MXPA97010410A - Quimioluminiscent assay of specific union sinseparac - Google Patents

Abstract

The assay described herein is to provide an observation of when the acridinium-acrylated ester tracer binds to its corresponding binding conjugate immobilized on a metal oxide solid phase, the emission of measurable chemiluminescent light from the labeled tracer bound to the solid phase is extinguished as compared to the free fraction tracer that is not free to the solid phase, according to the invention, a specific binding assay without separation is provided to detect or quantify the presence of an analyte, wherein the complete reaction mixture is vaporized (including the unreacted tracer) and the modulated signal (due to the extinction effect) is associated with a reference, thus determining the amount or presence of the analyte in the muestara. The inherent disadvantages of heterogeneous analyzes that employ separations can be avoided using this method without separation.

Description

QUIMIOLUMINISCENT ASSAY OF SPECIFIC UNION WITHOUT SEPARATION FIELD OF THE INVENTION This invention relates to a chemiluminescent specific binding assay without separation DEFINITIONS As used herein "analytes" is defined as a substance capable of undergoing a binding reaction with one or more specific binding patterns the term "analyte" encompasses but is not limited to serum proteins, hormones, antigen drugs, antibodies ( including monoclonal, polyclonal, and fragments thereof), pathogens, enzymes, metabolites, coenzymes and their binding patterns, pohnucleotides, hybrid oligonucleotides of polynucleotide or ohgonucleotides and metals and chelating agents thereof "Specific binding pattern" is defined as a substance capable of binding to the target analyte and an analogue of the analyte The term "specific binding standard" encompasses, but is not limited to, antibodies, antigens, avidin, biotin, thyroxine, thyroxine binding globulin, polysaccharide, phospho-phenol, residues of aminoethyl ester phosphate amino acid, intrinsic factor of vitamin B-12 binding proteins mixtures of the same and several of other protein s and peptides including oligonucleotides etc. "Analyte analogue" is defined as a substance that is capable of matching with the standard the specific binding pattern selected from the target analyte through a specific binding reaction "Specific binding complex" refers to either a complex of the standard specific target analyte binding or an analyte analog specific binding complex complex, or a first sandwich "specific sandwich" complex of specific binding pattern target analyte "wherein the first and second binding patterns can be the same or different) The "specific binding complex" can be formed through a number of specific binding reactions including immunological, chemical and complementary binding. "Tracker" is defined as a specific labeled binding pattern, a labeled analyte, or a labeled analyte analogue "Binding conjugate" is defined as a binding portion that can be selected of the analyte, analyte analog, or specific binding pattern of the analyte "Free fraction" is defined as the test components that are not bound to the solid phase.

BACKGROUND OF THE INVENTION As is well known in the art, compounds that provide a chemiluminescent signal are currently used as labels in specific binding assays. Both (heterogeneous) and non-separation (homogeneous) assays have been prepared using chemiluminescent compounds as labels. Heterogeneous assays generally refer to assays that require a separation of the specific binding complex formed on a solid phase from the free fraction prior to the activation and measurement of the chemiluminescent vaporization. Thus, typically, the heterogeneous assay protocol requires several separation steps of the solid phase reaction product from the free fraction, where decantation and resuspension in water are typically required. These separation steps associated with heterogeneous tests take up a significant amount of time and increase the chance of error by the operator Homogeneous assays generally refer to assays that typically do not require physical separation of the solid phase and free fraction reaction binding complex prior to activation and measurement of chemiluminescent vaporization. For example, US Pat. No. 5,017,473 des A solid-phase separation immunoassay is described using a light-absorbing material (dye) wherein the material is described as absorbing all chemiluminescence, except that it is associated with the binding tracer, so that only the emission due to the binding tracer is detected. Alternative assay systems that eliminate the need to separate the solid phase binding complex from the free fraction are necessary.

BRIEF DESCRIPTION OF THE INVENTION The assay described herein is proposed in an observation that when acridinium ester-tagged tracers bind to their corresponding binding conjugate immobilized on a metal oxide solid phase the measurable chemiluminescent light emission of the labeled tracer attached to the phase solid is reduced (referred to herein as "extinguished") as compared to the chemiluminescent signal generated by the tracer remaining in the free fraction separated from the solid phase. According to the invention, a specific binding assay without separation is provided to detect and quantifying the presence of an analyte in a sample said assay comprises contacting the mixture with a solid phase comprising a metal oxide having attached thereto a binding conjugate and a tracer comprising a labeled binding conjugate with acridinium ester of benzacridinium, allow the solid phase, the tracer and the sample to react to to form a reaction mixture comprising a specific binding complex bound to the solid phase and a free fraction separated from the solid phase, contacting the reaction mixture with an activating agent to vaporize the acridinium ester tracer or benzacpdinio and provide a modulated chemiluminescent signal; measure the modulated signal; and associating the modulated signal with a reference to determine the amount or presence of said analyte in the sample. Also provided in this invention is a diagnostic equipment to carry out the method described above. The inherent disadvantages in heterogeneous assays using multiple separations can be avoided using this method without separation, further, the automation of the assays is easier when the separation step of the assay can be eliminated or implied in a lesser manner, as is achieved by this invention.

