WO2000063238A1

WO2000063238A1 - Nonextractive method of assaying thymopoietin

Info

Publication number: WO2000063238A1
Application number: PCT/US2000/009486
Authority: WO
Inventors: Gideon Goldstein; Judy Haning; Cliff Asmus
Original assignee: Thymon LLC
Current assignee: Thymon LLC
Priority date: 1999-04-15
Filing date: 2000-04-11
Publication date: 2000-10-26
Anticipated expiration: 2001-10-15
Also published as: AU4222200A

Abstract

A method for measuring thymopoietin (TP) in a sample of mammalian plasma or serum includes the steps of (a) preparing the sample for use in a quantitative assay without exposure to extraction reagents or conditions by introducing into the sample reagents which minimize TP binding to plasma proteins without affecting binding to assay components, and (b) subjecting the sample (a) directly to the assay without further treatment. Preferred assays are binding or displacement ELISAs.

Description

NONEXTRACTIVE METHOD OF ASSAYING THYMOPOIETIN

Field of the Invention

The present invention relates generally to a method of measuπng thymopoietin levels in human plasma and serum samples, and more particularly to a method which can sensitively measure the protein at levels of less than 5 pmol/L without requiπng dissociation or extraction steps

Background of the Invention

The thvmic hormone thvmopoietm (TP) was originally isolated from bovine thymus as a 49 amino acid polypeptide [D H Schlesinger and G Goldstein, Cell. 5_ 361-365 (1975)] TP has been shown to play a regulatory role in immune, nervous, and endocrine functions and has been isolated from bovine and human thymus See, generally, G H Sunshine et al, J Immunol , 120 1594-1599 (1978), G Goldstein, Nature, 247 1 1-14 (1974), R H Brown et al, Brain Research, 381 237-243 (1986) A method of measuring TP proteins in plasma and serum was described in US Patent No 5,593,842, issued January 14, 1997, incorporated by reference herein, which employed an acidification step to dissociate TP from its binding protein This method was based on the fact that using a sandwich immunoassay, the inventors were unable to assay TP directly in plasma or serum They postulated that binding of TP to binding proteins in plasma interfered with the assay, hence the successful assay involved acidification of plasma to ensure dissociation, and subsequent extraction, preparation for assay and measurement by a sandwich immunoassay capable of detecting levels of TP amino acids 1 to 50 (TP,_₅₀) [amino acids 1-50 of Seq ID No 1] at less than 20 pg/ml This same extraction immunoassay method was utilized in US Patent No 5.591.588, issued Jan 7, 1997 for the measurement of elevated levels of TP in the plasma of patients with psychiatπcally documented, untreated major depression by comparison with levels in age and sex matched controls who were psvchiatπcallv documented as free from depression

In all TP assavs of the prior art. the isolated TP polypeptide of amino acids 1 - 50 or svnthetic TP, „, was subsequently used as the assay standard However, TP cDNAs were subsequently cloned [Harris et al, Proc Natl Acad Sc USA. 91_( 14):6283-6287 (Jul 1994)] Larger TP proteins were produced by alternative splicing from a single gene and shared a common N-terminal 1- 187 amino acid sequence TP,._S0 was the N terminal sequence of the larger polypeptides, but the 50 amino acid form could not be identified in Western blots of thymic extracts It was concluded that TP,.₅₀ was probably a fragment formed by proteolysis of a larger TP protein during extraction from the thymus (see also, US Patent No 5,472,856)

While these assays of the prior art were capable of accurately determining levels of TP,.₅₀ in the range of 5-200 pg/ml, the need for an extraction step introduced variability in the results obtained by the assay and did not readily lend itself to the automation required for useful high throughput assays

Thus, there remains a need in the art for assay methods and components adaptable to automation and capable of simply, accurately and reliably measuring low levels of TP in plasma

Summary of the Invention

In one aspect, the present invention provides a non-extractive method for measuring total thymopoietin (TP) levels in a human serum or plasma sample The method involves the steps of (a) obtaining a plasma or serum sample from a subject, (b) introducing into the sample reagents which minimize TP binding to plasma proteins without affecting binding to immunoassay antibodies; (c) measuring a binding value of the TP in the sample with an immunoassay, (d) generating a standard curve of TP binding values for a range of known amounts of a standard TP using the immunoassay; and (e) determining the amount of TP in the sample by comparing the binding value (c) with the standard curve (d) The determined amount of TP in the sample permits the diagnosis of conditions characterized by normal or abnormal levels ofTP

In one embodiment of this aspect, the immunoassay is an enzyme-linked sandwich immunoassay employing a detector labeled antibody which binds a first binding site on the TP amino acid sequence, and an immobilized capture antibody which binds a second binding site of the TP amino acid sequence which is distinct from the first site The label, e g , alkaline phosphatase, produces a signal which is amplified by an amplification system The amount of amplified signal is proportional to the TP binding value The amount of TP being sandwiched in the sample is determined by comparing the binding value with the standard TP binding curve

In another embodiment of this aspect, the immunoassay is an enzyme-linked displacement immunoassay which employs a limiting dilution of labeled TP, and an immobilized capture antibody which binds a binding site present on the labeled TP and on any TP naturally occurring in the sample The amount of signal generated by the labeled TP bound bv the immobilized capture antibody is measured with an amplification system, to generate a signal amount which is proportional to the binding of labelled TP The amount of TP in the sample is determined by comparing the binding value with the binding in the standard TP displacement curve, therebv providing the diagnostic information Preferably these embodiments of the invention are automated, and are useful to aid in the diagnosis of depression

