WO2001032916A2 - Automated lysophospholipid assay and methods of detecting cancer - Google Patents

Abstract

The present invention relates to an improved enzymatic diagnostic assay to detect carcinoma by measuring various lysophospholipids, including lysophosphatidic acid (LPA), in a patient. In a preferred embodiment, this assay measures the human plasma level of LPA in an automated format with a minimal number of reagents and with reduced incubation periods. The present invention also comprises several additional technical improvements to the current LPA assays disclosed in the prior art.

Description

IMPROVED AUTOMATED LPA ASSAY and METHODS of DETECTING CANCER

This application claims priority to the provisional application Serial No. 60/163,534 filed on November 4, 1999.

Introduction & Background of the Invention

Cancer is a major cause of death in the United States exceeded only by heart disease. In 1999 an estimated 563,100 Americans will die of cancer. Moreover, approximately 1 ,221 ,800 new cases of cancer are predicted in the US for the year. The major solid tumors in the US include those of the lung, breast, colon, prostate, and ovaries. Lung cancer is the most common cause of cancer death for both sexes with almost 159,000 lung cancer related deaths expected in 1999. Total colorectal cancer- related deaths are second only to lung cancer with over 56,000 expected. Breast cancer continues to be the most common form of cancer present in females in the US with an estimated 176,300 new cases projected to be diagnosed during the year. In males, prostate cancer is the most common form of cancer with projections of 179,300 new cases diagnosed and 37,000 prostate cancer related deaths occurring during 1999. Ovarian cancer is the leading cause of gynecologic death.

Procedures used for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining predisposition of diseases or conditions of these organs are of critical importance to the outcome of the patients. It is generally accepted that detection of a solid tumor at an early stage dramatically reduces disease-related mortality. For example, patients diagnosed with localized prostate cancer have greater than a 90% five-year relative survival rate compared to a survival rate of 25 to 31% for patients diagnosed with distant metastasis. Staging of the cancer is performed after its diagnosis is confirmed because it is a strong predictor of patient outcome and greatly influences patient treatment. In addition, patients are monitored after primary therapy to detect persistent disease and to detect early distant metastasis. New testing methods, however, which are more sensitive and specific than current standard procedures for the management of cancer patients are clearly needed. Women with gynecological cancers are especially in need of an accurate and early diagnostic, especially those with ovarian cancer. Patients with ovarian cancer have the highest mortality rate among women with gynecologic cancers, with an estimated 14,500 deaths from ovarian cancer in 1998 in the Unites States. More than two thirds of patients with ovarian cancer have widespread metastatic disease at initial diagnosis. The outlook for women with advanced disease remains poor, with a 5-year survival rate of no more than 15%. This dismal outcome is due, at least in part, to the failure to detect the disease at stage I, when the long-term survival rate may approach 90%. Methods for earlier detection are essential to improve prognosis and overall survival of patients with ovarian cancer.

Prior Art

It is generally known that detection of various lysophospholipids, such as lysophosphatidic acid ("LPA") is indicative of various types of disease, including carcinomas and especially ovarian carcinoma. Xu et al, "Lysophosphatidic Acid as a Potential Biomarker for Ovarian and Other Gynecological Cancers", JAMA , 1998 Aug.26;280 (8):719-723. Thus, LPA measurement can be used as a diagnostic to detect carcinomas and, especially to detect early stage ovarian cancer. The prior art generally describes a method of detecting LPA as follows. The lysophospholipid, such as LPA, is incubated with lysophospholipase to produce glycerol- 3-phosphate (G-3-P). G-3-P is then converted to dihydroxyacetone phosphate and hydrogen peroxide using G-3-P oxidase in the presence of oxygen and water. In the presence of NADH, G-3-P dehydrogenase converts dihydroxyacetone phosphate back to G-3-P and oxidizes NADH to NAD. The measurement of hydrogen peroxide correlates with LPA levels. Specifically, optical absorbance at 505nm indicates an accumulation of hydrogen peroxide and, thus, the presence of LPA in the test sample.

To achieve the aforementioned, the prior art teaches a multi-step process in order to measure LPA concentrations. First, the prior art procedure for measuring LPA in order to detect cancer involves an initial liquid: liquid organic phase extraction using a number of reagents in a multi-step procedure to a biological sample such as whole blood is collected from a patient. Because the sample, usually plasma, is presumed to contain materials which interfere with the assay, the analyte is extracted from the sample and reconstituted to its original concentration in a buffer compatible with the subsequent assay. The plasma sample is first vortexed with chloroform:methanol to precipitate protein. After centirfugation to pellet the protein, more chloroform and tris buffer are added and the mixture again vortexed and centrifuged. At this stage, most of the neutral phospholipids, including LPC are in the organic layer, which is separated and discarded. The aqueous layer is extracted with an additional portion of chloroform to remove any remaining neutral phospholipids, and the layers again separated. The aqueous layer is then subjected to a third chloroform:methanol extraction, this time with hydrochloric acid added to protonate and so neutralize the charge on LPA, resulting in its transfer to the organic layer. This time the aqueous layer, containing water soluble salts including G3P, is discarded. The organic layer is then mixed with a small amount of an aqueous tris buffer containing detergent and calcium chloride, and the solvent evaporated off, leaving the residue which is stored at -80° C until reconstitution and assay. Because of the numerous pipetting steps and multiple extractions there is great potential for loss and accumulation of error in this procedure. This, along with the use of the toxic, volatile solvent chloroform, greatly complicate its use in a clinical laboratory.

Following the aforementioned extraction and reconstitution of the sample to aqueous solution, the prior art dictates that LPA is hydrolysed in a separate single step to

G3P by exposure to a lysophospholipase for 60 minutes at 37° C. The G3P is then treated with a mixture of enzymes which consumes NADH and produces hydrogen peroxide at a rate dependent on the G3P concentration. One of the enzymes, G3P oxidase, oxidizes G3P to DHAP with the evolution of hydrogen peroxide. The other enzyme, Glycerophosphate degydrogenase, converts the DHAP back to G3P, this reverse reaction being driven by the conversion of NADH to NAD. The rate of this cycling reaction, i.e. the rate at which hydrogen peroxide is produced and NADH converted to NAD, is dependent (all other variables being held constant) on the concentration of G3P. According to the prior art the extent of reaction may be determined in either of two ways. The concentration of NADH may be monitored either continuously or at the end of the incubation, and its decrease determined by measuring the loss of absorbance at 340 nm. Alternatively, at the end of the incubation, set at 60 min in the prior art, the amount of hydrogen peroxide generated may be measured by a colorimetric reaction using perxidase and a colorigenic substrate

The disappearance (oxidation) of NADH is then monitored spectrophotometrically at OD ₄₀ (i.e. disappearance of OD₃₄₀). Alternatively, the production of hydrogen peroxide may be measured, for example colorimetrically by fluorometry or chemiluminescence. For a colorimetric assay any of a number of chromogenic substrates may be used including 4-aminoantipyrine (AAP), pyrogallol, 2- (2'-Azinobis (3-ethylbenzthiazoline-sulfonic acid) (ABTS) and 3,3',5,5'- tetramethylbernzidine) (TMB). As one can clearly see, this complicated LPA detection process calls for many separate reagents to be used in many separate steps in a specified chronological order. Further, several of the reagents must be mixed together just prior to use. Due to this complexity, it is difficult, if not impossible, to put this LPA detection assay on an automated format and, the lengthy incubation periods required for each step make it difficult to use this assay in a clinical format.

