METHOD FOR THE DIAGNOSIS OF DEPRESSION BASED ON MONITORING BLOOD LEVELS OF ARGININE VASOPRESSIN AND/OR THYMOPOIETIN Field of the Invention

The present invention relates generally to methods for diagnosis of disorders characterized by depression and stress. More particularly, the invention provides a novel means of making or confirming a diagnosis of an affective disorder, such as depression, based on the blood levels of arginine vasopressin and thymopoietin, either individually or in combination.

Background of the Invention A. Arginine vasopressin

Arginine vasopressin (AVP) , a neurohormone also known as anti-diuretic hormone, is characterized by a nine amino acid, partially cyclic structure. AVP has been reported to be associated in serum with a binding protein, called neurophysin [Brain Peptides, (D. T.

Krieger et al, eds.), John Wiley & Sons, New York, pp. 598-611 (1983)]. AVP is secreted from two major locations in the brain, from hypothalamic parvicellular neurons in the paraventricular nucleus, which also produce corticotropin releasing factor (CRF) , and from magnocellular neurons in the supraoptic and paraventricular nuclei [F. A. Antoni, in Frontiers in Neuroendocrinology, 14.(2) :76-122 (1993)]. CRF is known to synergize with AVP to stimulate ACTH release. It has been demonstrated that, following the application of chronic stress paradigms in laboratory animals, there is an increase in the level of AVP in the paraventricular nucleus of the hypothalamus. This AVP level is disproportionately large compared with the increase observed in CRF levels [Ant ⁰¹¹ !, cited above; D. C. De Goeij et al, Endocrinol.. 131.847 (1992)]. Following chronic stress, AVP levels within CRF-

containing neurons within the paraventricular nucleus of the hypothalamus of laboratory animals have been reported to increase by over 8 fold, while the increase in CRF levels was 1.5 fold. This disproportionate increase in the level of AVP in this portion of the hypothalamus as compared with CRF has also been observed to be maintained at the level of the median eminence, the terminal bed from which AVP and CRF are released to stimulate ACTH secretion from the pituitary. Previous attempts have been made to correlate altered plasma levels of both CRF and AVP with psychiatric disorders, such as depression, in humans. However, attempts to establish a diagnostic evaluation of such conditions by measuring blood levels of AVP have been unsuccessful. Such efforts have reported either no difference in AVP levels between depressed patients and normal controls, or a decrease in cerebrospinal fluid (CSF) AVP levels caused by depression. For example, P. S. Sorensen et al., J. Neurol. Neurosurα. Psvch.. 48;50- 57 (1985) found no difference in AVP levels in the plasma of depressed patients vs. normal controls.

In a study by P. W. Gold et al., Psvchopharmacol. Bull.. 12:426-431 (1983), decreases in AVP below the control levels in CSF were found in non- psychotic depressed patients. Other researchers have attempted to correlate AVP levels in CSF and in plasma for depressed patients and have found a decrease in CSF AVP, but no difference in plasma AVP [A. Gjerris et al, Brit. J. Psychiatry. 117:696-701 (1985)]. A more recent study of AVP levels in the CSF of depressed patients vs. normal controls also revealed a decrease in AVP levels for such patients [A. Gjerris, Acta Psvchiatr. Scand.. 78. Suppl. 345:21-24 (1988)]. To date, no one has ever reported an increase in plasma AVP levels in depression.

Blood levels of AVP have been studied most extensively in relation to water and electrolyte balance in the body. For example, a lack of AVP is associated with diabetes insipidus, which causes a failure to retain fluid by the kidneys and a resultant decrease in electrolytes. This condition is treated by the administration of AVP. There is also a rare syndrome of elevated AVP in blood, referred to as the syndrome of inappropriate antidiuretic hormone secretion (SIADH) . See, e.g., S. Hou, "Syndrome of Inappropriate

Antidiuretic Hormone Secretion" in Reichlin S., ed. , The Neurohypophysis, Plenum Press, New York (1984) pp. 166- 189. Occasionally, an excess of AVP has been found associated with certain cancers, such as malignant neoplasias, lymphomas, leukemias, thymomas and mesotheliomas. Inappropriate AVP levels have also been observed in rare cases of pulmonary disorders such as tuberculosis and pneumonia and in central nervous system disorders such as trauma. Abnormal AVP levels have also been noted as a consequence of drugs that enhance AVP release or action, e.g., diuretics.

However, the changes or elevations of AVP blood levels in these relatively rare conditions are accompanied by physiologic symptoms and changes in serum electrolytes, and are thus clearly distinguishable by the context in which such elevation is observed [See, e.g., Textbook of Endocrinology, 7th ed. (Wilson and Foster, eds) 1985 pp. 644-645]. Such increases in AVP have been detected by radioimmunoassay. B. Thymopoietin

The thy ic hormone thymopoietin (TP) has been shown to play a regulatory role in immune, nervous, and endocrine functions and has been isolated from bovine and human thymus. For additional general information on TP, see, also, G.H. Sunshine et al, . _r jηπmipo] . _f

12_p_:1594-1599 (1978); G. Goldstein, Nature. 247:11-14 (1974); D. H. Schlesinger and G. Goldstein, Cell. 1:361- 365 (1975); G. Goldstein et al . , Lancet 2:256-262 (1975). TP has also been found to be present in brain extracts [R. H. Brown, et al . , Brain Research 381:237-243 (1986)].

It has been found that as the thymus involutes with age, thymic hormone levels decrease, which is believed to be related to increased susceptibility to disease in aging [G. Goldstein and I. R. Mackay, The Human Thvmus. Wm. Heineman Med. Books Ltd., London

(1969)]. Additionally, hypersecretion of TP has been implicated in yasthenia gravis [G. Goldstein, Lancet. 2:1164-1167 (1966)], as being involved in the impairment of signal transmission from nerve to muscle. When this signal is interrupted, the result is generalized weakness.

Previous attempts to measure TP levels by bioassay have suggested various differences in TP levels in different pathological states. However, to date, no one has provided any correlation between affective disorders and TP.

Bioassays used to measure TP are cumbersome, inaccurate and unreliable. [J. J. Twomey, et al . , Proc. Natl. Acad. Sci. USA. 21:2541-2545 (1977); V. M. Lewis, et al . , J. Clin. Endo. Metab. 17:145-150

(1978); J. J. Twomey, et al . , Am. J. Med. §B 377-380 (1980)]. Immunoassays are the preferred format for measuring peptides and proteins in plasma or serum, but prior attempts to develop immunoassays to measure TP have not yielded clinically useful techniques. For example, a displacement radioimmunoassay (RIA) for measuring bovine TP was developed that detected TP concentrations greater than 5 ng/mL in tissue extracts. However this RIA is incapable of measuring TP levels in blood [see, e.g., G. Goldstein, J. Immunol. 117:690-692 (1976)].

The sensitivity of the TP RIA was subsequently increased to 20 picograms (pg) [see, e.g., P.J. Lisi et al , Clin. Chi . Acta. lflZ:lll-119 (1980)] using "human serum-based standards" and rabbit antisera. However, this assay has not proved effective or reproducible in practice. In addition, the present inventors have found that 20 picograms sensitivity is too poor to detect human blood levels of TP. A sandwich enzyme-linked immunoassay (ELISA) was later developed for bovine TP using a combination of polyclonal and monoclonal antibodies [A. Fuccello et al , Arch. Biochem. Biophys.. 228:292-298 (1984)]. Although the assay provided specificity in distinguishing bovine TP from bovine splenin, it proved ineffective in measuring TP in humans.

Direct measurement of TP in human plasma or serum has not been accurately reported. Possible reasons for this problem may include aggregation of TP with either itself or other blood proteins, complexing of TP with specific proteins, and too low a concentration of TP to be detected by standard assay methods.

There remains a need in the art for the development of reliable methods based on blood levels of AVP and/or TP, which enable the laboratory diagnosis or confirmation of affective disorders, such as depression and/or anxiety, in humans.

summary of the Invention

In one aspect, the present invention provides a method for the laboratory diagnosis (or confirmation of diagnosis) of affective disorders, including depression, anxiety and stress, which is based upon detection of an increase in blood levels of AVP, particularly plasma or serum levels. The increase is significant when compared to a normal AVP range for non-depressed subjects.

In another aspect, the present invention provides a method for the laboratory diagnosis (or confirmation of diagnosis) of affective disorders, including depression, anxiety and stress, which is based upon detection of an increase in blood levels of TP, particularly plasma or serum levels. The increase is significant when compared to a normal TP range for non- depressed subjects.

In yet a further aspect, the present invention provides a method for the laboratory diagnosis (or confirmation of diagnosis) of affective disorders, including depression, anxiety and stress, which is based upon detection of an increase in blood levels of both AVP and TP values, taken in combination, particularly plasma or serum levels. The increase is significant when compared to a normal range of combined AVP/TP values for non-depressed subjects.

In still another aspect, the invention provides a diagnostic kit for the diagnosis of depression, anxiety and stress, providing the components necessary for the detection of elevated blood levels of AVP and/or TP.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings

Fig. 1 is a graph plotting Hamilton Depression Scores for the subjects used in the examples. See, Example 1. Fig. 2A is a graph plotting AVP levels in pg/ml vs. time of day which illustrates the differences in mean (± SEM) AVP levels between patients diagnosed as clinically depressed (open circle) and age/sex matched normal subjects (closed circle) at two different times of day. See, Example 2.

Fig. 2B is a graph plotting the sensitivity (false negative index) and the specificity (false positive index) for afternoon levels of AVP used as a "cutoff" for a diagnosis of depression. See, Example 2. Fig. 3A is a graph plotting TP levels in pg/ml vs. time of day which illustrates the differences in mean (+ SEM) TP levels between patients diagnosed as clinically depressed (open circle) and age/sex matched normal subjects (closed circle) at two different times of day. See Example 3.

