier stage of type II diabetes and more reliably than can presently be done.

Surprisingly, it was found that the use of protein butyrylcholinesterase can overcome, at least in part, the problems known from the state of the art.

Butyrylcholinesterase is a serum esterase classified on the basis of its preference for butyrylcholine as a substrate rather than acetylcholine.

El Lakany et al. (Med. Sci. Res. 1997, 25, 393-395) investigated the relationship between serum butyrylcholinesterase activity to serum sensitivity in patients on maintenance haemodialysis and found an increase of butyrylcholinesterase activity; the difference of butyrylcholinesterase activity between insulin sensitive and insulin resistant patients on maintenance haemodialysis was only of low significance. Abbott et al. (Clinical Science 1993, 85, 77-81) investigated the serum activity of butyrylcholinesterase in patients with type 1 diabetes mellitus, type 2 diabetes mellitus and healthy controls, and found an elevation of serum butyrylcholinesterase activity in both types of diabetes. A correlation between insulin sensitivity and butyrylcholinesterase activity was of low significance.

Surprisingly, it was found that changes in the levels of secreted butyrylcholinesterase are found in Insulin Resistance. Therefore, the present invention provides a target for the treatment and/or prevention of Insulin Resistance, and a novel marker for the early diagnosis of Insulin Resistance in diabetes.

In preferred embodiments, the novel target and/or marker butyrylcholinesterase may be used for diagnostic, monitoring as well as for screening purposes.

When used in patient monitoring, the diagnostic method according to the present invention may help to assess efficacy of treatment and recurrence of Insulin Resistance in the follow-up of patients. Therefore, the present invention provides the use of protein butyrylcholinesterase for monitoring the efficacy of treatment of diabetes.

In a preferred embodiment, the diagnostic method according to the present invention is used for patient screening purposes. I.e., it is used to assess subjects without a prior diagnosis of diabetes by measuring the level of butyrylcholinesterase and correlating the level of butyrylcholinesterase to the presence or absence of Insulin Resistance.

The methods of the present invention are useful for monitoring progression of the disease through the different stages leading to diabetes, namely Insulin Resistance, Impaired Glucose Tolerance and Diabetes.

The present invention thus provides a method for monitoring the progression of diabetes, comprising the steps of (a) providing a liquid sample obtained from an individual, (b) contacting said sample with a specific binding agent for butyrylcholinesterase under conditions appropriate for formation of a complex between said binding agent and butyrylcholinesterase, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in Insulin Resistance.

The present invention also provides a method for monitoring the efficacy of treatment of diabetes, comprising the steps of (a) providing a liquid sample obtained from a patient treated against diabetes, (b) contacting said sample with a specific binding agent for butyrylcholinesterase under conditions appropriate for formation of a complex between said binding agent and butyrylcholinesterase, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in the absence of treatment.

The present invention provides an in vitro method of screening for a compound which interacts with butyrylcholinesterase, comprising the steps of a) contacting protein butyrylcholinesterase with a compound or a plurality of compounds under compositions which allow interaction of said compound or a plurality of compounds with butyrylcholinesterase; and b) detecting the interaction between said compound or plurality of compounds with said polypeptide.

The term "in vitro method" as used herein relates to methods performed with cell cultures or cell-free methods, but not with whole organisms.

The present invention provides an in vitro method of screening for a compound that prevents and/or inhibits and/or attenuates Insulin Resistance, comprising the steps of a) contacting a compound with protein butyrylcholinesterase; and b) measuring the

- A - activity of protein butyrylcholinesterase; wherein a compound which inhibits or stimulates the activity of protein butyrylcholinesterase is a compound that may prevent and/or inhibit and/or attenuate Insulin Resistance. Preferably, said method additionally comprises the step of immobilizing protein butyrylcholinesterase prior to step a) or between steps a) and b).

The term ,,activity" as used herein relates to butyrylcholinesterase activity. Assays to determine butyrylcholinesterase activity are well known in the art and are found e.g. in El-Lakany et al., Med. Sci. Res. 1997, 25, 393-395), and Abbott et al. (Clinical Science 1993, 85, 77-81).