In still another aspect, the invention provides a new use for the non-extractive TP assav method of this invention as well as a new use for the extractive method of the prior art The TP assays av be employed in methods for identifying depressed patients who are likely to be resistant to treatment with conventional pharmaceutics currently employed to treat depression

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 of a binding curve generated from a standard preparation of recombinant human (rh) TP, _{1 X7} expressed as pmol/L when used as a standard in the immunoassav experiment of Example 1 below The curve is described bv the formula log(v)= - 1 77 + 0 946 * log(x) The correlation coefficient is 1 0 Fig 2 is a graph of a binding curve generated from a standard preparation of the synthetic peptide TP,.₅₀ expressed as pg/ml when used as a standard in the immunoassay experiment of Example 1 below The curve is described by the formula log(y)= 0 813 + 0 972 *log(x) The correlation coefficient is 0 999 5 Fig 3 is a graph of the binding curves of the ethylene diamine tetraacetic acid

(EDTA) plasma dilutions (thick black dashed line) plotted against the TP,_.50 synthetic peptide standard curve (T PO 1-50; thin black line) at an OD₄₉₀ according to the assay described in Example 1 below Both curves are superimposed on each other

Fig 4 is a graph of the binding curves of the EDTA plasma dilutions (thick 10 black dashed line) plotted against rhTP,._I87 standard curve (TMPO1-187, thin continuous black line) according to the assay described in Example 1 below Both curves are superimposed upon each other

Fig 5 is a graph demonstrating the correlation of the prior art extractive assay (TP,.₅₀, pg/ml) vs the present invention's non-extractive assay (TP,.,,,_?, pmol/L) for 15 plasma TP in depressed patients according to the experiment described in Example 2 below Spearman r = 0 75 (p=0 0003)

Fig 6 is the sequence of rhTP,.₁₈₇ [SEQ ID NO 1]

Fig 7 is a scattergram of mean a.m /p.m pTP levels in 21 depressed patients and 21 matched controls in the assay of Example 3 The dotted line at 7 5 pg/ml 20 represents the cut-off value (P < 0 05 for values above this in controls)

Fig 8 is a column bar graph showing low incidence of response to antidepressant drugs in depressed patients with elevated pTP versus those with normal pTP (P < 0 05) in the assay of Example 3 Horizontal dotted line is response rate in the same population not sorted by pTP levels

i Detailed Description of the Invention

The method of the present invention permits measurement of total TP in a sample of human serum or plasma, or another body fluid or tissue, does not require extraction steps, and uses only reagents which minimize TP binding to plasma proteins without affecting binding to immunoassav antibodies This invention thus permits an automated assav methodology for detecting TP levels in a biological sample

According to this method, the blood sample removed from a mammalian subject mav be simply treated by introduction into an ethylenediamme tetraacetic acid (EDTA) medium or left to clot, and centπfuged to separate plasma or serum from other components in the blood Afterwards, certain reagents are introduced into the plasma or serum sample which minimize TP binding to plasma proteins without affecting binding to other assay components, such as antibodies For example, the assay diluents and washing fluids minimize TP binding to plasma proteins, without affecting binding to capture and/or detector antibodies This method employs mild conditions and does not require the rather harsh acidification, or other assay conditions which the prior art methods required to separate TP from its postulated binding protein Because such harsh acidification or other dissociation reagents interfere with the immunoassay antibody binding, prior art assays also required an additional extraction step to remove such reagents, and reconstitution of the TP in mild reagents, prior to measuring the TP See, US Patent No 5,593,842, particularly column 6 thereof Such additional steps introduced more variability in the results, more opportunity for error, and prevented the method from being readily automated In contrast, the method of the present invention includes neither dissociation or extraction reagents, but includes, without limitation, such mild reagents as sodium bromide, gamma globulins, normal non-human, mammalian immunoglobulins, and detergents For example, conventional useful mammalian immunoglobulins include normal mouse IgG, normal goat IgG, and normal rabbit gamma globulins Useful reagents which reduce non-specific binding include denatured alkaline phosphatase Useful detergents which reduce non-specific binding also include Tween® 20 polyoxethylene-sorbitan monolaurate, or other surfactants, detergents, and nonspecific binding reducing reagents known to those of skill in the art which do not interfere with binding between the sample TP and the immunoassav antibody reagents Such additional reagents include other detergents of the nonionic polvoxvethvlene ether type identified bv trademarks such as Triton X, Lubrol PX and NP40. or detergents of the zwitteπon tvpe, such as 3-[(3-cholamιdopropvl)-dιmethylammonιo]- 1-propanesulfonate (CHAPS) Additional reagents for the performance of an immunoassay can also be introduced to the sample, such as preservatives like sodium azide or ProClin 300™ preservative [Supelco], and buffers, such as phosphate buffered saline

The TP in the sample is then measured The presently preferred method of quantitating TP is the use of an enzyme-linked immunoassav (ELISA) In one embodiment of the method of this invention, the ELISA immunoassay is conducted in the following manner This assay is conducted by introducing the sample into a mixture containing a detector labeled antibody which binds a first binding site on the TP amino acid sequence in the sample and an immobilized capture antibodv which binds a second binding site of the TP amino acid sequence which is distinct from the first site A sample binding value is generated by measuring the amount of signal generated by the label in the sample, preferably employing an amplification system, which signal is proportional to the TP bound in the sandwich Also generated by the use of the immunoassay is a standard TP binding curve using known amounts of dilutions of a selected standard TP The sample binding value is correlated to the standard TP curve, and the correlation used to determine the amount of TP in the sample This value enables a diagnostic evaluation, I e , whether the sample TP value is in the normal or abnormal range of TP levels