DESCRIPTION OF THE DRAWINGS In the Figures included herein, Figure 1 is a graphic representation of TABLE I of Example 2 Figure 2 is a graphic representation of TABLE 2 of Example 3, showing both a-Theophylline PMP and a-LH PMP Figure 3 is a normal curve for the theophylline measurement, as used as in the reference of Example 4. Figure 4 is as described in Example 5. Figure 5 illustrates a competition hybridization assay as described in Example 6 DESCRIPTION DETAILED The present invention can be used to detect and / or quantify target analytes present in chemical or biological substances. Generally, any analyte interaction that is stable under chemiluminescent activation conditions can be prepared in this assay system. Although various types of chemiluminescent compounds having a type of acddinium, benzacpdinium or acpdan-type heterocyclic ring systems are preferred, they use the equivalent chemiluminescent compounds and means to activate the marks which do not depart from the scope of the invention. acridinium and benzacridinium are currently the most preferred chemiluminescent compounds, the preferred acridinium esters including those compounds having heterocyclic rings or ring systems containing a heterogeneous atom in a positive oxidation state, such as ring systems, acridinium, benz [a] acr? din? o, benz [b] acpd? n? o, benz [c] acridinium, a cation of benzimidazole, quinohnio, isoquinolinio quinolizinio, a quinolinio substituted cyclic, fenantpdinio, and quinoxalinío, as is well known in the art The tracker must be prepared by joining the selected conjugate of either direct or indirect binding. rightly with a reactive functional group present in the acpdinium or benzacpdinium ester, as well as those known in the art, for example Weeks and other Clinical Calc. 29 (8), 1474-1479, 1983. Particularly preferred compounds are acridinium and benzacridinium esters with an aryl ring leaving group and the reactive functional group present in either the para or meta position of the aryl ring. Particularly stable acpdinium and benzacpdinium esters are those which have a leaving group of aryl ring, the aryl ring having electron donating species (preferably a C1-C alkoxy, most preferably methyl group) present both at both ortho positions of the aryl ring and having the reactive functional group (preferably a -COOH which is converted to a group). N-succinimidyloxycarbonyl before joining the binding pattern) in the meta or para position, (most preferably for), as described in the US patent No. 4,745,181 and WO 94/21823, both incorporated herein by reference. The solid phase is preferably a metal oxide material, preferably chromium oxide, iron oxide, nickel oxide, or any mixture thereof. The solid phase must be insoluble in water and maintain a structural integrity when exposed to water or biological fluids and may be naturally-occurring particles (varying from a finely divided material, such as that in a magnetic ferrofluid, to a coarse granular material). , or a shaped article (such as beads, test tube trays, microtitre plates, membrane, film, filter paper, discs, etc.). Most preferably, the solid phase particles comprise an iron oxide core, as described in the U.S.A. 4,554,088 Whitehead, et al., 1985 (incorporated herein by reference). To assist in the binding of the binding conjugate, preferably the metal oxide solid phase has a bioaffinity substance present therein. Polamic silane coatings are particularly preferred bioaffinity substances and can generally be defined as organofunctional and functional compounds of silicon which are characterized in that the silicon portion of the molecule has an affinity for inorganic materials, while the organic portion of the molecule is designed to be combined with organic. Preferably, the chemical reactions for binding the conjugate to the solid phase include , but not limited to diazotization, carbodumide and glutaraldehyde couplings The coupling techniques that can be used are described in Method of Enzv .. 70, p. 159-165 (1985), and Groman, EV et al. In Bio Techniques, Vol 3 pp 156 (195, and in U.S. Patent 4,672,040 issued to Josephson in 1987 (each incorporated herein by reference) whether the present invention is practiced with gene rounds, nucleic acid hybridization can be obtained using the solid phase of the metal oxide, when hybridizations are performed by dispersing a solid phase coupled with nucleic acid), most preferably a DNA oligomer) in a reaction mixture containing molecules that will be isolated, allowing the solid phase coupled to the nucleic acid to hybridize to a complementary target sequence. The emission of chemiluminescent light from the acridinium ester compounds can be activated through known activation reagents, typically a base and H2O2 (including H2O2-producing compounds) or O2 present in a solvent including water, ethers, esters, alcohols and ketones, and mixtures thereof. In accordance with the invention, the activation reagents are contacted with the entire reaction mixture after the binding reaction has occurred (ie, the specific binding complex bound to the solid phase is not separated from the free fraction). Preferably, the activating agent is actually two separate