Patent Document

BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    This invention pertains to a competitive chemiluminescent assay for the detection of the presence of cyclic nucleotide monophosphates. In general, the cyclic nucleotide phosphates appear only as monophosphates, due to chemical structure considerations. Among these, cyclic adenosine monophosphate is perhaps the most widely known, implicated as a primary or secondary messenger in a variety of cellular, intracellular and extracellular pathways. This invention takes advantage of the high sensitivity of chemiluminescent 1,2-dioxetane reagents to overcome sensitivity and dynamic range problems encountered in the prior art.  
           [0003]    2. Background of the Prior Art  
           [0004]    A wide variety of metabolic responses are keyed to release of intracellular cAMP. In many cases, these responses are mediated by cAMP-dependent protein kinase which, in the presence of elevated cAMP levels, triggers a wide variety of activating reactions. Among the best known metabolic responses keyed to cAMP are the conversion of glycogen to glucose in the liver, as well as a variety of activities keyed to the glycogen/glucose energy cycle. The principle hormone in this cycle which induces a rise in cAMP is epinephrine. There are, however, a wide variety of other hormones which will also trigger cAMP release, which in turn keys a metabolic response mediated by the kinase. These include ACTH, FSH, LH, TSH, parathyroid hormone, vasopressin and prostaglandin I. Accordingly, it is clear cAMP levels in specific tissues of mammals, including humans, may be key indicators of a variety of hormonal functions and interactions.  
           [0005]    cAMP is only the best known of the cyclic nucleotides. In general, cyclic nucleotides appear as monophosphates. Guanosine monophosphate (cGMP), uridine monophosphate (cUMP) and cytidine monophosphate (cCMP) may all importantly bear on a wide variety of hormonal functions and intercellular interactions, that may be desirably measured. cAMP is the most studied of these “messenger” cyclic nucleotides.  
           [0006]    Assays, including competitive ELISA assays, for cAMP are known. A widely reported assay is available from Assay Design, and is a colorometric assay. Other immunoassay products are available from Amersham Med-Physics, Inc. (a radioimmunoassay) as well as IGEN and NEN. The Assay Design embodiment employs assay kits (also available under the mark Biomol) in a classic example of a competitive ELISA assay, in which the strength of the signal is inversely proportional to the concentration of the cyclic nucleotide present. The kit from Biomol is for measurement of light absorption. A fluorescent assay kit is available from Perseptive Biosystems.  
           [0007]    Because of the very low values of cyclic nucleotides that may need to be detected in a variety of tissue samples, high sensitivity is required. Many of the commercially available assays for cAMP itself do not offer this sensitivity, and require acetylation of the cAMP (to promote better antibody binding) for greater sensitivity.  
           [0008]    Accordingly, it remains a goal of those of skill in the art to find a competitive immunoassay which is highly sensitive, offers a broad dynamic range, and employs reagents which can be obtained through simplified procedures, including avoidance of acetylation.  