This assay method offers the advantages of specificity for TP, a high degree of sensitivity and precision, and the ability to selectively measure TP and not other proteins in the plasma or serum sample

In such a sandwich assay, the detector and capture antibodies are independently monoclonal antibodies to TP, chimeπc antibodies to TP, recombinant antibodies to TP, polvclonal antibodies to TP (see, e g , the high titer, anti-human/rat TP antibody of Example 3 of US Patent No 5,593,842), antibody fragments e g , Fab F(ab)₂ or F. , or binding antibody peptides or polypeptides produced by phage display For instance in the Examples below the detector antibodv is a monoclonal antibody that binds to TP amino acids 30 through 50, and the capture antibodv is from an affinity purified polyclonal antisera that binds to TP amino acids 1 through 20. However, clearly, other antibodies to other sites on the known TP amino acid sequence may be readily generated by conventional means and employed. Monoclonal antibodies with increased target specificity may be used in these assays. Synthetically designed monoclonal antibodies may be made by known genetic engineering techniques and employed in these immunoassay methods. For use in the assay, any monoclonal antibody, chimeric, or recombinant antibody or portion thereof generated by the now standard techniques of Kohler and Milstein, Nature. 256:495 ( 1975) and the myriad of improvements thereon which are known in the art may be used. Thus, the assays useful in this invention are not limited to any specific TP antibody, since many may be generated. It is presently preferable for each of the two antibodies employed in the assay to be a different monoclonal antibody, which recognizes a distinct site on TP. The choice of antisera, antibodies, and/or fragments utilized in the ELISA or other immunoassay to quantitate TP can be selected by one skilled in the art to render the assay specific for TP.

These assays may use a variety of conventional indicator or signal-generating molecules or labels which are known to those of skill in the art, including radioisotopes, colorimetric enzyme systems and the like. In such a sandwich immunoassay as described above, the label on the detector antibody may be selected from among many known diagnostic labels and labeling systems, e.g., radiolabeis, fluorescent labels, chemiluminescent labels, colorimetric labels, chemical labels or systems of cooperating labels and substrates, e.g., streptavidin and horseradish peroxidase or alkaline phosphatase, enzyme-linked biotin, avidin-alkaline phosphatase, and fluorescein-anti-fluorescein systems, green and blue fluorescent proteins, among others. Selection of such labels or systems capable of generating detectable signals enabling measurement of a target of the assay are well known to one of skill in the art. The particular indicator or label/signal systems used to measure the TP is not necessarily critical to the performance of this method.

However, it is presently preferred that a system capable of amplifying the signal without amplifying the background is employed in the method of this invention. In one such desirable method, the label on the detector antibodv is biotin and the amplification system comprises a streptavidin-alkaline phosphatase solution a substrate for alkaline phosphatase solution, and an alcohol dehvdrogenase and diaphorase amplifier solution These solutions are introduced into the sample following incubation of the sample with both antibodies Together with the label, these solutions react to form a colored product directly proportional to the amount of detector antibody TP capture antibody sandwich formed in the assay

In another embodiment of the assay of this invention, the immunoassav is an enzyme-linked displacement immunoassay This assay format involves introducing into the sample, which has an unknown amount of naturally occurring TP, a limiting dilution of labeled TP This sample containing unknown TP and labeled TP is then contacted with a limiting amount of an immobilized capture antibody which binds a binding site present on any TP in the sample Thus, the labeled TP and the naturally occurring TP compete for binding sites on the immobilized antibody The amount of signal generated by the labeled TP bound by the immobilized capture antibody is preferably measured with an amplification system similar to that described above to generate a value inversely proportional to the sample TP displacement value As with the ELISA binding assay, the sample displacement value is correlated with a standard displacement curve, to generate a sample TP amount for diagnostic evaluation The same immunoassav steps and components with a selected standard TP are emploved to generate a standard TP displacement curve Because the amount of labeled TP is known, a displacement curve is generated, wherein the greater the amount of naturally occurring TP in the sample, the lower the signal produced by the bound labeled TP The lower is the amount of naturally occurring TP in the sample, the greater is the signal produced by the bound labeled TP

In such a displacement assav, the capture antibody can be a monoclonal antibodv to TP, a chimeric antibody to TP, a recombinant antibody to TP, a polvclonal antibody to TP (see, e g the high titer, anti-human/rat TP antibodv of Example 3 of US Patent No 5,593 842), an antibodv fragment e g , Fab, F(ab)- or F , or a binding polypeptide produced by phage display For instance, the capture antibody is an affinity purified polvclonal antisera that binds to TP amino acids 1 through 20 However clearly, other antibodies to other sites on the known TP amino acid sequence may be readily generated bv conventional means and employed in these assavs Monoclonal antibodies with increased target specificity may be used in these assays Synthetically designed monoclonal antibodies may be made by known genetic engineering techniques and employed in these immunoassay methods As stated above, for use in the assay, any monoclonal antibody, chimeric, or recombinant antibody or portion thereof generated by the now standard techniques of Kohler and Milstein, Nature. 256 495 ( 1975) and the myriad of improvements thereon which are known in the art mav be used Thus, the assays useful in this invention are not limited to anv specific TP antibody, since many may be generated The choice of antisera antibodies/fragments utilized in the ELISA or other immunoassay to quantitate TP can be selected bv one skilled in the art to render the assay specific for TP Clearly, other antibodies or binding polypeptides to other sites on the known TP amino acid sequence may be readily generated by conventional means and employed It is desirable the immobilized antibody to be a monoclonal antibody