Fig. 3B is a graph plotting the sensitivity (false negative index) and the specificity (false positive index) for afternoon levels of TP used as a "cutoff" for a diagnosis of depression. See, Example 3. Fig. 4 is a graph plotting the diagnostic utility of "cutoff" levels of either 3.6 pg/ml for AVP alone (vertical line) or 6.4 pg/ml for TP alone (horizontal line) . A straight line corresponding to TP = 10.8 -1.1(AVP) (diagonal dashed line) is a linear discriminator used to predict clinical status as a function of the combined levels of TP and AVP. Subjects whose combined AVP and TP values are above the line are classified as depressed, while those whose values are below the line are designated normal. Control subjects are depicted by solid circles, depressed subjects are depicted by open circles. See, Example 4.

Fig. 5 is tabular evaluation of the discrimination of TP, AVP and combined measurements in depressed and control subjects when samples were obtained in the afternoon.

Detailed Description of the Invention

The present invention provides methods and laboratory kits which are useful for diagnosing affective disorders, such as depression, anxiety and stress, and/or

for confirming a diagnosis of such an affective disorder. As used herein, the term "affective disorder" has its conventional meaning in psychiatry and encompasses such illnesses as chronic or major depression, anxiety, stress, and eating disorders, among others.

The methods of this invention are based on the discovery by the inventors that in a subject with depression, the level(s) of arginine vasopressin and/or thymopoietin in plasma or serum detectably and significantly increases.

I. Methods based on AVP Measurement

Without wishing to be bound by theory, the inventors determined that increased levels of AVP in blood might be a useful clinical marker of chronic stress and related affective illnesses, such as major depression in humans. Despite the contradictory teachings of the art concerning the lack of utility of measuring levels of AVP in blood during depression or stress (see, e.g., Sorenson et al, Gold et al, and Gjerris et al, cited above) , the inventors created the novel diagnostic method of the present invention. As embodied in the examples below, the inventors have demonstrated by comparing AVP levels in clinically depressed subjects with normal subjects, that the detection of elevations in AVP blood levels beyond a normal range provides a reliable diagnostic indication of an affective disorder.

Further, while significantly elevated AVP levels were observed in depressed subjects both in the morning and in the afternoon, it was found that the afternoon levels were a more powerful predictor of affective illness. See, e.g., Fig. 5. Despite this observation, the invention is not limited to the detection of elevated AVP levels in the afternoon. Other times may also be advantageous in obtaining measurements

of AVP as a predictor of affective illness, for example, early morning or late evening AVP levels.

Thus, the invention provides a new method for confirming or initially diagnosing an affective disorder, which entails measuring AVP levels in human blood and comparing the measured levels to a laboratory range for normal human AVP levels. This measurement of the increase in AVP may be readily made by use of a suitable immunoassay, in concert with an extraction or dissociation technique to separate the AVP from its neurophysin prior to measurement. One such assay format for the measurement of AVP in plasma or in the cerebrospinal fluid is the radioimmunoassay disclosed by M. Hammer, Scand. J. Clin. Lab. Invest.. 38:707-716 (1978) , incorporated by reference herein.

According to the method of the invention, such an immunoassay is employed to evaluate a subject, preferably a human subject, for an affective disorder, e.g., depression. Briefly described, a plasma sample is prepared by taking a blood sample from a patient in the presence of an anticoagulent and separating the plasma from other components in the sample by conventional conditions. Such conditions may be selected by one of skill in the art, but can include, for example, centrifugation at a rate of about 1150 x g for about 20 minutes. The AVP in the sample is dissociated from its binding protein, neurophysin, by use of known dissociation methods, such as acidification or extraction of the plasma with ethanol. However, one skilled in the art may employ other conventional dissociation methods for the same purpose.

The AVP protein is then separated from the neurophysin by conventional techniques, which are determined by the method of dissociation employed. For example, in the case of ethanol extraction, the denatured

neurophysin is collected as a pellet by centrifugation using conventional conditions, such as centrifugation at about 2000 x g for about 15 minutes at 4°C. The supernatant containing the free AVP is removed and evaporated under nitrogen, resulting in a dried sample containing the free AVP. It should be noted that one skilled in the art may employ other conventional extraction methods for the same purpose.

This sample is then preferably assayed by one of several conventional AVP immunoassay methods, such as that described by M. Hammer, cited above and incorporated by reference herein. Similarly a commercial kit useful in this method is a radioimmunoassay from Diagnostic Systems Laboratories, Inc., Webster, Tx. Other similar AVP assays and kits may also be employed.

Using such a method, a sample of plasma, taken from the patient at one or more time points, is assayed for AVP levels. The AVP levels of the patient are compared to the range of AVP levels observed in normal subjects. A diagnosis of depression is indicated when the AVP levels of the test subject are statistically elevated above the AVP levels of the normal range. It should be readily understood by one skilled in the art that normally such ranges are established independently by each testing laboratory to provide for any fluctuations in values caused by the way one particular laboratory performs the test, the characteristics of the antibodies used and any slight alterations in the assay format used. For example, the afternoon level of plasma AVP observed in normal subjects in Example 2 in the inventor's laboratory, using a commercial AVP RIA kit (Diagnostic Systems Laboratories, Webster, TX) was about 2.7 + 1.3 pg/ml (mean + standard deviation) and ranged from about 0.5 to about 5.4 pg/ml.

As demonstrated by Example 2 below, a diagnosis of depression is indicated when the AVP levels of the test subject are greater than the statistically determined range of AVP levels for normal subjects as determined by the laboratory performing the test. For example, the afternoon level of plasma AVP observed in depressed subjects in the inventors' laboratory, using a commercial AVP RIA kit was about 5.0 ± 2.4 pg/ml (mean ± standard deviation) and ranged from about 1.4 to about 13.2 pg/ml. When analyzed by a logistic regression model, which is used to differentiate two populations [see, e.g., D. Hosmer and S. Lemeshow, "Applied Logistic Regression", Wiley & Sons, New York (1989)] a "cutoff" level of 3.6 pg AVP/ml plasma was determined. Thus, for this experiment in the inventors' laboratory and using the stated methods, an individual whose plasma AVP level is less than 3.6 pg/ml would be considered normal, while an individual whose plasma AVP level is greater than 3.6 pg/ml would be considered depressed. A slight overlap of the AVP levels in the normal and depressed populations represents potential false positive and false negative determinations. For Example 2, the false positive estimate is 24% and the false negative estimate is 19% for afternoon measurements of AVP.

This method may be performed as a primary diagnostic method or, preferably, it may be performed to confirm a preliminary diagnosis of a selected affective disorder based on psychological or behavioral symptoms. Because such symptoms can be related to other disorders than depression, the method of this invention provides a much needed laboratory diagnostic tool for depression and other affective disorders. This method has preliminarily been found to have at least an 81% success rate in diagnosing depression. It is further anticipated that

other affective disorders will correlate with analogous levels of AVP. Additionally the method of this invention may also be employed to monitor the progress of treatment of depression, i.e., to show that the subject is approaching normal AVP levels as treatment is ongoing.

II. Methods based on TP Measurement

In still another embodiment of this invention, a novel method for making or confirming a diagnosis of an affective disorder such as depression, entails measuring TP levels in human blood and comparing the measured levels to a laboratory range for normal human TP levels. The inventors have found that elevated plasma or serum levels of TP are also found in depressed subjects. Without wishing to be bound by theory, the inventors determined that increased levels of TP in blood might be a useful clinical marker of chronic stress and related affective illnesses, such as major depression in humans. Despite the dearth of teachings of the art concerning the any utility in measuring levels of TP in blood during depression or stress, the inventors have devised the novel diagnostic method of the present invention. As embodied in the examples below, the inventors have demonstrated by comparing TP levels in clinically depressed subjects with normal subjects, that the detection of elevations in TP blood levels beyond a normal range provides a reliable diagnostic indication of an affective disorder.

Further, while significantly elevated TP levels were observed in depressed subjects both in the morning and in the afternoon, it was found that the afternoon levels were a more powerful predictor of affective illness. See, e.g.. Fig. 5. Despite this observation, the invention is not limited to the detection of elevated TP levels in the afternoon. Other times may also be

advantagous in obtaining measurements of TP as a predictor of affective illness, for example, early morning or late evening TP levels.

Thus, the invention provides a new method for confirming or initially diagnosing an affective disorder, which entails measuring TP levels in human blood and comparing the measured levels to a laboratory range for normal human TP levels. This measurement of the increase in TP may be readily made by use of a suitable immunoassay which enables detection of TP at a sensitivity greater than 20 pg/ml. Such an immunoassay may include an optional extraction or dissociation technique to separate the TP from any other protein with which it may be bound in circulation prior to measurement, such as the techniques described in co- owned, concurrently filed, US Patent Application entitled "Method of Measuring Thymopoietin Proteins in Plasma and Serum" and incorporated by reference herein.

This sample is preferably assayed by a suitable assay for the measurement of TP. See, e.g., the above- referenced patent application. As described therein, TP may be measured in plasma and serum following dissociation of TP from any plasma protein complexes, and subsequent extraction. Alternatively, TP may be measured directly in plasma or serum by using the assay capable of detecting TP in human blood at levels well below 20 pg/ml.

According to the method of the invention, such an immunoassay is employed to evaluate a subject, preferably a human subject, for an affective disorder, e.g., depression. Briefly described, a plasma sample is prepared by taking a blood sample from a patient in the presence of an anticoagulent, e.g., EDTA or heparin, and separating the plasma from other components in the sample by conventional conditions. Such conditions may be

selected by one of skill in the art, but can include, for example, centrifugation at a rate of about 1150 x g for about 20 minutes.