reagents as described below, such as the "first" reagent (added first to the reaction mixture) and the "second" reagent (added to the reaction mixture rapidly after the first reagent) The first reagent is a solution of hydrogen peroxide, aqueous acid and the second reagent is a basic aqueous reagent and the chemiluminescent light is measured quickly ("rapidity" quickly defined here as a period not exceeding several minutes, most preferably less than one minute, preferably from 0 1 seconds to a little after the second reagent is added to the reaction mixture) The first acid Reagent can be any suitable acid including nitric acid, hydrochloric acid, sulfuric acid and mixtures thereof, and the like. Most preferably, the acid in the first reagent is nitric acid present in a concentration of about 0.05 N to about 05 N (most preferably about 0.1 NJ in an aqueous solution having from 0 1% to about 10% (v / v) of hydrogen peroxide present, the percentage based on the total volume of the aqueous solution The base present in the second reagent is any suitable base, including sodium hydroxide, potassium hydroxide, lithium hydroxide, and mixtures thereof, and the like Generally, sodium hydroxide is preferred as the base in the second reagent, and can be used at a level of correlation of from about 025 N to about 0.25 N. (most preferably about 025 NJ) In addition, the usual constituents known to those experts in the art can be included in the activation reagents, such as pH-regulating substances (including phosphate pH regulator, regulates citrate pH buffer, borate pH buffer, etc.), various surfactants and / or preservatives, such as those described in (US Patent 4 927,726 and pending US Patent 08 / 339,870, filed 11/14/94 , both commonly assigned to Ciba Corning Diagnostics Corp.) and proteins (including bovine serum albumin, gelatin, casein, etc.) Very preferred activation reagents are First reactive an aqueous solution of nitric acid (preferably 0 1) in about 0 1 % to about 10% (preferably from 0 5% to 1%) of hydrogen peroxide; and the Second Reagent: an aqueous solution of about 0.25 N. to about 1 25 NaOH (preferably 0.25 N_) in water containing from about 1 to about 1% (v / v) of surfactant (preferably N- chloride). alkyl trimethylammonium), the percentage based on the total volume of the aqueous reagent solution. As used herein, the modulated chemiluminescent signal is inclusive of the total chemiluminescent signal provided by the reaction mixture. This includes the chemiluminescent extinction signal provided by the tracer bound to the solid phase, as well as any tracer that is in the solution of free fraction once the binding reaction has transpired on the solid phase The extinction percentage of the chemiluminescent signal is calculated through the following equation: Modulated signal accounts extinction% = (1 -) x 100% signal not extinguished The non-extinguished signal counts are the measured accounts of the chemiluminescent vaporization of a given amount of unbound tracer, taken in the absence of the solid phase. The given quantity is defined as the same amount of tracer needed for the assay. Extinction can then be associated with a reference, (including, for example, a DNA of synthetic target sequence etc), to determine the amount or presence of the analyte in the sample) It has been observed that the chemiluminescence is extinguished in proportion to the mass of the solid phase of the present during vaporization when the solid phase is coated with an irrelevant binding pattern The signal modulation described herein is in addition to magnitude greater than the extinction due to the mass of the solid phase, and is due to the specific binding reaction of the anahto labeled with acddinium ester and the binding pattern covalently bound to the solid phase evaluation and measurement of the emission of light generated by the activation, (for example, vaporization) of the chemiluminescent label in all modalities, can be achieved through techniques known to those skilled in the art. For example, luminometric instruments that can be used to measure chemiluminescence signals are commercially available, such as the MAGIC LITE analyzer (MLAI) manufactured by Ciba Corning Diagnostic Corp. Medfield, Mass The assay can also be prepared through the use of an automatic system that includes a luminometer with one or more photomultiplier tubes, with multiple photomultiplier tubes described in WO 94/22002, priority date 03/19/93, commonly assigned and incorporated herein by reference. The method of the invention can be practiced in various test systems and formats through techniques known to those skilled in the art. Both competitive assay formats as non-competitive (eg sandwich) can be practiced with immunological binding, chemical binding, complementary binding, and combinations thereof. In accordance with the invention, the following are illustrative immunological formats that can be used Competitive Antigen Marked in this format, the sample containing the target antigen to be measured is incubated with a solution comprising (1) a limited amount of antibody coupled to the metal oxide solid phase and (2) a tracer comprising an antigen or antigen