Further problems in the prior art involve poor calibrator stability. In the past, calibrators were stored at -70° C until time for use. One disadvantage is that the requirement of -70° C storage is both difficult and costly. Further, easy spoilage of the calibrator due to a lapse in -70° C conditions leads to false patient read-outs. The present inventors have invented a calibrator which is stable at 4° C as well as room temperature. This novel calibrator has calcium completely eliminated from the calibrator storage matrix. It was previously believed that calcium was critical in complexing LPA away from the sides of storage containers. The present inventors found that the presence of calcium actually was responsible for reduced stability or unavailability of the LPA itself once the storage was at a temperature greater than -70° C.

Other problems in the prior art involve separate hydrolysis and cycling steps, as mentioned previously. In the past at least two separate steps - one hydrolysis step and one cycling step - were used. The disadvantage was that many different reagent formulations were used and two separate 1 hour incubations were required. Specifically, the prior art methodology teaches keeping the various enzymes as separate reagents and storing each at a different temperature. Lysophospholipase is stored at -80° C and kept as a separate hydrolysis reagent for use in the 1 hour hydrolysis step. The cycling enzymes were broken out into a multitude of reagents. The reason for the aforementioned steps and multi-reagent format is that it was previously thought that a complete conversion of LPA to G3P, prior to the cycling reaction, was necessary in order to obtain an accurate measurement. The inventors have combined hydrolysis and cycling compounds into a single reagent and further optimized the assay such that the reaction time can be reduced from over 2 hours to 15 minutes or less. Specifically, the inventors have combined the lipase, G3P oxidase, G3P dehydrogenase, CaCl₂ and Tris into a single reagent stable at 4° C. The NADH is stored separately at -20° C. The inventors found, quite unexpected, that the generation of G-3-P from LPA did not interfere with the cycling steps performed by G-3-P oxidase and G-3-P-dehydrogenase and that measurements were highly accurate. Short incubation periods are crucial to automating an assay as well as providing an effective assay format to clinicians, both of which this invention allows.

Yet another problem in the prior art involves severe lysophosphotidyl choline (LPC) cross-reactivity. In the past, a mandatory lipid extraction step mentioned above was used to separate LPA from LPC and/or G3P. Since the novel single step-hydrolysis and cycling format does not even detect LPC, the cumbersome extraction step can be completely eliminated. With a lipid extraction step eliminated from the assay, it is possible to put this assay on an automated format due to the elimination of the extraction's complexity and difficulty.

As with the assay of the prior art, sample handling remains critical. Blood sampling must be collected so as to prevent lysis of platelets, centrifuged sufficiently to remove platelets as well as erythrocytes. If not tested promptly after collection/centrifugation, they must be frozen at -20° C to prevent changes in LPA concentration. Additionally, if free G3P is present, it may be necessary to measure this separately and subtract to get actual LPA concentration.

Still another problem in the prior art involves the lack of stabilized detection reagents, i.e. peroxidase and chromophore precursor solutions. In the past, the components of this formulation were stored separately and then mixed just minutes prior to use. Again, this multi-step format makes automation of this assay impossible, or impractical. What the present inventors have discovered is a combined formulation of peroxidase and chromophore precursor solutions that are stable at both room temperature and 37° C. Specifically, the inventors have combined the chromophore precursors i.e. phenol, phenol derivatives and phenazones together as one stable reagent. Further, the inventors have added sodium azide (an antimicrobial) TritonX-100, and FG-10 anti-foam to further improved stability and shelf-life. Also, with the aforementioned improved formulation, the inventors found the chromophore precursor solution also stabilizes added HRPO, further reducing reagent number. The improved simplicity and stability enhances the assay considerably and makes automation possible and reliable.

Summary of the Invention

The present invention relates to an improved enzymatic diagnostic assay to detect carcinoma by measuring various lysophospholipids, including lysophosphatidic acid (LPA), in a patient. In a preferred embodiment, this assay measures the human plasma level of LPA in an automated format with a minimal number of reagents and with reduced incubation periods. The present invention comprises several additional technical improvements to the current LPA assays disclosed in the prior art as described below.

The inventors have also shown that the cross-reactivity of LPC is significantly reduced in their improved single step assay versus the prior art multi-step assay. This completely eliminates the need for the extraction step currently used.

The inventors also illustrate that the hydrolysis and cycling enzymes and related solutions could be combined and that this single reagent remained stable and efficacious.

The inventors have shown that the stability of the LPA calibrators could be significantly improved by eliminating calcium, previously thought to be essential to the calibrator's efficacy, from the calibrator matrix.

Further, the inventors have formulated an LPA assay in which unextracted plasma is actually the sample.

The inventors have further shown an improved formulation of a detection reagent, which contains both peroxidase and chromophore precursors, that has significantly improved stability compared with the prior art reagents. The present invention not only combines these precursors into a single reagent format, but also stabilizes these reagents so that they are efficacious at room temperature, thereby eliminating the need for refrigeration and further lengthening shelf-life. This also allows simplification of the assay and enables it to be put onto an automated format.

Further, the inventors have actually automated the assay and have incorporated it onto an automated machine by incorporation of a fluorophore that enables conversion of the assay from an absorbance readout to a fluorescence readout. This fluorescent read out has a higher sensitivity than the prior art colorgenic method.

The invention differs from prior art due to improved assay specificity, improved formulation stability, the elimination of numerous steps, reduced incubation time of the assay and actual assay automation. Also, the present invention differs in that it eliminates the extraction step and can use plasma as the actual sample.

Detailed Description of the Invention

The lysophospholipase used in this assay can be any lipase that hydrolyzes the fatty acids (ester bonds) from either position 1 or 2 of lysoglycerophospholipids (i.e. sn-1 or sn-2 positions). Examples include phospholipase B, phospholipase C, phospholipase D, lysophospholipase, phospholipase Aj, and phospholipase A , lecithinase B and lysolecithinase.

Cycling enzymes used are any enzymes or combination of enzymes used to convert the glycerol -3-phosphate (G3P) intermediate to and from DHP and in the process increase the production of hydrogen peroxide, which is, preferably, the actual species that is detected. The two enzymes that are preferred are glycerol-3-phosphate oxidase which converts G3P to dihydroxyacetone phosphate (hydrogen peroxide is also generated in this step) and glycerol-phosphate dehydrogenase, which in the presence of NADH, converts the dihydroxyacetone phosphate back to G3P. G3P then goes through the same cycle generating additional hydrogen peroxide. Other cycling enzymes which can be used include serine dehydrogenase, serine deaminase, aldehyde dehydrogenase, ethanolamine deaminase, glycerokinase and glycerol dehydrogenase.

The NADH is preferably stabilized, and the methods to stabilize NADH are described in the U. S. Patent 4,704,365, issued November 3, 1987 entitled "Composition and Method for Stabilization of Dinucleotides", herein incorporated by reference. The formulation described includes propylene glycol (polyhydroxyl alkyl solvent) at 50 %, boric acid and buffers. Specifically, this patent discloses a reduced dinucleotide, preferably nicotinamide adenine dinucleotide (NADH), stabilized in an aqueous base liquid containing propylene glycol, boric acid and a buffer capable of buffering within a pH range of 8-11. The stabilized liquid contains greater than 50% (v/v) water. The remaining volume contains propylene glycol, which has been chemically treated to remove oxidants. The accuracy of this stabilizer is dependent on pH and the amount of glycerol as well as the sample volume.