According to one aspect of this method, the conditions for the first step, i.e., dissociating the TP from any complex, involves acidifying the serum or plasma sample sufficiently to effect the dissociation of bound TP from a protein complex. Generally, the sample is acidified with a selected acid to provide a sample pH of less than about 3. However, it is anticipated that partial and, eventually, complete dissociation may be achieved at other sample pH levels less than neutral. A presently preferred acid for use in this acidification/extraction step is trifluoroacetic acid (TFA) . Other acids which may be substituted for TFA include, without limitation, mineral acids, such as hydrochloric, nitric and sulfuric acid.

Once the TP is so dissociated, it may be extracted from the sample in order to prevent reaggregation that may potentially interfere with subsequent assay of TP content. A presently preferred method for the extraction of free TP is through the use of a C-18 reverse phase cartridge. The presently preferred method utilizes C-18 SepPak® cartridges (™Waters, a Division of the Millipore Corp.). The

SepPak® cartridge is activated by the sequential passage through the cartridge of deionized, distilled water, a 20:80 ratio of 0.1% TFA:acetonitrile for TP extraction, and 0.1% TFA alone in sufficient quantities to achieve full activation. Desirably, the free TP, now bound to the C-18 matrix, is eluted with an appropriate ratio of TFA:acetonitrile containing 0.01% Tween® 20 reagent. The elution is preferably performed using a 20:80 ratio of 0.1% TFA:acetonitrile and the eluted, free TP is contained in the 20:80 mixture. Alternatively, other

methods to extract free TP from potentially interfering plasma substances may include without limitation, high-performance liquid chromatography (HPLC) , size-exclusion chromatography, ion exchange chromatography, dye-ligand chromatography, affinity chromatography, lectin-carbohydrate binding matrixes, solvent extractions, ultrafiltration, dialysis, etc.

The TFA and acetonitrile present in the sample following the extraction step described above must be separated from the TP prior to assay in order to prevent interference of these solvents with the assay reagents. The presently preferred method of removing acetonitrile is to evaporate the eluted solvent in a heated block (about 40°C) under a stream of nitrogen gas. The evaporation is preferably performed to at least about 20% of the original, eluted volume. Alternatively, other conditions known to those of skill may be employed to prepare the extracted TP for assay.

The free TP now substantially free of protein complexes, and interfering materials, e.g., solvents and reagents used for extraction, are placed into a medium suitable for use in the assay of choice. The presently preferred assay medium is 0.05M phosphate buffered physiologic saline (PBS) containing 0.025M ethylenediaminetetraacetic acid (EDTA) , 0.04% sodium azide, 1% bovine serum albumin, and 0.1% Tween® 20 reagent. Alternatively, other media known to those of skill in the art may be employed.

The extracted plasma/serum sample now suspended in an appropriate assay medium can be assayed for the mass of total TP, a specific TP form (e.g. [SEQ ID NO: 2], β [SEQ ID NO: 4], or γ [SEQ ID NO: 6]), or a circulating TP molecule derived from one of these proteins. The presently preferred method of quantitating TP is the use of an enzyme-linked immunoassay. However,

other appropriate assay methods may be selected, e.g. fluorescence assays such as those employing europium, electrophoretic methods, receptor assays, bioassays, and mass spectroscopy. One particularly desirable and sensitive assay for the measurement of TP in the serum/plasma extract is enzyme-linked "sandwich" immunoassay (ELISA) .

The sample is then preferably assayed by a conventional TP immunoassay method. The presently preferred method of quantitating TP is by an enzyme- linked immunoassay [C. P. Price et al (eds.), "Principles and Practice of Immunoassay", Stockton Press, New York, New York (1991)]. Other appropriate assay methods may be selected, e.g., fluorescence assays such as those employing europium, electrophoretic methods, receptor assays, bioassays, and mass spectroscopy.

Using such a method, a sample of plasma, taken from the patient at one or more time points, is assayed for TP levels. The TP levels of the patient are compared to the range of TP levels observed in normal subjects. A diagnosis of depression is indicated when the TP levels of the test subject are statistically elevated above the TP levels of the normal range. It should be readily understood by one skilled in the art that normally such ranges are established independently by each testing laboratory to provide for any fluctuations in values caused by the way one particular laboratory performs the test, the characteristics of the antibodies used and any slight alterations in the assay format used. For example, the afternoon level of plasma TP observed in normal subjects in Examples 3 and 3A in the inventor's laboratory ranged from about 2.5 to about 7.9 pg/ml, with a mean ± standard deviation of 5.3 ± 1.5 pg/ml.

As demonstrated by Example 3 below, a diagnosis of depression is indicated when the TP levels of the test subject are greater than the statistically determined range of TP levels for normal subjects as determined by the laboratory performing the test. For example, the afternoon level of plasma TP observed in depressed subjects in the inventors' laboratory, using the immunoassay described above ranged between about 3.1 and 13.1 pg/ml, with a mean (± standard deviation) level of 7.8 ± 2.5 pg/ml. When analyzed by the logistic regression model referred to above, which is used to differentiate two populations, a "cutoff" level of 6.4 pg TP/ml plasma was determined.

Thus, for this experiment in the inventors' laboratory and using the stated methods, an individual whose plasma TP level is less than 6.4 pg/ml would be considered normal, while an individual whose plasma TP level is greater than 6.4 pg/ml would be considered depressed. A slight overlap of the TP levels in the normal and depressed populations represents potential false positive and false negative determinations. For Example 3, the false positive estimate is 19% and the false negative estimate is 24% for afternoon measurements of TP. This method may be performed as a primary diagnostic method or, preferably, it may be performed to confirm a preliminary diagnosis of a selected affective disorder based on psychological or behavioral symptoms. Because such symptoms can be related to other disorders than depression, the method of this invention provides a much needed laboratory diagnostic tool for depression and other affective disorders. This method has preliminarily been found to have at least a 76% success rate in diagnosing depression. It is further anticipated that other affective disorders will correlate with analogous

levels of TP. Additionally the method of this invention may also be employed to monitor the progress of treatment of depression, i.e., to show that the subject is approaching normal TP levels as treatment is ongoing.

JJJ. Method Combining the AVP/TP Scores

As yet another embodiment of this invention, a novel method for making, or confirming a diagnosis of an affective disorder, such as depression, involves utilizing the AVP measurements, as described above, and the TP measurements, as described above, in the plasma or serum of a subject. While both measurements of AVP and TP levels independently show elevation in major depression, together they provide even greater discrimination in distinguishing subjects with depression from normal controls. Thus, this method can be an even more accurate predictor of an affective disorder.

Subjects were scored as depressed according to this invention when the AVP and TP results obtained in the individual assays described above were combined by a conventional statistical formula for logistical regression analysis using two parameters, i.e., both AVP and TP scores [Hosmer et al, cited above].

For example, using the scores obtained in Examples 2 and 3 discussed herein, a linear discriminant analysis, which confirmed the results of the logistic regression analysis, produced a straight line corresponding to the appropriate statistical formula. According to this analysis combining the individual AVP and TP scores, a subject was categorized as depressed with a combination score of greater than about 10.0 pg/ml. See, e.g.. Fig. 4.

This combined method may be performed as a primary diagnostic method or, preferably, it may be performed to confirm a preliminary diagnosis of a

selected affective disorder based on psychological or behavioral symptoms. Because such symptoms can be related to other disorders than depression, the method of this invention provides a much needed laboratory diagnostic tool for depression and other affective disorders. This method of using combined AVP and TP levels in this formula has preliminarily been found to have at least 81% success in correctly identifying depressed subjects and 90% success in correctly identifying normal controls. It is further anticipated that other affective disorders will correlate with analogous evaluations of the combined levels of AVP and TP. Additionally the method of this invention may also be employed to monitor the progress of treatment of depression, i.e., to show that the subject is approaching normal AVP and TP levels as treatment is ongoing.

IV. Diagnostic Kits of the Invention

Another embodiment of this invention is a diagnostic laboratory kit containing the essential components necessary for the performance of the assays of this invention. Such a kit includes reagents and hardware necessary to extract AVP from its neurophysin, an i-AVP antibodies and conventional immunoassay components for the measurement of AVP and a normal range of AVP levels in controls. Also included may be other conventional components, e.g., means for withdrawal of blood and vials for the collection and separation of blood components. Additionally, such a kit may include reagents and hardware necessary to extract TP, anti-TP antibodies and conventional immunoassay components for the measurement of TP and a normal range of TP levels in controls. Further, such a kit may include reagents and equipment necessary to measure both AVP and TP levels in

plasma or serum, as well as a normal range for both hormones, either alone or in combination.

The following examples illustrate preferred methods for diagnosing depression based on the method of this invention. The examples are illustrative only and do not limit the scope of the invention.

Example 1 - Identification. Enrollment and Blood Sampling of Patients Suffering from Maior Depression and Normal Age/Sex Matched Control Subjects

Normal (control) and depressed individuals who were age matched (+ 2 years) and sex matched formed the study population of interest. Patients were diagnosed as moderately to severely depressed based on criteria that fulfill the DSM-III-R (Diagnostic and Statistical Manual of Mental Disorders - Third Edition - Revised; American Psychiatric Association, 1987) criteria for current major depressive episodes, including a Hamilton Depression Rating > 20 and a Raskin Depression Score > 7. Normal subjects were also screened using the

DSM-III criteria to verify the absence of affective illness. The Hamilton Depression Ratings obtained for both the depressed and normal subjects used in the examples are illustrated in Fig. 1. Symptoms must have been present for a minimum of one month prior to entry in the study. Patients with current or past diagnosis of psychotic disorders, mania or hypomania, organic brain syndrome, mental retardation or dysthymia were excluded. The diagnosed patients were untreated for depressive symptoms prior to and during inclusion in the study. A total of 22 age/sex matched pairs of depressed patients and normal controls were obtained for the study.