In a preferred embodiment, the above in vitro screening assays are cell-free assays. Such assays involve contacting a form of butyrylcholinesterase (e.g., full-length polypeptide, a biologically active fragment of said polypeptide, or a fusion protein comprising all or a portion of said polypeptide) with a test compound and determining the ability of the test compound to bind to said polypeptide. Binding of the test compound to said polypeptide can be determined either directly or indirectly as described above. In one embodiment, the assay includes contacting the said polypeptide with a known compound which binds said polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with said polypeptide, wherein determining the ability of the test compound to interact with said polypeptide comprises determining the ability of the test compound to preferentially bind to the said polypeptide as compared to the known compound.

The cell-free assays of the present invention are amenable to use of either a membrane-bound form of a polypeptide or a soluble fragment thereof. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include non- ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-IOO, Triton X- 114, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl-N, N-dimethyl-3-ammonio-l -propane sulfonate.

In various embodiments of the above assay methods of the present invention, it may be desirable to immobilize a polypeptide to facilitate separation of complexed from uncomplexed forms of the polypeptide with a binding molecule, as well as to accommodate automation of the assay. Binding of a test compound to a polypeptide, or interaction of a polypeptide with a binding molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed binding protein or polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of a polypeptide hereinbefore described can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a polypeptide hereinbefore described or its binding molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with a polypeptide or binding molecules, but which do not interfere with binding of the polypeptide of the invention to its binding molecule, can be derivatized to the wells of the plate. Unbound binding protein or polypeptide of the invention are trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with a polypeptide hereinbefore described or binding molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with a polypeptide or binding molecule.

The present invention also provides a method of screening for a compound that prevents and/or inhibits and/or delays Insulin Resistance, comprising the step of detecting soluble butyrylcholinesterase secreted from a host in the presence or absence of said compound, wherein a compound that prevents and/or inhibits and/or delays Insulin Resistance is a compound with which the level of butyrylcholinesterase secreted from a host is changed.

A host may be a model cell representing beta-cells in culture, or an animal which can be used as a model for Insulin Resistance.

The present invention also provides for a use of protein butyrylcholinesterase as a target and/or as a marker for screening for a compound that prevents and/or inhibits Insulin Resistance.

The diagnostic, monitoring or patient screening methods according to the present invention are based on a liquid sample which is derived from an individual. Unlike to methods known from the art butyrylcholinesterase is specifically measured from this liquid sample by use of a specific binding agent.

A specific binding agent is, e.g., a receptor for butyrylcholinesterase or an antibody to Butyrylcholinesterase. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for butyrylcholinesterase. A level of less than 5% cross- reactivity is considered not significant.

A specific binding agent preferably is an antibody reactive with butyrylcholinesterase. The term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.

Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam). For the achievements as disclosed in the present invention polyclonal antibodies raised in rabbits have been used. However, clearly also polyclonal antibodies from different species, e.g. rats or guinea pigs, as well as monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in

any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine. The generation and use of monoclonal antibodies to butyrylcholinesterase in a method according to the present invention is yet another preferred embodiment.

As the skilled artisan will appreciate now, that butyrylcholinesterase has been identified as a marker which is useful in the diagnosis of Insulin Resistance, alternative ways may be used to reach a result comparable to the achievements of the present invention. For example, alternative strategies to generate antibodies may be used. Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of butyrylcholinesterase for immunization. Alternatively, DNA immunization also known as DNA vaccination may be used.

For measurement the liquid sample obtained from an individual is contacted with the specific binding agent for butyrylcholinesterase under conditions appropriate for formation of a binding agent butyrylcholinesterase-complex. Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.

As a final step according to the methods disclosed in the present invention the amount of complex is measured and correlated to the diagnosis of Insulin Resistance or to a respective control, as hereinbefore described. As the skilled artisan will appreciate there are numerous methods to measure the amount of the specific binding agent butyrylcholinesterase-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996) Immunoassay, Academic Press, Boston).

Preferably butyrylcholinesterase is detected in a sandwich type assay format. In such assay a first specific binding agent is used to capture butyrylcholinesterase on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable, is used on the other side.

As mentioned above, it has surprisingly been found that butyrylcholinesterase can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker butyrylcholinesterase in the diagnosis of Insulin Resistance.

In a preferred embodiment the method according to the present invention is practiced with serum as liquid sample material.

In a further preferred embodiment the method according to the present invention is practiced with plasma as liquid sample material.

