MXPA96004646A

MXPA96004646A - Functionalized hydrofilic acridine sterriesnovedo

Info

Publication number: MXPA96004646A
Application number: MXPA/A/1996/004646A
Authority: MX
Inventors: Law Sayjong; Sotiriouleventis Chariklia; Natrajan Anand; Jiang Qingping; B Connoly Peter; P Kilroy Jhon; R Mccudden Constance; M Tirrell Stephen
Original assignee: Chiron Diagnostics Corporation
Priority date: 1994-04-08
Filing date: 1996-10-07
Publication date: 1998-02-01

Abstract

Novel acridinium esters are disclosed which are either singly or when incorporated into liposomes as chemical phosphorescence agents in binding analysis (v .gr, immunoassay and gene test analysis) with improved sensitivity. In addition, the synthesis of these esters and their use to detect an analyte is described. In particular, the analyzes for testosterone and rubella virus are described

Description

NON-PROCESSED, NOVELED FIELD OF ACTION OF THE INVENTION Field of the Invention The present invention relates to a novel method for the detection of an analyte. The present invention relates to the detection of an analyte using acridinium esters as markers of chemical phosphorescence, which can be encapsulated within liposome vesicles without significant leakage of vesicle esters. The present invention also relates to the synthesis and use of novel functionalized hydrophilic acridinium esters, which are useful as chemical phosphorescence labels, and surprisingly, give a much higher quantum yield, than the previous acridinium ester compounds. The present invention also relates to novel conjugates formed from said functionalized hydrophilic acridinium ester compounds.

The present invention further relates to analyzes using these novel functionalized hydrophilic acridinium esters, and conjugates thereof. The present invention relates to immunoassays using the compounds of the present invention. Background of the Invention The use of acridinium esters as chemical phosphorescence labels is known in clinical analyzes. For example, that described in European Patent 0 082 636. The use of an aryl acridinium ester activated with a portion of N-hydroxy-succinimidyl as a chemical phosphorescence label in immunoassay. The patents of E. U.A. No. 4, 745, 181, (see European Patent 0 263 657); 4.918, 192; 5,241, 070, (see European Patent 0 361 817), discloses polysubstituted aryl acridinium esters (EAAP) which are useful in immunoassay and nucleic acid hybridization assays. The Patent of E. U.A. No. 5,227,489 (see European Patent 0 353 971) and the Patent of E. U.A. 5,449,556, which is the mother of the present application, discloses hydrophilic polysubstituted aryl acridinium esters, and lumisome conjugates thereof, useful in clinical analyzes, particularly those involving liposomes. European Patent 0273 1 15 describes acridinium and phenanthridium salts of chemical phosphorescence. Zomer et al. (227 Analytica Chimica Acta (1989) 1 1) discusses aspects for chemical phosphorescence labels for immunoassays. The 2'-6'-dimethyl-4 '- (N-succycidydoxycarbonyl) phenyl-10-methyl 9-acridinocarboxylate methylsulfate (DMAE-NHS) as described in the U.A. No. 4,745, 181, requires the use of a phenoxy group substituted with a benzyloxycarbonyl group as an intermediate to form the acridine ester via a long synthetic route. It is desirable to develop new and efficient methods for synthesizing the useful acridinium ester labels of the present invention. The unexpected ability to form 2,6-dimethyl-4 '- (N-succinimidyloxycarbonyl) phenyl 9-acridincarboxylate (DMAeE-NHS) by the simplified procedure of combining a solution of 3,5-dimethyl-4-hydroxybenzoate and 4-dimethylaminopyridine with 6-acridinocarbonyl chloride hydrochloride was not easily predicted in view of the coexistence of two reactive leaving groups in the same reaction, an acid chloride of 9-acridinocarbonyl chloride and a succinimidyl ester of 3.5 -dimethyl-4-hydroxybenazoate of succinimidyl. For example, in the case where N-succinimidyl 3 (4-hydroxyphenyl) -propionate is reacted, the 9-acridinocarbonyl chloride, referred to above, the condensation can be carried out under average conditions (approximately ambient temperature) due to the absence of the two methyl groups in the ortho positions. (See, for example, U.S. Patent No. 4,946,958, columns 4-5, wherein the reagent does not contain the methyl groups substituted with ortho). On the other hand, when the two methyl groups are present, much more drastic conditions are required (100 degrees C. for 2 hours), so that the condensation reaction takes place, due to the spherical obstacle caused by the methyl groups. Although it should be noted, that there is a benefit provided by the presence of the methyl groups, namely, the added stability of the resulting acridinium ester, as discussed, for example, in the U.S. Patent. No. 4,745, 181. The lipophysical nature of the acridinium esters of the prior art and other chemical fluorescence compounds renders them unsuitable for encapsulation within liposomes since their rapid leakage through the wall of the liposomes. Additionally, the limited water solubility of prior art acridinium esters and other chemical fluorescence compounds only allow the encapsulation of few marker molecules per liposome vesicle, resulting in a relatively low signal amplification. The novel functionalized hydrophilic acridinium esters of the present invention are useful in immunoassay, the results produced are superior in sensitivity to previous methods, and do not need to use hazardous radiolabels, or organic substrates / enzymes. The use of chemical fluorescence labels of this type, resulted in an improvement not expected during previous methods, and can lead to the functional improvement of previous analysis methodology, inoperable or inaccurate. The novel discovery that the hydrophilic acridinium esters of the present invention could be used to label biomolecules and compounds directly without reducing the solubility of the complex, allows many applications in the field of immunoassay. The novel compounds of the present invention will allow for sensitive immunoassay without the need for excessive use of reagents in the reaction mixture, and are therefore designed to reduce non-specific interactions, as long as it does not interfere with the specific interactions desired. Another unexpected benefit of having an EAAP carrying a hydrophilic moiety, particularly as a substituent on the nitrogen atom of the acridinium nucleus, is the significant improvement of the quantum yield of chemical phosphorescence relative to that of acridinium esters that simply have an alkyl group (Patent of US Pat. No. 4,745, 181) or carboxymethyl group (G. Zomer et al., 1989, Anal. Chem. Acta 227: 11-19) substituted in the same position. The area of immunoassay is well developed, and it is the discovery of unique tagging systems that can drive a quantum advance in the field of immunoassay. In the field of immunoassay, it is convenient to have analyzes that are highly specific, and highly sensitive to low concentrations, and still produce detectable signals and distinguish for measurement. The present invention allows the specific detection of analytes at low concentration, without losing solubility. The present invention provides means for making many specific marker agents, which can be used to detect low levels of analytes. The present invention also teaches someone with ordinary skill in the art, novel and useful means to carry out the immunoassay that easily leads to automation and marketing as analytical equipment and reagent equipment. The application of novel acridinium esters of the present invention, in conjugates with bioactive proteins such as avidin, antibodies, DNA binding proteins, histones, and ribosomes, and others, is possible because of the hydrophilic properties of the synthesized compounds. These novel acridinium esters are also useful for labeling RNA, DNA, proteins, peptides, inactivated protein, neurotransmitters, hormones, viruses, viral antigens, bacteria, bacterial antigens, toxins, cytokines, antibody fragments, receptor proteins, isolated or intact, and others, such as whites both in vitro, and in vivo. Sampling of tissue samples, serum samples, or other samples such as biological samples, and the detection of specific analytes difficult to measure previously in rapid analyzes, is possible by the compositions and methods of the present invention. A specific application of the present invention is in the field of pathogenic detection. While there have been analyzes developed to detect viral pathogens, such as Rubella, the present invention provides a unique and highly sensitive analysis that is unexpectedly superior to previous methods of chemical phosphorescence. The methods of the present, are comparable with conventional analyzes in sensitivity, are still more efficient in the time required to carry out the analysis. Another application of the present invention is in the detection area of hormones or haptens or other biologically active small molecules in biological samples. When the hormone or hapten labels are low and temporary, it is useful to have a sensitive and rapid method to test, and to measure the levels of hormones or haptens in said biological samples. One of the possible examples is the detection of levels of steroid hormones such as testosterone. The detection of testosterone using homologous and heterologous hapten conjugates is known. In this methodology, the antibodies are generated to a form of * _ »• testosterone immunogen. Then, said antibodies are used to detect testosterone in a sample, while a conjugated competitive hapten indicator is added, which has a different form to that of the immunogen. Homologous analyzes are loaded with unacceptably high cross-reactivity such as when conjugated haptens of C4-testosterone-B-al, and conjugates of T-3-O-CMO-glucoamylase are used. Somewhat lower cross-reactivity has been obtained with 11-substituted hapten-HRP, and T-3-O-CMO hapten-penicillinase (Rao et al., 1992, Steroids 57: 154-162). The The present invention provides superior detection on analyzes of previous heterologous haptens, in that the hydrophilic acridinium ester labels of the present invention contribute to the reduced nonspecific interactions. Accordingly, the purpose of the present invention is provide novel functionalized hydrophilic acridinium esters and conjugates thereof to be used as indicators of chemical phosphorescence. It is also a purpose of the present invention to provide novel methods for detecting an analyte using functionalized hydrophilic acridinium esters and conjugates of the same. It is also a purpose of the present invention to provide a new improved synthetic process for the efficient production of acridine esters. Compendium of the Invention Novel acridinium esters are described that are useful, since either alone or when incorporated into liposomes, as chemical phosphorescence agents in binding analysis (eg, analysis and analysis and analysis of genetic tests) with improved sensitivity. In addition, the synthesis of these esters and their use in analysis to detect an analyte are described. In particular, analyzes for testosterone and Rubella virus are described. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram illustrating the improved synthesis of DMAE-NHS. Figure 2 is a diagram illustrating the old method of synthesizing DMAE-N HS. Figure 3 is a normal curve for a testosterone phosphorescence immunoassay using an NSP-DMA E-19-HCMT indicator, which uses the compounds of the present invention.