analogue that has an acddinium ester label attached thereto During an incubation of the reaction mixture, the antigen in the sample competes with the labeled antigen (or antigen analog) for binding to the antibody bound to the solid phase after the incubation period , there are up to three components present in the reaction solution (1) a complex comprising the sample antigen u nested with the antibody bound to the solid phase (producing no signal after activation), (2) a complex comprising a tracer antigen (or antigen analogue) bound with the antibody bound to the solid phase (producing an extinguished signal after of the activation; and possibly (3) an unreacted tracer (producing a non-extinct signal) The amount of labeled antigen (or antigen analog bound to the solid phase is inversely proportional to the amount of antigen in the sample. Competitively labeled antibody: In this format, the sample containing the target antigen to be measured is incubated with a solution comprising (1) a limited amount of antigen (or antigen analogue) coupled to the metal oxide solid phase and (2) a tracer comprising an antibody having an acpdinium ester label attached thereto During an incubation of the reaction solution, the antigen in the mixture competes with the solid phase antigen, or antigen analogue) to bind to the tracer antibody After the incubation period, there are three components present in the reaction mixture (1) a complex comprising a sample antigen bound to the tracer antibody (producing a non-quenched signal after activation), (2) a complex comprising the tracer antibody bound to the antigen (or antigen analogue) bound to the solid phase) producing an extinguished signal after activation, and, possibly (3) an unreacted tracer antibody (producing a non-quenched signal) the antigen in the sample and the antigen (or analogue in the solid phase compete for the labeled antibody The amount of labeled antibody that binds to the solid phase is inversely proportional to the amount of antigen in the sample. Sandwich of labeled antibody in this format, which is typically performed on antigens which are large enough to bind two antibodies (same or different) simultaneously, the sample was incubated with an excess of an antibody bound to the solid phase together with an excess of another tracer antibody that is labeled with an acdmidium ester The antigen binds to the solid phase through its antigenic determinants and the labeled antibody in turn binds to the antigen a through a different determinant The complex formed on the solid phase (ie, the tracer antibody-antigen-solid-phase antibody) produces a chemiluminescent signal extinguished after activation and the unreacted tracer produces a non-extinct signal The amount of antibody Marking that binds to the solid phase is directly proportional to the amount of antigen in the sample. With respect to hybridization assays (eg, gene zonda assays), competitive and sandwich formats can be practiced. When used with assays of hybridization, the assay of the invention may have the advantage of the solid phase that is already present in the assay as part of the r of the initial hybridization-capture step In order for the hybridization extinction phenomenon to be used for an assay for specific nucleic sequences, preferably the extinction effect is modulated by dividing the labeled oligonucleotide between the immobilized and solution phase sequences. competitive format the immobilized oligomer and the target sequence, therefore, preferably share a substantially common sequence which is capable of hybridizing to at least a portion of the labeled oligonucleotide When the invention is practiced in ester zonda assays the ester Acidium can be placed on any desired position of the ohgonome, but most preferably the label is placed on the 5 'end of the molecule. Gene zonda assays preferably incorporate an amplification step using a replica of DNA or RNA to generate copies of DNA or RNA for each target nucleic acid sequence in the shows, through techniques well known to those skilled in the art, such, for example, those described in EP-A-0 481 704 (commonly assigned and incorporated herein by reference) After or during this amplification step, it is added to the sample shows an oligomer labeled with the ester of the actidmium. The polynucleotide sequence allows it to hybridize specifically to a portion of the sequence, which is being amplified, as well as to the same sequence immobilized to a solid phase of metal oxide, which has been added earlier in the assay procedure The amount of labeled ohomer that will be captured by the solid phase will depend on the relative amounts of the complementary sequence present in solution or immobilized in solid phase. The amount of target amplified sequence produced from the rephcase reaction , it is expected that it is in excess of that immobilized on the solid phase. Using the chemo detection method luminescent without separation, samples containing nothing of or a threshold less than the target threshold amount will result in an extinction of the light emission of the labeled ohgomero, whereas for the samples containing targets, the chemiluminescence of the labeled oligomer