Blood samples were withdrawn from the depressed and normal control subjects between 8 and 9 a.m. and again between 4 and 6 p.m. Blood samples were collected into ethylenediaminetetraacetic acid (EDTA) containing Vacutainer ¹ " (Becton-Dickinson) tubes and placed on ice. Plasma was separated by centrifugation at 1150 x g for 20 minutes and stored frozen (-70°C) until assay.

Example 2 - Demonstration of Elevated AVP in Clinically Depressed Subjects

AVP was extracted from each plasma sample of each subject of Example 1 by mixing 800 μl plasma with 5 ml 4 ^β C ethanol and inverting the tube end-over-end for 30 minutes at 4°C. The sample was centrifuged for 15 minutes at 2000 x g at 4°C to pellet the denatured protein. The supernatant was decanted and evaporated under a stream of nitrogen in a heated block.

The dried sample was resuspended in 0.8 ml assay buffer and the quantity of AVP was determined using a commercial AVP radioimmunoassay kit [Diagnostic Systems Laboratories, Inc., Webster, TX] .

Fig. 2A plots the AVP levels in pg/ml vs. time of day, illustrating the differences in mean (+ SEM) AVP levels between patients diagnosed as clinically depressed (open circle) and age/sex matched control subjects (closed circle) at two different times of day. The results demonstrate elevated AVP levels in patients diagnosed as clinically depressed at both morning (a.m.) and afternoon (p.m.) time points. The symbol, *, denotes a significant difference from control subjects drawn at the same time point.

Measurable levels of AVP were detected in all subjects. The mean AVP measurement obtained from normal control subjects was 2.7 ± 1.3 pg/mL (mean ± SD) and ranged between 0.5 and 5.4 pg/mL. However, the levels of

AVP in the depressed subjects were significantly higher than those in control subjects at both the morning (p<0.02) and afternoon (p=0.001) time points. The mean AVP in depressed patients was 5.0 ± 2.4 pg/ml (mean ± SD) and ranged from 1.4 to 13.2 pg/mL.

A logistic regression model [see, Hosmer et al, cited above] was used to differentiate control and depressed individuals. The model determines the probability that an individual falls into either group. The logistic equation is solved for the AVP level which provides the greatest discrimination between the two groups. In the present example, this level was determined to be 3.6 pg AVP/mL for afternoon values. Using 3.6 pg/mL as the diagnostic criterion, individuals whose AVP levels are greater than 3.6 pg/mL are classified as 'depressed' and those whose AVP levels are less than 3.6 pg/mL are classified as 'normal'.

Using this diagnostic criterion, this analysis demonstrated that afternoon AVP levels accurately predicted the status of 81% of the depressed patients and 76% of the control subjects (Fig. 2B) . This indicates a false negative diagnosis error rate of 19% and a false positive diagnosis error rate of 24%.

Example 3 - Demonstration of Elevated TP in Clinically Depressed Subjects

The same study population of Example 1 was also evaluated for thymopoietin levels.

TP was extracted from the plasma samples collected from depressed and age/sex matched control subjects of Example 2 and assayed for TP content by the methods described in co-owned, concurrently filed, US Patent Application entitled "Method of Measuring Thymopoietin Proteins in Plasma and Serum". Briefly, this method was performed as follows.

The samples of human plasma were acidified and allowed to incubate at room temperature for 1 hour to achieve dissociation and loaded onto an activated SepPak® C-18, reverse phase cartridge. To achieve dissociation, the sample had been acidified by dilution in 0.1% trifluoroacetic acid containing 0.01% Tween® 20 reagent to prevent non-specific absorption of free TP onto laboratory vessels and equipment. The SepPak® cartridge had been activated through the sequential passage of distilled, deionized water, 0.1% TFA:acetonitrile at a ratio of 20:80 and, finally, 0.1% TFA alone. The dissociated sample was passed through the SepPak® cartridges and the material passing through the cartridges was discarded. Each cartridge was then washed with 0.1% TFA containing 0.01% Tween® 20 reagent to completely remove all unbound material. Air was then passed through the cartridge to completely push all of the wash solution through the cartridge. The bound TP was eluted with the 20:80 ratio of 0.1% TFA:acetonitrile. The eluting solvent contained 0.01% Tween. Air was then passed through the cartridge to completely push all of the eluting solution through the cartridge. The eluting material was collected and saved. The acetonitrile in the collected material was evaporated, with warming, under a stream of nitrogen. The remaining aqueous phase was frozen and lyophilized to dryness.

The dried sample was then resuspended in 500μL TP assay buffer and added at a volume of 200 μL/well to Immulon 4® polystyrene microtiter plates, that had been previously coated with rabbit anti-hTP 1-19 [amino acids 1-19 of SEQ ID NOS: 2, 4, 6] and post-coated with 1% BSA to prevent non-specific absorption. Synthetic hTP 1-52 [SEQ ID NO: 7] was added to wells at concentration ranging from 100 to 1.56 pg/mL at a volume of 200 μL/well to serve as a standard curve.

The samples/standards were incubated for 3 hours at 37 degrees C. The wells were then washed 3X with ELISA buffer and 200 μL biotinylated monoclonal antibody against hTP 29-50 [amino acids 29-50 of SEQ ID NOS: 2, 4, 6] added at an appropriate dilution in ELISA buffer containing 1% horse serum and incubated for 2 hours at 37 degrees C. The wells were then washed 3X with ELISA buffer. Streptavidin-polyhorseradish peroxidase was added at a dilution of 1:5000 (200 μL volume) and the incubation continued at room temperature for 30 minutes. The wells were then washed 5X with ELISA buffer and Ultra-Blue® substrate added for an additional 30 minutes incubation at room temperature. The optical density of each well was determined as a direct index of the amount of TP present.

Measurements were taken at the same time points as described above for AVP. Fig. 3A illustrates the TP levels (pg/ml) vs. time of day which illustrates the differences in mean (± SEM) TP levels between patients diagnosed as clinically depressed (open circle) and age/sex matched control subjects (closed circle) at two different times of day.

Measurable levels of TP were detected in all subjects; however, the levels of TP in the depressed subjects were significantly higher than those in control subjects at both the morning (p<0.02) and afternoon (p>0.001). In control subjects, the mean TP level was 5.3 ± 1.5 pg/mL (mean ± SD) and ranged from 2.5 to 7.9 pg/mL. In depressed patients, the mean TP level was 7.8 ± 2.5 pg/mL (mean ± SD) and ranged from 3.1 to 13.1 pg/mL.

A logistic regression model [Hosmer et al., cited above] was used to discriminate control and depressed individuals. The model determines the probability that an individual falls into either group.

The logistical equation is solved to determine the level of TP which provides the greatest discrimination between the two groups. In the present example, this level was determined to be 6.4 pg TP/ l for afternoon TP values. Using 6.4 pg/mL as the diagnostic criterion, individuals whose afternoon TP levels are greater than 6.4 pg TP/mL are classified as 'depressed' and those whose TP levels are less than 6.4 pg TP/mL are classified as 'normal'. With this diagnostic criterion, this analysis demonstrated that afternoon TP levels accurately predicted the status of 76% of the depressed patients and 81% of the control subjects (Fig. 3B) . This indicates a false negative diagnosis error rate of 24% and a false positive diagnosis error rate of 19%.

Example 4 - Diagnostic Use of Combined TP and AVP Levels in Depressed Patients

The combined results of AVP and TP levels, measured as described in Examples 2 and 3, provide better discrimination than either AVP or TP levels alone. A stepwise logistic regression model was used to preliminarily assess whether both hormones together provide improved discrimination over either AVP or TP levels when considered separately. Results indicated significant improvement by using both AVP and TP.

A logistic regression model [Hosmer et al, cited above] was then fit to these data, and the model parameters were used to differentiate depressed and normal individuals. Further substantiation was provided using a conditional logistic regression model, in which the age and sex matching were incorporated into the model. In addition, a linear discriminant analysis [see, D. Kleinbawm and L. Kupper, "Applied Regression Analysis and Other Multivariant Methods", Ducksbury Press, Ducksbury, Mass. (1978)] confirmed the results obtained

by the logistic model. The solution to the linear discriminant analysis is a line which provides the maximum separation between depressed and control subjects. To illustrate this concept, Fig. 4 displays the TP and AVP levels in normal (solid circles) and depressed individuals (open circle) . A dotted line (TP = 10.8 - (1.1 X AVP)) is the linear discriminant function which best separates the data. Values above the line are classified as 'depressed ¹ and values below the line are classified as 'normal'. Also indicated in Fig. 4 are the cutoff values for either hormone, when used separately. The AVP cutoff level is a vertical line (at 3.6 pg/mL) , and the TP cutoff is a horizontal line (at 6.4 pg/mL) .

Fig. 5 demonstrates the discriminating ability of afternoon measurements of AVP alone, TP alone, and AVP and TP in combination, as performed in this example, as predictors of depression. Clinical status is scored depressed (D) or normal (N) . Classification rate is scored by the number of positives over the total tested and rated in percent sensitivity.

With this diagnostic criterion, the analysis demonstrated that combined afternoon AVP and TP levels accurately predicted the status of 81% of the depressed patients and 90% of the normal subjects (Fig. 4) . This indicates a false negative diagnosis error rate of 19% and a false positive diagnosis error rate of 10% (Fig. 5).