Figure 4 is a comparison of binding curves for heterologous and homologous indicators in a chemical phosphorescence immunoassay for testosterone, using the compounds of the present invention. The figure also shows a comparison between the use of hydrophilic versus non-hydrophilic acridinium esters. Figure 5 is a graph showing the correlation of results of a testosterone immunoassay of ACS using the compounds of the present invention with radioimmunoassay "Coat-a-Cou nt DPC" (Diagnostic Products Corp., Los Angefes, CA). Figure 6 is a bar graph showing a population study comparing the positive to negative relationship of the test results using the compounds of the present invention compared to the rubella analysis of DMAE. Figure 7 shows the analysis architecture for a Testosterone analysis of ACS, which operates on the "Ciba Corning ACS-180" instrument. Figure 8 shows the architecture for an estradiol analysis of ACS, which is carried out in the "Ciba Corning ACS-180" instrument. Figure 9 shows the analysis architecture for a specific IgE analysis of aCS, which is carried out in the "Ciba Corning ACS-180" instrument. Description of the invention The following terms, as used in the specification and claims, should have the following meanings: Analyte: the compound or composition to be measured which may be a ligand that is mono- or polypeptide, antigenic, or haptenic . The analyte can be a piece of DNA or RNA. The analyte can be found in liquid or on solid support. Conjugates: the combination of chemical phosphorescence compound with a second compound or molecule. Said conjugates may have several molecules of chemical phosphorescence per second compound. Indirect conjugate: the combination of the chemical phosphorescence compound with a second compound or molecule. The chemical phosphorescence compound combined with a second compound or molecule, is then able to be combined with a third compound or molecule. Such conjugates can have many chemical phosphorescence molecules per complex. Homologous: the same compound bound by the same ligand in the same position. Heterologist: the compound bound by a different ligand in a different position, a different linker in the same position, the same ligand in a different position. Ligand: a bridge, binding, physical or chemical connection, between two compounds or molecules; bifunctional or multifunctional ligature. The binding of a ligand to a compound may allow a reactive site to be available for further reaction. Hapten: an incomplete antigen, unable on its own to elicit an immune response, but when properly bound to another molecule, it becomes capable of producing antibodies that will specifically recognize the hapten. The acridinium esters modalized by the present invention, and useful in the methods of the present invention, can be any acridinium ester which is a hydrophilic acridinium ester, a functionalized form of said compound, or conjugate thereof, and which can generate a chemical phosphorescence signal. The EAAP of the present invention, with an additional electrophilic or protruding group, is useful for the conjugation of many targets. The hydrophilic portion of said EAAP (comprising an alkyl separator carrying a highly ionizable group such as sulfonate, phosphonate, sulfate, phosphate) is responsible for increasing the aqueous solubility, thus reducing the hydrophobic nature of the indicators, particularly those formed from the conjugation of EAAP with small organic biomolecules such as steroids, therapeutic drugs, and other controlled chemical substances. When applied in macrobiomolecules of marking such as proteins, nucleic acids, and polysaccharides, the hydrophilic EAAP allows the increase in the incorporation ratio (defined as the number of EAAP molecules covalently linked to a single macrobiomolecule) without any problem of solubility. . Therefore, the increase in activity and wash capacity specific to the indicator would be a factor to increase the sensitivity of the analysis of union and improved signal / noise ratio. The lipophilic nature of the acridinium esters of the prior art and other chemical phosphorescence compounds makes them difficult to use when conjugated to certain biological compounds since there was a significant amount of nonspecific binding due to the hydrophobic nature of the indicator conjugate . Therefore, the acridinium esters of the prior art, and other chemical phosphorescence compounds, were not efficient to be used when detection required fine distinction between the specific binding signal against non-specific, and resulted in an increased risk of results. False positives or false negatives.

Preferred functionalized acridinium esters include acridinium esters of the following formula: wherein Ri is alkyl, alkenyl, alkynyl, aryl or aralkyl, having up to 24 carbons and up to 20 heleroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur; R2, R3, Rs, and R7, are hydrogen, amino, hydroxyl, halide, nitro, -CN, -SO3H, -SCN, -R, -OR, -NHCOR, -COR, -COOR, or -CONHR, wherein R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, having up to 24 carbons and up to 20 carbon atoms; heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur; R4 and R1 are alkyl, alkenyl, alkynyl, aralkyl or alkoxy having up to 8 carbons, without branching wherein the side chain groups have more than 2 carbons; R6 represents the following substitutions: wherein R9 is not required but optionally may be an alkyl, or aralkyl, group having up to 5 heteroatoms selected from the group of Phosphorus, Sulfur, Nitrogen, Oxygen, and R 10 is an electrophile, a leaving group , or a group with these two natures combined, as shown in the following examples: ^ N. ^. S / N ^ ^ ° - "N« 3 ,,. A halide -COOH wherein Y is a halide and R is alkyl, aryl, aralkyl, and; wherein the substituent positions R3, R6, and R7 are interchangeable in the phenoxy ring. More preferably, R1 is a sulfopropyl or sulfoethyl group; R2 is hydrogen, methoxy, ethoxy, nitro or halogen; R3, Rs, and R7 are hydrogen; R and R8 are methyl, ethyl or isopropyl groups; R6 is N-succinimidyloxycarbonyl; N-succinimidyloxycarbonylalkyl, or carboxylate. Unexpectedly, EAAP with the substituted hydrophilic moiety (e.g., 10- (3'-sulfopropyl) -acridinium-9-carboxylate of 2 \ 6'-dimethyl-4'-8N-succinimidylsycarbonyl) phenyl (NSP) was found. -DMAE-NHS)) is able to generate chemical phosphorescence quantum yield approximately 1.5 times higher, compared to the corresponding EAAP with simple alkyl substitution in the same position (eg, 10-methyl-9- methyl-sulfate). 2 ', 6'-dimethyl-4'-8N-succinimidyloxycarbonyl) phenyl acridinocarboxylate (DMAE-NHS)). (See Table I). The increase in the phosphorescence quantum yield of EAAP, is a very desirable and essential factor to improve the signal to noise ratio (S / N) of the chemical phosphorescence indicators and the sensitivity of the chemical phosphorescence binding analysis. detection schemes. In particular, wherein the signal increase of the indicator is mediated through multiple EAAP tags of a ligand / vehicle biomolecule conjugate, such as a protein, an improvement of 1.5 sc see or more in chemical phosphorescence quantum yield of EAAP will mean that less EAAP is required to be united to achieve the same signal amplitude. Also, if the same levels of EAAP binding to vehicle conjugates are achieved, it may be possible to detect lower concentrations of an analyte. TABLE I Relative Quantum Performance of DMAE-NHS versus NSP-DMAE-NHS Ratio of AE Type Weight Mol. RLU / 4.2 fmol AE R L U DMAE-NHS 595 690,187 1.0 644,587 743,587 average 693,787 SD 49,551 15% C.V. 7.2, ~ ** NSP-DMAE. NHS 591 1,122,017 1.7 1,204,017 1,122,017 mean 1,149,350 20 SD 47.343% C.V. 4.1 In Table I, DMAE-NHS was dissolved in acetonitrile, while NSP-DMAE-NHS in 50% (v / v) of acetonitrile in water to generate stock solutions of 1.0 mg / ml. The serial dilutions were carried out at 107 with scintillating pH buffer diluent (10 mM Phosphate, 150 mM NaCl, 0.1% ASB, 0.05% NaN3, pH 8.0). The dilution method consists of three steps of dilution in 100 times, using transfer volumes of 50 uL and in the dilution step in 10 times, using transfer volume of 100 uL. The light emission of the samples was determined in Analyzer "Ciba Corning Diagnostics MLA1" under normal conditions (light collection time of 2 sec.) Using 25 uL of dilution solutions at 10"7. There are many modifications that someone with experience ordinary in the art will recognize, which are within the scope of this specification. A number of spacers can be attached, which will facilitate the connection of the hydrophilic group to the nitrogen atom in the acridinium core, and an electrophilic or leaving group that will facilitate the covalent attachment of a hydrophilic EAAP to other compounds or biomolecules. The novel acridinium esters of the present invention are highly soluble in water and can be encapsulated in liposomes at high concentrations. Once the liposomes are in high concentrations, the novel acridinium esters remain encapsulated for extended periods and do not leak appreciably. It will be appreciated that while the novel acridinium esters of the present invention are useful for encapsulation within liposomes, the novel acridinium esters of the present invention are also useful in other applications where acridinium esters, such as ligands, are used. or analyte markers (such as antigens); labeling the specific binding partners of the ligands or analytes (such as the corresponding antibodies); or by labeling nucleic acids and molecules comprising nucleic acids. In particular, the novel functionalized hydrophilic acridinium esters of the present invention will be useful for labeling biological materials for chemical phosphorescence analysis where it is preferable to reduce the non-specific binding of indicator to biological material. The novel functionalized hydrophilic acridinium esters of the present invention will allow a reduction in signal / noise interference (S / R ratio), which will allow greater accuracy in making measurements and for diagnostic analyzes, since the hydrophilic nature of the Novel compounds of the present invention will be less prone to non-specific interactions compared to other chemical phosphorescence compounds. The synthesis of acridinium esters has been described in the patent of E.U.A. No. 4, 745, 181, and the process is shown in Figure 2. This method involved the use of a phenoxy group substituted with a benzyloxycarbonyl group to form the acridine ester. The desired succinimidyloxycarbonyl was then introduced through the removal of the benzyl protecting group and the condensation of the product with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.

In addition, the present invention provides a new and improved method of synthesis. This method has the advantage of unexpectedly discovering that the core, under appropriately selected conditions, can be directly condensed with the phenoxy group carrying succinimidyloxycarbonyl, without any problem, this is illustrated in Figure 1. Using this new and simpler process, two synthetic steps of the old process are eliminated, for the preparation of hydrophilic EAAP with the appropriate electrophilic or outgoing group. The generalized reaction scheme of Figure 1 also applies to the preparation of substituted acridine acid chlorides, such as and N-hydroxysuccinimide ester of substituted p-hydroxybenzoates, such as wherein R 2, R 3, R 5, R 7, are hydrogen, halide, nitro, -R, -OR, -CN, -N HCOR, -COR, -COOR, or -CON H R; wherein R is alkyl, alkenyl, alkynyl, or aralkyl, and wherein R4 and R8 are R, and R is as defined above. In this way, the present invention provides hydrophilic EAAP with an additional electrophilic or leaving group, which are useful for conjugation with mono- or polynucleophilic compounds or biomolecules. The present invention thus provides the covalent attachment of functionalized hydrophilic EAAP with nucleophilic compounds such as hexyl-1, 6-diamine, ethylenediamine, and aminocaproic acid. The present invention also provides the covalent attachment of functionalized hydrophilic EAAP with biomolecules such as aminoglycosides, proteins, viral antigens, peptides, and oligonucleotides functionalized with aminoalkyl. These conjugates will be useful for sensitive immunoassays and nucleic acid binding assays. In addition, all attributes of the present invention are similarly applicable to related acridinium compounds, including, but not limited to, benzacridinium esters, such as those claimed in co-pending application EP 0617 288. An example of a sensitive chemical phosphorescence immunoassay , useful, which uses the compounds and conjugates of the present invention, is one that can detect specific steroid hormones in serum samples. An improved analysis using the conjugates and methods of the present invention is an accelerated assay for testosterone. This analysis combines the use of a highly specific antibody, developed for testosterone and a heterologous indicator formed by conjugating a C19-mono or C19-bi-substituted testosterone, with a hydrophilic EAAP, functionalized. The benefit of using the heterologous chemical phosphorescence indicator is observed through the improvement of sensitivity of testosterone analysis at 0.1 ng / mL, which can not be achieved , ... quickly somehow, (that is, approximately 5 minutes) with the use of the corresponding homologous indicator, or using hydrophobic EAAP. Therefore, the present invention also provides the use of functionalized hydrophilic EAAPs, which were formed run conjugated by novel C-type of C19 bound, testosterone derivation (as described in the following Examples). These novel heterologous indicators, can be ^ formed, optionally with additional olefinic ligation at C19, or additional heteroatom attached at C19, such as the following O-linked derivative, of C 19, of testosterone (NSP-DMAE-HD-19-CMET).