In such a displacement assay, a vaπety of conventional indicator or signal- generating molecules or labels which are known to those of skill in the art, including radioisotopes, colorimetric enzyme systems and the like may be used The label on the capture antibody may be selected from among many known diagnostic labels and labeling systems, e g , radiolabels. fluorescent labels, chemiluminescent labels, colorimetric labels, chemical labels or systems of cooperating labels and substrates, e g , streptavidm and horseradish peroxidase or alkaline phosphatase, enzyme-linked biotin-avidin, alkaline phosphatase, and fluorescein-anti-fluorescein systems, green and blue fluorescent proteins, among others Selection of such labels or systems capable of generating detectable signals enabling measurement of a target of the assay are well known to one of skill in the art The particular indicator or label/signal systems used to measure the TP is not necessarily critical to the performance of this method However, it is presently preferred that a system capable of amplifying the signal without amplifying the background is employed in the method of this invention In one such desirable method, the label on the detector antibody is biotin and the amplification system comprises a streptavidin-alkahne phosphatase solution, a substrate, and an alcohol dehvdrogenase and diaphorase amplifier solution These solutions are introduced into the sample following incubation of the sample with the immobilized antibodies Together with the label, these solutions react to form a colored product directly proportional to the amount of labelled TP capture antibody formed in the assay Selection of such labels or systems capable of generating detectable signals enabling measurement of a target of the assay are well known to one of skill in the art

In both embodiments of the immunoassays described above, the sample binding or displacement values generated by the assays are compared with the "'standard" TP displacement and/or binding curves In performance of these methods, it is presently preferred to use TP,._lg7 as the standard, as this is most closely related to circulating native TP in plasma To determine the molecular size of the TP-like immunoreactivity in human plasma, a plasma sample was fractionated by size exclusion chromatography and the fractions assayed for TP by the sandwich immunoassay described above TP-like immunoreactivity appeared as a single peak in fractions corresponding to a molecular weight of approximately 20,000 dalton Human TP_M87, produced recombinantly, co-eluted with the plasma TP-like immunoreactivity Hence recombinant human (rh) TP_M87 (see Fig 6) was used as the standard in the present assay development The rhTP ₈₇ most closely resembles the naturally occurring protein and when employed in the methods of this invention permits the detection of total TP at a level of less than 5 pmol/L

Alternatively the TP,_.50 polypeptide or other TP polypeptides may be employed as the "standard" TP However, Applicant has determined that for accuracy, the alternate TP standard must be calibrated with the TP_ι.,₈₇ standard to correctly interpret the displacement and/or binding curves generated using the standard TP See. for example, US Patent No 5.593.842. Examples 1 and 2 The synthetic or rhTP, ₅₀ when employed in the methods of this invention permits the detection of total TP at a level of less than 1 pg/ml 1 e , 7 3 pmol/L This should be contrasted with the prior art assay, in which the TP,.,₀ standard was useful at measuring samples TP at less than 20 pg/ml, which is equivalent to less than 146 ^ pmol/L of TP,__]87 in the present non-extraction assay

The methods of the present invention are readily adapted to automation because they avoid use of the additional dissociation and extraction steps of the prior art The elimination from TP assays of the requirement for a dissociation step is surprising and not predictable, because if the affinity of the TP binding protein taught

10 by the prior art was greater than the antibodies used in the immunoassays, the method of the present invention would not operate successfully Prior to applicants invention, there was no way to predict that a simplified assay would be able to detect accurately and efficiently sensitive levels of TP in a sample without dissociation and separation steps

I 5 The methods of the present invention thus provide additional and more desirable methods for use in measuring TP levels in serum or plasma, thereby aiding in the diagnosis of pathologies in which TP levels are altered directly or indirectly as an indicator of dysfunction For example, the methods of the present invention provide a readily automated clinical test useful in the diagnosis of depression or other TP- 0 associated conditions, such as HIV infection, mvasthenia gravis, immune system disorders, among others These methods may also be useful in the measurement of TP as a direct or indirect index of changes in TP levels induced by drugs or other biologically active reagents in human clinical studies and trials

The following examples illustrate preferred methods for performing the 5 methods of the invention or employing such methods for diagnostic evaluation These examples are illustrative only and do not limit the scope of the invention Example 1 - Measurement of TP Levels in Volunteers

Blood samples from 40 male and female human volunteers of varying ages and no obvious illness were collected between 8 and 9 a m into ethylenediamme tetraacetic acid (EDTA) containing Vacutainer™ tubes (Becton Dickinson) and placed on ice Plasma was separated by centrifugation at 1 150Xg for 20 minutes and stored frozen (-70 °C) until assay These samples were assayed for TP levels using the non-extractive method of this invention, employing a quantitative sandwich enzyme immunoassay technique The mean ±SEM was 1 l±O 07 pmol/L and the median (range) was 10 (4-23) pmol/L The non-extractive method, immunoassay and signal generation techniques employed in these experiments are described in detail below