will remain unaffected. The extinction percentage of the labeled oligomer chemiluminescence that occurs after hybridization to an oligomer bound to the solid phase can be calculated through the following formula Reaction RLU containing the oligomer - the solid phase% Q = x 100 RLU of less solid phase control - oligomer where Q represents the extinction and RLU represents the units of relative light As shown in the calculation, the hybridization reaction and the control reaction are preferably incubated under substantially equal conditions, since the chemiluminescence of the labeled oligomer generally vanes with time , Temperature and pH Regulator Conditions According to the invention, hybridization of the labeled ohomer to the oligomer bound to the solid phase will result in a maximum reduction of the chemiluminescent signal when the solid phase capacity is in excess of the labeled or labeled gum. In addition, an excess of labeled oligomer relative to the solid phase will result in a minor extinction (ie, an excess of labeled oligomer that is not hybridized will be detected with superior efficiency generating more chemiluminescence relative to the control). The invention can be practiced using complementary substances attached to the tracer and the solid phase conjugate. Preferred complementary substances include biotin and avidin, illustrative biotin compounds include, for example, biocytin (ie biotin-eN-lysine), biocytin hydrazide. , derivatives of amine or sulfhydryl of 2-? m? nob? ot? na and hydrazide of biotinyl-e-aminocaprioic acid and biotin derivatives, including, for example, biotin-N-hydroxysuccinimide ester, of b? ot? n acid ? le-am? nocapró? co, sulfosuccinimidil 6 - (bio 11 n am? do) -bromoacet? l? draz? da, p-diazobenzoil biocitina and 3- (N-male? m? dopropo? on? l) b ? oc? t? na, which can bind binding proteins (preferably attached to the solid phase polymeric coating), as well as is well known to those skilled in the art. Avidin compounds that can be used include streptavidin, succinylated avidin. , monomeric avidin, and the like The method p to bind avidin, and biotin (or derivative of both) either directly or indirectly to the specific or solid phase conjugate tracer can be achieved through techniques known to those skilled in the art, for example, through the reaction of amino or sulfidyl groups of avidin or biotin With respect to immunological assays, the invention is particularly useful for detecting or quantifying theophylline and dinitrophenol protein (DCNP) and derivatives thereof. With respect to hybridization assays, the invention is particularly effective to detect or quantify enteric pathogens, such as, for example, Salmonella and Campylobacter species. It should be understood that various modifications to the invention will be apparent and can be readily made by those skilled in the art, giving the description herein, without departing from the scope and materials of this invention. It is noted that the following examples given in present, are intended to illustrate and not limit the invention to the same EXAMPLES The solid phase in all the examples consisted of paramagnetic particles (PMP) having a silane polymeric coating around the iron oxide core (purchased from Advanced Manetics Ine, Cambridge, MA). The PMP is activated through glutaraldehyde and conjugated with the various specific binding patterns according to the two-step procedure described by E V., et al. (Bio Technique, 70, p 159-165) The acridinium ester (AE) used in the examples was of the following structure: R, wherein X is CH3SO4, Rj is methyl, R2, R3, R5 and R7 are hydrogen, R4 and Rs are methyl, Rg = to COOH converted to an N-succinimidyloxycarbonyl group to aid in the binding of the specific binding pattern A description of the technique that was used to bind acpdinium esters with binding conjugates can be found in Weeks et al., Clinical Chemistry, 29 (8), 1474-1479 (1983) and EP-A-0 537 994 (each of which is incorporated herein by reference) The assays were vaporized with two reagents as follows Vaporization Reagent 1 = approximately 0 3 ml of 0 1 HNO 3 in approximately 0 5% aqueous H2O2 solution - Vaporization Reagent 2 = approximately 0.3 ml of 0.25 N. of NaOH in approximately 0.5% aqueous solution of ARQUADR, N-alkyltrimethylammonium chloride 16-50 (50% active purchased from AKZO Chemical Inc., Chicago IL). The vaporization reagent 1 was added to the reaction mixture first, followed immediately by the vaporization reagent 2. The relative light units (RLU's) were measured during a 2 second interval after injection of the vaporization reagents 1 and 2 using a luminometer (MAGICR lite Analyzer "MLA I", Ciba Corning Diagnostic Corp., Medfiled, Mas). The extinction percentage was calculated through the following equation: Modulated signal accounts% extinction = (1 x 100% Signal accounts not extinguished The modulated signal counts were a measurement of the total signal provided by the reaction mixture after the reaction between the specific binding patterns transpired The non-extinguished signal counts were a measurement of the light emitted from a quantity of tracer added to the assay , and vaporized in the absence of the solid phase.