Therefore, the present invention also provides testosterone derivatives of the following structures. (Seven examples are shown below, along with a model that identifies the numerical system used for the nomenclature). wherein A is a functionalized group selected from the group consisting of -OH, -NfR Rz, -NHRi, nitro, halide, -OR ,, -OCORi, -tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, SR1, nitro, -SH, -SSR ,, -phosphonate, -sulfonate, -NHCOOR ,, -NHCOR1, -NHCONH2, hydrazines, cyanide, and -R ^ wherein R, is an alkyl, alkenyl, alkynyl, aralkyl, contains up to 10 carbons and up to 5 heteroatoms; where B is - (CH2) n where n = 1-4 or -C =; when B is -C =, preferably A is omitted; wherein X is a carboxylate, -COOR2, -CONHR2, (wherein R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl containing up to 15 carbons and up to 5 heteroatoms), or a single carbonyl with appropriate leaving groups, including, but not limited to halide, N-hydroxysuccinimide, and imidazole. The improved utility of the hydrophilic EAAP of the present invention in immunoassays of therapeutic drugs or hormones such as testosterone, illustrates one aspect of the invention modalized in the present specification. Specifically, the use of testosterone derivatives wherein the bound bridges of C19-B (as shown in the previous figure) are C19-C and an important element for the effective use of the compounds of the present invention. It has been found that the use of a C 19-C binding, balances the reduction of the indicator that binds to the anti-testosterone specific antibody, and the ease of its displacement from the antibody by the competent sample testosterone. A preferred embodiment of the testosterone indicators of the present invention, which will be used for conjugation to functionalized hydrophilic EAAP, via bifunctional crosslinkers, encompasses the forms represented by formula I above. More specifically, it is preferred that A is hydroxy, B is -CH2-, and X is carboxylate. These compounds are novel, being distinguishable from those known in the prior art. For example, the compounds cited in Rao (Patent of E. U.A. No. 4, 197,286) contain ligatures of C19-O (ie, C19 to oxygen). Using the teachings of the present specification, one of ordinary skill in the art will recognize the use of bifunctional ligatures for conjugation. A preferred embodiment encompasses a functionalized hydrophilic EAAP, binds n-hexyl-1, 6-diamine at one end through the formation of amide ligature, and subsequently to the target biomolecule, such as the testosterone derivative by forming of another amide ligature with the other end, as exemplified by the following (NSP-DMAE-HD-19-HCMT).

Another preferred embodiment of the testosterone indicator is a form of! compound of formula I above, where A, B is omitted is -CH = forming an olefinic bond with C 19, and X is carboxylate, as shown below (NSP-DMAE-H D-19-CVNT).

A further aspect of the present invention encompasses the unexpected improved results of using functionalized hydrophilic EAAP to label viral antigen as used in various chemical phosphorescence immunoassays of infectious diseases. For example, in an IgG capture analysis format, wherein the immobilized solid phase of mouse anti-human IgGFc and the viral antigen indicator, are the reagents used to analyze antiviral antibodies in the serum sample, it was found that the greatest improvement in the signal to noise ratio of the analysis can be achieved when a functionalized hydrophilic EAAP is used, instead of conventional DMAE-N HS, to prepare the viral antigen indicator. These results most likely occur via better binding activity that results in increased immunocomplex formation between indicator and white antibodies, and decreased nonspecific binding due to the use of hydrophilic acridinium ester labels. In this way, someone with ordinary skill in the art could take the teachings of the present invention and apply them to the detection of a variety of infectious or pathogenic agents. These infectious agents such as Rubella, Hepatitis of various kinds and subtypes, Toxoplasma, Cytomegalovirus (CMV), HIV, Chlamydia, to name but a few, are targets for early detection that will require highly sensitive analyzes and low incidence of positive indication false. A normal architecture of said analysis consists of, but is not limited to, three components, an indicator, a sample containing the analyte, and means for dividing the binding of the unbound analyte. For example, such analyzes could combine the hydrophilic functionality, with appropriate antigen or agglutinant specific for an analyte, to form an indicator, incubation with a biological sample (containing the analyte), and capture of the indicator by an immobilized antibody in solid ase . Other architectures will be practical with the application of the teachings of the present invention, and are readily apparent to those with ordinary experience in the immunoassay technique. The present invention encompasses the use of the compositions and methods of the present invention for allergen immunoassays. The hydrophilic EAAPs of the present invention will be useful for the detection of IgE Ab specific for specific allergens. In this application, hydrophilic EAAPs are unique to streptavidin, and can be used to detect specific biotin-allergen molecules, linked to allergen-specific antibodies. (See an example for the architecture of a specific IgE analysis for ACS, Figure 9). Thus, the present invention encompasses methods for measuring the amount of analyte comprising a sample and detecting an analyte specifically and in proportion to its concentration in a sample, and measuring a signal that is generated by a chemical phosphorescence label of hydrophilic acridinium ester, which is directly or indirectly proportional to the concentration of the analyte in a sample. Said methods for detecting an analyte in a sample, comprise contacting a sample with a hydrophilic acridinium ester-labeled detector molecule, sequestering the bound detector and analyte, washing the detector in excess, and measuring a signal from a detector attached to the detector. analyte In addition, methods for detecting an analyte in a sample comprising, contacting the sample with a competitive indicator marked hydrophilic acridinium ester, and a specific binder for an analyte, recovering the specific binder, and determining a signal generated by the united indicator. (See an example for the analysis architecture of a testosterone analysis of ACS, Figure 7). Also encompassed are methods for detecting an analyte in a sample comprising contacting a sample with a labeled competitive indicator of hydrophilic acridinium ester, and a specific binder for an analyte, recovering the specific binder, and determining a signal generated by the indicator not united. Methods for detecting an analyte in a sample comprising, contacting a sample with a specific first binder, and a second specific binder labeled with a hydrophilic acridinium ester, and detecting a signal are included. These analysis architectures may have both the form of sandwich analysis or analyzes in which the second binder (or antibody) is reactive with the first binder. Different variations of these architectures will be evident, for those with ordinary experience in the art. The present invention encompasses methods for detecting an analyte in a sample comprising, contacting a sample with a hydrophilic acridinium ester labeled detector and a competitive binder for the detector, sequestering the detector bound to the competitive binder, washing the detector in excess and the analyte bound to the detector, and measuring a detector signal bound to the competitive binder. (See an example for the analysis architecture of an estradiol analysis of ACS, Figure 8). The methods of the present invention can be practiced with or without the addition of release agents prior to the detection of a signal, or other agents that will increase the signal generated.

A further aspect of the present invention encompasses the use of the compositions and methods of the present invention for gene test analyzes. For example, the low concentration of the target nucleic acids (the analyte) can be detected by hybridizing to a first nucleic acid test, which is conjugated to a macromolecular carrier, such as bovine serum albumin, glycoproteins, etc. , labeled with multiple hydrophilic EAAP and optionally, multiple hydrophilic polymers, such as polyethylene glycol (PEG), and capturing with a second nucleic acid probe, which is immobilized on a solid phase such as paramagnetic particles. The two nucleic acid probes may be preferentially ligated with ligase after the hybridization step, but before the separation step (which removes the unbound probe indicator remaining in the supernatant) to increase the specificity of the assay. Due to the greater number of EAAP incorporated in the indicator due to the hydrophilic nature, good solubility is maintained. Said nucleic acid detection can be achieved using various schemes that one of ordinary skill in the art can conceive, and using the teachings of the present invention, develops the appropriate indicators of hydrophilic EAAP for sensory detection. The following examples are presented to illustrate the present invention, and are not intended to limit the scope of the present invention in any way. EXAMPLE 1 A Enhanced Synthesis of 2'-6'-dimethyl-4 '- (N-succinimidyloxycarbonyl) phenyl 9-acridinocarboxylate (DMAeE-NHS, precursor of DMAE-NHS and NSP-DMAE-NHS) 3,5-dimethyl- Succinimidyl 4-hydroxybenzoate A solution of 3,5-dimethyl-4-hydroxybenzoic acid (12.0 g, 71.21 mmol) in 400 ml of tetrahydrofuran anhydride and 200 ml of anhydrous N, N-dimethylformamide was cooled in a water bath. ice treated with N-hydroxysuccinimide (8.31 g, 71.21 mmol) with stirring for 15 minutes, followed by the addition of dicyclohexylcarbodiimide (17.88 g, 86.65 mmol). After 16 hours of stirring at 15 ° C under nitrogen, the mixture was filtered; and the wet cake was washed with 10 ml of N, N-dimethylformamide. The combined filtrate was evaporated to dryness under reduced pressure. The residue, after washing with anhydrous diethyl ether (2 x 50 ml), was suspended in 80 ml of boiling ethyl acetate for 5 minutes.The cooling of the suspension at 25 ° C gave 1 1 .95 g (63% yield). yield) of the off-white product, which was homogeneous in LC (Rf 0.6, silica gel, ether). 9-Acetidinocarbonyl chloride hydrochloride A mixture of 9-acridinocarboxylic acid hydrate (10.0 g, 44.80 mmol) in 80 ml of thionyl chloride, heated to reflux at 100 ° C under nitrogen for 2 hours, and then cooled to 25 ° C. The resulting solution was reduced to about half the original volume under reduced pressure, and then poured into 500 ml. of anhydrous diethyl ether The yellow precipitate was collected, washed with ether (3 x 100 ml), and dried under reduced pressure for 2 hours, to yield 12.95 g (-100% yield) of the product, which was used immediately for the next reaction 9-acridine 2 ', 6'-dimethyl-4' - (N-succinimidyl-carboninphenyl) carboxylate (DMAEeE-N HS) A solution of 3, 5-dimethyl-4-hydroxybenzoate of succinimidyl (1.29 g, 42.88 mmol) and 4-dimethylaminopyridine (2.19 g, 17.93 mmol) in 200 ml of anhydrous pyridine, was cooled in an ice water bath, followed by the addition of 9-acridine-carbonyl chloride hydrochloride (12.95 g, 44.80 mmol). The solution was heated with stirring under nitrogen at 100 ° C for 2 hours, and then at 25 ° C for 16 hours. After removal of the solvent under reduced pressure, the residue was flash chromatographed on a column of silica gel (Baker silica gel, Cat # 7124-1; F7 x 50 cm) packed with 50% diethyl ether / hexane, and was eluted with 75% diethyl ether / hexane (2.5 L), diethyl ether (2 L), ethyl acetate / 5% diethyl ether (3 L) and ethyl acetate / methylene chloride % (5 I). The fractions containing the product (Rf 0.4; LC silica gel, ether), was collected from the eluent of ethyl acetate / methylene chloride at 25%, combined, and evaporated to dryness under reduced pressure, to collect the light yellow product (DMAeE-N HS) in 10.03 g (50% yield of 9-acetylcarboxylic acid hydrate). EXAMPLE 1 B Improved Synthesis of 2 ', 6'-dimethyl-4' (N-succinimidyloxycarbonyl) phenyl 10-methyl-9-aceidinocarboxylate (DMAE-NHS), Using Toluene as Solvent. This procedure uses toluene as a solvent in methylation of DMAeE-NHS, to replace the reduced reagent with ozone 1,1-trichloroethane, which was previously used as the solvent in the methylation process as described in the Patent of E. U .A. # 4.45, 181. A suspension of DMAeE-N HS (1.0 g, 2.13 mmol) in 50 l of toluene anhydride was heated at 10 ° C to give a homogeneous solution, which was cooled to room temperature. environment followed by the addition of 11.0 ml (16.25 mmol) of distilled dimethyl sulfate.