In a further preferred embodiment the method according to the present invention is practiced with whole blood as liquid sample material.

Whereas application of routine proteomics methods to tissue samples, leads to the identification of many potential marker candidates for the tissue selected, the inventors of the present invention have surprisingly been able to detect protein butyrylcholinesterase in a bodily fluid sample. Even more surprising they have been able to demonstrate that the presence of butyrylcholinesterase in such liquid sample obtained from an individual can be correlated to the diagnosis of Insulin Resistance.

Antibodies to Butyrylcholinesterase with great advantage can be used in established procedures, e.g., to Insulin Resistance in situ, in biopsies, or in immunohistological procedures.

Preferably, an antibody to butyrylcholinesterase is used in a qualitative (butyrylcholinesterase present or absent) or quantitative (butyrylcholinesterase amount is determined) immunoassay.

Measuring the level of protein butyrylcholinesterase has proven very advantageous in the field of Insulin Resistance and diabetes. Therefore, in a further preferred embodiment, the present invention relates to use of protein butyrylcholinesterase as a marker molecule in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.

The term marker molecule is used to indicate that changes in the level of the analyte butyrylcholinesterase as measured from a bodily fluid of an individual marks the presence of Insulin Resistance.

It is preferred to use the novel marker butyrylcholinesterase in the early diagnosis of type II diabetes.

It is especially preferred to use the novel marker butyrylcholinesterase in the early diagnosis of glucose intolerance.

It is also especially preferred to use the novel marker butyrylcholinesterase in the monitoring of disease progression in diabetes.

The use of protein butyrylcholinesterase itself, represents a significant progress to the challenging field of Insulin Resistance diagnosis. Combining measurements of butyrylcholinesterase with other known markers for diabetes, like insulin, or with other markers of Insulin Resistance yet to be discovered, leads to further improvements.

Therefore in a further preferred embodiment the present invention relates to the use of butyrylcholinesterase as a marker molecule for diabetes, preferably for Insulin Resistance, in combination with another marker molecule for diabetes, preferably for Insulin Resistance, in the diagnosis of diabetes, preferably of Insulin Resistance from a liquid sample obtained from an individual. Preferred selected other diabetes markers with which the measurement of Insulin Resistance may be combined are insulin, pre-insulin, and/or C-peptide.

Diagnostic reagents in the field of specific binding assays, like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay. The present invention therefore also relates to an immunological kit comprising at least one specific binding agent for butyrylcholinesterase, recombinant butyrylcholinesterase as a standard antigen, binding buffer and the reagents required to detect bound butyrylcholinesterase. Preferably, said kit comprises a first specific binding agent for butyrylcholinesterase, recombinant butyrylcholinesterase as antigen standard, a second specific binding agent which is coupled to a detection system, and detection reagents, for measurement of butyrylcholinesterase. Preferably, said first and second binding reagents are antibodies which recognize different epitopes on butyrylcholinesterase and which preferably do not crossreact upon detection. Said detection system may be an enzyme bound to the second specific binding reagent, such as, as a non-limiting example, horseradish peroxidase, or in another embodiment, said second specific binding agent is covalently linked to biotin, and the signal is detected with enzyme-labeled avidin/streptavidin. In another embodiment, the detection system is not directly coupled to the second specific binding agent, but to a third binding agent, such as an antibody, which specifically bind the second specific binding agent.

One way of assessing clinical utility of the novel marker butyrylcholin esterase is by measuring its levels in 17 patients that were diagnosed as being insulin resistant by measuring the glucose disposal rate with the EHC method and comparing the levels with those measured in 17 patients with demonstrated normal glucose disposal rate as determined by the same methodology. For statistical analysis, standard Student's t-test evaluation is performed with values < 0.05 being taken as significant.

Accuracy of a test can be described by its receiver-operating characteristics (ROC) (see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed.

The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease.

In each case, the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true- positive test results) (number of true-positive + number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 - specificity [defined as (number of false-positive results)/(number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. (If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the

criterion for "positivity" from "greater than" to "less than" or vice versa.) Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measure is the area under the ROC plot. By convention, this area is always > _. 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0).

Also claimed are the methods, uses and kit substantially as hereinbefore described, especially with reference to the examples below.