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

27 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Immunobiology Research, Institute Inc.

(ii) TITLE OF INVENTION: Method for the Diagnosis of Depression Based on Monitoring Blood Levels of Arginine Vasopressin and/or Thymopoietin

(iϋ) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: 321 Norristown Road, Box 457

(C) CITY: Spring House

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19477

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version#1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/309,420

(B) FILING DATE: 20-SEP-1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(C) REFERENCE/DOCKET NUMBER: IRI46PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 540-9206

(B) TELEFAX: (215) 540-5818

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2490 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 205..2286

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTTCGTAGTT CGGCTCTGGG GTCTTTTGTG TCCGGGTCTG GCTTGGCTTT 50

GTGTCCGCGA GTTTTTGTTC CGCTCCGCAG CGCTCTTCCC GGGCAGGAGC 100

CGTGAGGCTC GGAGGCGGCA GCGCGGTCCC CGGCCAGGAG CAAGCGCGCC 150

GGCGTGAGCG GCGGCGGCAA AGGCTGTGGG GAGGGGGCTT CGCAGATCCC 200

CGAG ATG CCG GAG TTC CTG GAA GAC CCC TCG GTC CTG ACA AAA 243 Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys 1 5 10

GAC AAG TTG AAG AGT GAG TTG GTC GCC AAC AAT GTG ACG CTG 285 Asp Lys Leu Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu 15 20 25

CCG GCC GGG GAG CAG CGC AAA GAC GTG TAC GTC CAG CTC TAC 327 Pro Ala Gly Glu Gin Arg Lys Asp Val Tyr Val Gin Leu Tyr 30 35 40

CTG CAG CAC CTC ACG GCT CGC AAC CGG CCG CCG CTC CCC GCC 369 Leu Gin His Leu Thr Ala Arg Asn Arg Pro Pro Leu Pro Ala 45 50 55

GGC ACC AAC AGC AAG GGG CCC CCG GAC TTC TCC AGT GAC GAA 411 Gly Thr Asn Ser Lys Gly Pro Pro Asp Phe Ser Ser Asp Glu

60 65

GAG CGC GAG CCC ACC CCG GTC CTC GGC TCT GGG GCC GCC GCC 453 Glu Arg Glu Pro Thr Pro Val Leu Gly Ser Gly Ala Ala Ala 70 75 80

GCG GGC CGG AGC CGA GCA GCC GTC GGC AGG AAA GCC ACA AAA 495 Ala Gly Arg Ser Arg Ala Ala Val Gly Arg Lys Ala Thr Lys 85 90 95

AAA ACT GAT AAA CCC AGA CAA GAA GAT AAA GAT GAT CTA GAT 537

Lys Thr Asp Lys Pro Arg Gin Glu Asp Lys Asp Asp Leu Asp 100 105 110

GTA ACA GAG CTC ACT AAT GAA GAT CTT TTG GAT CAG CTT GTG 579

Val Thr Glu Leu Thr Asn Glu Asp Leu Leu Asp Gin Leu Val

115 120 125

AAA TAC GGA GTG AAT CCT GGT CCT ATT GTG GGA ACA ACC AGG 621

Lys Tyr Gly Val Asn Pro Gly Pro lie Val Gly Thr Thr Arg

130 135

AAG CTA TAT GAG AAA AAG CTT TTG AAA CTG AGG GAA CAA GGA 663

Lys Leu Tyr Glu Lys Lys Leu Leu Lys Leu Arg Glu Gin Gly 140 145 150

ACA GAA TCA AGA TCT TCT ACT CCT CTG CCA ACA ATT TCT TCT 705

Thr Glu Ser Arg Ser Ser Thr Pro Leu Pro Thr lie Ser Ser 155 160 165

TCA GCA GAA AAT ACA AGG CAG AAT GGA AGT AAT GAT TCT GAC 747

Ser Ala Glu Asn Thr Arg Gin Asn Gly Ser Asn Asp Ser Asp 170 175 180

AGA TAC AGT GAC AAT GAA GAA GGA AAG AAG AAA GAA CAC AAG 789

Arg Tyr Ser Asp Asn Glu Glu Gly Lys Lys Lys Glu His Lys

185 190 195

AAA GTG AAG TCC ACT AGG GAT ATT GTT CCT TTT TCT GAA CTT 831

Lys Val Lys Ser Thr Arg Asp lie Val Pro Phe Ser Glu Leu

200 205

GGA ACT ACT CCC TCT GGT GGT GGA TTT TTT CAG GGT ATT TCT 873

Gly Thr Thr Pro Ser Gly Gly Gly Phe Phe Gin Gly lie Ser 210 215 220

TTT CCT GAA ATC TCC ACC CGT CCT CCT TTG GGC AGT ACC GAA 915

Phe Pro Glu lie Ser Thr Arg Pro Pro Leu Gly Ser Thr Glu 225 230 235

CTA CAG GCA GCT AAG AAA GTA CAT ACT TCT AAG GGA GAC CTA 957

Leu Gin Ala Ala Lys Lys Val His Thr Ser Lys Gly Asp Leu 240 245 250

CCT AGG GAG CCT CTT GTT GCC ACA AAC TTG CCT GGC AGG GGA 999

Pro Arg Glu Pro Leu Val Ala Thr Asn Leu Pro Gly Arg Gly

255 260 265

CAG TTG CAG AAG TTA GCC TCT GAA AGG AAT TTG TTT ATT TCA 10 1

Gin Leu Gin Lys Leu Ala Ser Glu Arg Asn Leu Phe lie Ser

270 275

TGC AAG TCT AGC CAT GAT AGG TGT TTA GAG AAA AGT TCT TCG 1083 Cys Lys Ser Ser His Asp Arg Cys Leu Glu Lys Ser Ser Ser 280 285 290

TCA TCT TCT CAG CCT GAA CAC AGT GCC ATG TTG GTC TCT ACT 1125 Ser Ser Ser Gin Pro Glu His Ser Ala Met Leu Val Ser Thr 295 300 305

GCA GCT TCT CCT TCA CTG ATT AAA GAA ACC ACC ACT GGT TAC 1167 Ala Ala Ser Pro Ser Leu lie Lys Glu Thr Thr Thr Gly Tyr 310 315 320

TAT AAA GAC ATA GTA GAA AAT ATT TGC GGT AGA GAG AAA AGT 1209 Tyr Lys Asp lie Val Glu Asn lie Cys Gly Arg Glu Lys Ser 325 330 335

GGA ATT CAA CCA TTA TGT CCT GAG AGG TCC CAT ATT TCA GAT 1251 Gly lie Gin Pro Leu Cys Pro Glu Arg Ser His lie Ser Asp

340 345

CAA TCG CCT CTC TCC AGT AAA AGG AAA GCA CTA GAA GAG TCT 1293 Gin Ser Pro Leu Ser Ser Lys Arg Lys Ala Leu Glu Glu Ser 350 355 360

GAG AGC TCA CAA CTA ATT TCT CCG CCA CTT GCC CAG GCA ATC 1335 Glu Ser Ser Gin Leu lie Ser Pro Pro Leu Ala Gin Ala lie 365 370 375

AGA GAT TAT GTC AAT TCT CTG TTG GTC CAG GGT GGG GTA GGT 1377 Arg Asp Tyr Val Asn Ser Leu Leu Val Gin Gly Gly Val Gly 380 385 390

AGT TTG CCT GGA ACT TCT AAC TCT ATG CCC CCA CTG GAT GTA 1419 Ser Leu Pro Gly Thr Ser Asn Ser Met Pro Pro Leu Asp Val 395 400 405

GAA AAC ATA CAG AAG AGA ATT GAT CAG TCT AAG TTT CAA GAA 1461 Glu Asn lie Gin Lys Arg lie Asp Gin Ser Lys Phe Gin Glu

410 415

ACT GAA TTC CTG TCT CCT CCA AGA AAA GTC CCT AGA CTG AGT 1503 Thr Glu Phe Leu Ser Pro Pro Arg Lys Val Pro Arg Leu Ser 420 425 430

GAG AAG TCA GTG GAG GAA AGG GAT TCA GGT TCC TTT GTG GCA 1545 Glu Lys Ser Val Glu Glu Arg Asp Ser Gly Ser Phe Val Ala 435 440 445

TTT CAG AAC ATA CCT GGA TCC GAA CTG ATG TCT TCT TTT GCC 1587 Phe Gin Asn lie Pro Gly Ser Glu Leu Met Ser Ser Phe Ala 450 455 460

AAA ACT GTT GTC TCT CAT TCA CTC ACT ACC TTA GGT CTA GAA 1629 Lys Thr Val Val Ser His Ser Leu Thr Thr Leu Gly Leu Glu 465 470 475

GTG GCT AAG CAA TCA CAG CAT GAT AAA ATA GAT GCC TCA GAA 1671 Val Ala Lys Gin Ser Gin His Asp Lys lie Asp Ala Ser Glu

480 485

CTA TCT TTT CCC TTC CAT GAA TCT ATT TTA AAA GTA ATT GAA 1713 Leu Ser Phe Pro Phe His Glu Ser lie Leu Lys Val lie Glu 490 495 500

GAA GAA TGG CAG CAA GTT GAC AGG CAG CTG CCT TCA CTG GCA 1755 Glu Glu Trp Gin Gin Val Asp Arg Gin Leu Pro Ser Leu Ala 505 510 515

TGC AAA TAT CCA GTT TCT TCC AGG GAG GCA ACA CAG ATA TTA 1797 Cys Lys Tyr Pro Val Ser Ser Arg Glu Ala Thr Gin lie Leu 520 525 530