         SUMMARY OF THE INVENTION  
         [0009]    The above objects, and others discussed in more detail below, are met by a chemiluminescent competitive ELISA assay which relies on the high chemiluminescent sensitivity of 1,2-dioxetanes. These dioxetanes, developed by the common assignee herein, Tropix, Inc., a division of P.E. Biosystems, are the subject of a wide variety of United States patents. The 1,2-dioxetanes that are useful in the claimed invention typically have a generic structure of Formula I:  
                         
 
           [0010]    In Formula I, Y 1 , Y 2  and X are variously electron-active moieties, in that they are either electron donating or electron withdrawing. Exemplary groups include halogens, particularly chlorine, alkoxies, particularly methoxy, amine, alkyl, etc. In the alternative, these groups are hydrogen. Any one or more of Y 1 , Y 2  or X may be other than hydrogen, or they may all be hydrogen. Substituent R is an alkyl, aralkyl, cyclic alkyl, heteroalkyl comprising an O, N, P or S moiety, in general of less than 20 carbon atoms. Desirably, R is alkyl. R may be substituted with groups intended to enhance solubility as well as reactivity, which may include halogen substituents such as one or more fluorine atoms, carboxy (COO) substituents, sulfoxy substituents, etc. The same substituents to enhance solubility may also be present on Y 1 , Y 2  or X. Ar is an aryl moiety, typically phenyl or naphthyl, most preferably phenyl. Z is a moiety which includes a bond which is cleaved by an enzyme, which, when cleaved, leaves either O or N attached to the aryl moiety. This anion destabilizes the dioxetane, leading to its decomposition. On decomposition, the dioxetane releases light. For the purposes of this invention, Z is a phosphate moiety, preferably disodium phosphate. Dioxetanes of this type are disclosed in U.S. Pat. Nos. 4,962,192; 4,931,569; 5,112,960; 5,145,772 and 5,654,154, as well as a variety of others. All of the foregoing patents are incorporated herein by reference. As disclosed, e.g., in U.S. Pat. No. 5,112,960, an enzyme-triggerable dioxetane such as 3-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1 3,7 ]decan-4-phenylphosphate and its salts (AMPPD) is a highly effective reporter molecule of this type. Derivitisation of this “unsubstituted” 1,2-dioxetane with substituents on the adamantyl ring, such as a chlorine atom (CSPD) can dramatically improve performance. Similarly, substituents on the phenyl ring besides the enzyme-cleavable substituent, particularly at the 3 or meta position, give further improved yields. These reporter molecules, which are chemiluminescent in nature, are referred to as enzyme-triggerable dioxetanes. The particular enzyme acting in the claimed invention is alkaline phosphotase, selecting therefore, Z as a phosphate moiety.  
           [0011]    As set forth in U.S. Pat. No. 5,145,772 and U.S. Pat. No. 5,336,595, also incorporated herein by reference, the use of polymeric onium salts (ammonium, phosphonium and sulfonium) as enhancer molecules results in a heightened degree of light emission from the dioxetane on decomposition. This is due to the tendency of these macromolecular polymers to sequester the dioxetane anion, which is highly hydrophobic in non-aqueous “micro environments”, which maximizes chemiluminescent emission. As further disclosed in U.S. Pat. No. 5,145,772, these dioxetanes may be combined with energy accepting fluorescent molecules, such as fluorescein, such that the energy released by the dioxetane on decomposition is transferred to the fluorescent receiver, the fluorescence being detected. The assay of this invention is particularly suited for chemiluminescent emission.  
           [0012]    Neither the 1,2-dioxetanes, nor their preparation or use, per se, of enhancement agents, alone, or with secondary enhancement agents such as those taught in U.S. Pat. No. 5,547,836, constitute an aspect of the invention, per se.  
           [0013]    In the assay of this invention, a microtiter well or similar “reaction chamber” is coated with a capture antibody, which is exemplified by goat anti-rabbit IgG (available from a wide variety of sources, including American Qualex).  
           [0014]    According to the invention, a conjugate of cAMP and alkaline phosphatase is prepared and added to the well with an antibody for cAMP (or other cyclic nucleotide) and the sample to be inspected (or standard). The reaction mixture is incubated, and washed. Thereafter, an alkaline phosphatase-triggerable 1,2-dioxetane, such as CSPD, is added, preferably in the presence of an enhancer, such as poly(vinylbenzyltributyl ammonium chloride) or other enhancement agent. The dioxetane is incubated, and the reaction chamber inspected, preferably with a luminometer, or some other type of light-sensitive device, for chemiluminescent signal. The stronger the signal, the lower the cyclic nucleotide concentration in the sample. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0015]    [0015]FIG. 1 is a flowchart illustration of the method of manufacturing coated plates or reaction chambers of the invention.  
         [0016]    [0016]FIG. 2 is a flowchart illustration of the steps of the assay of the invention.  