The following examples, references, sequence listing and figure are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Examples

Example 1

Identification of candidate protein markers by determination of differences in protein levels between insulin resistant and insulin sensitive individuals

We established a method to search for Insulin Resistance markers in human plasma by applying proteomics technologies. Plasma is a difficult to analyse by Proteomics techniques because it includes ca. ten high- abundance proteins, which represent approximately 98% of the total protein mass. We removed the high-abundance proteins, albumin and antibody chains, by applying chromatographic techniques and fractionated the flow through fraction over an ion exchanger. The scheme described comprises three chromatography steps, matrix blue, protein G and ion exchange, and is highly

reproducible. All chromatographic steps were performed on an FPLC System (Pharmacia).

Removal of albumin by affinity chromatography on Mimetic blue and removal of immunoglobulins by affinity chromatography on Protein G

Human plasma was received from three control individuals and three patients with diabetes type II. Protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany) was added to the plasma (one tablet to 50 ml). Plasma was diluted three-fold with 25 mM MES, pH 6.0, to reduce the salt concentration and adjust the pH to about 6.0. The two columns, Mimetic blue SA P6XL (50 ml, ProMetic BioSciences Ltd.) and HiTrap Protein G HP (5 ml, Amersham Biosciences) were connected in series and equilibrated with 25 mM MES, pH 6.0. The volume corresponding to approximately one g of plasma protein(15 ml, 66 mg/ml) was filtered through a 0.22 μm filter and applied onto the Mimetic blue column at 5 ml/min. The flow through of this column was directly loaded onto the Protein G column and the flow-through fraction from the latter column was collected (about 120 mg). The two columns were washed with 100 ml of 25 mM MES, pH 6.0 and then they were separated. The Mimetic blue column was eluted with a step gradient of 2 M NaCl in 50 mM Tris-HCl, pH 7.5 and the Protein G was eluted with 100 mM glycine-HCl, pH 2.8 and the eluate was neutralized with 1 M Tris base. The flow through fraction and the two eluates were analyzed by two-dimensional gels and the proteins were identified by MALDI-MS. In the eluate from Mimetic blue, mainly full- length and fragmented albumin were detected. In the eluate from the Protein G column, mainly heavy and light Ig chains were detected. Most of the other plasma proteins were recovered in the flow through fraction.

Protein fractionation by ion exchange chromatography

The flow through and the wash fractions from the Mimetic blue and Protein G columns were combined, adjusted to pH 8.0 with 2 M Tris base and were applied onto a HiTrap Q HP column (5 ml, Amersham Biosciences), equilibrated with 50 mM Tris-HCl, pH 8.0 at 5 ml/min. The column was eluted with a liner gradient of increasing salt concentration from 0 to 1 M NaCl in 50 mM Tris-HCl, pH 7.5. Five-ml fractions were collected and analyzed by 1-D gels. Approximately 50 mg of total protein were recovered from this column. On the basis of the gel analysis, the fractions were pooled to form eight pools, so

that each pool included about 5 mg of total protein. The pools were concentrated with Ultrafree-15 Centrifugal Filter (5k MWCO, Millipore) and each of the eight pools was analyzed by 2-D gels. About 400 spots from each gel were excised and analyzed by MALDI-MS.

Two-dimensional Electrophoresis

Immobilized pH gradient (IPG) strips were purchased from Amersham Biosciences (Uppsala, Sweden). Acrylamide was obtained from Biosolve (Valkenswaard, The Netherlands) and the other reagents for the polyacrylamide gel preparation were from Bio-Rad Laboratories (Hercules, CA, USA). CHAPS was from Roche Diagnostics

(Mannheim, Germany), urea from Applichem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland) and dithioerythritol from Merck (Darmstadt).