The hydrolysis/cycling mixture may also contain compounds which prevent degradation or production of lysophospholipids. Reagents for inhibiting production or hydrolysis of lysophospholipids include specific PLA2 inhibitors such as Aristolic Acid

(9-methoxy-6-nitrophenanthro-(3,4-d)-dioxole-5-carboxylic acid, Biomol Research Laboratories, Plymouth Meeting, PA); ONO-R-082 (2-(p-Amylcinnamoyl)amino-4- chlorobenzoic acid, Biomol); OBAA (3-(4-Octadecyl)-benzoylacrylic acid, Biomol), 4- Bromophenacyl Bromide (Sigma); Quincrine (6-Chloro-9-(4-diethylamino)-l- methylbutyl)amino-2-methoxycridine, Mepacrine, Sigma); Manoalide (Biomol) and

HELSS (Haloenol lactone suicide substrate, Biomol); phosphodiesterase inhibitors such as IBMX (3-Isobutyl-l-methylxanthine, CalBiochem, La Jolla, CA); Ro-20-1724 (CalBiochem); Zaprinast (CalBiochem) and Pentoxifylline (CalBiochem); general protease inhibitors such as E-64 (trans-Epoxysuccinyl-L-leucylamido-(4- guanidino)butane, Sigma); leupeptin (Sigma); pepstatin A (Sigma); TPCK (N-tosyl-L- phenylalanine chloromethyl ketone, Sigma); PMSF (Phenylmethanesulfonyl fluoride, Sigma); benzamidine (Sigma) and 1,10-phenanthroline (Sigma); organic solvents including chloroform and methanol; detergents such as SDS; proteases that would degrade phospholipases such as trypsin (Sigma) and thermostable protease (Boehringer Mannheim Biochemicals, Indianapolis, IN); and metal chelators such as EDTA

(Ethylenediaminetetracetic acid, Sigma) and EGTA (Ethylene glycol-bis-(beta- aminoethyl ether), Sigma). These reagents are characterized by their ability to preserve lysophospholipid levels in a sample by either reducing lysophospholipid production or degradation. The peroxidase solution contains peroxidase which is a hemoprotein catalyzing the oxidation by hydrogen peroxide of a number of substrates such as ascorbate, ferrocyanide, cytochrome c and the leuco form of many dyes. In short, peroxidases are heme-binding enzymes that carry out a variety of biosynthetic and degradative functions using hydrogen peroxide as the electron acceptor. The function of the peroxidase is to catalyze the reaction of a suitable substrate to a detectable colored oxidized state species. Examples for a liquid assay include 3,3',5,5'-tetramethylbenzidine, 5-aminosalicylic acid (5AS), o-dianisidine, o-toluidine, o-phyeylenediamine, 2,2'-azinodi-(3- ethylbenzothiazoline-6-sulfonate) (ABTS) and those for a strip assay include 3,3'- diaminobenzidine (DAB), 3-amino-90-ethylcarbazole, 4-chloro-l-naphthol, 3,4- diamihnotoluene, 4,5-dimethyl-l,2-phenylenediamine, 4-chloro-l,2-phenylenediamine, 4,5-dichloro- 1 ,2-phenylenediamine. The chromophore precursor solutions are the mixtures of compounds, that when oxidized, result in color. Any chromophore that develops a color that corresponds to the spectra of the fluorophore partner could be used in this technology. In other words, the preferred chromophore must be able to absorb light resulting in an attenuation of the fluorescence of the fluorphore used in the assay. The chromophore precursors act as electron donors and as they donate electrons, they are oxidized and thereby generate color.

Fluorescent compounds are those compounds that when irradiated with UV or visible light, re-emit some of this light as longer wavelength light. The fluorescent compounds in the present invention are used to measure, via the Radiant Energy Attenuation (REA) method, the amount of signal generated. Alternative fluorescent compounds that could be used include those that are characterized by excitation and/or emission spectra that coincide with the absorption spectra of the generated chromophores. The generic REA method is better described in US Patent No. 4,495,293, issued January 22, 1985, entitled "Fluorometric Assay", herein incorporated by reference. Specifically, this patent provides a method to fluorometrically determine a ligand in an assay solution containing the ligand, reagent system and a fluorescer wherein the intensity of the fluorescer emitted by the assay solution is related to the change in the transmittive properties of the assay solution produced by the interaction of the ligand to be determined and a reagent system capable of producing a change in the transmittive properties of the assay solution in the presence of the ligand. In addition, novel reagent compositions are provided which may be utilized to either spectrophotometrically or fluorometrically determine the concentration of a ligand in an assay solution. Examples of fluorphores include R-Phycoerythrin, TexasRed, Oregon Green, Fluorescein, Rhodamine Red, Tetramethylrhodamine, BODIPY FL, BODIPY TR, BODIPY TMR, YOYO-f, DAPI, Indo-1. cascade Blue, Fura-2, Amino methylcoumariln, Carboxy-Snarf, Lucifer Yellow, Dansyl Derivitive.

Cations contemplated in this invention are positively charged ions such as Na⁺, Ca ^, Zn⁺⁺, etc. They are used in the present invention to activate the glycerol-3- phosphate oxidase and any cation which activates glycerol-3 -phosphate oxidase is suitable. Others include those disclosed in Table 1.

TABLE 1

This Table illustrates the effect of metal ions on 1-a-glycerophosphate oxidase activity.

1-a-Glycerophosphate oxidase activity was measured in 1 mM potassium phosphate buffer, pH 7.0, and at 10 mM DL-a-glycerophosphate in the presence of the salts below by the peroxidase-linked system described under "Experimental Procedures". Effector L-a-Glycerophosphast Activation oxidase activity

Units/ml

None 9.7 1

CaCl₂

10 mM 74.5 7.7 MgCl₂

10 mM 66.2 6.8

ZnC

I mM 60.7 6.3 10 mM 8.3 0.9

MnCL₂* b

10 mM 73.6 7.6

CoCl₂*

10 mM 99.4 10.2

NaCl

10 mM 12.9 1.3 100 mM 36.8 3.8

KC1 lO mM 25.8 2.7 100 mM 56.1 5.8

"The rate in the presence of effector was divided by the rate in the absence of effector. *Rate in presence of this salt was linear for only a short time. Esders, T. W. and Michrina, C.A. Purification and Properties of L-alpha— Glycerophospate Oxidase from Streptococcus faecium ATCC 12755. Journal of Biological Chemistry 254, 2710-2715, 1979.

Chelators contemplated within the scope of this invention are multidentritic species (e.g. citrate, EDTA, EGTA) that bind to positively charged metal ions. Chelators may preferably be used to stabilize calibrators or to temporarily lower the availability of divalent cations in our system.

Phenol and phenazones are used as electron donors. Phenol as defined is hydroxybenzene sometimes referred to as carbolic acid. Derivitives would include compounds that have substituants on the positions of the benzene other than at the phenolic hydroxyl. Examples of phenazones include antipyrenes.

LPA is the compound preferably detected, but other lysohospholipids are also contemplated within the scope of this invention in order to detect cancer, including but not limited to LysoPC, lysophosphatidyl serine (LysoPS), lysophosphatidyl inositol

(LysoPI), lysophosphatidyl ethanolamine (LysoPE) and lysophosphatidyl glycerol (LysoPG).

Manual test kits useful for detecting LPA in a test sample are also provided which comprise a container containing the necessary enzymes and other reagents for conducting the assay described herein. These test kits further comprise containers with tools useful for collecting test samples (such as, for example, food, urine, saliva and stool). Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; and cups for collecting and stabilizing urine or stool samples. Collection materials, such as papers, cloths, swabs, cups, and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample. The collection materials also may be treated with or contain preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens.