         [0017]    [0017]FIG. 3 provides graphic illustration and tabular data comparing the signal obtained by shaking the reaction mixture, as opposed to not shaking it, during the incubation period.  
         [0018]    [0018]FIG. 4 is a schematic illustration of the formation of the alkaline phosphatase/cyclic nucleotide conjugate of the invention.  
         [0019]    [0019]FIG. 5 is omitted.  
         [0020]    [0020]FIG. 6 is a graphic illustration of standardized signals for a variety of concentration of cAMP.  
         [0021]    [0021]FIG. 7 is a table comparing sensitivity and dynamic range of the claimed invention compared with other commercial assays for cAMP.  
         [0022]    [0022]FIG. 8 compares the data obtained for two different luminometers according to the invention of this application. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    The assay protocol of this invention resembles other competitive immunoassays, and provides an inverse relationship between strength of signal detected and cyclic nucleotide concentration. Although the invention is generically applicable to the cyclic nucleotides, including cAMP, cGMP, cCMP, cUMP, cIMP and cTMP, cAMP and cGMP, which frequently appear in a tandem, inverse relationship in a wide variety of biophysical pathways, receive the most attention. cAMP is clearly the most well studied and characterized of the cyclic nucleotides, and accordingly, this invention is exemplified using cAMP and cAMP detection reagents. The substitution of reagents, in particular, primary antibodies, preferably monoclonal antibodies, for the other cyclic nucleotides is straight forward and easily arrived at by one of skill in the art, given the disclosure herein.  
         [0024]    This invention is also exemplified using CSPD, where, in the general Formula I given above, the adamantyl group is substituted with 1 chlorine atom, X is hydrogen, R is methyl and Z is phosphate. Other alkaline phosphatase-triggerable 1,2-dioxetanes corresponding to the general Formula I may be used in its place.  
         [0025]    This invention is also exemplified using an enhancement agent, commercially available from Tropix, Inc. As an exemplary enhancement agent, poly(vinylbenzyltributylammonium chloride) is selected. An enhancement agent is preferred, but not required for the practice of the invention. Other quaternary onium polymers, as well as hydrophobic macromolecules such as large proteins, including bovine serum albumin may be similarly employed.  
         [0026]    In the practice of the invention, the wells of conventional microtiter plates, or similar reaction chambers, are first coated with capture antibody. In the exemplification of this invention, the capture antibody is a goat anti-rabbit antibody. The wells of the plates are coated with a preparation of the capture antibody, incubated over night, and then washed. To avoid non-specific binding, the rest of the plate may be treated, so as to suppress any interaction therewith. Accordingly, after washing, each plate is treated with SuperBlock blocking buffer (available from Pierce) or similar agent. The plate is thereafter dried. This process is reflected in FIG. 1.  
         [0027]    The prepared plates are now ready to perform as reaction chambers, or wells, for the competitive assay. The source of cAMP is either standards prepared to specific dilutions, in order to establish standardized values, or cells that have been subjected to some type of stimulus, and are then lysed, so as to provide sample material for inspection. Thus, the cell lysate or cAMP standard is added to the wells, together with a conjugate of cAMP and alkaline phosphatase, described herein below. Final addition to the reaction mixture is the primary antibody, an antibody for cAMP. Both polyclonal and monoclonal antibodies are available against cAMP. Either may be used effectively. Rabbit anti-cAMP antibodies are available from Zymed Laboratories, Inc. as well Immunogen. Other antibodies are similarly available. The reactants are mixed in the coated well, and then incubated to insure adequate antibody binding. Typically, incubation is at room temperature for a period of about one hour. Subsequently, the incubated reaction mixture is washed repeatedly, with a wash buffer which may be 0.05 M carbonate bicarbonate and 0.05% Tween-20 at a pH of about 8.0-11.0, an exemplary value being about 9.6. While Applicants do not wish to be bound by this theory, the alkaline character of the wash buffer may improve alkaline phosphatase performance. The assay buffer itself is BSA (0.02%) with sodium acetate (0.05 molar) at a pH convenient for the assay, between about 5.5 and 6.0, preferably about 5.8. Those of skill in the art will appreciate that the identities of the blocking buffer, the wash buffer and the assay buffer, as well as the buffer used to coat the microtiter wells, are not rigid features of the invention, per se, but rather can be varied by those of ordinary skill in the art and still employ the claimed invention.  