Samples of 0.5 mg total protein were applied on 3-10 NL IPG strips, in sample cups at their basic and acidic ends. Focusing started at 200 V, and the voltage was gradually increased to 5000 V at 3 V/min, using a computer-controlled power supply and was kept constant for a further 6 h. The second-dimensional separation was performed either on 12% constant SDS polyacrylamide gels (180x200x1.5 mm) at 40 mA per gel. After protein fixation for 12 h in 40% methanol that contained 5% phosphoric acid, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 24 h. Excess dye was washed from the gels with H2O, and the gels were scanned in an Agfa DUOSCAN densitometer (resolution 400). Electronic images of the gels were recorded with Photoshop (Adobe) software. The images were stored in tiff (about 5 Mbytes/file) and jpeg (about 50 Kbytes/file) formats. The gels were kept at 4 ⁰ C until used for MS analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI- TOF-MS)

Selected spots of 1.2 mm diameter were excised with a homemade spot picker, placed into 96-well microtiter plates and each gel piece was destained with 100 μl of 30% acetonitrile in 50 mM ammonium bicarbonate in a CyBi™-Well apparatus (Cybio AG, Jena,

Germany). After destaining, the gel pieces were washed with 100 μl of H ₂ O for 5 min, and dried in a speedvac evaporator without heating for 45 min. Each dried gel piece was rehydrated with 5 μl of 1 mM ammonium bicarbonate, which contained 50 ng trypsin (Roche Diagnostics, Mannheim, Germany). After 16 h at room temperature, 20 μl of 50% acetonitrile, that contained 0.3% trifluoroacetic acid was added to each gel piece. The gel pieces were incubated for 15 min with constant shaking. A peptide mixture (1.5 μl) was simultaneously applied with 1 μl of matrix solution, that consisted of 0.025% α-cyano-4- hydroxycinnamic acid (Sigma), and that contained the standard peptides des-Arg- bradykinin (Sigma, 20 nM, 904.4681 Da) and adrenocorticotropic hormone fragment 18- 39 (Sigma, 20 nM, 2465.1989) in 65% ethanol, 32% acetonitrile, and 0.03% trifluoroacetic acid, to the AnchorChip™. The sample application was performed with a CyBi-WeIl apparatus. Samples were analyzed in a time-of-flight mass spectrometer (Ultraflex TOF-TOF, Bruker Daltonics) in the reflectron mode. An accelerating voltage of 20 kV was used. Proteins were identified on the basis of peptide-mass matching.

Peak annotation for MALDI mass spectra

Mass spectrometric data is two times filtered using a low-pass median parametric spline filter in order to determine the instrument baseline. The smoothed residual mean standard deviation from the baseline is used as an estimate of the instrument noise level in the data. After baseline correction and rescaling of the data in level-over-noise coordinates, the data point with the largest deviation from the baseline is used to seed a non-linear (Levenberg-Marquardt) data fitting procedure to detect possible peptide peaks. Specifically, the fit procedure attempts to produce the best fitting average theoretical peptide isotope distribution parameterized by peak height,- resolution, and monoisotopic mass. The convergence to a significant fit is determined in the usual way by tracking sigma values. After a successful convergence, an estimate for the errors of the determined parameters is produced using a bootstrap procedure using sixteen repeats with a random exchange of 1/3 of the data points. The resulting fit is subtracted from the data, the noise level in the vicinity of the fit is adjusted to the sum of the extrapolated noise level and the deviation from the peak fit, and the process is iterated to find the next peak as long as a candidate peak more than five times over level of noise can be found. The process is stopped when more than 50 data peaks have been found. The zero and first order of the time-of flight to mass conversion are corrected using linear extrapolation from detected internal standard peaks, and confidence intervals for the monoisotopic mass values are estimated form the mass accuracies of the peaks and standards.

Probabilistic matching of spectra peaks to in-silico protein digests: Peak mass lists for mass spectra are directly compared to theoretical digests for whole protein sequence databases. For each theoretical digest, [1-11(1- N P(pi))] ^cMatches is calculated, where N is the number of peptides in the theoretical digest, P(pi) is the number of peptides that match the confidence interval for the monoisotopic mass of the peak divided by the count of all peptides in the sequence database, and cMatches is the number of matches between digest and mass spectrum. It can be shown that this value is proportional to the probability of obtaining a false positive match between digest and spectrum. Probability values are further filtered for high significance of the spectra peaks that produce the matches. After a first round of identifications, deviations of the identifications for mass spectra acquired under identical conditions are used to correct the second and third order terms of the time-of- flight to mass conversion. The resulting mass values have mostly absolute deviations less than 10 ppm. These mass values are then used for a final round of matching, where all matches having a P _mι$m less than 0.01/NProteins (1% significance level with Bonferoni correction) are accepted.