The diseases correlated with altered concentrations of these lysophospholipids include conditions associated with platelet activation such as, inflammatory conditions.

Altered phospholipid metabolism has been reported in many diseases and can lead to altered lysophospholipid and phospholipid levels in biological fluids, such as blood. These diseases include, but are not limited to, Alzheimer's, diabetes, heart disease, ischemia, liver disease, lung disease, malaria, muscular dystrophy, Parkinson^'s, sickle cell anemia, and various cancers. In these diseases, defective cellular functions may contribute to changes in levels of phospholipids. Other diseases include bleeding disorders including those associated with abnormal platelet function resulting in coagulopathy.

Various automated instruments can be used in conjunction with this assay, including, but not limited to Abbott IMx, Abbott Alcyon, Abbott AxSym, and the Toshiba Aeroset.

Examples

The following examples are presented to demonstrate the methods of the present invention and to assist one of ordinary skill in using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure or the protection granted by Letters Patent granted hereon. Example 1

Reagent Combination/Step Reduction

This example illustrates how an LPA assay can be reduced to only two steps and only two reagents.

Lysophospholipase was combined with the cycling enzymes (i.e. glycerol-3- phosphate oxidase and glycerophosphate dehydrogenase) and NADH to form one reagent in the following way. One hundred uL's of lysophosphatidic acid (Atairgin, Irvine CA) calibrators were added to the wells of a 96 well microtiter plate. Fifty uL's of a solution that contained lysophospholipase (Atairgin) at 5 units/mL, glycerol phosphate dehydrogenase (Atairgin) at 17 units mL, glycerol-3 -phosphate oxidase (Atairgin) at 134 units/mL, NADH, 1.25 mM, calcium chloride, 20 mM, and Tris, 50 mM pH 8.0 were added to the wells and the contents of the wells were mixed.

Following a 60 minute incubation at 37° C, 50 uL of 50 mM Tris, pH 8.0 and 50 uL of a solution that contained 0.5% 3,5-dichloro-2-hydroxy benzenesulfonic acid, 0.15% 4 aminoantipyrene, 10 units/mL horse radish peroxidase (HRPO, Atairgin), luM fluorescein, 50 mM Tris pH 8.0 were added to the wells and the contents of the wells were mixed. The absorbances at 490 ran were then read using a microtiter plate reader. The results shown in Figure 1A and IB demonstrate that LPA could be detected by combining the lipase and cycling enzymes. Figure 1 A shows the results as an absorbance read while Figure IB shows that the results can also be read as fluoresence at 520 ran by incorporation of fluorescein in to the chromaphore mixture and utilizing the REA ("Radiant Energy Attenuation") method. As one can see, the fluorescent readout has improved sensitivity as compared to the absorbance read-out.

Example 2

Novel Calibrators LPA calibrators were prepared as in the prior art by adding LPA (Sigma) to 2.5%

Triton X-100, 50mM CaCl_2.50 mM Tris, pH 8.0. Novel calibrators were prepared using the same solution but only this time the calcium chloride was omitted. Calibrators were then stored for approximately 72 hours at both room temperature and 4° C. After 72 hours fresh calibrators were prepared (+/-) calcium. The fresh and stored calibrators were then evaluated in the LPA assay using the microtiter format as follows. One hundred uL of sample was added to the wells of a microtiter plate. Fifty uL's of the lipase cycling solution that contained 2 units/mL of Lysophospholipase, 80 units/ mL of glycerol 3-phosphate dehydrogenase, 40 units/mL of glycerol-3- phosphate oxidase in 20mM calcium chloride, 50 mM Tris. PH 8.0 was added to the sample followed by 50 uL of a 1.5 mM solution of nicotinamide adenine dinucleotide reduced (NADH). The mixture was mixed and then incubated at 37° C for 20 minutes. Fifty uL of a color development solution which contained 0.5% 3,5-dichloro-2-hydroxy benzenesulfonic acid, 0.15% 4 aminoantipyrene, lOunits/mL horse radish peroxidase(HRPO) in 50 MM Tris pH 8.0 was then added. Following mixing the absorbances were read at 490 ran. The results in Figure 2 show that the calibrators prepared without calcium are more stable than those prepared in the presence of calcium.

Example 3

Improved Detection Reagent The REA Chromophore/Fluorophore (C/F) reagent that contained glycine 1.0M;

3,5-dichloro-2-hydroxy benzensulfonic acid, 0.22M; 4 aminoantipyrene, 0.05M; dimethyl sulfoxide 50%; sodium azide, 0.1 %; Triton X-100, 5.4%; fluorescein , 4.5 xl0E-6M and FG-10 anti foam (Dow Corning) at 0.01%, pH 7.0 was prepared . Eight microliters of an HRPO solution (2.5u/uL) was added to 500uL's of the C/F-reagent. Eight microliters of the HRPO solution was also added to C/F reagent that did not contain azide or Triton. Portions of the mixtures were stored at either room temperature of 37° C for 24 hours. Fresh solutions were made for comparison to the ones stored for 24 hours. Fifty microliters of 1/20 dilutions (PBS) of the test solutions were added to microtiter plate wells. Fifty microliters of peroxide calibrators were added to the wells and the resulting absorbances at 490 nm were read. The results shown in Figure 3 indicate that that HRPO is stable in the C/F reagent (_+/- azide and Triton) and suggest that GRPO could be incorporated as a reagent into the C/F reagent formulation. In other words, this data clearly shows an improved formulation of a detection reagent, which contains peroxidase and chromophore precursers, which has significantly improved stability compared with the prior art reagent.

Further, this aspect of the invention is critical in achieving an automated assay and incorporate it onto an automated machine, such as an Abbott Imx.

Example 4

Automated Assay on Alcyon Analyzer

LPA was detected on the Abbott Alcyon analyzer using the "Dual Reagent End-Point

Chemistry" method. Briefly, 180ul of a 0.50 mM solution of NADH (Boehringer Mannheim, Indianapolis, IN) in 50mM Tris, pH 8.0 was added to a reaction cuvette. Two measurements of the absorbance at 340nm were recorded at 12 second intervals. Thirty microliters of the sample were added to the NADH solution and the reaction mixture was mixed. Another absorption measurement at 340nm was made. One hundred eighty microliters of a solution that contains phospholipase B (Sigma, St.Louis, MO) at 1 unit mL, glycerol phosphate dehydrogenase (Boehringer Mannheim, Indianapolis, IN) at 40 units/mL, and glycerol phosphate oxidase (Shinko American, New York, NY) at 600 units/mL in 20mM CaCl and 50mM Tris, pH 8 were added to the reaction cuvette. As the reaction proceeds the absorbance at 340 nm decreases. After an incubation of 12 minutes 48 seconds at 37° C, a final absorbance measurement was made. The absorbance difference that occurs as a result of the reaction was automatically calculated by the Alcyon analyzer using an input value of the absorption of the enzyme reagent and the formulas contained in the Alcyon operations manual. A typical calibration curve is shown in Figure 4. Example 5

One-Step Assay vs. Multi-Step Assay Comparison A comparison between the prior art two step (a separate lipase digestion prior to a combined lipase/cycling) microtiter format and a one step lipase/ cycling format was made. The sample size, incubation times and reagent quantities were kept identical so that a direct comparison can be properly made.