After stirring for 20 hours at 1 10 ° C under nitrogen, the solution was cooled to room temperature, and then at 4 ° C. 1 hour. The mixture was filtered, and the yellow wet cake was washed with toluene (2 x 5 ml) followed by anhydrous methylene chloride, heated to boiling for 2-3 minutes, and filtered. The volume of the filtrate was reduced to approximately 50 ml on a hot plate. Cooling the concentrate to room temperature gave yellow crystals, which were collected and washed with diethyl ether (3x20 ml), to give 500 mg (39%) of the pure product. FAB / MS (483, M +). EXAMPLE 2 Synthesis of 10- (3'-sulfopropyl) acridinium-9-carboxylate 2 ', 6'-dimethyl-4' - (N-succinimidyloxycarbonyl) phenyl (NSP-DMAE-NHS) DMAeE-NHS (500 mg, 1 .07 mmoles) and 1,3-propanesultone (6.5g, 53.3 mmoles) were heated at 150 ° C under nitrogen in a sealed tube for 20 h. After cooling, the excess 1, 3-propan-sultone was removed by trituration with toluene (3x5 ml). The crude product was purified by RP-CLAR using a C 18, 30 x 500 mm, 10 μm, preparative column, and eluted with a mixture of 65% of solvent A and 35% of solvent B. Conditions: solvent A: 0.05 % (v / v) TFA / H2O, solvent B: 0.05% (v / v) TFA / CH3CN; flow rate 25 ml / min; UV detection at 260 nm. The product was eluted at 26.8 min; yield: 60 mg (25%); FAB / MS (591, M + H). EXAMPLE 3 Synthesis of 19-carboxymethyl-17β, 19-dihydroxy-4-androsten-3-one (19-h id roxi-19-carboxy methyl-testosterone, 19-HCMT) 3, 17-Bis (ethylenedioxy) 19-ethoxycarbonylmethyl-19-hydroxy-5-androstene A 2-neck flask equipped with a rubber septum Magnetic stirring bar and N2 inlet was charged with lithium bis (trimethylsilyl) amide (12.9 ml of 1.0 M solution, 12.9 mmol). The solution was cooled to -78 ° C under N 2 in an acetone bath on dry ice, and ethyl acetate anhydride (1.2 ml, 12.9 mmol) was added dropwise from a syringe. The reaction was stirred at -78 ° C for 30 minutes and then a solution of 3,17-bis (ethylenedioxy) -5-androsten-19al (1 g, 2.58 mmol) was added dropwise (Lovett, J.A. others, 1984, J. Med. Chem 27: 734-740) in tetrahydrofuran anhydride (15 ml), to the enolate solution. After the addition was complete the reaction was stirred for an additional 1.5 hours in the acetone bath on dry ice under nitrogen. The reaction was then quenched with the addition of saturated ammonium chloride solution (-30 ml) and the resulting suspension was extracted three times with ethyl acetate (3 x 25 ml). The combined ethyl acetate extract was washed once with brine (-30 ml) and then dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure to produce an oil which was used as such in the following reaction: yield 1.67 g (crude); mass spectrum (chemical ionization) m / z 477 (M + H +). 3, 17-dioxo-19-ethoxycarbonylmethyl-19-hydroxyl-5-androstene The crude bis-ketal of the above (1.6 g) was dissolved in tetrahydrofuran (20 ml) and 3N perchloric acid (10 ml) was added. The reaction was stirred at room temperature for 3 hours and diluted • v * 34 then with water (40 ml). The resulting suspension was extracted twice with ethyl acetate (2 x 40 ml). The combined ethyl acetate extract was washed once with water (-40 ml) and then dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure to give the crude product, which was purified by preparative CL using ethyl acetate / hexanes 1: 1 as eluent (Rf = 0.17): yield of 0.46 g (46 % in two steps) of white foam; mass spectrum (chemical ionization) m / z 389 (M + H +). 10 19-Ethoxycarbonyl-17β.19-dihydroxy-4-androsten-3-one In a 50 ml round bottom flask equipped with a magnetic stir bar and a nitrogen inlet, 3,17-dioxo-19- was placed. ethoxycarbonylmethyl-19-hydroxy-5-androstene (50 mg, 0.129 mmol) and anhydrous methanol (5 ml). The steroid solution was stirred and cooled on ice under nitrogen and sodium borohydride (6.4 mg, 0.168 mmol) was added as a solution in methanol (1 ml). The reaction was stirred on ice for 8 minutes and then quenched with the addition of acetone (1 ml) and acetic acid (2 ml). The reaction mixture was then diluted with water (-25 ml) and the The resulting suspension was extracted with ethyl acetate (-35 ml). The ethyl acetate extract was dried over magnesium sulfate, filtered and then concentrated under reduced pressure to give a white powder; yield of 51 mg (cant.) which was homogeneous in CL (MeOH / CHCl3 1:19) (Rf = 0.26). This material was used as such in the next reaction. 19-carboxymethyl-17b, 19-dihydroxy-4-androsten-3-one (19-HCTM) The ethyl ester of the above (51 mg, 0.129 mmol) was dissolved in methanol (5 ml) and treated with KOH (0.25). g). The reaction was stirred at room temperature for 1 hour and then quenched with the addition of water (25 ml) and acetic acid until the pH of the solution was adjusted to 6. The resulting mixture was further acidified with the addition of 0.5 HCl. N (1 ml) and the resulting suspension was extracted three times with ethyl acetate (3 x 15 ml). The combined ethyl acetate extract was dried over magnesium sulfate, filtered and concentrated. The crude acid was purified by preparative CL using 2% acetic acid in ethyl acetate as eluent (Rf = 0.4) and isolated as a white powder, yield: 10 mg (22%); mass spectrum (chemical ionization) 363 (M + H +). Example 4 Synthesis of 10-carboxyvinyl-17β-hydroxy-19-nor-4-androsten-3-one (19-carboxyvinyl-nor-testosterone; 19-CVNT) 3,17-bis (ethylenedioxy) -10-ethoxycarbonylvinyl-19 -nor-5-andostene A 50 ml two-necked flask equipped with a magnetic stir bar, reflux condenser with a nitrogen inlet and a rubber septum, was charged with triethylphosphonaoacetate (0.35 g, 1.7 mmol) and anhydrous tetrahydrofuran. (5 ml). The solution was cooled in a salt bath on ice at about -10 ° C under nitrogen and n-butyl lithium (1.24 ml of 1.6 M solution, 1.7 mmol) was added. The reaction was stirred at -10 ° C for 30 minutes and then a solution of 3.17bis (ethylenedioxy) -5-androsten-19-al (0.22 g, 0.57 mmol) in tetrahydrofuran anhydride (5 ml) was added dropwise from a syringe The reaction was then heated to room temperature and then heated to reflux under nitrogen for 24 hours. After cooling to room temperature, the reaction mixture was then partitioned between a 1: 1 mixture of water and ethyl acetate. The ethyl acetate layer was separated and washed with brine. It was then dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative CL using 2: 3, ethyl acetate / hexanes as eluent (Rf = 0.34) and isolated as a white flabby solid: yield 0.1 1 g (43%); mass spectrum (chemical ionization) 459 (M + H +). 3117-dioxo-10-ethoxycarbonylvinyl-19-nor-4-androstene A solution of the bis-ketal (0.1 1 g) of the above in acetone (10 ml) was treated with p-toluenesulfonic acid (20 mg). The reaction was stirred at room temperature for 16 hours and then concentrated under reduced pressure. The resulting residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated, dried over magnesium sulfate and then concentrated under reduced pressure. The product was purified by preparative CL using 1: 1, ethyl acetate / hexanes as eluent (Rf = 0.51) and isolated as a white powder, yield 20 ml (23%). This material was used as such for the next reaction.