Microtiter plates (96 wells) were prepared by coating them with affinity purified rabbit polyclonal ("capture") antibodies to synthetic peptides corresponding to human TP(aa 1 -20), the plates were then blocked with sucrose/Tween 20 detergent in phosphate buffered saline at pH7 3 As assay solution was prepared and about 100 μL were pipetted into each well This solution included a cocktail of chaotropic assay diluents and reagents for reducing non-specific binding, but which was sufficiently mild so as not to interfere with the binding between the TP in the samples and the assay antibodies, and the detector antibodies The chaotropic reagents were 0 75 M sodium bromide to inhibit negative interference by plasma proteins, 1 mg/ml rabbit gamma globulins to reduce non-specific binding, 0 3 mg/ml acidified normal mouse IgG to reduce non-specific binding, 0 5 mg/ml normal goat IgG to reduce non-specific binding, and 0 3% Tween 20 detergent to reduce non-specific binding The detector antibody was 0 46 μg/ml biotinylated mouse monoclonal antibody to synthetic human TP (amino acids 30-50) Additionally, the assay solution includes as the preservative, 0 1% sodium azide and 0 1% ProClin300 preservative (Supelco)

About 50 μL of a calibrator, I e . recombinant human TP ₈₇ in delipidated bovine serum (range 3 75-240 pmol/L) or EDTA plasma sample as described above is added to each well The solutions and samples in each well are incubated for about 2 5 hours at room temperature on an orbital shaker Any TP present in the sample is thus sandwiched bv the immobilized capture antibodv and the biotin-linked monoclonal detector antibodv specific for TP The wells are then washed four times with Tπs/Tπton X- 100 wash buffer to remove any unbound substances

To detect the amount of TP captured by the immunoassav. an amplification system is employed to enhance the signal of the labeled detector antibodv In this system, the alkaline phosphatase reaction is amplified by a cofactor that activates a redox cycle leading to the formation of a colored product Specifically, the amplification protocol is as described in US Patent Nos 4,446,231 , 4,595,655, and 4,598,042, incorporated by reference herein A streptavidin-alka ne phosphatase solution (200 μL) is added to detect immobilized biotin-linked monoclonal antibody The solution contains Hepes buffered saline at pH7 6, two enzyme cofactors 2 mM MgCl- and 0 ImM ZnCU, 0 3% Tween 20 detergent to reduce non-specific binding, 1 ing/mL rabbit gamma globulins to reduce non-specific binding, 0 3 mg/ml acidified normal mouse IgG to reduce non-specific binding, 0 1% sodium azide and 0 1% ProClιn300 preservative (Supelco), 0 1% bovine serum albumin to improve stability and streptavidin-alkahne phosphatase The solutions and samples in each well are incubated for about 45 minutes at room temperature on an orbital shaker The wells are then washed four times with Tπs/Tπton X-100 wash buffer to remove any unbound substances The substrate, i e , 50 μL nicotinamide adenine dinucleotide phosphate

(NADPH), is added to each well and incubated for about 20 minutes at room temperature Thereafter 50 μL of amplifier, l e , alcohol dehydrogenase, lodonitrotetrazolium (INT) violet, ethanol, and diaphorase, is added to each well and the wells incubated 20 minutes at room temperature The alkaline phosphatase dephosphorylates the reduced form of NADPH to reduced nicotinamide adenine dinucleotide, NADH The NADH subsequently serves as a specific cofactor that activates a redox cycle driven bv the amplifier In the reaction catalvzed bv diaphorase, NADH is used to reduce a tetrazolium salt, i e INT violet, to produce an intensely colored formazan dve and NAD^ NAD+ in turn is reduced in an alcohol dehvdrogenase-catalvzed reaction to regenerate NADH. which can re-enter the redox cycle The rate of reduction of the tetrazolium salt and thus the amount of colored product formed is directly proportional to the amount of TP bound

The color development is stopped by adding 50 μL sulfuπc acid, and the intensity of the color is measured at an optical density of 490nm

Example 2 - Use of Assay to Diagnose Depression

Normal (control) and depressed individuals who were age matched (±2 years) and sex matched formed the experimental population Patients were diagnosed as moderately to severely depressed based on criteria that fulfill the DSM-III-R [Diagnostic and Statistical Manual of Mental Disorders - 3^rd edition, revised. American Psychiatric Association ( 1987)] criteria for current major depressive episodes, including a Hamilton Depression Rating > 7 Normal subjects were also screened using the DSM-III criteria to verify the absence of affective illness The Hamilton Depression Ratings obtained from both the depressed and normal subjects are shown in Fig 1 of US Patent No 5,591,588 Symptoms must have been present for a minimum of one month prior to entry in the experiment Patients with current or past diagnosis of psychotic disorders, mania or hypomania, organic brain syndrome, mental retardation or dysthymia were excluded The diagnosed depressed patients were untreated for depressive symptoms prior to and during inclusion in the experiment Blood samples were withdrawn from 18 depressed patients and 12 matched normal controls between 8 and 9 a m into EDTA containing Vacutainer™ tubes (Becton Dickinson) and placed on ice Plasma was separated by centπfugation at 1 150Xg for 20 minutes and stored frozen (-70°C) until assay These samples were assayed by the non-extractive method detailed above in Example 1 and the results were analyzed and compared with the results of the extractive method performed on the same samples, as described in US Patent No 5 593 842

Depressed subjects (n=18) in the prior art extractive method, using TP, ,„ as the standard had a median of 6 (range 3-21 ) pg/ml compared to control subjects (n= 12) with a median of 5 (range 1-7) pg/ml This difference was statistically significant (P< 0 004 using the Mann- Whitney rank sum test)