The following reagents were prepared: Reagent A that contained lysophospholipase at 5 U/mL, glycerol dehydrogenase at 34 U/mL and glycerol oxidase at 134 U/mL in 10 mM calcium chloride, 50 mM Tris pH 8. Reagent B that contained 25 mM NADH in 50mM Tris pH 8. Reagent C, similar to reagent A, but also containing

12.5 mM NADH. A color development solution, reagent D, that contained 0.5% 3,5- dichloro-2-hydroxy benzenesulfonic acid, 0.15% 4 aminoantipyrene, lOunits/mL horse radish peroxidase (HRPO, Atairgin), luM fluorescein in 50 mM Tris pH 8.0 was prepared immediately before use. Prior Art Assay(Multi Step)

Fifty uL's of Lysophosphatidic acid (LPA, Atairgin, Irvine CA) calibrators were added in duplicate to the wells of a 96 well microtiter plate. One hundred uL's of reagent A was added to the calibrators. The plates were mixed, covered and incubated for 15 minutes at 37° C. At that time 50 ul's of reagent B was added to the wells. The addition of this reagent initiated the cycling. The plates were mixed then incubated for another 15 minutes at 37° C. Fifty uL's of the color development reagent (D) was added to all wells. The contents of the wells were mixed and the absorbances at 490 nm were read. The results are shown in Figure 5. Novel Assay (One Step) Fifty uL's of Lysophosphatidic acid (LPA, Atairgin, Irvine CA) calibrators were added in duplicate to the wells of a 96 well microtiter plate. One hundred 100 uL's of reagent C was added. The plates were mixed, covered and incubated for 30 minutes at 37° C. Following this incubation 50 uL's of 50mM Tris pH 8.0 was added to the wells to adjust the volume to be the same as the prior art. Fifty uL's of the color development reagent (D) was added to all wells. The contents of the wells were mixed and the absorbances at 490 nm were read. The results shown in Figure 5 demonstrate the enhanced performance of the single step (Novel Assay) format relative to the two step prior art format that utilizes a separate lipase digestion.

Example 6 Novel Colorgenic Reagent

A comparison was made between the stabilities of the novel REA chromophore/fluorophore reagent and horseradish peroxidase (HRPO) mixture and the prior art color development reagent and HRPO mixture. The novel REA chromophore/fluorophore (C/F) reagent contained glycine, 0.1 M; 3,5-dichloro-2- hydroxy benzensulfonic acid, 0.22M; 4 aminoantipyrene, 0.05M; dimethyl sulfoxide

50%; sodium azide, 0.1%; Triton X-100, 5.4%; fluorescein , 4.5 xlOE-6M and FG-10 anti foam(Dow Corning) at 0.01%, pH 7.0. A modified C/F solution that contained 0.1M Tris, pH 8 instead of glycine was also made.

The prior art color development solution contained 0.5% 3,5-dichloro-2-hydroxy benzenesulfonic acid, 0.15% 4 aminoantipyrene, in 50 mM Tris pH 8.0. To one mL of the C/F and prior art color development solutions 4uL of a HRPO solution (2500 U/mL) was added. These solutions as well as a solution that contained only HRPO were incubated at room temperature for three days. The spectra of these were recorded and compared with C/F solutions that did not have HRPO added. The results shown in Figure 6 A show that the absorption for the C/F solution (pH 7) + HRPO at 512 nm (spectra 1) is slightly lower than that seen for the C/F(pH 8) + HRPO (spectra 2) while the absorption for the prior art color development reagent + HRPO (spectra 3) is significantly higher. The absorption band near 512 nm is also red shifted for the prior art mixture. Figure 6B shows the spectra (5) of the C/F (pH 7) reagent, the spectra (6) of the C/F (pH 8) reagent and of HRPO only (spectra 4). After three days the C/F peroxidase mixtures are still the same yellow color as they were two hours after addition of the HRPO, while the prior art mixture has gone from clear to red. The absorption at 512 nm of these solutions as a function of time is shown in Figure 6C. For the prior art color development reagent HRPO mixture we observe an initial low absorbance at 512 nm followed by a steady increase in absorbance at 512 nm. For the C/F mixtures we see an initial increase in absorbance at 512 nm upon addition of HRPO. However this quickly decreases (after 2 hours) and then remains constant. Although not shown in this experiment the absorption of the prior art color development solution, in the absence of HRPO, at 512 nm is insignificant.

These data along with those shown in Example 3 demonstrate that C/F peroxidase mixture has enhanced stability compared to the prior art color development peroxidase mixture.

Example 7

Automated Assay for LPA using IMx Instrument. The IMx instrument was designed to perform immunoassays in both microparticle and fluorescence polarization formats. The fluorescence polarization format can be adapted to perform "Radiative Attenuation Assay", which permits measurements based on optical absorbance. By adding peroxidase and appropriate dyes - fluorescein and a colorigenic peroxidase substrate for example — to the mixture of Example 1 after the cycling reaction has proceeded to a sufficient degree the concentration of G3P, and therefore, sample LPA can be determined.

Reagents:

Lipase (hydrolvsisVCvcling Enzyme Reagent: lU/mL lysophospholipase, 200U/mL glycerophosphate dehydrogenase, 500uL glycerol-3 -phosphate oxidase, 40mM calcium chloride, 50mM Tris, 5mM sodium benzoate, 20% glycerol pH 8.0.

Chromophore/Fluorophore Reagent: 220mM 3,5 dichloro-2-hydroxy benzene sulfonic acid, 50mM 4-aminoantipyrene, lOOmM glycine, 4.5uM Fluorescein, 0.1 % sodium azide, 5.4% Triton X 100, 50% dimethyl sulfoxide, pH 8.5.

HRPO Mixture: 20U/mL horseradish peroxidase in 50mM tris pH 8.0. NADH Solution: 1.5mM NADH in 50mM tris pH 8.0.

Protocol:

In a preferred embodiment, a plasma sample can be prepared by the following method. Blood is collected in presence of a stabilizer such as EDTA or citrate. It is then centrifuged sufficiently to sediment erythrocytes and platelets (15 min at 3000XG) at 4°

C or is filtered to remove these components. lOOuL NADH Solution, 5uL sample and 20uL Lipase/Cycling Enzyme Reagent are aspirated by the sample probe. 70uL of this is dispensed to the cuvette and the remaining NADH Solution in the probe dispensed to waste (This is to prevent contamination of the cycling mixture by the required line diluent, which contains phosphate buffer, which would slow the reaction by complexing the calcium). The mixture is incubated for 30 min at 35° C in the instrument, then 40uL Chromophore/FIuorphore Reagent and 690uL line diluent are added. The mixture is incubated for 4 min then the fluorescence intensity is measured and immediately thereafter 20uL Chromophore/FIuorphore Reagent, 40uL HRPO mixture and 340uL line diluent are added. The mixture is incubated an additional 4 min during which the color is formed. Finally the fluorescence intensity is measured again and the data transferred to a file for analysis. The ratio of the final fluorescence intensity to the initial fluorescence intensity decreases with increasing peroxide-generated color, and so can be used with appropriate calibrators to determine the amount of glycerol-3 -phosphate, and by extension the amount of LPA originally in the sample. When the test is performed under identical conditions, but without lysophospholipase in the Lipase/Cycling Enzyme Reagent, the LPA does not react and only the free glycerol-3 -phosphate is measured. For samples collected under conditions in which free glycerol-3-phosphate may be present, performing this assay both with and without lysophospholipase can provide the information needed to determine the actual LPA concentration.

Table 2 shows the results of applying the above protocol to LPA standards ranging from 0 to 5uM. Initial and final fluorescence intensities and their ratio are shown along with the LPA concentration of the sample. The first 12 positions are duplicates of LPA standards in buffer, then replicates of 4 each of the zero and 2.0 uM standards. A 4-parameter log-logit curve fitting algorithm was used with these results to generate the curve in Figure 7.