EXAMPLE 1 This example demonstrates the antibody-dependent extinction phenomenon. Monoclonal antibodies were prepared to 2,4-d? N? Trofenol (DNP) through normal methods, after immunizing mice with a DNP conjugate with tproglobu (DNP-TG) This conjugate was prepared by the reaction of equal weights thyroglobulin and 2,4-din-tobene-sulfonyl acid at 0 15 M of a Na2C03 solution for 18 hours, followed by exhaustive dialysis against 0.001 M sodium phosphate buffer, pH 74. The antibody (anti-DNP) was purified from acites by precipitation of unwanted protein with caprylic acid, followed by dialysis of the supernatant against 0 1 M pH regulator of sodium phosphate pH 7.4 For the immobilization of the antibody, 10 mg of PMP with 6.25% glutaraldehyde in 0 1 M phosphate, pH 74, were activated for 2 hours After washing to remove the excess glutaraldehyde, the PMP was mixed with 1 ml of an antibody solution (diluted to 15 mg / ml) and allowed to stand overnight. The precipitate was then washed several times in phosphate buffer , and end ally was resuspended at a concentration of 10 mg / ml in 005 M sodium phosphate, pH 74, 0 15 M NaCl, 1 mg / ml bovine serum albumin (PBS / BSA) DNP-TG was labeled ( prepared as mentioned above) and a fluorescence isothiocyanate conjugate with bovine serum albumin (FITC-BSA) (purchased from Sigma Chemical Co., ST. Louis, MO) with acdmidium ester (EA) as follows: 2 mg of conjugate in 1 mol of 0 1 M. sodium phosphate, 0 15 M NaCl, pH 8 0 was mixed with 80 μl of EA (1 mg / ml in dimethylformamide) and incubated for 1 hour at room temperature. Then, the mixture was added to 05 ml of a 10 mg / ml solution. of DL-lysine and incubated for 15 minutes The labeled conjugate was then purified through gel filtration on a 20 cm column of Sephadex G 25 DNP-β-alanine was prepared, as one gram of β- was dissolved alanine in 50 ml of 1 M NaHC? 3 Seven milliliters of 2,4-d? n? rofluorobenzene (DNFB) was added to 100 ml of ethanol and this was added to the? -alanine and stirred at room temperature for 2 hours The ethanol was removed by rotary evaporation and the remainder was extracted with ether to remove excess DNFB. Normal HCl was added to the aqueous part until a precipitate formed. The latter was recovered through filtration and washed with ether, and It was dried in the air. The material was recrystallized twice first from water with the addition of 1 N HCl after NaHC? 3 / aC? 3, pH = 9, washed with ether and dried. One hundred microliters of the anti-DNP PMP (diluted a 1 30 with PBS / BSA) either with 100 μl of AE-DNP-TG (diluted to 15,000 with PBA / BSA) or with 10Oμl of AE-FITC-BSA (diluted to 1: 60000 with PBA / BSA). After one hour, the emission of chemiluminescent light from each mixture was measured and compared with that of the same amount of each conjugate labeled with AE in the absence of the anti-DNP PMP. For the AE-DNP-TG conjugate, 52% of the light output was extinguished by the particles carrying the specific anti-DNP antibody. For the AE-FITC-BSA conjugate, which did not bind through this antibody, only 26% of the light was quenched by the particles. These data support that the extinction increases after the formation of the binding reaction complex. specific on the solid phase. These findings are contrary to the expectation that the amount of light emission extinction of a given amount of tracer marked by a given mass of the solid phase could be equal.