-Ethoxycarbonylvinyl-1 7β-hydroxy-19-nor-4-androsten-3-one A solution of 3,17-dioxo-10-ethoxycarbonylvin-19-nor-4- androstene (19.5 mg, 0.053 mmol) in methanol (3 ml) was cooled on ice under nitrogen and treated with sodium borohydride (2.7 mg). the reaction was stirred on ice under nitrogen for 30 minutes and then quenched with the addition of acetone (1 ml) and acetic acid (1 ml). The resulting solution was concentrated and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated, dried over magnesium sulfate, filtered and then concentrated under reduced pressure. The residue obtained (10 mg) was dissolved in anhydrous chloroform (5 ml) and treated with activated MnO2 (100 mg). The black suspension was stirred 30 minutes at room temperature under nitrogen, and then diluted with ethyl acetate and filtered. The filtrate was concentrated under reduced pressure to give a powder white which was homogeneous in LC (methanol / chloroform at 1:19, Rf = 0.28): yield of 18.2 mg (92%). 10-carboxyvinyl-17β-hydroxy-19-nor-4-androsten-3-one (19-carboxyvinyl-nor-testosterone; 19 CVNT) A solution of 10-ethoxycarbonylvinyl-17β-hydroxy-19-nor-4 20 androsten-3-one (17 mg) in methanol (3 ml) was treated with 5% KOH (2 ml). The reaction was stirred at room temperature for 30 minutes and then acidified with 1 N HCl. The resulting suspension was extracted twice with ethyl acetate (2x25 ml). The combined ethyl acetate extract was dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a white powder which was homogeneous in LC (2% acetic acid in ethyl acetate, Rf = 0.25): yield 15.2 mg (97%); mass spectrum (chemical ionization) 345 (M + H +). Example 5 Synthesis of NSF-DMAE / 19-HCMR Conjugate entangled with n-hexyl-1,6-diamine (NSP-DMAE-HD-19-HCTM) First, NSP-DMAE-NHS was crosslinked with n-hexyl-16- diamine to produce NSP-DMAE-HD. Therefore, a yellow solution of NSP-DMAE-NHS (60 mg, 0.1 mmol) in DMF anhydride / CH 3 OH (1: 4, 10 ml) was added n-hexyl-1,6-diamine (116.2 mg, 1 mmol) ). The resulting colorless solution was stirred at room temperature under nitrogen for 15 hours, and concentrated to -1 ml. The crude product was purified by CL using a preparative silica gel plate (2000 μm, 20x20 cm) developed with CHCl3 / CH3OH / H2O (55/40/5). The product had Rf = 0.5; yield: 50.2 mg (84%); FAB / MS (593, M + H). Subsequently, a solution of 19-hydroxy-19-carboxymethyl-testosterone (19-HCMT, 4 mg, 0.011 mmol) in anhydrous DMF (0.1 ml) was diluted with CHCl3 anhydride (0.4 ml), cooled in an ice bath, and activated with a solution of DCC (2.7 mg, 9.13 mmol) in CHCl3 anhydride (0.1 ml). The reaction mixture was stirred at 0 ° C for 10 minutes, followed by the addition of NSP-DMAE-HD (6 mg, 0.01 mmol) in DMF anhydride (0.2 ml) and the mixture of fresh reaction was stirred at room temperature for 15 hours. The crude product was purified by R P-CLA R using a C 18, 7.8 x 300 mm, 10 μm, semi-preparative column, solvent A: 0.05% (v / v) TFA / H2O, solvent B: 0.05% (v / v) ) TFA / CH3CN, flow rate: 2.5 ml / min, UV detection at 260 nm. Initially 30% of solvent B was used for 15 minutes followed by a linear gradient to 50% solvent B in 5 minutes. The product was eluted at 24.5 minutes yield: 2.3 mg (25%); FAB / MS (936, M + H). Example 6 Synthesis of NSP-DMAE / 19-CVNT Conjugate Interlaced with n-hexyl-1,6-diamine (NSP-DMAE-HD-19-CVNT). NSP-DMAE-H D-19-CVNT was prepared from 19-CVNT (2 mg, 0.006 mmol) and NSP-DMAE-H D (3 mg, 0.005 mmol) as described in example 5. The crude product was purified by RP-CLAR using a C18, 10 μm, 7.8 x 300 mm semi-preparative column. Initially solvent used at 30% for 15 minutes s'eguido by a linear gradient to 60% solvent B in 5 minutes, where solvent A: 0.05% (v / v) TFA / H2O and solvent B: 0.05% ( v / v) TFA / CH3CN; flow rate 2.5 ml / min; UV detection at 260 nm. The product was eluted at 23.2 minutes. Product yield 0.72 mg (16%); FAB / MS (918, M + H). Example 7 Synthesis of NSP-DMAE / Testosterone-19-CME Conjugate, Interlaced with n-hexyl-1, 6-diamine (NSP-DMAE-HD-19-CMET) NSP-DMAE-HD-19-CMET was prepared from testosterone-19-CME (4 mg, 0.011 mmol) and NSP-DMAE-HD (3 mg, 0.005 mmol) as described in example 5. The crude reaction mixture was first separated on a preparative LC plate ( silica gel, 1000 μm, 20% MeOH / CH 2 Cl 2). two bands were collected at Rf = .2 by RP-CLAR using a C18, 10 μm, 7.8 x 300 mm semi-preparative column. Initially 30% solvent B was used for 15 minutes followed by a linear gradient to solvent B to 60% in 5 minutes, where solvent A: 0.05% (v / v) TFA / H2O and solvent B: 0.05% (v / v) TFA / CH3CN; flow rate 2.5 ml / min; UV detection at 260 nm. The product was eluted at 24.1 minutes. Product yield: 0.3 mg (6.4%); FAB / MS 936, M + H). EXAMPLE 8 Synthesis of DMAE / 10-HCMT conjugate, entangled with n-hexyl-1,6-diamine (DMAE-HD-19-HCMT) First, DMAE-NHS was derived with n-hexyl-1,6-diamine to produce DMAE-HD as described in Example 5. Therefore, to a yellow solution of MAE-NHS (50 mg, 0.084 mmol) in DMF / CH3OH anhydride (1: 4, 10 ml) was added n-hexyl- 1,6-diamine (98 mg, 0.84 mmol). The resulting solution was stirred at room temperature under nitrogen for about 4 hours and evaporated. The crude product was purified by CL using preparative silica gel plates (2000 μm, 10 x 20 cm) developed with CHCl3 / CH3OH / H2O (65/25/4). The product band at Rf = 0.4, was separated, eluted with the same solvent system. The eluent was evaporated to a residue which was triturated with chloroform, filtered and the filtrate was evaporated to give purified DMAE-HD (20 mg, 41%). The DMAE-H D-19-HCMT was prepared from 19-HCMT (4.75 mg, 0.013 mmol) and DMAE-DH (7 mg, 0.012 mmol) as described in Example 5. One third of the crude product was purified by RP-CLAR using a C 18, 10 μm, 7.8 x 300 mm, semi-preparative column. Initially 30% of solvent B was used for 15 minutes followed by a linear gradient to 60% of solvent B in 5 minutes, in which solvent A: 0.1% (v / v) TFA / H2O and solvent B: 0.1% ( v / v) TFA / CH3CN, flow rate 2.3 ml / min; UV detection at 260 mm. The product eluted at 23 minutes. Product yield: 1.5 mg (45%); FAB / MS (828.5 M +). EXAMPLE 9 Analysis for Testosterone This example teaches a sensitive and rapid competitive chemical phosphorescent immunoassay for testosterone, using the hydrophilic indicator of NSP-DMAE-H D-19-HCMT, which is heterologous to the antibody immunogen in the bridge. The data show the clear advantage of this new hydrophilic indicator compared to the conventional hydrophobic indicator of DMAE-H D-19-HCMT allowing the development of a fast, high-sensitivity testosterone assay. A) Production and characterization of polyclonal and monoclonal antibodies: Commercially available rabbit polyclonal antibodies, which are Human Serum Albumin of anti-testosterone-19-o-carboxymethyl ether RAT-19), were purchased from the OEM, (Tom's Rive, NJ). The production of this antibody was previously described by Rao (Patent of E. U.A. # 4, 197, 286). A mouse monoclonal antibody against rabbit IG was produced by a modified version of the technique described by Kohler and Misltein (Natura London) 256, 495,497 (1975)). The mice were immunized with purified rabbit IgG, to produce a mouse monoclonal anti-rabbit IgG (MAR-IgG). Normal antibody screening techniques were used to select a hybridoma with high specificity for rabbit IgG. Ascytes were produced using HM F 1 -1 1 B5-3F 1 -5H7. The MAR-IgG antibody was purified from the ascites using affinity chromatography of A-sepharose protein (Pharmacia Fine Chemicals, Piscataway, N.J.). B) Development of an I nmunoanalysis of Chemical Phosphorescence of Testosterone. The optimal analysis performance for a rapid chemical phosphorescence analysis was achieved with the MAR-IgG (described above) covalently coupled to a solid phase and preincubated with the RAT-19 antibody, using HSP-DMAE-H D- 19- HCMT as the indicator. The analysis used paramagnetic particles (PMP) (Advanced Magnétics Inc., Cambridge, MA) as the solid phase. The MAR-IgG (160 mg per gram of PMP) in 0.1 M sodium phosphate, pH 7.4, was covalently bound to the PM P after activation of the PMP with 0.5% glutaraldehyde in 0.1 N sodium phosphate (modified method of Groman et al., 1985, BioTechniques 3, 156-160). This was followed by incubation with RAT-19. Excess RAT-19 was removed by further washes of the reaction mixture with phosphate buffer. Twenty-five microliters (μl) of a normal serum or testosterone sample (0.1 to 24 ng / ml or 2.5 to 600 pg / tube), 50 μl of NSP-DMAE-HD-19-HCTM indicator (6x106 Light Units) Relative / test) and 300 μl of solid phase with MARlgG / RAT-19 antibody were added simultaneously, incubated at 37 ° C for 5 minutes, washed and the chemical phosphorescence measured in an automated chemical phosphorescence immunoassay system ACS: 180 (Ciba Corning Diagnostics, Medfield, MA). The normals of the analysis consisted of pure testosterone in human plasma separated with charcoal, free of steroids. The normal testosterone binding curve (percentage of RLU from normal to Rlu of normal of zero) for the analysis described, is shown in Figure 3. The testosterone doses of the serum samples were generated in the ACS: 180 using two calibration points outside of a master curve. Three other testosterone indicators were evaluated, namely, NSP-DMAE-HD-19-CVNT (heterologous for the antibody immunogen), NSP-DMAE-H D-19-CM ET (homologous to the antibody immunogen) , and DMAE-H D-19-HCMT (a non-hydrophilic version of the NSP-DMAE-H D-19-H CMT) in the same analysis format for its effect on the binding curve and the 50% maximum binding ( DE50) of the normal binding curve. The DMAE-H D-19-HCTM indicator was evaluated to demonstrate the advantage of employing a hydrophilic EAAP label (NSP-DMAE) against a hydrophobic EAAP label (DMAE). C) Conventional Radioimmunoassay for Testosterone The results (n = 145) of a conventional testosterone radioimmunoassay (12Syodide) were compared in serum samples of male and female with the chemical phosphorescence analysis of ACS: 180 using the heterologous testosterone indicator, NSP -DMAE-HD- 19-HCMT. The commercial Testosterone system of "Coat-A-Count Total" (DPC, Los Angeles, CA) was used. This analysis has an incubation time of three hours with a sample size of 50 μl. The minimum detectable dose reported is approximately 2 picograms per tube as calculated from the packaging insert of manufacturers V116 dated January 27, 1992. This analysis was carried out in accordance with the manufacturer's instructions. D) Competitive Chemical Phosphorescence Analysis The minimum detectable dose (Rodbard, 1978, Anal. Biochem. 90_, 1-12) of the rapid, automated testosterone immunoassay, which uses the heterologous testosterone indicator NSP-DMAE-H D-19-HCTM is 1.25 pg / tube, an increase of 1.6 times over the 3-hour conventional radioimmunoassay described above. As shown in Figure 5, the results of the patient sample for chemical phosphorescence analysis correlate very well with radioimmunoassay. The cross-reactivity profile of the analysis is shown in Table 2. These data suggest that the new chemical phosphorescence analysis is of useful value in the analytical determination of testosterone in human serum samples. TABLE 2 Specificity of chemical phosphorescence immunoassay of Testosterone from ACS. using NSP-DMAE-H indicator D- 19- HCMT Dose Specifications Cross Reactivity% Cross Reagent% Androstenedione 100 ng / ml 1 .55 < 1 Androsterone 1 ug / ml 0.03 < 0. 1 Cortisol 1 ug / ml 0.03 < 0. 1 Corticosterone 1 ug / ml 0.02 < 0. 1 1 1 -deoxycortisol 1 ug / ml 0.05 < 0.1 5A-dihydrotestosterone 100 ng / ml 8.3 < 1 5 Estradiol-17B 100 ng / ml 0.12 < 0.1 Estrone 100 ng / ml 0.07 < 0. 1 Progesterone 1 ug / ml 0.09 < 0. 1 Danazol 1 ug / ml 0.06 < 0. 1 Dexamethasone 1 ug / ml 0.01 < 0. 1 A comparison of the normal binding curves and ED50 calculated for each chemical phosphorescence indicator under the same conditions are shown in Figure 4. The highest ED50 for the NSP-DMAE-HD-19-CMET indicator it is homologous to the antibody immunogen. The ED50 for both heterologous indicators, NSP-DMAE-HD-19-CVNT and NSP-DMAE-HD-19-HCMT, are lower than the homologous indicator that indicates that heterologous indicators are more easily displaced by testosterone than the homologous indicator . Little displacement is observed with DMAE-HD-19-HCMT indicating the clear advantage of the hydrophilic NSP-DMAE-HD-19-HCMT version of the indicator in testosterone immunoassay. EXAMPLE 10 RUBELOUS IgG CAPTURE ANALYSIS Rubella IgG capture analysis was used to evaluate the NSP-DMAE-NHS and DMAE-NHS as reagents for direct rubella labeling. The intact rubella virus was labeled with either NSP-DMAE-NHS or DMAE-NHS as described below. Both of these indicator preparations were subjected to a titration study and a population study. The performance of each rubella indicator can be determined by analyzing the positive sample / negative sample ratio generated by each indicator for a given sample population. In this rubella IgG capture assay, the positive signal / negative signal (P / N) ratio is also reflected on the ability to distinguish precisely small differences in the rubella-specific IgG level. The preferred rubella indicator is one that generates appreciable differences in p / N ratios for samples covering a broad scale of levels of lU / mL, since this leads to greater sensitivity in rubella-specific igG detection. A) Preparation of Rubella Virus Indicators Rubella virus (grade IV, 1 ml at 0.5 mg / ml) purchased from Viral Antigens, Inc. (Memphis, TN) was dialysed into labeling buffer (10 nM Phosphate, 0.125 N NaCl, pH 8) using a centricon-30 microconcentrator (Amicon, Danvers, MA). The virus sample was brought to a volume of 1 ml in buffer pH buffer, then concentrated to 10 times by turning the centricon-30 in a centrifuge at the top of the table, for 30 minutes, at 2600 rpm. The concentrate was brought back to 2 ml with pH buffering solution. This concentration and reconstitution process was repeated 3 times. The sample was then divided into 2 x 1 ml aliquots for use in subsequent labeling procedures. For each rubella virus aliquot (0.25 mg in 1 ml of pH buffer), a solution of NSP-DMAE-NHS (50 μg in 25 μl of dimethylformamide) or DMA-N HS (50 μg in 25 μl of dimethylformamide). The labeling reaction mixture was vortexed briefly, and incubated at room temperature for 15 minutes and stopped by adding 5 mg of Usin (Sigma, St. Louis, MO) in 50 μl of water. The reaction mixture was vortexed briefly, incubated at room temperature, for another 5 minutes and subjected to the following purification steps. The reaction mixture was first purified through the "PD10 Sephadex G25M" sizing column (Pharmacia, Pisctaway, NJ), pre-equilibrated and eluted with labeling buffer containing 0.1% ASB. The flow velocity of the elution, it was mained at 1 ml / min and fractions of 20 x 0.8 minutes were collected. The indicator that appeared in the vacuum volume of the first RLU peak (fractions 6-9) was located by taking a sample of 10 μl of each fraction, diluting it 200 times with pH buffer, and counting 10 μl of the sample diluted in an "MLA II" luminometer (Ciba Corning Diagnostics, Medfield, MA). The desired combined fractions were concentrated with a 30-centricon to approximately 300 μl. The concentrate was subjected to additional CLAR gel filtration. The Waters CLAR system (Waters Associate, Mildford, MA) mounted with a HPLC gel filtration column of Protein "Pak 300SW" (Waters # 1 1878) was first equilibrated with buffer solution in a CLAR column containing 50 nM of Tris pH 8, 0.25 M MgCl2, 0.1% ASB, and 0.01% Triton X-100. The labeled virus concentrate from the PD10 column was loaded onto the 300sW column. The elution was mained at a flow rate of 0.5 ml / min and fractions of 40 x 0.25 ml were collected starting from 1 1 minutes after the injection of the sample. The marked virus peak (retention times of 13-17 minutes) was again identified in the same way as above, looking for the first peak of RLU through counting samples of the collected fractions. Fractions containing the first RLU peak were combined and represented the indicator of purified labeled virus. B) Preparation of solid phase of antihuman IgG. The mouse anti-human IgGFc monoclonal antibody was covalently bound to the PMP with glutaraldehyde (in 20 mM sodium phosphate, pH 7). This is a modification of the method described in "BioTechniques" 3, 156 (1985). C) Preparation of monoclonal anti-human IgGFc (clone 157.8D6.5d -). Fusion Procedure: The method uses polyethylene glycol to fuse isolated mouse spleen cells, from donors immunized with mouse myeloma cells SP2 / 0 Ag4-1 (Galfrie and Milstein, (1981), "Methods in Enzimology" 73: 3 ). Screening Procedure: The secretion of specific antibody was tested by the ability of the cells supernatant to bind human IgG labeled with acridinium ester to human IgG immobilized on paramagnetic particles. The monoclonal antibody was shown to be specific for human IgGFc and non-reactive for Fab and Fab2 fragments of human IgG, IgA, IgM, IgE, and human IgD, when tested by the "western" dot plot (as Proc. Nat. Acad. Sci. USA, 76: 4350 (1979)).