In addition to plasma samples, the standards AS 1 and AS 1 extracted are also measured. The LPA concentration measured for AS1 extracted is 0.53 uM, consistent with values determined using the microtiter format. The LPA concentration of the unextracted sample is 2.04 uM, about 4-fold higher than the extracted sample. LPC cross-reactivity is ruled out as a cause for reasons shown in Table 3. Most likely, the difference results from losses of LPA during the extraction process. Table 3 shows the application of the method to human plasma. Blood collected from normal volunteers in EDTA tubes was cooled in an ice bath immediately after collection. Within 80 min of collection it was centrifuged 15 min at 3000XG in a refrigerated centrifuge at 4° C. The clear supernate was tested by the above protocol both with and without lysophospholipase. Standards consisting of 0 to 5uM glycerol-3- phosphate in buffer were used for both conditions. The same curve-fitting algorithm was used to generate the results. The results in Table 3 illustrate the importance of obtaining background measurement in order to accurately measure LPA concentration.

Table 4 shows the effect of sample handling and storage, and demonstrates that lysophosphatidylcholine, which interferes in the microtiter formatted assay, does not interfere in the IMx configured assay if samples are stored at -20° C. Blood was collected with EDTA anticoagulant, cooled 10 min in an ice bath 5 min after drawing, then centrifuged 15 min at 500XG at 2° C. Of the 12 mL resulting turbid plasma, portions were stored at -20° C, 4° C and room temperature = 37° C. The remainder was centrifuged 30 min at 3100XG at 2° C, and the clear supernate aspirated from the pellet.

500uL portions of the clear supernate were subjected to the following treatments, then aliquoted and stored as above at -20° C, 4° C and room temperature: Set B: no treatment Set C: 2.0uL sample buffer (2.5% Triton X 100 in 50mM tris pH 8.0) added to 500uL clear plasma.

Set D: 2.0uL l.OmM LPA in sample buffer added to 500uL clear plasma = 4.0uM LPA. Set E: lOuL lOmM LPC in sample buffer added to 500uL clear plasma = 200uM LPC. The samples were stored 18 hours at the indicated conditions, then brought to room temperature and assayed as above, both with and without lysophospholipase. It is immediately apparent that the insufficiently centrifuged turbid plasma contains a high background of G3P, most of which is removed by adequate centrifugation. The LPA concentration increases with storage at higher temperatures, indicating that samples should be stored frozen. Addition of tris/triton buffer makes little difference at the concentrations added. The samples spiked with 4uM LPA showed recovery of most of the spike, but no change with storage conditions. An increase of 1.5uM would be expected from the results of the unspiked sample. One possibility for its absence is that the increase in LPA is compensated by a process which destroys it, such as some background phosphatase activity. Most interesting here is that spiking the sample with 200uM LPC increases the measured LPA by only about 0.1 uM when the sample is stored at -20° C, and by extension when the sample is fresh. Evidently LPC interferes much less with the IMx formatted assay than with the microtiter format of the prior art, possibly due to decreased exposure to the lysophospholipase. However, when the sample is stored at 4° C or room temperature, the LPC spiked sample shows an increasing signal for LPA. A possible explanation is the presence of a phospholipase C activity in the plasma, which cleaves choline from LPC, leaving LPA.

Table 2 pos uMLPA lo I l/lo uMLPA

1from curve

1 0.0 23098 22440 0.972 0.00

2 0.0 23935 22793 0.952 0.05

3 0.5 23386 19709 0.843 0.46

4 0.5 23411 19487 0.832 0.50

5 1.0 24340 16841 0.692 1.05

6 1.0 23672 16614 0.702 1.01

7 2.0 23922 12063 0.504 1.96

8 2.0 24086 11995 0.498 1.99

9 3.0 23623 8391 0.355 2.92

10 3.0 24732 8211 0.332 3.09

11 5.0 24103 3346 0.139 5.01

12 5.0 24495 3439 0.140 5.00

13 0.0 24901 22751 0.914 0.20

14 0.0 24113 22712 0.942 0.09

15 0.0 24218 22476 0.928 0.14

16 0.0 24598 22729 0.924 0.16

17 2.0 24220 11754 0.485 2.07

18 2.0 23981 11759 0.490 2.04

19 2.0 24384 11633 0.477 2.12

20 2.0 24563 11699 0.476 2.12

Example 8

Detection of LPA Using a Strip Assay

This assay can also be formatted on a strip. Specifically, whole blood is collected in the presence of a stabilizer, such as EDTA or citrate. It is then placed on a strip, which wicks the plasma away from the solid components. The plasma, preferably, passes through a portion of the strip containing calcium and the solid components are removed by continued passage through the strip. The lipase and cycling enzymes are located downstream on a conjugate pad along with detection reagents or labels.

Example 9

Effect of NADH concentration on rate of H7O7 production. 50uL of solutions containing 0-l.OuM LPA in 50mM tris pH 8.0 with 2.5% Triton X 100 and 5mM CaCl were added to the wells of a microtiter plate. To this was added 50uL of a mixture containing 5U/mL lysophospholipase, lOU/mL glycerophosphate dehydrogenase, lOOU/mL glycerol-3 -phosphate oxidase, lOmM CaCl and various concentrations of NADH in 50mM tris pH 8.0. After 20min at 37° C 50uL of a mixture of 19mM DHBS, 7.5mM 4AAP, lOU/mL HRPO in 50mM tris pH 8.0 was added to one set of the duplicate samples and the absorbance read at 490nm (see Fig. 8A). After 60min at 37° C the other set of samples was treated the same way (see Fig. 8B). Figures 8A-B shows the absorbances for each NADH concentration plotted vs LPA concentration. For the shorter reaction time lower concentrations of NADH result in more signal for the same LPA concentrations, suggesting an inhibiting effect of NADH on the overall reaction. The results shown for the longer reaction time demonstrate that, while higher NADH concentrations decrease the rate of H O₂ production, when the NADH is completely consumed no further reaction can take place. These results point to the need to limit the NADH concentration in the cycling reaction. Example 10

Automated assay for LPA using IMx instrument with combined peroxidase and color reagent.

This is similar to Example 7 (above) except the peroxidase and color generating reagents are combined into a single reagent, simplifying the assay.

Reagents:

Lipase (hydrolysis)/Cycling: lU/mL lysophospholipase, 200U/mL glycerophosphate dehydrogenase, 500U/mL glycerol-3-phosphate oxidase, 40mM calcium chloride, 50mM tris, 5mM sodium benzoate, 20% glycerol pH 8.0.

Chromophore/Fluorophore/HRPO Mixture: 20U/mL horseradish peroxidase, 220mM 3,5-dichloro-2-hydroxy benzene sulfonic acid, 50mM 4-aminoantipyrene, lOOmM glycine, 4.5uM Fluorescein, 0.1% sodium azide, 5.4% Triton X 100, 50% dimethylsulfoxide, pH 8.5.

NADH solution: 1.5mM NADH in 50mM Tris pH 8.0.