The non-extractive method of the present invention showed results for the same samples using the synthetic TP, ₁₈₇ standard of median 18 (range 8-85) pmol/L for the depressed subjects versus median 1 1 (range 4-15) pmol/L for control subjects

These results were similarly statistically significant (P< 0 004 using the Mann-Whitnev rank sum test) Using paired observations in the 18 depressed subjects, the Spearman non-parametric correlation coefficient was 0 75, which was highly statistically significant (P=0 0003) It is noteworthy that of the differences in values determined by the prior art extractive method with svnthetic TP, ,„ standard and the non-extractive method of the invention with the recombinant TP_M87 standard, the latter is likely to be the more accurate measure, since this recombinant protein approximates more closelv to the form present in plasma The parallel binding curves seen in Figs 3 and 4 show similar discriminative power in distinguishing depressed versus control samples, and the highly significant correlation all argue that the tests are measuring the same moiety in plasma

In keeping with the similar values for TP in the psychiatπcally tested controls

(n= 12) and the volunteer control group of Example 1 (n=40), the depression group (n=18) was statistically different (P< 0 001, Mann-Whitney rank sum test) from the volunteer group controls These results are summarized for comparison in Table I below

TABLE I

While both the prior art and present methods are apparently measuring the same moiety, i e . total TP in the sample, without wishing to be bound by theory, the inventor believes that the combination of the increased sensitivity achieved by the substrate amplification system and the addition of the non-specific proteins and salts in the assay diluents listed above eliminates the need for the extraction step of the prior art, and thus enables this system to function efficiently and accurately and to be automated

Example 3 - Novel Use of TP Assay to Identify Patient Non-Responsiveness to Antidepressant Drugs

This example demonstrates a novel use of a TP assay, including both the extractive TP assay of the prior art and the non-extractive TP assay of this invention Early adverse life events and chronic stress predispose human subjects to major depression [Heim et al, Psvchopharmacol Bull.. 33 185-192 ( 1997)], a highly prevalent disease causing extensive morbidity [Lopez and Murray, Nat Med . 4 1241 - 1243 ( 1998); Kessler et al, Arch Gen Psychiatry. 51 8- 19 ( 1 94) Although antidepressant drugs are widely used and effective in the treatment of depression, non- response to antidepressant treatment of adequate dose and duration is observed in 19 to 34% of patients [Keller et al. Psvchopharmacol Bull 3 1 205-212 ( 1995), Fava and Davidson, Psvchiat Clin North Am . \ 9 179-200 ( 1996)] The synthetic TP-deπved peptide thvmopentin an active fragment of thvmopoietin [Goldstein et al. Science, 204 1309-1310 ( 1979)], which circulates as a 20 kDa protein corresponding to the N-terminal 187 amino acids of larger intracellular forms of TP, is involved in stress responses associated with depression It reduces endocrine and behavioral responses to experimental stress, possibly by lowering plasma TP (pTP) levels

In this example, pTP and the stress-activated, hypothalamic-pituitary-adrenal (HP A) axis hormones argin e vasopressin (pAVP), adrenocorticotropic hormone (pACTH), and plasma cortisol were used, as possible biological correlates of depression and/or responsiveness to antidepressant drugs Levels of these hormones and TP were measured in 21 untreated depressed patients and 21 matched controls Clinical responses to antidepressants were evaluated in 17 depressed patients A Patient Population

Twenty-one untreated patients (1 1 males, 10 females) fulfilling the DSM-III-R criteria for a current major depressive episode lasting at least one month were studied along with 21 age (within 2 years) and sex matched normal controls Depressed patients were free of antidepressant drugs for one month prior to obtaining the plasma samples Routine hematology blood chemistry and uπnalysis tests were performed A medical history, complete physical exam and psychiatric evaluation with the structured clinical interview for DSM-III-R (SCID-P) were obtained for all subjects plus the 17 point Hamilton Depression Rating (HamD) HamD greater than 16 was required at entry The protocol was approved by Institutional Review Boards and all subjects signed Informed Consent forms The 17 depressed study subjects from the Massachusetts General Hospital were entered into a prospective treatment protocol and response data from these patients was utilized to determine, in a blinded fashion, the clinical responses to drug treatment, using an intent-to-treat analysis A full response required a decline of HamD > 50% to < 7 and a partial response required a 25 to 50% decline in HamD B Immunoassay

Two 90 mL blood samples, the first drawn between 8-9 a.m and the second between 4-6 p m , were drawn into pre-chilled EDTA-vacutainer tubes, centrifuged in the cold, and the plasma aliquoted into chilled polypropylene tubes and stored at 70 to 80 °C Commercial radioimmunoassay kits from Diagnostic Systems Laboratories, Inc , Webster, TX (AVP and ICN Biomedicals, Inc , Cosa Mesa, CA) (cortisol) were used in accordance with the manufacturer's instructions and ACTH was measured as described [Nicholson et al, Clin Chem . 30 259-265 (1984)], using reagents from IgG Corp , Nashville, TN Plasma TP was measured by ELISA according to the prior art method described in US Patent No 5,593,842 Briefly, plasma was extracted using a C- 18 SepPak® column and assayed with a sandwich ELISA using affinity purified rabbit antibody to synthetic TP,.,<- for capture and mouse monoclonal antibody to synthetic TP₃₀.₅₀ (6E10) as the detector antibody Synthetic TP,.₅₀ was used as a standard The direct high sensitivity plasma TP assay described in the examples above may also be employed herein C. Statistical Analyses