TCA GTT CCA AAA GTA GAT GAT GAA ATC CTA GGG TTT ATT TCT 1839 Ser Val Pro Lys Val Asp Asp Glu lie Leu Gly Phe lie Ser 535 540 545

GAA GCC ACT CCA CTA GGA GGT ATT CAA GCA GCC TCC ACT GAG 1881 Glu Ala Thr Pro Leu Gly Gly lie Gin Ala Ala Ser Thr Glu

550 555

TCT TGC AAT CAG CAG TTG GAC TTA GCA CTC TGT AGA GCA TAT 1923 Ser Cys Asn Gin Gin Leu Asp Leu Ala Leu Cys Arg Ala Tyr 560 565 570

GAA GCT GCA GCA TCA GCA TTG CAG ATT GCA ACT CAC ACT GCC 1965 Glu Ala Ala Ala Ser Ala Leu Gin lie Ala Thr His Thr Ala 575 580 585

TTT GTA GCT AAG GCT ATG CAG GCA GAC ATT AGT CAA GCT GCA 2007 Phe Val Ala Lys Ala Met Gin Ala Asp lie Ser Gin Ala Ala 590 595 600

CAG ATT CTT AGC TCA GAT CCT AGT CGT ACC CAC CAA GCG CTT 2049 Gin lie Leu Ser Ser Asp Pro Ser Arg Thr His Gin Ala Leu 605 610 615

GGG ATT CTG AGC AAA ACA TAT GAT GCA GCC TCA TAT ATT TGT 2091 Gly lie Leu Ser Lys Thr Tyr Asp Ala Ala Ser Tyr lie Cys

620 625

GAA GCT GCA TTT GAT GAA GTG AAG ATG GCT GCC CAT ACC ATG 2133 Glu Ala Ala Phe Asp Glu Val Lys Met Ala Ala His Thr Met 630 635 640

GGA AAT GCC ACT GTA GGT CGT CGA TAC CTC TGG CTG AAG GAT 2175 Gly Asn Ala Thr Val Gly Arg Arg Tyr Leu Trp Leu Lys Asp 645 650 655

TGC AAA ATT AAT TTA GCT TCT AAG AAT AAG CTG GCT TCC ACT 2217 Cys Lys lie Asn Leu Ala Ser Lys Asn Lys Leu Ala Ser Thr 660 665 670

CCC TTT AAA GGT GGA ACA TTA TTT GGA GGA GAA GTA TGC AAA 2259 Pro Phe Lys Gly Gly Thr Leu Phe Gly Gly Glu Val Cys Lys 675 680 685

GTA ATT AAA AAG CGT GGA AAT AAA CAC TAGTAAAATT AAGGACAAAA 2306 Val lie Lys Lys Arg Gly Asn Lys His

690

AGACATCTAT CTTATCTTTC AGGTACTTTA TGCCAACATT TTCTTTTCTG 2356

TTAAGGTTGT TTTAGTTTCC AGATAGGGCT AATTACAAAA TGTTAAGCTT 2406

CTACCCATCA AATTACAGTA TAAAAGTAAT TGCCTGTGTA GAACTACTTG 2456

TCTTTTCTAA AGATTTGCGT AGATAGGAAG CCTG 2490

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 694 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys 1 5 10 15

Leu Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly

20 25 30

Glu Gin Arg Lys Asp Val Tyr Val Gin Leu Tyr Leu Gin His Leu

35 40 45

Thr Ala Arg Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys

50 55 60

Gly Pro Pro Asp Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro

65 70 75

Val Leu Gly Ser Gly Ala Ala Ala Ala Gly Arg Ser Arg Ala Ala 80 85 90

Val Gly Arg Lys Ala Thr Lys Lys Thr Asp Lys Pro Arg Gin Glu 95 100 105

Asp Lys Asp Asp Leu Asp Val Thr Glu Leu Thr Asn Glu Asp Leu 110 115 120

Leu Asp Gin Leu Val Lys Tyr Gly Val Asn Pro Gly Pro He Val 125 130 135

Gly Thr Thr Arg Lys Leu Tyr Glu Lys Lys Leu Leu Lys Leu Arg 140 145 150

Glu Gin Gly Thr Glu Ser Arg Ser Ser Thr Pro Leu Pro Thr He 155 160 165

Ser Ser Ser Ala Glu Asn Thr Arg Gin Asn Gly Ser Asn Asp Ser 170 175 180

Asp Arg Tyr Ser Asp Asn Glu Glu Gly Lys Lys Lys Glu His Lys 185 190 195

Lys Val Lys Ser Thr Arg Asp He Val Pro Phe Ser Glu Leu Gly 200 205 210

Thr Thr Pro Ser Gly Gly Gly Phe Phe Gin Gly He Ser Phe Pro 215 220 225

Glu He Ser Thr Arg Pro Pro Leu Gly Ser Thr Glu Leu Gin Ala 230 235 240

Ala Lys Lys Val His Thr Ser Lys Gly Asp Leu Pro Arg Glu Pro 245 250 255

Leu Val Ala Thr Asn Leu Pro Gly Arg Gly Gin Leu Gin Lys Leu 260 265 270

Ala Ser Glu Arg Asn Leu Phe He Ser Cys Lys Ser Ser His Asp 275 280 285

Arg Cys Leu Glu Lys Ser Ser Ser Ser Ser Ser Gin Pro Glu His 290 295 300

Ser Ala Met Leu Val Ser Thr Ala Ala Ser Pro Ser Leu He Lys 305 310 315

Glu Thr Thr Thr Gly Tyr Tyr Lys Asp He Val Glu Asn He Cys 320 325 330

Gly Arg Glu Lys Ser Gly He Gin Pro Leu Cys Pro Glu Arg Ser 335 340 345

His He Ser Asp Gin Ser Pro Leu Ser Ser Lys Arg Lys Ala Leu 350 355 360

Glu Glu Ser Glu Ser Ser Gin Leu He Ser Pro Pro Leu Ala Gin 365 370 375

Ala He Arg Asp Tyr Val Asn Ser Leu Leu Val Gin Gly Gly Val 380 385 390

Gly Ser Leu Pro Gly Thr Ser Asn Ser Met Pro Pro Leu Asp Val 395 400 405

Glu Asn He Gin Lys Arg He Asp Gin Ser Lys Phe Gin Glu Thr

410 415 420

Glu Phe Leu Ser Pro Pro Arg Lys Val Pro Arg Leu Ser Glu Lys 425 430 435

Ser Val Glu Glu Arg Asp Ser Gly Ser Phe Val Ala Phe Gin Asn

440 445 450

He Pro Gly Ser Glu Leu Met Ser Ser Phe Ala Lys Thr Val Val 455 460 465

Ser His Ser Leu Thr Thr Leu Gly Leu Glu Val Ala Lys Gin Ser 470 475 480

Gin His Asp Lys He Asp Ala Ser Glu Leu Ser Phe Pro Phe His 485 490 495

Glu Ser He Leu Lys Val He Glu Glu Glu Trp Gin Gin Val Asp 500 505 510

Arg Gin Leu Pro Ser Leu Ala Cys Lys Tyr Pro Val Ser Ser Arg 515 520 525

Glu Ala Thr Gin He Leu Ser Val Pro Lys Val Asp Asp Glu He 530 535 540

Leu Gly Phe He Ser Glu Ala Thr Pro Leu Gly Gly He Gin Ala 545 550 555

Ala Ser Thr Glu Ser Cys Asn Gin Gin Leu Asp Leu Ala Leu Cys

560 565 570

Arg Ala Tyr Glu Ala Ala Ala Ser Ala Leu Gin He Ala Thr His 575 580 585

Thr Ala Phe Val Ala Lys Ala Met Gin Ala Asp He Ser Gin Ala 590 595 600

Ala Gin He Leu Ser Ser Asp Pro Ser Arg Thr His Gin Ala Leu 605 610 615

Gly He Leu Ser Lys Thr Tyr Asp Ala Ala Ser Tyr He Cys Glu 620 625 630

Ala Ala Phe Asp Glu Val Lys Met Ala Ala His Thr Met Gly Asn 635 640 645

Ala Thr Val Gly Arg Arg Tyr Leu Trp Leu Lys Asp Cys Lys He 650 655 660

Asn Leu Ala Ser Lys Asn Lys Leu Ala Ser Thr Pro Phe Lys Gly 665 670 675

Gly Thr Leu Phe Gly Gly Glu Val Cys Lys Val He Lys Lys Arg 680 685 690

Gly Asn Lys His

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1743 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 238..1599

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGTTGGTGCG AGCTTCCAGC TTGGCCGCAG TTGGTTCGTA GTTCGGCTCT 50

GGGGTCTTTT GTGTCCGGGT CTGGCTTGGC TTTGTGTCCG CGAGTTTTTG 100

TTCCGCTCCG CAGCGCTCTT CCCGGGCAGG AGCCGTGAGG CTCGGAGGCG 150

GCAGCGCGGT CCCCGGCCAG GAGCAAGCGC GCCGGCGTGA GCGGCGGCGG 200

CAAAGGCTGT GGGGAGGGGG CTTCGCAGAT CCCCGAG ATG CCG GAG TTC 249

Met Pro Glu Phe

1

CTG GAA GAC CCC TCG GTC CTG ACA AAA GAC AAG TTG AAG AGT 291 Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys Leu Lys Ser 5 10 15

GAG TTG GTC GCC AAC AAT GTG ACG CTG CCG GCC GGG GAG CAG 333 Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly Glu Gin 20 25 30

CGC AAA GAC GTG TAC GTC CAG CTC TAC CTG CAG CAC CTC ACG 375 Arg Lys Asp Val Tyr Val Gin Leu Tyr Leu Gin His Leu Thr 35 40 45