Protocol: lOOuL NADH solution, 5uL sample and 20uL Lipase/Cycling Enzyme reagent are aspirated by the sample probe. 70uL of this is dispensed to the cuvette and the remaining NADH reagent in the probe dispensed to waste (This is to prevent contamination of the cycling mixture by the required line diluent which contains phosphate buffer which would slow the reaction by complexing the calcium). The mixture is incubated for 15 min at 35° C in the instrument, then 40uL Chromophore/Fluorophore/HRPO reagent and

690uL line diluent are added. The mixture is incubated for 4 min then the fluorescence intensity is measured and the data transferred to a file for analysis. The measured fluorescence intensity decreases with increasing peroxide-generated color, and so can be used with appropriate calibrators to determine the amount of glycerol-3 -phosphate, and by extension the amount of LPA originally in the sample. As with the test of Example 7 with separate Chromophore/Fluorophore and Peroxidase reagents, the test can be run without lysophospholipase in the Lipase/Cycling Enzyme reagent so as to determine the background glycerol-3-phosphate.

Figure 9 shows the results of applying the above protocol to LPA standards ranging from O to lOuM.

Example 1 1

The effects of pH on the stability of chromogen/peroxidase mixtures has been studied. Two, one mL solutions of 0.03% 4 aminoantipyrene, 1.0 % 3,5-dichloro-2- hydroxy-benzenesulfonic acid were prepared. One contained 35mM Tris at pH 8 while the other contained 70mM sodium phosphate at pH 7. To each of these solutions 4uL of horse radish peroxidase (2.5U/uL, HRPO) was added. Control solutions that did not contain peroxidase were also prepared. The solutions were allowed to sit overnight at room temperature. The absorbance at 512 nm of solutions was then measured. The results shown in Figure 10 demonstrate that the solution that contained HRPO prepared at pH 7 is more stable than the solution that contained HRPO prepared at pH 8.

Example 12

The efficiency of cycling may be increased by covalently linking the cycling enzyme, G3P oxidase and G3P dehydrogenese. Since the product of one is the substrate of the other, the linkage of the two would assure availability of the appropriate enzyme in the vicinity of its substrate. Covalent linkage of the two may be carried out by methods well known in the art.

Claims

CLAIMSWhat is claimed is:

1. A diagnostic kit for detecting the concentration of lysophospholipids in a sample of bodily fluid taken from a test subject, said kit comprising: (i) at least one lipase enzyme that digests sn-1 and/or sn-2 lysophospholipids combined with at least one cycling enzyme to form a single reagent ; and (ii) NADH.

2. The diagnostic kit of claim 1 wherein said lipase enzyme is selected from the group consisting of phospholipase B, phospholipase C, phospholipase D, lysophospholipase, phospholipase Aj, and phospholipase A , lecithinase B and lysolecithinase; and said cycling enzyme is selected from the group consisting of glycerol- 3 -phosphate dehydrogenase, glycerol-3 -phosphate oxidase, serine dehydrogenase, serine deaminase, aldehyde dehydrogenase, ethanolamine deaminase, glycerokinase and glycerol dehydrogenase.

3. The diagnostic kit of claim 1, further comprising preferably nicotinamide adenine dinucleotide (NADH), stablized in an aqueous base liquid containing propylene glycol, boric acid and a buffer capable of buffering within a pH range of 8-11.

4. The diagnostic kit of claim 3, further comprising a reagent for inhibiting production or hydrolysis of lysophospholipid in said test sample selected from the group consisting of Aristolic Acid (9-methoxy-6- nitrophenanthro-(3,4-d)- dioxole-5-carboxylic acid; ONO-R-082 (2-(p- Amylcinnamoyl)amino-4-chloro9benzoic acid); OBAA (3-(4-Octadecyl)- benzoylacrylilc acid), 4-Bromophenacyl Bromide; Quincrine (6-Chloro-9- (4-diethylamino)- 1 -methylbutyl)amino-2-methoxycridine, Mepacrine; Manoalide and HELSS (Haloenol lactone suicide substrate); phosphodiesterase inhibitors such as IBMX (3-Isobutyl-l-methylxanthine; Ro-20-1724; Zaprinast and Pentoxifylline; general protease inhibitors such as E-64 (trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane); leupeptin; pepstatin A; TPCK (N-tosyl-L-phenylalanine chloromethyl ketone); PMSF (Phenylmethanesulfonyl fluoride); benzamidine and 1,10- phenanthroline; organic solvents including chloroform and methanol; detergents such as SDS; proteases that would degrade phospholipases such as trypsin and thermostable protease; and metal chelators such as EDTA (Ethylenediaminetetracetic acid) and EGTA (Ethylene glycol-bis-

(beta-aminoethyl ether). Phenylmethylsulonylfluoride, Mn2+, Co2+, Zn2+, Cu2+, Hg2+, Fe2+, Fe3+, Ca2+, Mg2+, A13+, 3(Cis,cis-7,10) hexadecadienyl-4-hydroxy-2-butenolide, Diisopropylfluorophosphate, Dithiothreitol, Sulfhydryl reagents (e.g. N- ethylmaleimide, iodoacetate), p-Chloromercuribenzoate,

Sodiumdeoxycholate, Detergents (e.g. Triton X-100), L- Palmitoylcarnitine, N-Bromosuccinimide, 2-Hydroxy-5- nitrobenzyl bromide, Phenylglyoxal, Glutathione, SDS and

Bis-(p-nitrophenyl)phosphate.

5. The diagnostic kit of claim 1, further comprising a peroxidase solution, and a chromogenic peroxidase substrate.

6. The diagnostic kit of claim 5, wherein said chromogenic peroxidase substrate further comprises a fluorescent compound.

7. The diagnostic kit of claim 6, wherein said peroxidase solution, said chromogenic peroxidase substrate, and a fluorescent compound are combined to form a single reagent.

8. The diagnostic kit of claim 2, wherein the lysophospholipid is selected from the group consisting of LysoPA, LysoPS, LysoPE, LysoPI and LysoPG.

9. The diagnostic kit of claim 1, wherein said first enzyme comprises a combination of enzymes.

10. The diagnostic kit of claim 1, wherein said lipase enzyme, said cycling enzyme and said NADH are combined to form one reagent.

11. The diagnostic kit of claim 1 , which further comprises a cation.

12. The method of claim 1 in which said cycling enzyme is comprised of two different cycling enzymes covalently linked together to form one entity.

13. A lysophospholipid detection system calibrator having improved stability comprising:

LPA in a calcium-free, non-phosphate buffer solution.

14. The calibrator of claim 13, which further comprises a chelator.

15. The calibrator of claim 13, wherein said chelator is selected from the group consisting of citrate, EDTA and EGTA.

16. A lysophospholipid detection system calibrator having improved stability comprising: G3P in a calcium-free, non-phosphate buffer solution.

17. The calibrator of claim 16, which further comprises a chelator.

18. The calibrator of claim 17, wherein said chelator is selected from the group consisting of citrate, EDTA and EGTA.

19. A lysophospholipid detection system peroxidase solution having improved stability comprising: peroxidase, an antimicrobial agent and glycerol.

20. A lysophospholipid detection system chromogen solution having improved stability comprising: a pH 7 buffer, a phenol or phenol derivative, and a phenazone.

21. The chromogen solution of claim 20, wherein said phenol is 3,5 dichloro-

2-hydroxybenzene sulfonic acid and said phenazone is 4- aminoantipyrene.

22. An improved assay to detect the concentration of one or more lysophospholipids in a sample of bodily fluid taken from a test subject comprising:

(a) simultaneously contacting a sample of bodily fluid taken from a test subject with a first enzyme that digests lysophospholipids and a second cycling enzyme to produce a product; and (b) determining the concentration of at least one lysophospholipid present in the sample by measuring the product by using a calibrator selected from the group consisting of G3P in a calcium- free, non-phosphate buffer solution, LPA in a calcium-free, non- phosphate buffer solution, and combinations thereof, as a reference measurement.