Statistical analyses were performed on Prism™ Version 3 0, GraphPad Software, Inc , San Diego, CA, unless otherwise specified The significance of depression control and a m /p m differences was assessed by repeated measured two- way ANOVA using SAS® (SAS Institute Inc , Cary. NC) With no significant differences in a m /p m values, the mean a.m /p m values in each subject were used for subsequent comparisons Between-group differences were assessed with a two- tailed T test with Welch^'s correction Correlation of TP and AVP values in depression was assessed with the Spearman correlation coefficient pTP and pAVP distributions were tested as Gaussian controls, and cutoff values of mean + 1 645 SD in controls were used to define significant elevations (P < 0 05) Fisher's exact test, two tailed, was used to analyze 2 x 2 contingency tables P > 0 05 was considered non-significant (NS) D. Results

In summary, pTP was elevated in depression (P < 0 002), in 8/21 (38%o) depressed patients having significant elevations (P < 0 03) For 17 patients whose antidepressant responses were evaluated, response failures occurred in 6/7 (86%) with elevated pTP (7 5 pg/ml) versus 3/10 (30%) with normal pTP (P < 0 05) The significant association in depressed patients of non-responsiveness to antidepressant drugs with elevated pTP may signify a distinct pathogenesis for the depression of patients with elevated pTP

Specifically, 21 depressed subjects (22 males, 20 females) aged 40±12 (mean ± standard deviation) and 21 matched control subjects with identical age and gender distributions were entered into the study HamD scores at entry were 23 ± 4 in depressed patients and 1 ± 1 in controls (mean ± SD) (see Table II)

Comparisons were made between depression/control subjects and between a.m. and p m. differences as follows. pTP (P < 0 002) and pAVP (P < 0 002) levels were significantly elevated in depressed subjects, but there were no significant between-group differences in pACTH or plasma cortisol levels. No hormone showed significant a.m /p m differences nor any significant group-by-time interaction The mean a m /p m. values for each parameter similarly reflected these differences (see Table II below) The incidence of elevation of pTP and pAVP levels in depression was determined Using cut-off values of 7 5 pg/mL for pTP and 5 0 pg/mL for pAVP, pTP was elevated in 8/21 (38%) depressed subjects versus 1/21 (5%) controls (P < 0 03) (Fig 7) and pAVP was elevated in 6/21 (29%) depressed subjects versus 1/21 (5%) controls (NS) A decreased clinical response rate was observed in subjects with elevated pTP Forty-one percent (7/17) of depressed subjects treated with fluoxetine ( 13) or nortryptiline (4) had full or partial responses Eighty-six percent (6/7) of patients with elevated pTP were non-responders versus 30% (3/10) non-responders in patients with normal pTP (P < 0 05) (Fig 8) The response rates for subjects divided on the basis of p AVP levels did not differ statistically The relationship between pAVP and pTP in depressed subjects was revealed pAVP elevations were found in 3/8 (38%) subjects with elevated pTP versus 3/13 (23%) subjects with normal pTP (NS) There was no significant correlation between pTP and pAVP levels in depressed subjects (Spearman r 0 39, NS)

These results demonstrate that early morning and late afternoon plasma levels of the HP A hormones ACTH and cortisol did not prove useful diagnostic measures in major depression AVP secretion is pulsatile [Redekopp et al, Endocπnol . 1 18 1410-1416 (1986). Wood et al. Scand J Urol Nephrol Suppl . 163 93- 101 ( 1994)] HP A hvperactivity in depression is related to a greater frequency of episodic hormone release, which can be gauged by frequent sampling over 24 hours [Deuschle et al. J Clin. Endocπnol Metab . 82 234-238 (1997)] or by measuring 24 hour urinary cortisol secretion [Carroll et al, Psychol Med . 6 43-50 ( 1976)] However, these methods have not found widespread acceptance in clinical practice The finding reported in this example of elevated pAVP in 29% of depressed subjects using infrequent samples is in agreement with van Londen et al, Neuropsychopharmacol , 17 284-292 (1997), and probably reflects an increased statistical probability of sampling higher levels during the more frequent secretion pulses of AVP Accordingly, there was no significant correlation of pAVP elevation with the clinical response status of patients treated with antidepressant drugs

In contrast, pTP was elevated in 7/17 depressed subjects whose clinical responses to antidepressant drugs were evaluated and most of the subjects with elevated pTP failed to respond to conventional drug treatment with fluoxetine or nortrypty ne pTP elevations were present in 6/9 (67%) non-responders but in only 1/8 (13%) full or partial responders Fluoxetine and nortryptyhne modify CRF/AVP release by regulating serotonin and/or catecholamme pathways However CRF/AVP release can be triggered through alternative pathways It is anticipated that the identification of TP as a serotonin and catecholamme independent stress mediator explains this finding of poor therapeutic responses in depressed patients with elevated pTP TABLE II

HamD scores and plasma TP, AVP, ACTH and cortisol levels in 21 depressed and 21 healthy matched control subjects

* Two tailed T test with Welch's correction.

Animal data provide some additional clues. Thymopentin pre- treatment in mice and rats inhibited stress-induced behavioral changes, alterations in hippocampal GAB A and benzodiazepine receptor densities and elevations of plasma corticosterone levels, but did not affect these parameters when administered alone

[Klusa et al, Reg. Peptides. 27:355-365 (1990)]. IRI-514, an analog of thymopentin, was similarly stress-protective [Menzaghi et al, Physiol. & Behav.. 60:397-401 ( 1996)]. IRI-514 dose-dependently reversed the behavior induced by social stress if administered at least one day earlier and the effect lasted for several days. Since small peptides are eliminated rapidly, direct action of the peptide could not account for these long-lasting modulatory effects on behavior. pTP was measured in normal human volunteers during a phase I study of an orally active thymopentin analog, IRI- 695 [Adsumali et al, Biopharmac. & Drug Dispos.. 17:25-41 (1996)], and statistically significant decreases in pTP were found starting 12 hours after a single oral dose and persisting through 2 days, the last time point measured (unpublished observations).