         [0028]    After washing, the dioxetane, with the enhancer, if any, is added. A further incubation, to permit a glow discharge from the chemiluminescent dioxetane to reach a constant level, approximately 30 minutes at room temperature, follows, and then the chemiluminescent signal is read in a detection device, such as the Tropix 717 or North Star luminometer detection device (CCD). The assay protocol itself is set forth in FIG. 2.  
         [0029]    It is clear that the assay is optimized for automated robotic assay systems, providing high throughput opportunities. Incubation for one hour, as reflected in FIG. 2, is the standard protocol. Results, in terms of sensitivity and dynamic range, may significantly improve if the microtiter plates, or other reaction chambers, are shaken during the incubation period. The relative performance of shaken and non-shaken assays is reflected in FIG. 3 hereto.  
         [0030]    This assay is suitable for use in a wide variety of conditions. The heightened sensitivity, and broadened dynamic range, makes application consistent with a wide variety of variables. When standards have been established, the samples assayed according to this invention can be obtained by lysing mammalian adherent and non-adherent cell lines. Optimal performance can be achieved across a wide range of cell densities, ranging from 1,000-100,000 cells per well, depending on cell type in a 96-well plate. Other plates with a higher number of wells, may be used. The use of luminometers to detect the chemiluminescent signal (light emission) is recommended. If a luminometer is used, an appropriate measure is 1 second per well. In the alternative, a scintillation counter, such as that available under the trademark Top Count may be used a substitute for a luminometer. Sensitivity may be reduced, and it is necessary to turn off the coincident circuit to measure chemiluminescence directly.  
         [0031]    The “competitive” basis of the assay is the use of a conjugate of cAMP and alkaline phosphatase (cAMP-AP conjugate). It is the alkaline phosphatase of this cAMP-AP conjugate, captured by the antibody specific for cAMP, that cleaves the alkaline phosphatase-cleavable dioxetane. Thus, the lower the cAMP concentration in the lysed sampled, the more cAMP/AP conjugate is captured by the primary antibody, and the larger the signal. Preparation of the conjugate is straight forward, and comprises combining NHS activated cAMP with alkaline phosphatase (in a ratio of 4 moles activated cAMP to 1 mole AP). This preparation is reflected in FIG. 4.  
         [0032]    As noted above, it is necessary to establish standards, so that actual concentrations in lysed samples can be determined, on the basis of established concentration. A typical standard curve collection is reflected in FIG. 6, which shows the superior performance of this assay.  
         [0033]    Sensitivity and dynamic range for a variety of cAMP detection assays have been published. Sensitivity and dynamic range of a variety of detection systems are set forth in FIG. 7. It is clear that the unexpected improvements in sensitivity and dynamic range obtained by combining dioxetane chemiluminescent technology with cAMP-specific immunoassay techniques has led to an unexpectedly superior performance, even in the absence of acetylation.  
         [0034]    As previously noted, the cyclic AMP chemiluminescent assay of the invention is preferably detected by the luminometer. Two exemplary luminometers are compared in FIG. 8, TR717 verses Orion CCD. Both give the improved performance advantangeously provided by this invention.  
         [0035]    This invention has been disclosed generically, and in terms of specific examples. The specific examples are not intended, and should not be construed, as limiting, unless so indicated. In particular, variations in the identity of the dioxetane, buffer compositions, signal detecting apparatus, protocol time, temperatures and conditions and the like will occur to those of ordinary skill in the art without the exercise of inventive faculty. Unless excluded by the recitation of the claims set forth below, these variations remain within the scope of the invention.

Technology Category: g