Isotope of Antibodies: The isotope of antibodies is IgG 1, identified using the Zuned MonoAb ID EIA equipment (Zymed Lab Inc., San Francisco, CA). D) Preparation of anti-Rubella monoclonal antibody (clone ML109.10E7.4A3) Fusion procedure: the same as the previous one. Screening Procedure: The secretion of specific antibody was tested for the ability of the cell supernatant to bind rubella virus. The monoclonal binding to the antigen was detected by ELISA (according to J.R. Coligan, "Current Protocols in Immunology Vol. 1" (1991) section 2.1.3). The clones were shown to be specific without reactivity for controls of non-infected cultures. E) Immunoassay of Chemical Phosphorescence of Rubella IgG Capture The performance of the different rubella virus indicators was evaluated in a "Magic LiteR" manual (Ciba Corning Diagnostics, Medfield, MA) analysis format in triplicate. All serum samples were diluted five times with the diluent (IxDPBS, BioWhittaker, Waikersville, MG) containing 50% calf serum, 1% Triton X-100, 1% normal mouse serum, and 0.1% Sodium azide. The diluted sample (50 μl), solid phase of anti-human IgG, and 0.1% sodium azide. The diluted sample (50 μl), solid phase of anti-human IgG (80 μg in 250 μl), and indicator of the diluted rubella virus (100 μl) were incubated together for 7.5 minutes at 37 ° C in a polystyrene tube " Sarstedt "(Newton, NC). The reaction mixture was separated, aspirated and washed twice with 1 ml 1 x DPBS ("BioWhittaker", Waikersville, MG). The final pellet was reconstituted with 100 μl of water and its chemical phosphorescence was measured in an MLA-II luminometer (Ciba Corning Diagnostics, Medfield, MA). Since the two rubella virus indicators (prepared as described above) varied in their specific chemical phosphorescence activity, the titration study was carried out on different scales of input counts, using nine sample of rubella IgG as shows in Table 3. TABLE 3 Rubal IgG Capture Analysis Results from the Rubella Titration Study NAP-DMAE Sample Rubella from NSP-DMAE (RLU.% CV.P / N) 1x1065.95x1013.3x10ß 24.3x10646. 1x10ß Diluent 1233 1793 2473 2757 4253 9360 % cv 1.9 2.6 9.4 3.3 5.5 17.2 P / N 1.0 0.9 1.0 0.8 i.i 146 Negative R? B 17 1233 2037 2447 3367 4023 5987 % CV 3.3 20.1 3.9 23 8.5 7.5 P / N 1.0 1.0 1.0 1.0 1.0 1.0 R? B 36 1383 1897 25B7 2880 6357 8357 % CV 13.3 13.2 4.7 0.9 16.3 21.7 P / N 1.1 0.9 1.1 0.9 1.6 1.4 Low + C? P 10 1393 2903 4247 5613 8100 14830 % cv 2.7 6.1 2.4 6.4 9.1 7.6 P / N 1.1 1.4 2.2 2.1 2.6 3.3 C? P 15 1473 3170 5283 7003 10263 J9737 * CV 2.7 3 3.2 2.7 6.3 7.9 P / N 1.2 1.6 2.2 2.1 2.6 3.3 Medium-High + Tßl 3156 3547 14420 32143 40403 66857 142657 % CV 3.8 3.3 4 3.2 1.3 1 P / N 2.9 7.1 13.1 12.0 16.6 23.8 RAB 67 10970 52003 120987 151000 262317 536050 % CV 2.7 6.4 5 2.4 1.06 2.3 P / N 8.9 25.5 49.4 44.8 65.2 89.5 TSI 3167 7357 35337 75027 94530 167653 342447 % CV 1.9 1.9 8 3.4 4.6 3.5 P / N 6.0 17.3 30.7 28.1 41.7 57.2 T8I 3163 11270 56443 111327 142290 249450 514440 % CV 2.2 3.3 4.9 1.7 2.2 10.6 P / N 9.1 27.7 45.5 42.3 62.0 85.9 D 1-3150-388 11477 55093 113783 146473 263267 542483 % CV 1.4 6.5 12.3 3.2 3.2 3.4 P / N 9.3 27.0 46.5 43.5 65.4 90.6 The labeled rubella indicator from DMAE was titrated on a broader scale due to a 4-5 times higher degree of DMAE incorporation in the labeled virus indicator compared to NSP-DMA E. In order to compare the conditions of the analysis using a comparable mass of rubella indicators, the scale of the total entry of the DMAE rubella indicator must be several times higher than that of the rubella status of NSP-DMAE. The results of the titration study showed improved P / N ratios as the increased NSP-DMAE rubella indicator level, while the P / N ratios generated by the DMAE rubella indicator, plaque formed at 10 x 106 RLU / test. The selection of 24 x 106 R LU / test as the optimal entry level for the rubella indicator of NSP-DMAE, was based on the results of higher ratios of P / N, while maintaining non-specific low binding with the diluent of control. The comparable analysis conditions using rubella indicator of DMAE, is 125 x 106 RLU / test. When these two conditions are compared, it is clear that the rubella indicator of NSP-DMAE is the superior reagent with P / N ratios that vary from 2 to 4 times more than those generated by the DMAE rubella indicator. The test of these two rubella indicators was expanded to a population of twenty rubella IgG samples (ranging from 0.3-500 lU / ml) and 4 potential cross-reaction samples (all identified as rubella IgG negative by the analysis of Rubazyme (Abbott Labs, Chicago, IL) (Table 4A) The study was carried out using the following total inputs for each reagent; NSP-DMAE rubella indicator (24 x 106 RLU / test) and rubella indicator DMAE (125 x 106 RLU / test). The population data suggested that the sensitivity of rubella IgG capture analysis was notably affected by the class of EAAP used to directly label the virus. The P / N ratios (result of positive to negative), generated from the use of the rubella indicator of NSP-DMAE, are considerably higher than those generated by the rubella indicator of DMAE (Figure 6). These results reflect the ability of the analysis carried out by the rubella indicator of hydrophilic NSP-DMAE; to more clearly distinguish positive samples from negative samples than the conventional DMAE indicator. This is particularly important in the low positive, diagnostically critical population.