EXAMPLE 2 The following example illustrates the effect of the addition of a DNP derivative (not labeled with acpdinium ester) to the system described in Example 1. 100 microliters of anti-DNP PMP (diluted 1:30 with PBS / BSA) were incubated. with 50 μl of AE-DNP-TG (diluted to 1: 7500 with PBS / BSA) and with 50 μl of a solution of DN P-β-alanine at concentrations of 50 to 500 ng / ml. After 1 hour, the chemiluminescent of each sample was measured. The results are presented in Table 1 below and graphically in Figure 1 TABLE 1 EFFECT OF ADDITION OF A DNP DERIVATIVE DNP-ß-Alanma (ng / ml chemiluminescence (RLU) 0 545,000 50 566,000 100 802,000 250 983,000 500 1,040,000 The data shows that the addition of concentration is increased by an unlabeled DNP derivative resulted in a progressive increase in light production. This effect is presumably due to unlabelled DNP competing for the available binding sites of the immobilized antibody giving as result less than the binding of DNP labeled with AE to the particles, and thereby minimizing the quenching of chemiluminescent light emission EXAMPLE 3 Monoclonal antibodies to theophylline and luteinizing hormone (LH) were generated by normal techniques after immunization of mice with 8-carboxypropylteophylline-thyroglobulin and LH respectively. A polyclonal antibody to theophylline was produced by immunization of rabbits with 8-carboxypropylteophylline-thyroglobulin. The monoclonal and polyclonal antibody (anti-LH) were purified as described above for Examples 1 and 2, except that activation in coupling was performed at 0.01 M. sodium acetate pH buffer (pH 5.5). The particles were finally resuspended at 25 mg / ml in a pH buffer of PBS / BSA. Theophylline standards were prepared by diluting a theophylline supply solution (Sigma) in PBS / BSA for the normal-based pH regulator or serum serum paranormal drug serum RIA theophylline equipment standards obtained from Clinical Assays PMP were used immobilized with either the theophylline monoclonal antibody (THEO) or the LH monoclonal antibody that was serially diluted from 2.5 mg / ml to 0.2156 mg / ml in a PBS / BSA regulator. Five hundred microliters of PMP were supplied to the test tubes, the pH regulator was removed after the magnetic separation of the particles and 100 μl of water was added to re-suspend the AE-THEO particles (04 million RLU / tube) diluted in PBS / BSA was added to each tube. The emission of chemiluminescent light was measured immediately using MLA I. RLU observed for the solution of AE-THEO with the anti-theophylline PMP and the anti-LH PMP (total modulated signal counts) were compared with the RLU measured in the absence of PMP (total accounts without extinguishing). The results are summarized in TABLE 2 below.

TABLE 2 Extinction of AE-theophylline in the presence of anti-thiofilin antibody of PMP (Anti-THEO) and PMP of anti-Luteotropic hormone antibody (Anti-LH) Antibody in (PMP) mg / tube RLU Percentage PMP 0 33597 (UTC) 0 Anti-LH 0.075 294570 13 Anti-LH 0.156 244583 27 Anti-LH 0.312 215243 36 Anti-LH 0.625 157693 53 Anti-LH 1.25 105153 69 0 412450 (UTC) 0 Anti-THEO 0.075 39865 90 Anti-LH THEO 0156 30915 92 Anti-THEO 0312 24900 94 Anti-THEO 0625 19635 95 Anti-THEO 125 13255 97 TABLE 2 is graphically represented in Figure 2, where the separated lines represent PMP of a-teofi na and PMP of a-LM, as shown in the data shown in TABLE 2 and in Figure 2, illustrate that the percentage of extinction observed when anti-THEO PMP was present appeared relatively independent of the concentration of the PMP mass on this scale. In contrast, extinction of beads was observed in relation to the mass of particles when the particles were coupled with an "irrelevant" antibody (anti-LH) obtained EXAMPLE 4 Patient samples were obtained from a clinical laboratory (Metpath, Ine) Theophylline levels were obtained for patient samples using a commercially available assay, TDX theophylline assay (Abbott Laboratories Abbott Park, 111) Theophylline assays were performed without separation as follows normal controls or samples were incubated with AE-theophylline and PMP aptiteof monoclonal mine at room temperature for 10 minutes. Chemiluminescence was measured in MLA I without further manipulation of the tubes. At a low concentration of analyte, most of the tracer joined the solid phase and did not vaporize, thus giving low counts. As the concentration of analytes increased, more tracker remained unbound, thus giving a higher signal. A normal sample curve is presented in Figure 3. The results of an assay of 125 patient samples are given in TABLE 4. A commercially available theophylline assay (TDX assay purchased from Abbott) was used as a control with the data from the control shown in TABLE 4 TABLE 3 Theophylline assay without separation PATIENT Theophylline, μg / ml INVENTION TEST CONTROL TDX WITHOUT SEPARATION (μg / ml) (μg / ml) 1 5.2 8.3 2 16.0 13 0 3 7.1 68 4 11.1 12.9 5 13.2 9 3 6 4.6 4 8 7 232 198 8 146 146 9 50 64 10 159 