23. An improved assay to detect the concentration of one or more lysophospholipids in a sample of bodily fluid taken from a test subject comprising: (a) simultaneously contacting a sample of bodily fluid taken from a test subject with a first enzyme that digests lysophospholipids and a second cycling enzyme to produce a product; and (b) determining the concentration of at least one lysophospholipid is by incubating said product produced in step (a) in a peroxidase solution, said peroxidase solution comprising peroxidase, an antimicrobial agent and glycerol; and (c) a chromogenic peroxidase substrate, said chromogenic peroxidase substrate comprising a pH 7 buffer, a phenol or phenol derivative, and a chromogenic substrate, such that hydrogen peroxide is detected.

24. The improved assay of claim 23, wherein said phenol is selected from the group consisting of phenol is 3,5 dichloro-2-hydroxybenzene sulfonic acid and said chromogenic substrate is 4- aminoantipyrene.

25. The improved assay of claim 23, wherein determining the concentration is done by measuring the concentration of hydrogen peroxide by electrochemical detection.

26. The improved assay of claim 23, wherein said chromogenic peroxidase substrate further contains a fluorescent compound and the concentration of hydrogen peroxide is performed by measuring the residual fluorescence.

27. The improved assay of claim 23, wherein said bodily fluid is selected from the group consisting of whole blood, serum, plasma, ascites, urine, saliva, cerebral spinal fluid and pleural fluid.

28. The improved assay of claim 23, further comprising the step of comparing the concentration of lysophospholipid determined in step (b) from a test subject with the concentration of that lysophospholipid in samples from normal subjects to detect the presence of a disease condition associated with altered levels of lysophospholipid in the test subject, wherein an increase or decrease in the concentration of the lysophospholipid in the sample from the test subject relative to the concentration of that lysophospholipid in samples from normal subjects indicates the presence of the disease condition in the test subject.

29. The improved assay of claim 23, wherein the disease condition is cancer associated with alteration in the level of at least one lysophospholipid relative to the level in normal subjects.

30. The improved assay of claim 23, wherein the disease condition is a gynecological cancer.

31. The improved assay of claim 23, wherein the disease condition is ovarian cancer and the lysophospholipid detected is LysoPA.

32. The improved assay of claim 23, wherein the disease condition is breast cancer.

33. The improved assay of claim 23, wherein the disease condition is a blood disorder associated with alteration in the level of at least one lysophospholipid relative to the level in normal subjects.

34. An automated assay to detect the concentration of one or more lysophospholipids in a sample of bodily fluid taken from a test subject comprising: (a) on an automated format simultaneously contacting a sample of bodily fluid taken from a test subject with lysophospholipase, glycerolphosphate dehydrogenase and glycerol-3 -phosphate oxidase to form a mixture; (b) subsequently contacting said mixture with NADH to produce a detectable product; and (c) determining the concentration of at least one lysophospholipid present in the sample by measuring the concentration of the detectable product produced.

35. The automated assay of claim 34, wherein step (b) is eliminated and step

(a) comprises said sample of bodily fluid taken from a test subject being simultaneously contacted with with lysophospholipase, glycerolphosphate dehydrogenase , glycerol-3-phosphate oxidase and NADH .

36. The automated assay of claim 34, wherein said step C is done by measuring NAD.

37. The automated assay of claim 34, wherein said concentration is determined by incubating said detectable product after step (a) with a peroxidase solution and a chromogenic peroxidase substrate, such that hydrogen peroxide is produced and the absorbance of a chromophore indicates said lysophospholipid concentration.

38. The automated assay of claim 34, wherein said bodily fluid is selected from the group consisting of whole blood, serum, plasma, ascites, urine, salisa, cerebral spiral fluid and pleural fluid.

39. The automated assay of claim 34, wherein said peroxidase solution further contains a fluorescent compound and said lysophospholipid concentration is measured by fluorescence.

40. A method for measuring LPA in a test sample, said method comprising: (a) combining to form an assay solution:

(i) a test sample; (ii) an effective amount of a fluorescent compound selected from the group consisting of fluorscein and rhodomine; (iii) an effective amount of lysophospholipase, glycerolphosphate dehydrogenase, glycerol-3-phosphate oxidase, and NADH to produce a solution capable of providing a change in the transmitive properties of the assay solution within a wavelength band that overlaps the excitation and/or emission wavelength band of the fluorescer; (b) irradiating the assay solution with light having a wavelength within the excitation wavelength band of the fluorescer; and (c) measuring the intensity of the fluorescence emitted by the assay solution as a measure of the concentration of LPA in the sample.

41. The method according to claim 40, wherein said solution of step iii further comprises a peroxidase solution and a chromogenic peroxidase substrate such that hydrogen peroxide is produced and the absorbance of a chromophore indicates said lysophospholipid concentration.

42. The method according to claim 40, wherein said LPA is measured by determining the amount of NAD.

43. The method according to claim 40, wherein the absorption wavelength band associated with the change in the transmitive properties of the assay solution overlaps the excitation wavelength band of the fluorescent compound.

44. A method according to claim 40, wherein the absorption wavelength band associated with the change in the transmitive properties of the assay solution overlaps the emission wavelength band of the fluorescent compound.

45. A method according to claim 40, wherein the absorption wavelength band associated with the change in the transmitive properties of the assay solution overlaps the excitation and emission wavelength bands of the fluorescent compound.

46. A method of reducing the incubation time necessary for an assay used to detect lysophospholipids comprising:

(a) providing a combination of a first enzyme which digests lysophospholipids and a second amplification enzyme together;

(b) contacting said combination to a bodily fluid of a test sample to produce a detectable product wherein the combination period necessary to produce a detectable product is less than or equal to 1 hour; and

(c) determining the concentration of at least one lysophospholipid present in the sample by measuring the detectable product produced.

47. The method of claim 46, wherein said incubation time is 15 minutes or less.

48. The method of claim 46, wherein said incubation time is 30 minutes or less.

49. A method of eliminating cross-reactivity between LPA and LPC comprising:

(a) obtaining a bodily fluid from a test subject; (b) simultaneously hydrolyzing and amplifying said test sample; and

(c) detecting the presence of LPA.

50. An improved method for diagnosing the presence of carcinoma in a subject comprising the following steps: (a) preparing a plasma sample from a blood specimen collected from the subject; (b) contacting said plasma sample to a single reagent comprised of a first enzyme which digests lysophospholipids and a second amplification enzyme;

(c) testing for the presence of lysophosphatidic acid in said plasma sample; and

(d) correlating the presence of lysophosphatidic acid in said plasma sample with the presence of said carcinoma in said subject.

51. The method of claim 50, wherein said carcinoma is selected from the group consisting of ovarian carcinoma, peritoneal carcinoma, endometrial carcinoma, cervical carcinoma, and combinations thereof.

52. The method of claim 50, wherein the blood specimen is from a patient who has not been diagnosed as having Surgical Stage III or Surgical Stage IV carcinoma.

53. The chromogen solution of claim 20 further comprising, dinethyl sulfoxide, Triton X-100 and anti-foam.

54. The chromogen solution of claim 52 further comprising HRPO.

55. A method for measuring lysophospholipids from a non-extracted sample comprising:

(a) measuring all species capable of generating a detectable signal from a cycling reaction in the absence of lysophospholipase to obtain a background reading;

(b) repeating step (a) in the presence of lysophosolipase to obtain a measurement of the lysophospholipid concentration plus background reading; and (c) taking the difference between said lysophospholipid concentration and background reading to obtain a total lysophospholipid concentration.

6. The method of claim 55 wherein said non-extracted sample is stored at - 20° C.