The impact of the hydrophilic NSP-DMAE rubella indicator on the specificity of potentially cross-reactive samples was evaluated. Using a reduction in the P / N ratio that corresponds to the CAP10 sample, the results do not indicate false positive identifications, which result from the use of rubella indicators with these types of samples. (Table 4B). The results of both of these studies clearly suggest that NSP-DMAE-NHS is a superior reagent for the direct labeling of the rubella virus. This reagent results in a rubella preparation which binds specifically to a broad scale of rubella IgG levels to produce specific values of RLU which are clearly distinguishable from those of a negative population of rubella IgG. In addition, these conditions exist without incurring false positive results, potentially with cross-reactive samples. Table 4A Population Study using Rubella Indicators of NSP-DMAE and Rubella of DMAE Rubella IgG Capture Analysis (ratio P / N) Rubella Sample NSP-DMAE Rubella DMAE R? B17 1.0 1.1 AP05874-4 1.0 1.0? S04912-9 1.1 1.1 R? B 36 1.1 1.2? S14837-8 1.1 1.0 C? P 10 1.8 1.2? S01721-3 2.1 1.0 AP27771-3 2.4 1.0? R99B60-4 2.5 0.8 C? P 15 2.5 1.0 TSI 3164 5.8 1.8? S06137-4 8.0 1.3 GP1-27 23.9 2.8? P41719-0 24.9 3.5 GP2-9 26.2 3.1 GP2-18 27.7 3.4? R86731-7 51.1 5.6? R96863-2 58.7 5.9 AS15758-2 68.5 6.9? R74744-4 88.9 9.8 TABLE 4B Interference Samples Crubeleol Analysis (Ratio of P / N) Rubazima (Index) (All Rubella IgG-) NSP-DMAE DMAE > = 1.0 is + EBV + E9-3204-467 1.5 1.1 0.725 EBV + E8-2405-99 1.10 0.98 0.171 VZV + E02606-135 1.4 1.1 0.183 ANA + 12615M-910513 1.7 1.1 0.793 F) Comparison with the Control Analysis of "Ciba Corning Magic LiteR "and Analysis of" Abbott IMx. "The SNP-DMAE rubella indicator assessment was further extended to include an investigation of how this" Magic LiteR "rubella IgG test using rubella indicator of NSP-DMAE, is compared to (1) the analysis of "Ciba Corning Diagnostic Magic Lite®" (which uses an unlabeled rubella complex and antirubella monoclonal antibody labeled with NSP-DMAE 10E7) and (2) Rubella IMx IgG (Abbott, Chicago, 11) The sample population tested by both "Magic LiteR" analyzes included thirty-nine IgG samples identified as positive or negative for rubella-specific IgG by Imx (Table 5). Positive / negative results (P / N) ratios generated by the rubella indicator NSP-DMAE are significantly higher than those observed with the "Magic LiteR" control analysis format.This is particularly evident in the High positive samples of rubella IgG and may reflect competition between rubella-specific IgG and the NSP-SMAE-MAb10E7 indicator to link sites in unlabeled rubella viruses. Based on a cut-off ratio corresponding to the CAP10 sample, the "Magic Lite" rubella IgG assay using the rubella indicator of NSP-DMAE demonstrated 97.4% according to the rubella control analysis of "Magic LiteR" and 92.3% according to rubella IgM from IMx.

TABLE 5 Study of expanded population using Rubella Indicator of NSP-DMAE Sample Rubella Rubella + NSP-DMAE NSP-DMAE 10E7 Imx R? B 17 1.0 1.0 negative ? P05874-4 1.0 0.9 negative ? S04912-9 1.1 0.9 negative R? B 36 1.1 1.0 negative ? S14B3 / -8 1.3 0.9 negative C? P 10 1.8 1.1 positive AS0172Í-3 2.1 1.05 negative ? P27771-3 4 1.1 negative ? R99860-4 1.1 negative C? P 15 1.2 positive? S00 41-9 B 1.2 positive ? R97400-3 2 1.3 positive AP41598-Í 4.0 1.5 ~ positive TSI 3164 5.8 2.0 positive ? S06137-4 8.0 1.7 positive ? S00407-3 9.9 2.4 positive TSI 3156 13.6 3.0 positive GP2-3 23.4 3.7 positive GP1-27 23.9 ~ 76 ~ positive ? P41719-0 24.9 2.6 positive GP2-9 26.2 3.9 positive GP1-1 26.3 3.7 positive GP2-11 26.9 4.1 GP2-18 positive 27.7 4.1 positive GP2-4 28.1 4 ~ .4 ~ positive TSI 3167 33.4 6.2 positive GP2-22 39.7 5.4 positive GP2-14 42.0 4.1 positive GP1-17 43.3 7.4 positive GP2-35 44.6 7.6 positive TSI 3163 48.7 13.1 positive ? S15788-7 49.3 5.3 positive DLl-3510-388 51.0 11.6 positive ? R86731-7 51.1 9.6 AR9G863-2 positive 58.7 10.6 positive AP35925-6 62.0 7.9 positive HG6B085-5 G3.0 16.5? S15758-2 positive 68.5 11.3 AR74744-4 positive 88.9 27.9 positive Example 1 1. Dialing of DMAE & Protein NSP-DMAE This example shows that a large amount of NS-DMAE can be covalently bound to a protein, such as ASB (Bovine Serum Albumin) given the hydrophilic nature of N DP-DMAE. A solution of ASB (10 mg, 150 nmol) in 0.1M carbonate pH buffer solution, pH 9, (2.7 ml) was treated with a solution of 50 equivalents both - DMAE-N HS ester or NSP-DMAE ester -N HS (4.5 mg coate one, 7.5 μmol) in DMF (300 μl). The reaction using DMAE-NHS remained completely clear during the reaction. The reactions were stirred at room temperature for 24 hours and the labeled proteins were isolated by size exclusion chromatography on a "Sephadex G-25" column (3.7 x 42 cm) eluted with 10 nM phosphate pH buffer, pH 8. The protein fraction was collected in each case, concentrated and repurified on the same Sephadex G-25 column. After the second concentration, the amount of the labeled protein recovered from each reaction and the degree of Acridinium ester (AE) labeling was calculated by carrying out a protein analysis (Bio-Rad protein analysis) and measuring the activity of chemical phosphorescence of the labeled protein, respectively. From these measurements, it was found that the protein was labeled with approximately 14 molecules of DMAE and 24 molecules of NSP-DMAE.

EXAMPLE 12 Conjugation of ASB (Bovine Serum Albumin) Marked to NSP-DMAE for 508.CF-3'-Maleimide 5'-32p-labeled: This example shows how the protein carrier previously labeled with plurality of NSP-DMAE may be covalently linked to a 24-mer oligonucleotide, which was named 508.CF-NH2. The oligonucleotide was first radiolabelled with 32-P in the 5 'end phosphate, using the conventional radiolabelling technique, commonly known to those skilled in the art. The purpose of labeling 32P of the oligonucleotide was to provide a way to quantify the oligonucleotide once conjugated with the protein vehicle. The 3 'end of the oligonucleotide carries an aminoalkyl group to allow reaction with a bi-functional interleaver, as described below, such that a group of reactive maleimido with sulfhydryl can be attached. The methods for preparing the oligonucleotide with the aminoalkyl group at the 3 'end, is well known (See User Manual on DNA Modification Reagents for Use in Automated DNA Synteresis, Doc. No. PB022789-1, CLONTECH Laboratories, Inc., Palo Alto, CA, 1989). Preparation of maleimide 508.CF 32R-labeled: A solution of 32P-508.CF-NH2 (4 nmol) in 50 nM carbonate (600 μl, pH 8.4), was treated with 4- (N-maleimidomethyl) cyclohexane-1 -sulfosuccinimidocarboxylate (sulfo-SMCC, 10 mg, 22.9 μmol). The reaction was stirred at room temperature for 30 minutes, after which the product was purified directly by preparative H PLC on a C8 column (0.7 x 25 cm) using a gradient of 8% to 20% acetonitrile in buffer solution of pH of 0.1 M triethylammonium acetate pH 7.0, for 20 minutes at a flow of 2.3 ml / min and UV detection at 260 nm (product, t R = 14.8 min, starting material t R = 12.7 min,). The eluent that came out of the HPLC, containing the product, was lyophilized to dryness: yield of 25 nmol (60%). Conjugation of the NSP-DMAE marked ASB to SSP-doOd.CF-maleimide: Prepared (NSP-DMAE) 21 -ASB, reacting ASB with 30 equivalents of NSP-DMAE-NHS as described above. The labeled protein (6 mg, 90 nmol) in 100 nM phosphate buffer, containing 5 mM EDTA, pH 8, was treated with 2-iminothiolane (50 equivalents, 0.6 mg, Pierce, Rockford, I I). The reaction was stirred under nitrogen at room temperature for 1 hour. The titrated protein was then isolated by size exclusion chromatography on a Sephadex G-25 column (3.7 x 42 cm) using 20 mM phosphate, 1 mM EDTA, pH 6.8, as eluent. The protein fraction outside the column was concentrated to a volume of approximately 2 ml of an acceleration vacuum. The protein solution was then mixed with 508. 5'-32P-labeled CF-3'-maleimide (8.9 nmoies) and the resulting solution was stirred at room temperature under nitrogen for 16 hours. The reaction was then quenched by the addition of 600 nmoles of bromoacetic acid. After stirring for an additional 2 hours at room temperature, the reaction mixture was concentrated to a volume of approximately 0.5 ml by centrifugal ultrafiltration. A mixture of conjugated and unconjugated protein was separated from 508. Unreacted CF-maleimide by Polyacrylamide Gel Electrophoresis (PAGE) in a 7.5% density gel (3% interlaced) in the TBE pH buffer , pH 8, operated at a constant current of 10 mA at 4 degrees C. The mixture of conjugated and unconjugated protein was eluted from the gel matrix by a crushing and soaking method in pH buffer of PPBS (130 mM NaCl, 8.1 mM Na2H PO4, 2.7 M KCl, 1.2 mM KH2PO4, pH 7.4). To further separate the conjugate which incorporates the oligonucleotide from the unconjugated protein, an affinity purification is required. A typical example of the separation of the conjugate with the incorporated oligonucleotide from the contaminating unconjugated protein is the use of a solid phase (e.g., functionalized polystyrene or controlled pore glass) immobilized with the complementary oligonucleotide. By carrying out a step of hybridization, separation, and washing, the desired conjugate can be captured on the solid phase, washed of the conjugated protein, and continued with a release step to re-obtain the purified conjugate from the solid phase. Said purification / enrichment steps can be easily performed by those skilled in the art of nucleic acid hybridization. Example 13. Preparation of antibody-DNA conjugate (anti-TSH-508.CF) This example further illustrates the scale of uses for the compounds of the present invention. In this example, the appropriate antibody-DNA conjugate is generated to carry out a large number of chemical phosphorescence labels. The conjugate can be prepared in three steps using an alternative thiol-maleimide coupling chemistry. Preparation of thiolated anti-TSH and conjugation to melimide-508.CF: A solution of anti-TSH (20 mg, 133 nmoles) in 2 ml of 0.2 M phosphate pH buffer, pH 8, containing 150 nM NaCl and 5 mM EDTA was treated with 2-iminothiolane (Traut reagent 0.37 mg, 20 equivalents) under nitrogen. The reaction was stirred at room temperature for 1 hour under nitrogen and the titled protein was isolated by size exclusion chromatography on a "Sephadex G-25" column (2.7 x 42 cm) using 10 mM phosphate, pH 6.8, containing 0.5 mM EDTA as eluent. The protein fraction eluting from the column (yield of 17 mg, 13 nmol) was concentrated under reduced pressure in an accelerating vacuum to a volume of about 2 ml. This solution was then mixed with maleimide-508.CF (12 nmol). The reaction was stirred at room temperature for 24 hours under nitrogen at that time the solution became slightly cloudy.