Example 2

Identification of candidate endothelial marker proteins for insulin resistance by analysis of mRNA expression in umbilical vein endothelial cells

Culture of endothelial cells, mRNA extraction, reverse transcription, labeling and hybridization to DNA microarrays

Based on an analysis of the protein levels in plasma from patients with insulin resistance compared to plasma from controls it became evident that markers of endothelial activation can be detected at increased levels in insulin resistant individuals. Recent literature supports these findings (e.g. Meigs et al., JAMA 291 (2004) 1978 ff). Therefore, highly expressed endothelial proteins carrying a signal sequence targeting them for excretion or Type 1 membrane proteins with a large extracellular domain anchored to the membrane by a single transmembrane sequence are potential novel marker proteins for endothelial activation. In order to identify candidate proteins an mRNA expression dataset was analyzed in silico for extracellular proteins.

Human Vascular Endothelial Cells (HUVECs) were prepared following standard procedures. Briefly, the veins were washed free of blood before filling them with Dispase- solution (Roche, Cat. No 295 825, diluted 1:10 in DMEM). The blood vessels were closed

on both ends and incubated for 30 min at 37°C. The detached endothelial cells were then collected and the umbilical vein washed once with PBS. The cells were centrifuged for 10 min at 320 x g, resuspended in PO Culture medium: M199 (Sigma Cat. No. M7528 + 20% Fetal Calf Serum + 1% Pen/Strep + 1% Glutamine + lOOμg/ml ECGS (Sigma Cat. No E2759) + lOOμg/ml Heparin (Sigma Cat. No. H3149) + 1/500 Vol. Gentamicin

(Roche Cat. No. 1 059467). After 24 h, the erythrocytes were washed off and the medium was replaced with fresh PO medium. After incubation for another 24 h, the medium was replaced with pi medium: M199 (Sigma Cat. No. M7528 + 20% Fetal Calf Serum + 1% Pen/Strep + 1% Glutamine + 50μg/ml ECGS (Sigma Cat. No E2759) + lOOμg/ml Heparin (Sigma Cat. No. H3149).

The HUVECs was cultured in pi medium for 48h. After 48h the cells were harvested by scraping and the total cellular RNA was extracted with RNA-Bee™. From each sample 10 μg of total cellular RNA were reverse transcribed (Invitrogen, U.S.), labelled (Ambion, U.S.) and processed by using commercial kits according to the supplier's instructions. The methods of the alkaline heat fragmentation and the following hybridization of the cDNA with the U133 A and B GeneChip arrays were standard procedure provided by the manufacturer of the microchips (Affymetrix, U.S.).

The cell intensity values of the arrays were recorded with a confocal laser scanner (Hewlett Packard, U.S.) and data were analyzed using GeneChip v3.1 software (Affymetrix, U.S.). The expression level for each gene was calculated as normalized average difference of fluorescence intensity as compared to hybridization to mismatched oligonucleotides, expressed as average difference (A.D.). This experiment was performed in triplicate in order to account for biological variation.

Identification of highly expressed extracellular proteins by in silico analysis of an mRNA expression data set

The 200 genes that showed the highest mRNA levels in the above mentioned dataset (highest A.D. values) were selected. The corresponding protein sequences were analyzed by a custom software tool that predicts signal and membrane anchor sequences in a probabilistic manner. Briefly, this software tool displays signal peptide or membrane anchor predictions for proteins from SwissProt, or predicted in the eukaryotic genomes. It is based on a set of specialized, manually curated Hidden Markov Models (HMMs) that attempt to recognize the sequence features common to signal peptides or anchors, respectively (Sean R. Eddy, HMMER 2.3.2, http://hmmer.wustl.edu) . As these sequence signals cannot be reliably predicted, the "signal" and "anchor" scores that any input sequence is assigned are fed into a Support Vector Machine (SVM) in a second analysis

step (Cristianini N, Shawe-Taylor J. An Introduction to Support Vector Machines and other Kernel-based Learning Methods. Cambridge University Press, Cambridge, England, 2000). The SVM was trained on a set of bonafide examples for both classes. On this training set, the SVM obtained the following results on three training sets (signal - anchor - neither). The proteins predicted as extracellular ("signal" or "anchor") were further evaluated for organ specificity. A search for public domain expressed sequence tags encoding the candidate proteins was carried out and grouped according to tissue source. Only those protein were retained that were expressed in blood vessels and that did not show a strong expression in other secretory organs (e.g. liver, pancreas).