GCT CGC AAC CGG CCG CCG CTC CCC GCC GGC ACC AAC AGC AAG 417 Ala Arg Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys 50 55 60

GGG CCC CCG GAC TTC TCC AGT GAC GAA GAG CGC GAG CCC ACC 459 Gly Pro Pro Asp Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr

65 70

CCG GTC CTC GGC TCT GGG GCC GCC GCC GCG GGC CGG AGC CGA 501 Pro Val Leu Gly Ser Gly Ala Ala Ala Ala Gly Arg Ser Arg 75 80 85

GCA GCC GTC GGC AGG AAA GCC ACA AAA AAA ACT GAT AAA CCC 543 Ala Ala Val Gly Arg Lys Ala Thr Lys Lys Thr Asp Lys Pro 90 95 100

AGA CAA GAA GAT AAA GAT GAT CTA GAT GTA ACA GAG CTC ACT 585 Arg Gin Glu Asp Lys Asp Asp Leu Asp Val Thr Glu Leu Thr 105 110 115

AAT GAA GAT CTT TTG GAT CAG CTT GTG AAA TAC GGA GTG AAT 627 Asn Glu Asp Leu Leu Asp Gin Leu Val Lys Tyr Gly Val Asn 120 125 130

CCT GGT CCT ATT GTG GGA ACA ACC AGG AAG CTA TAT GAG AAA 669 Pro Gly Pro He Val Gly Thr Thr Arg Lys Leu Tyr Glu Lys

135 140

AAG CTT TTG AAA CTG AGG GAA CAA GGA ACA GAA TCA AGA TCT 711 Lys Leu Leu Lys Leu Arg Glu Gin Gly Thr Glu Ser Arg Ser 145 150 155

TCT ACT CCT CTG CCA ACA ATT TCT TCT TCA GCA GAA AAT ACA 753 Ser Thr Pro Leu Pro Thr He Ser Ser Ser Ala Glu Asn Thr 160 165 170

AGG CAG AAT GGA AGT AAT GAT TCT GAC AGA TAC AGT GAC AAT 795 Arg Gin Asn Gly Ser Asn Asp Ser Asp Arg Tyr Ser Asp Asn 175 180 185

GAA GAA GAC TCT AAA ATA GAG CTC AAG CTT GAG AAG AGA GAA 837 Glu Glu Asp Ser Lys He Glu Leu Lys Leu Glu Lys Arg Glu 190 195 200

CCA CTA AAG GGC AGA GCA AAG ACT CCA GTA ACA CTC AAG CAA 879 Pro Leu Lys Gly Arg Ala Lys Thr Pro Val Thr Leu Lys Gin

205 210

AGA AGA GTT GAG CAC AAT CAG AGC TAT TCT CAA GCT GGA ATA 921 Arg Arg Val Glu His Asn Gin Ser Tyr Ser Gin Ala Gly He 215 220 225

ACT GAG ACT GAA TGG ACA AGT GGA TCT TCA AAA GGC GGA CCT 963 Thr Glu Thr Glu Trp Thr Ser Gly Ser Ser Lys Gly Gly Pro 230 235 240

CTG CAG GCA TTA ACT AGG GAA TCT ACA AGA GGG TCA AGA AGA 1005 Leu Gin Ala Leu Thr Arg Glu Ser Thr Arg Gly Ser Arg Arg 245 250 255

ACT CCA AGG AAA AGG GTG GAA ACT TCA GAA CAT TTT CGT ATA 1047 Thr Pro Arg Lys Arg Val Glu Thr Ser Glu His Phe Arg He 260 265 270

GAT GGT CCA GTA ATT TCA GAG AGT ACT CCC ATA GCT GAA ACT 1089 Asp Gly Pro Val He Ser Glu Ser Thr Pro He Ala Glu Thr

275 280

ATA ATG GCT TCA AGC AAC GAA TCC TTA GTT GTC AAT AGG GTG 1131 He Met Ala Ser Ser Asn Glu Ser Leu Val Val Asn Arg Val 285 290 295

ACT GGA AAT TTC AAG CAT GCA TCT CCT ATT CTG CCA ATC ACT 1173 Thr Gly Asn Phe Lys His Ala Ser Pro He Leu Pro He Thr 300 305 310

GAA TTC TCA GAC ATA CCC AGA AGA GCA CCA AAG AAA CCA TTG 1215 Glu Phe Ser Asp He Pro Arg Arg Ala Pro Lys Lys Pro Leu 315 320 325

ACA AGA GCT GAA GTG GGA GAA AAA ACA GAG GAA AGA AGA GTA 1257 Thr Arg Ala Glu Val Gly Glu Lys Thr Glu Glu Arg Arg Val 330 335 340

GAA AGG GAT ATT CTT AAG GAA ATG TTC CCC TAT GAA GCA TCT 1299 Glu Arg Asp He Leu Lys Glu Met Phe Pro Tyr Glu Ala Ser

345 350

ACA CCA ACA GGA ATT AGT GCT AGT TGC CGC AGA CCA ATC AAA 1341 Thr Pro Thr Gly He Ser Ala Ser Cys Arg Arg Pro He Lys 355 360 365

GGG GCT GCA GGC CGG CCA TTA GAA CTC AGT GAT TTC AGG ATG 1383 Gly Ala Ala Gly Arg Pro Leu Glu Leu Ser Asp Phe Arg Met 370 375 380

GAG GAG TCT TTT TCA TCT AAA TAT GTT CCT AAG TAT GTT CCC 1425 Glu Glu Ser Phe Ser Ser Lys Tyr Val Pro Lys Tyr Val Pro 385 390 395

TTG GCA GAT GTC AAG TCA GAA AAG ACA AAA AAG GGA CGC TCC 1467 Leu Ala Asp Val Lys Ser Glu Lys Thr Lys Lys Gly Arg Ser 400 405 410

ATT CCC GTA TGG ATA AAA ATT TTG CTG TTT GTT GTT GTG GCA 1509 He Pro Val Trp He Lys He Leu Leu Phe Val Val Val Ala

415 420

GTT TTT TTG TTT TTG GTC TAT CAA GCT ATG GAA ACC AAC CAA 1551 Val Phe Leu Phe Leu Val Tyr Gin Ala Met Glu Thr Asn Gin 425 430 435

GTA AAT CCC TTC TCT AAT TTT CTT CAT GTT GAC CCT AGA AAA 1593 Val Asn Pro Phe Ser Asn Phe Leu His Val Asp Pro Arg Lys 440 445 450

TCC AAC TGAATGGTAT CTCTTTGGCA CGTTCAACTT GGTCTCCTAT 1639 Ser Asn

TTTCAATAAC TGTTGAAAAA CATTTGTGTA CACTTGTTGA CTCCAAGAAC 1689

TAAAAATAAT GTGATTTCGC CTCAATAAAT GTAGTATTTC ATTGAAAAGC 1739

AAAC 1743

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 454 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys 1 5 10 15

Leu Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly

20 25 30

Glu Gin Arg Lys Asp Val Tyr Val Gin Leu Tyr Leu Gin His Leu

35 40 45

Thr Ala Arg Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys

50 55 60

Gly Pro Pro Asp Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro

65 70 75

Val Leu Gly Ser Gly Ala Ala Ala Ala Gly Arg Ser Arg Ala Ala

80 85 90

Val Gly Arg Lys Ala Thr Lys Lys Thr Asp Lys Pro Arg Gin Glu

95 100 105

Asp Lys Asp Asp Leu Asp Val Thr Glu Leu Thr Asn Glu Asp Leu

110 115 120

Leu Asp Gin Leu Val Lys Tyr Gly Val Asn Pro Gly Pro He Val

125 130 135

Gly Thr Thr Arg Lys Leu Tyr Glu Lys Lys Leu Leu Lys Leu Arg

140 145 150

Glu Gin Gly Thr Glu Ser Arg Ser Ser Thr Pro Leu Pro Thr He

155 160 165

Ser Ser Ser Ala Glu Asn Thr Arg Gin Asn Gly Ser Asn Asp Ser

170 175 180

Asp Arg Tyr Ser Asp Asn Glu Glu Asp Ser Lys He Glu Leu Lys

185 190 195

Leu Glu Lys Arg Glu Pro Leu Lys Gly Arg Ala Lys Thr Pro Val

200 205 210

Thr Leu Lys Gin Arg Arg Val Glu His Asn Gin Ser Tyr Ser Gin

215 220 225

Ala Gly He Thr Glu Thr Glu Trp Thr Ser Gly Ser Ser Lys Gly

230 235 240

Gly Pro Leu Gin Ala Leu Thr Arg Glu Ser Thr Arg Gly Ser Arg

245 250 255

Arg Thr Pro Arg Lys Arg Val Glu Thr Ser Glu His Phe Arg He

260 265 270

Asp Gly Pro Val He Ser Glu Ser Thr Pro He Ala Glu Thr He

275 280 285

Met Ala Ser Ser Asn Glu Ser Leu Val Val Asn Arg Val Thr Gly 290 295 300

Asn Phe Lys His Ala Ser Pro He Leu Pro He Thr Glu Phe Ser 305 310 315

Asp He Pro Arg Arg Ala Pro Lys Lys Pro Leu Thr Arg Ala Glu 320 325 330

Val Gly Glu Lys Thr Glu Glu Arg Arg Val Glu Arg Asp He Leu 335 340 345

Lys Glu Met Phe Pro Tyr Glu Ala Ser Thr Pro Thr Gly He Ser 350 355 360

Ala Ser Cys Arg Arg Pro He Lys Gly Ala Ala Gly Arg Pro Leu 365 370 375

Glu Leu Ser Asp Phe Arg Met Glu Glu Ser Phe Ser Ser Lys Tyr 380 385 390

Val Pro Lys Tyr Val Pro Leu Ala Asp Val Lys Ser Glu Lys Thr 395 400 405

Lys Lys Gly Arg Ser He Pro Val Trp He Lys He Leu Leu Phe 410 415 420

Val Val Val Ala Val Phe Leu Phe Leu Val Tyr Gin Ala Met GlU 425 430 435

Thr Asn Gin Val Asn Pro Phe Ser Asn Phe Leu His Val Asp Pro 440 445 450

Arg Lys Ser Asn

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2392 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 241..1275