The reaction mixture was then loaded onto a DEAE-Cellulose column (1 x 15 cm) equilibrated with 20 mM phosphate, pH 7. The column was eluted with a gradient of 0 to 200 mM NaCl in 20 mM phosphate, pH 7, to elute the excess of the unreacted protein. Elution with 500 mM NaCl in the same pH buffer eluted a mixture of the conjugate and unreacted DNA. After the concentration of the outside fraction with high salt content of the ion exchange column by ultrafiltration, separation of the unreacted DNA conjugate was achieved by size exclusion chromatography on a "Sephadex G-75!" Column. 1.5 x 40 cm) using water as eluent at 0.5 ml / min.The solution of the conjugate isolated by this procedure was lyophilized to dryness.The characterization of the conjugate and calculation of the conjugate yield was carried out by UV spectrophotometry. The UV spectrum of the conjugate isolated by the procedure described above was identical in appearance to that of a 1: 1 mixture of 508. CF and Anti-TSH This UV spectrum was characterized by a maximum absorption at -270 nm and a 1: 1 correspondence between the values of A2eo and A2ßo- The value of A2ßo and A ßo, in the UV spectrum of the conjugate, was increased by a factor of 1.5 when compared with DNA alone and protein alone, respectively. From this result, the conjugate yield of 1: 1 of 508. CF and anti-TSH, was calculated to be 1.6 nmol (13% overall maleimide 508. CF).

The conjugates thus produced can be covalently bound to the chemical phosphorescence label of N HS-DMAE, by the methods previously described, among others.

Claims

R EI VI N DICATION IS 1. An acridinium ester of the following formula: wherein R \ is alkyl, alkenyl, alkynyl, aryl or aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur; R2, R3. Rs, and R7, are hydrogen, amino, hydroxyl, halide, nitro, -NC, -SO3H, -SCN, -R, -OR, -NHCOR, -COR, -COOR, or -CONHR, wherein R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus, and sulfur; R4 and Ra are alkyl, alkenyl, alkynyl, aralkyl or alkoxy having up to 8 carbons, without branches wherein the side chain groups have more than 2 carbons; R6 represents the following substitutions: wherein R9 is not required but, optionally it may be an alkyl, or aralkyl group, having up to 5 heteroatoms selected from the group of Phosphorus, Sulfur, Nitrogen, Oxygen, and R10 is an electrophile, a leaving group , a group with these two natures combined, or selected from the following structures: wherein Y is a halide and R is alkyl, aryl, aralkyl group, and wherein a selected position of Rx, Ry and Rz are R6, while the other two positions are R5 and R7.
2. An acridinium ester of claim 1, wherein R1 is a sulfopropyl or sulfoethyl group; R2 is hydrogen, methoxy, ethoxy, nitro or halogen; R3, R5, and R7 are hydrogen; R4 and R8 are methyl, ethyl or isopropyl groups; R6 is N-succinimidyloxycarbonyl; N-succinimidyloxycarbonylalkyl, or carboxylate.
3. An acridinium ester conjugate of claim 1, wherein the acridinium ester is directly or indirectly conjugated to a compound or macromolecule.
4. An acridinium ester conjugate of claim 3, wherein the conjugation occurs by a bifunctional interleaver.
5. An acridinium ester conjugate of claim 3, wherein the conjugation is presented by hexyl-1, 6-diamine, ethylenediamine, or aminocaproic acid.
6. An acridinium ester conjugate of claim 3, wherein the macromolecule is selected from the group consisting of protein, peptide, inactivated protein, DNA, RNA, ologonucleotide, polysaccharide, neurotransmitter, hormone, steroid hormone, virus, bacterium , toxin and cytokine.
7. An acridinium ester conjugate of claim 6, wherein the protein is selected from the group consisting of antibody, antibody fragments, avidin, streptavidin, allergen, receptor protein, DNA binding protein, inactivated protein, neurotransmitter , hormone, viral antigen, bacterial antigen, toxin and cytokine.
8. An acridinium ester conjugate of claim 3, wherein the compound is a hapten or biologically active small molecule.
9. An acridinium ester conjugate of claim 8, wherein the hapten is a steroid hormone.
10. An acridinium ester conjugate of claim 9, wherein the steroid hormone is testosterone, and the bridge and linker arm, are connected by a C-19-C ligature, olefinic ligature of C-19, or ligation. of C19-0. eleven .
An acridinium ester conjugate of claim 9, wherein the steroid hormone is testosterone, and wherein conjugation is present by hexyl-1, 6-diamine, ethylenediamine, or aminocaproic acid.
12. An acridinium ester conjugate of claim 9, wherein said steroid is a testosterone derivative, selected from the group consisting of Structures I to VII wherein A is a functionalized group, selected from the group consisting of - OH,, -N (R?) R2, -NH Ri, nitro, halide, -ORi, -OCOR L -tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, -SRi, nitro, -SK, -SSRi , -phosphonate, -sulfonate, -NHCOORi, -NHCORi, -NHCONH2, hydrazines, cyanide, and -Ri; wherein R T is an alkyl, alkenyl, alkynyl, aralkyl, containing up to 10 carbons and up to 5 heteroatoms; where B is - (CH2) p where n = 1 -4 or -C =; when B is -C =, preferably A is omitted; wherein X is a carboxylate, -COOR2, -CONH R2, (wherein R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl containing up to 15 carbons and up to 5 heteroatoms). or a single carbonyl with appropriate leaving groups, including, but not limited to, halide, N-hydroxysuccinimide, and imidazole.
13. An acridinium ester conjugate of claim 3, wherein said macromolecule is an infectious or pathogenic agent.
14. A method of synthesis of the acridinium ester precursor of claim 1, wherein it is reacted with a compound of the formula wherein R2, R3, R5, R7 are hydrogen, halide, nitro, -R, -OR, -CN, -NHCOR, -COR, -COOR, or -CONHR, wherein R is alkyl, alkenyl, alkynyl, or aralkyl , and where R 4 and R 8 are R and R is as defined above.
15. A method of claim 14, for the synthesis of DMAeE-NHS, where R4 and R8 are methyl and R, R3, Rs and R7 are hydrogen.
16. A method for measuring the amount of an analyte in a sample comprising: a. detect an analyte specifically and in proportion to its concentration in a sample, and b. measuring a signal that is generated by the chemical phosphoresis hydrophilic acridinium ester of claim 1, which is directly or indirectly proportional to the concentration of an analyte in a sample.
17. A method of claim 16 for detecting an analyte in a sample comprising: a. contacting a sample with a detector molecule, said detector molecule, optionally being a complex of two or more molecular entities, b. sequester the bound detector and analyte, c. wash the detector in excess, and c. measure a signal from a bound analyte detector.
18. A method of claim 16 for detecting an analyte in a sample, comprising a. contact the sample with a competitive indicator marked hydrophilic acridinium ester, and a specific binder for an analyte, b. recover the specific binder, and determining a signal generated by the united indicator.
19. A method of claim 16 for detecting an analyte in a sample comprising: a. contacting a sample with a competitive indicator labeled with hydrophilic acridine ester, and a specific binder for an analyte, b. recover the specific agglutinnate, and c. determine a signal generated by the unlinked indicator.
20. A method of claim 16, for detecting an analyte in a sample comprising: contacting a sample with a specific first binder, and a second specific binder labeled hydrophilic acridinium ester, and b. detect a signal.
21. A method of claim 16 for detecting an analyte in a sample comprising a. contacting a sample with a hydrophilic acridinium ester labeled detector and a competitive binder for the detector, b. sequester the detector bound to the competitive binder, c. wash the detector in excess and the analyte attached to the detector, and d. Measure a detector signal attached to the competitive binder.
22. A method of claim 21, wherein a release agent is used.
23. A method of claim 21, wherein a release agent is not used.
24. An acridinium ester conjugate of claim 3, wherein an oligonucleotide is conjugated to a macromolecule, said macromolecule being further conjugated to multiple acridinium esters and optionally to multiple hydrophilic polymers.
25. An acridinium ester conjugate of claim 24, wherein said oligonucleotide is a gene test.
26. The acridinium ester of claim 1, which contains a hydrophilic portion, said hydrophilic portion comprising a highly ionizable group.
27. The acridinium ester of claim 16, wherein said ionizable group is sulfate, sulfonate, phosphate, or phosphonate.
28. The acridinium ester of claim 1, wherein said R 1 group is sulfopropyl or sulfoethyl.
29. The acridinium ester of claim 1, wherein said R2 or R3 is a substituted or unsubstituted aryl fused to said acridinium portion.
30. The acridinium ester of claim 1, wherein said aryl in R2 or R3, can be a benzene ring fused to the acridinium nucleus to form a benz [a] -acridinium ester, benz ester [b] -acridinium or benz [c] -acridinium ester.
31 The acridinium ester conjugate of claim 8, wherein said hapten is an aminoglycoside.
32. The acridinium ester conjugate of claim 8, wherein said hapten is a therapeutic drug, steroid, or a controlled chemical substance.
33. The acridinium ester conjugate of claim 13, wherein said infectious agent is selected from the group consisting of Rubella, Hepatitis of various classes and subtypes, Toxoplasma, Cytomegalovirus, HIV, and Chlamydia.
34. The acridinium ester of claim 24, wherein said hydrophilic polymers are polyethylene glycol.
35. A testosterone derivative selected from the group consisting of Structures I to VI, said derivative being an intermediate in the synthesis of the compound of claim 12, wherein A is a functionalized group, selected from the group consisting of -OH, -N (R1) R2, -NHRL nitro, halide, -OR ?, -OCORi, -tosylate, -mesylate, -azide, -isocyanate, -thioisocyanate, -SR1, nitro, -SH, -SSR1, -phosphonate, sulfonate, -NHCOOR1, -NHCOR1, -NHCONH2, hydrazines, cyanide, and -R ^ wherein Rt is an alkyl, alkenyl, alkynyl, aralkyl, containing up to 10 carbons and up to 5 heteroatoms; where B is - (CH2) n where n = 1 -4 or -C =; when B is -C =, preferably A is omitted; wherein X is a carboxylate, -COOR2, -CON HR2, (wherein R2 is an alkyl, alkenyl, alkynyl, aryl, aralkyl containing up to 15 carbons and up to 5 heteroatoms), or a single carbonyl with appropriate leaving groups, including , but not limited to halide, N-hydroxysuccinimide, and imidazole.