Thus, the behavioral effects of thymopentin and its analogs are mediated via reduction of pTP levels, and pTP is a mediator or regulator of stress-induced behavioral changes.

: i The high incidence of non-responders in subjects with elevated pTP was statistically significant This example demonstrated that a pTP assay was useful in the identification of patients with elevated TP who are likely to be non-responsive to standard antidepressant drugs, and can provide an indication that alternative treatment is necessary

All references, patents and patent applications recited above are incorporated herein by reference 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

Claims

WHAT IS CLAIMED IS

1 A method for measuring thymopoietin (TP) in a sample of mammalian plasma or serum comprising

(a) preparing said sample for use in a quantitative assay without exposure to extraction reagents or conditions by introducing into said sample reagents which minimize TP binding to plasma proteins without affecting binding to assay components, and

(b) subjecting said sample (a) directly to said assay without further treatment

2 The method according to claim 1, further comprising

(a) obtaining a plasma or serum sample from a subject,

(b) introducing into said sample reagents which minimize TP binding to plasma proteins without affecting binding to immunoassay antibodies,

(c) measuring a binding value of said TP in said sample with an immunoassay,

(d) generating a standard curve of TP binding values for a range of known amounts of a standard TP using said immunoassay, and

(e) determining the amount of TP in said sample bv comparing the binding value (c) with said standard curve (d), wherein said determined amount of TP in said sample aids in the diagnosis of conditions characterized by normal or abnormal levels of TP

3 The method according to claim 2 wherein said reagents are selected from the group consisting of sodium bromide, gamma globulins, normal non-human serum proteins, mammalian immunoglobulins, and detergents 4 The method according to claim 2 wherein said immunoassay is an enzyme-linked sandwich immunoassay which comprises the steps of

(a) introducing into said sample a detector labeled antibody which binds a first binding site on said TP amino acid sequence,

(b) contacting said sample (a) with an immobilized capture antibody which binds a second binding site of said TP amino acid sequence, which is distinct from said first site;

(c) measuring the amount of signal produced by said label in said sample with an amplification system, said amount of signal proportion to the TP binding value in said sample,

(e) determining the amount of TP in said sample by comparing the binding value (c) with said standard TP binding curve (d)

5 The method according to claim 4 where said detector and capture antibodies are independently selected from the group consisting of monoclonal antibodies to TP, chimeric antibodies to TP, recombinant antibodies to TP, and polyclonal antibodies to TP, Fab, Fab-, and F_v fragments, and antibody fragments generated by phage display

6 The method according to claim 5 wherein said detector antibody binds to TP amino acids 30 through 50

7 The method according to claim 6 wherein said capture antibody binds to TP amino acids 1 through 20

8 The method according to claim 4 wherein said label is biotin 9 The method according to claim 4 wherein said label is biotin and said amplification system comprises a streptavidin-alkaline phosphatase solution and an alcohol dehydrogenase and diaphorase amplifier solution, which upon reaction forms a colored signal directly proportional to the amount of detector antibody. TP capture antibody sandwich formed in the assay

10 The method according to claim 2 wherein said immunoassay is an enzyme-linked displacement immunoassay which comprises the steps of

(a) introducing into said sample a limiting dilution of labeled TP,

(b) contacting said sample (a) with a limiting amount of an immobilized capture antibody which binds a binding site present on said labeled TP and on any TP naturally occurring in said sample,

(c) measuring the amount of labeled TP bound by said immobilized capture antibody by amplifying the signal produced by said label with an amplification system to generate a sample TP binding value;

(d) generating a standard curve of TP displacement values for a range of known amounts of a standard TP using said immunoassay;

(e) determining the amount of TP in said sample by comparing the displacement value calculated from step (c) with said standard TP displacement curve ( ), wherein said determined amount of TP in said sample aids in the diagnosis of conditions characterized by normal or abnormal levels of TP

1 1 The method according to claim 10 where said immobilized antibody is selected from the group consisting of monoclonal antibodies to TP, chimeric antibodies to TP, recombinant antibodies to TP, polyclonal antibodies to TP, Fab, Fab_: and F. fragments, and antibody fragments generated by phage display

12 The method according to claim 10 wherein said label is biotin 13 The method according to claim 12 wherein said labei is biotin and said amplification system comprises a streptavidin-alkaline phosphatase solution and an alcohol dehydrogenase and diaphorase amplifier solution, which upon reaction forms a colored signal directly proportional to the amount of labeled TP- immobilized antibody formed in the assay

14 The method according to claim 2 wherein said standard TP curve is generated using TP amino acids 1-187, and wherein said method measures total TP at a level of less than 5 pmol/L

15 The method according to claim 2 wherein said standard TP curve is generated using TP amino acids 1-50, and wherein said method measures total TP at a level of less than 7 pmol/L

16 The method according to claim 2 wherein said immunoassay is employed to generate a sample TP curve which is parallel to said standard TP curve

17 The method according to any of claims 1-16, wherein said steps are automated

18 The method according to any of claims 1-17, which is performed to aid in the diagnosis of depression