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CCCTGCTACC AAGGCCCAGC TATGGCCCCA GGGTTGAAAA GTTATGAGGG 50

TCAGGGGTCT TTTGTGTCCG GGTCTGGCTT GGCTTTGTGT CCGCGAGTTT 100

TTGTTCCGCT CCGCAGCGCT CTTCCCGGGC AGGAGCCGTG AGGCTCGGAG 150

GCGGCAGCGC GGTCCCCGGC CAGGAGCAAG CGCGCCGGCG TGAGCGGCGG 200

CGGCAAAGGC TGTGGGGAGG GGGCTTCGCA GATCCCCGAG ATGCCGGAGT 250

TCCTGGAAGA CCCCTCGGTC CTGACAAAAG ACAAGTTGAA GAGTGAGTTG 300

GTCGCCAACA ATGTGACGCT GCCGGCCGGG GAGCAGCGCA AAGACGTGTA 350

CGTCCAGCTC TACCTGCAGC ACCTCACGGC TCGCAACCGG CCGCCGCTCC 400

CCGCCGGCAC CAACAGCAAG GGGCCCCCGG ACTTCTCCAG TGACGAAGAG 450

CGCGAGCCCA CCCCGGTCCT CGGCTCTGGG GCCGCCGCCG CGGGCCGGAG 500

CCGAGCAGCC GTCGGCAGGA AAGCCACAAA AAAAACTGAT AAACCCAGAC 550

AAGAAGATAA AGATGATCTA GATGTAACAG AGCTCACTAA TGAAGATCTT 600

TTGGATCAGC TTGTGAAATA CGGAGTGAAT CCTGGTCCTA TTGTGGGAAC 650

AACCAGGAAG CTATATGAGA AAAAGCTTTT GAAACTGAGG GAACAAGGAA 700

CAGAATCAAG ATCTTCTACT CCTCTGCCAA CAATTTCTTC TTCAGCAGAA 750

AATACAAGGC AGAATGGAAG TAATGATTCT GACAGATACA GTGACAATGA 800

AGAAGACTCT AAAATAGAGC TYAAGCTTGA GAAGAGAGAA CCACTAAAGG 850

GCAGAGCAAA GACTCCAGTA ACACTCAAGC AAAGAAGAGT TGAGCACAAT 900

CAGGTGGGAG AAAAAACAGA GGAAAGAAGA GTAGAAAGGG ATATTCTTAA 950

GGAAATGTTC CCCTATGAAG CATCTACACC AACAGGAATT AGTGCTAGTT 1000

GCCGCAGACC AATCAAAGGG GCTGCAGGCC GGCCATTAGA ACTCAGTGAT 1050

TTCAGGATGG AGGAGTCTTT TTCATCTAAA TATGTTCCTA AGTATGTTCC 1100

CTTGGCAGAT GTCAAGTCAG AAAAGACAAA AAAGGGACGC TCCATTCCCG 1150

TATGGATAAA AATTTTGCTG TTTGTTGTTG TGGCAGTTTT TTTGTTTTTG 1200

GTCTATCAAG CTATGGAAAC CAACCAAGTA AATCCCTTCT CTAATTTTCT 1250

TCATGTTGAC CCTAGAAAAT CCAACTGAAT GGTATCTCTT TGGCACGTTC 1300

AACTTGGTCT CCTATTTTCA ATAACTGTTG AAAAACATTT GTGTACACTT 1350

GTTGACTCCA AGAACTAAAA ATAATGTGAT TTCGCCTCAA TAAATGTAGT 1400

ATTTCATTGA AAAGCAAACA AAATATATAT AAATGGACTT CATTAAAATG 1450

TTTTTGAACT TTGGACTAGT AGGAGATCAC TTTGTGCCAT ATGAATAATC 1500

TTTTTTAGCT CTGGAACTTT TTGTAGGCTT TATTTTTTTA ATGTGGGCAT 1550

CTTATTTCAT TTTTGAAAAA ATGTATATGT TTTTTGTGTA TTTGGGAAAC 1600

GAAGGGTGAA ACATGGTAGT ATAATGTGAA GCTACACATT TAAATACTTA 1650

GAATTCTTAC AGAAAAGATT TTAAGAATTA TTCTCTGCTG AATAAAAACT 1700

GCAAATATGT GAAACATAAT GAAATTCAGT AAGAGGAAAA GTAACTTGGT 1750

TGTACTTTTT GTAACTGCAA CAAAGTTTGA TGGTGTTTAT GAGGAAAAGT 1800

ACAGCAATAA TCTCTTCTGT AACCTTTATT AATAGTAATG TTGTTGTAGC 1850

CCTATCATAC TCACTTTTTA AGACACAGTA TCATGAAAGT CCTATTTCAG 1900

TAAGACCCAT TTACATACAG TAGATTTTTA GCAGAGATCT TTTAGTGTAA 1950

CATACATATT TTAGAGAATT GTTGGCTAGC TGTACATGTT TTGAAAAGCT 2000

GTTTAGCTAG CTATAAGGCT ATAATTGGAA ATTTGTATTT TTTATTTACA 2050

GCAAAACATT TATTCAGTCA TCCAGTTTGC TACCAAAATA TGTTTTAGAT 2100

AAGTGTGTGT ATGTTTGTTT AGAAGTTAGA AATTGTAAAC ACTGGTCTTA 2150

TGTTTCATTT GGATTCATTA TTGCATTGTC TTGTTACCAG AAACAAATTT 2200

TGCCGAGCTT TTTTTGCCCT ATATTTCCCA GCATAATTTG ATTAGAAAGT 2250

ACAAAAAGGG CCGGGCGCGG TGGCTTACGC CTGTAATCCC AGCACTTTGG 2300

GAGGCCAGGG CGGGTGGATC ACGAGGTCAG GAGATCGGGA CCATCCTGGC 2350

CAACATGGTG AAACCCCGTC TCTACTAAAA AAAAAAAAAA AA 2392

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 345 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: pe tide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys

1 5 10 15

Leu Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly

20 25 30

Glu Gin Arg Lys Asp Val Tyr Val Gin Leu Tyr Leu Gin His Leu

35 40 45

Thr Ala Arg Asn Arg Pro Pro Leu Pro Ala Gly Thr Asn Ser Lys

50 55 60

Gly Pro Pro Asp Phe Ser Ser Asp Glu Glu Arg Glu Pro Thr Pro

65 70 75

Val Leu Gly Ser Gly Ala Ala Ala Ala Gly Arg Ser Arg Ala Ala

80 85 90

Val Gly Arg Lys Ala Thr Lys Lys Thr Asp Lys Pro Arg Gin Glu

95 100 105

Asp Lys Asp Asp Leu Asp Val Thr Glu Leu Thr Asn Glu Asp Leu

110 115 120

Leu Asp Gin Leu Val Lys Tyr Gly Val Asn Pro Gly Pro He Val

125 130 135

Gly Thr Thr Arg Lys Leu Tyr Glu Lys Lys Leu Leu Lys Leu Arg

140 145 150

Glu Gin Gly Thr Glu Ser Arg Ser Ser Thr Pro Leu Pro Thr He

155 160 165

Ser Ser Ser Ala Glu Asn Thr Arg Gin Asn Gly Ser Asn Asp Ser

170 175 180

Asp Arg Tyr Ser Asp Asn Glu Glu Asp Ser Lys He Glu Leu Lys

185 190 195

Leu Glu Lys Arg Glu Pro Leu Lys Gly Arg Ala Lys Thr Pro Val

200 205 210

Thr Leu Lys Gin Arg Arg Val Glu His Asn Gin Val Gly Glu Lys 215 220 225

Thr Glu Glu Arg Arg Val Glu Arg Asp He Leu Lys Glu Met Phe 230 235 240

Pro Tyr Glu Ala Ser Thr Pro Thr Gly He Ser Ala Ser Cys Arg 245 250 255

Arg Pro He Lys Gly Ala Ala Gly Arg Pro Leu Glu Leu Ser Asp 260 265 270

Phe Arg Met Glu Glu Ser Phe Ser Ser Lys Tyr Val Pro Lys Tyr 275 280 285

Val Pro Leu Ala Asp Val Lys Ser Glu Lys Thr Lys Lys Gly Arg 290 295 300

Ser He Pro Val Trp He Lys He Leu Leu Phe Val Val Val Ala 305 310 315

Val Phe Leu Phe Leu Val Tyr Gin Ala Met Glu Thr Asn Gin Val 320 325 330

Asn Pro Phe Ser Asn Phe Leu His Val Asp Pro Arg Lys Ser Asn 335 340 345

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Pro Glu Phe Leu Glu Asp Pro Ser Val Leu Thr Lys Asp Lys Leu 1 5 10 15

Lys Ser Glu Leu Val Ala Asn Asn Val Thr Leu Pro Ala Gly Glu

20 25 30

Gin Arg Lys Asp Val Tyr Val Gin Leu Tyr Leu Gin His Leu Thr

35 40 45

Ala Arg Asn Arg Pro Pro Leu

50