Example 3

Generation of antibodies to the Insulin Resistance marker butyrylcholinesterase

Polyclonal antibody to the Insulin Resistance marker butyrylcholinesterase is generated for further use of the antibody in the measurement of serum and plasma and blood levels of butyrylcholinesterase by immunodetection assays, e.g. Western Blotting and ELISA.

Recombinant protein expression in E. coli

In order to generate antibodies to butyrylcholinesterase, recombinant expression of the protein is performed for obtaining immunogens. The expression is done applying a combination of the RTS 100 expression system and E.coli. In a first step, the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY" system. This is a commercial web based service (www.proteoexpert.com). Using the recommended primer pairs, the "RTS 100 E. coli Linear Template Generation Set, His-tag" (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No. 3186237) system to generate linear PCR templates from the cDNA and for in-vitro transcription and expression of the nucleotide sequence coding for the butyrylcholinesterase protein is used. For Western-blot detection and later purification, the expressed protein contains a His-tag. The best expressing variant is identified. All steps from PCR to expression and detection are carried out according to the instructions of the manufacturer. The respective PCR product, containing all necessary T7 regulatory regions (promoter, ribosomal binding site and T7 terminator) is cloned into the pBAD TOPO ^® vector

(Invitrogen, Karlsruhe, Germany, Cat. No. K 4300/01) following the manufacturer's instructions. For expression using the T7 regulatory sequences, the construct is transformed into E. coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression.

Purification of His-butyrylcholinesterase fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-Butyrylcholinesterase fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidinium chloride, imidazole and thioglycerole, followed by homogenization using an Ultra-Turrax ^® . Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acidic conditions.

Production of monoclonal antibodies against the protein butyrylcholinesterase

a) Immunization of mice

12 week old A/J mice are initially immunized intraperitoneally with 100 μg butyrylcholinesterase. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 μg Butyrylcholinesterase adsorbed to aluminum hydroxide and 10 ⁹ germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 μg butyrylcholinesterase in PBS buffer for each.

b) Fusion and cloning

Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C., Methods in Enzymology 73 (1981) 3-46. In this process ca. 1* 10 ⁸ spleen cells of the immunized mouse are mixed with 2xlO ⁷ myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 g and 4°C). The cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 g in a 50 ml conical tube. The supernatant is discarded, the cell sediment is gently loosened by tapping, 1 ml PEG (molecular weight 4000, Merck,

Darmstadt) is added and mixed by pipetting. After 1 min in a water-bath at 37°C, 5 ml RPMI 1640 without FCS is added drop-wise at room temperature within a period of 4-5 min. Afterwards 5 ml RPMI 1640 containing 10% FCS is added drop-wise within ca. 1 min, mixed thoroughly, filled to 50 ml with medium (RPMI 1640 + 10% FCS) and subsequently centrifuged for 10 min at 400 g and 4 ⁰ C. The sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (100 mmol/1 hypoxanthine, 1 μg/ml azaserine in RPMI 1640 + 10% FCS). Interleukin 6 at 100 U/ml is added to the medium as a growth factor. After approx. 10 days the primary cultures are tested for specific antibody, butyrylcholinesterase- positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.

c) Immunoglobulin isolation from the cell culture supernatants

The hybridoma cells obtained are sown at a density of IxIO ⁵ cells per ml in RPMI

1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS- 104XL, Order No. 144-050). On average concentrations of 100 μg monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Bruck, C, et al., Methods in Enzymology 121 (1986) 587-695).

Generation of polyclonal antibodies

a) Immunization

For immunization, a fresh emulsion of the protein solution (100 μg/ml protein butyrylcholinesterase) and complete Freund's adjuvant at the ratio of 1:1 is prepared.

Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90.

Blood is drawn and resulting anti-butyrylcholinesterase serum used for further experiments as described in examples 3 and 4.

b) Purification of IgG (immunoglobulin G) from rabbit serum by sequential precipitation with caprylic acid and ammonium sulfate

One volume of rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 μl/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected.

The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered

(0.2 μm).

The immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.

The precipitated immunoglobulins are collected by centrifugation (8000 x g, 15 min,

4°C).

The supernatant is discarded. The pellet is dissolved in 10 mM NaH ₂ PO ₄ /NaOH, pH 7.5,

30 mM NaCl and exhaustively dialyzed. The dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 μm).

Biotinylation of polyclonal rabbit IgG

Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH ₂ PO ₄ /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 μl Biotin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH ₂ PO ₄ /NaOH, pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies have been biotinylated according to the same procedure.

Digoxygenylation of polyclonal rabbit IgG

Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH ₂ PO ₄ /NaOH, 30 mM NaCl, pH 7.5. Per ml IgG solution 50 μl digoxigenin-3-O-methylcarbonyl-ε- aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 ( 10 mM

NaH ₂ PO ₄ ZNaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.

Example 4

Western Blot

Protein samples enriched and isolated from the medium by Heparin columns (mentioned above) were solved in sample buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05 % Tween 20, 1 % SDS, and centrifuged at 12,000 g for 10 min at 4°C. The protein concentration of the supernatant was measured by Bradford using a standard curve constructed from a range of known bovine serum albumin standards. After mixing samples with sample buffer (60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8) and incubation at 70 ⁰ C for 5 min, samples were separated by 12.5% homogenous ExcelGel SDS gels (Amersham Bioscience) and electro transferred onto Nitrocellulose membranes. After incubation in blocking solution (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% non-fat dry milk), membranes were incubated with rabbit anti-rat antibody for 2 hrs at room temperature, respectively. After washing 3 times for 10 min with washing solution (0.3% Tween 20 in tris-buffered saline), membranes were incubated with a horseradish peroxidase conjugated anti-rabbit IgG (H+L), anti-mouse IgGi and anti-mouse IgG2a (Southern Biotechnology Associates, Inc., Birmingham, AL), respectively, for 1 hr at room temperature. Membranes were washed 3 times for 10 min and antigen-antibody complexes were visualized by an enhanced chemiluminescence's reagent (Western Lightning TM, PerkinElmer Life Sciences, Inc., Boston, MA) on an X-ray film according to the manufacturer's protocol.

Example 5

ELISA for the measurement of butyrylcholinesterase in human serum and plasma samples.

For detection of butyrylcholinesterase in human serum or plasma, a sandwich ELISA was developed. The following reagents were used: Mouse anti-butyrylcholinesterase Mab ( Accurate, prod.No.HAH 002-01), Rabbit anti-cholinesterase, IgG ( Cortex Biochem.Prod.No. CR 6030RP ), Goat anti-Rabbit-HRP ( Jackson Imm.Research, Prod.No. 711-036-15), 3,3',5,5'- tetramethylbenzidine (TMB), ( Sigma, Prod.No. T-4444 )•

The assay was performed as follows: A plate (Immuno Plate MaxiSorp Surface ( NUNC)) was coated with lOOμl anti.buturylcholinesterase Mab at 0.1 μg/ ml in PBS was incubated at +4°C overnight. The plate was washed 2x prior to blocking for Ih at room temperature with 1% BSA in washing buffer ( PBS + =.05% Tween-20 ). The plate was then washed 2x. 0.1-0.5 μl/ well plasma in 0.1% BSA in washbuffer was incubated for 1 h at room temperature on a shaker. The wells were then washed 3x. 50μl rabbit anti- butyrylcholinesterase at a dilution of l:10'000 in wash buffer were added to each well and incubated for 1 h at room temperature on a shaker. The plate was then washed again 3x. 50μl of goat anti-rabbit- HRP at a dilution of l:15'000 in wash buffer were added to each well and incubated for 1 h as above. Then the plate was washed again 3x. 50μl TMB of a 59% solution in water was added to each well and left for 5 mins on a shaker. The reaction was stopped with 50μl/ well of 0.5M phosphoric acid and absorbance read at 450 nm.

Example 6

Statistical analysis of patient data:

Clinical utility of the novel marker butyrylcholinesterase is assessed by measuring its levels in 10 diabetic patients depending on injections of exogenous insulin and comparing the levels with those measured in 10 patients with demonstrated normal beta cell function. Statistical analysis is performed by standard Student's t-test evaluation with values <0.05 taken as significant.