105 11 87 68 12 56 63 13 11 4 77 14 144 90 Correlation Test without separation = 2 42 + 0656 TDX R = 0874 EXAMPLE 5-7 Hybridization Assays The nucleic acid sequences used in the hybridization assays described in Examples 5-7 are provided in the sequence listing herein, wherein the following abbreviations are used to present the hybridization assays adenine (A), tnmine (T ), uracil (U), guanine (G), cytosine (C) PM979 shown as SEQ NO 1, where bases 1-10 constitute a separating arm, bases 11-46 constitute the 5 'sequences of the nanovaper template ( +) and bases 47-70 are complementary to a specific target sequence of Salmonella MASA5 shown as SEQ NO 2, where the bases 1-10 constitute a spacer arm, bases 11-71 constitute the 5 'sequences of the midivapant template (+) and bases 72-95 are complementary to a specific target sequence of Salmonella PM1076 shown as SEQ NO 3, wherein half of the Salmonella target, complementary to PM979 and MASA5 was used PM2058 shown as SEQ NO 4, where the Salmonella target, complementary to PM979 and MASA5 was used Some versions of this sequence have a 5'-ammo group for conjugation with acddinium ester, as described in the present SA7 objective shown as SEQ NO 5 MD24 shown as SEQ NO 6, where a complementary zonda was used, the bases 34-57 of the midivapant (+) template, that is, were used bases 44-67 of MASA 5 Some versions of this sequence have a 5 'amino group for conjugation with this of acpdinium, as described in the present MDV-SA2 RNA transcript shown as SEQ NO. 7, wherein the bases 1-61 consist of the 5 'end of the midivapant template (+), the bases 62-69 and 118-123 are plasmid slugging sequences, the bases 70-117 are complementary to the specific target sequence of Salmonella, and the bases 124 -282 consist of the 3 'end of the midivapant template (+) The solid phases consisted of PMP to which one of the amplification oligomer beads was covalently linked (through the 5'-thermnal amino group using a coupling reagent heterobifunctional for PM979, or by activation of glutaraldehyde for MASA5) The sequences AE-Pm1076 and 5 '32p_p 2C.58 are complementary to the anti-target portion of the immobilized zondas, (therefore, hybridization and capture occurred simultaneously ) PM979-PMP was prepared using oligomer PM979 (obtained from Promega Corp, Madison, Wl) and a uncle-terminated PMP (purchased from Advanced Magnetics, Cambpde, MA) PM1076 labeled with AE (oligomer AE) was prepared using the dimethylacpdinium ester label and the PM1076 oligomer obtained from Promgega Corp MASA5-PMP was prepared using the MASA5 oligomer (obtained from Promega Corp) and a PMP Uncle-finished purchased from Advanced Magnetics PM 2058 oligomer was also obtained from Promega Corp Sodium citrate was purchased at Mallinckrodt, Ine, St Louis, MO NaCI, Tris EDTA and Tween-20 were all purchased from Sigma Chemical Co, St. Louis, MO) and BSA (Fraction V) of Miles, Ine EXAMPLE 5 This example illustrates the quenching of the AE-oligomer chemiluminescence through hybridization to the PMP oligomer. Hybridization reactions containing PMP979 immobilized in PMP (PM979-PMP) and AE-PM1076 (oligomer AE) were performed on ice by adding 40 μl of 60 mM sodium citrate, 600 mM sodium chloride, 10 mM tris (hydroxymethyl) amino methane (Tris) HCl, 1 mM ethylene diaminotetraacetic acid (EDTA), 0 1% (w / v) BSA, and 0.02% (v / v) polyoxyethylene sorbitan mololaurate (20) (TWEEN-20) at a pH 7.5 (hereinafter "pH regulator") to ten tubes. Second, 5 μl of pH buffer containing 50 μg of PM979-PMP was added to five of the ten tubes, and 5 μl of pH buffer alone was added to the remaining five tubes for control reactions. Finally, 5 μl of pH buffer containing 10, 32, 100, 320 and 1000 (fmol (1 fmol = 1 x 10'15 mol) of AE-PM1076 was added to the wall of tubes, a group containing PMP and a group of control without PMP (control for AE decomposition during hybridization) All reactions were incubated at 56 ° C and samples of 5 μl were removed at 0, 75, 120 and 180 minutes, added to 100 μl of water, vaporized with reagents 1 and 2 in a luminometer to determine the chemiluminescence activity The extinction percentage was calculated from the measured signals of the various tubes and are presented in Figure 4 As shown in Figure 4, the chemiluminescence of all the reactions containing the oligomer-PMP were dramatically reduced relative to the corresponding control without oligomer-PMP, up to a maximum of 74%, after hybridization Also, the data demonstrate conditions where the relative input of AE-oligomer and oligomer PMP gene The reaction conditions having 100 fmoles of input of the AE oligomer, where the solid phase capacity and five times the input of the AE oligomer, were chosen to test the reliability of the competitive assay of Example 6 EXAMPLE 6 Hybridization reactions were performed on ice by adding 10 μl of pH buffer (described in Example 5) alone or 10 μl of pH buffer containing 10 μg of PM979-PMP to each of the five tubes. Then, 10 μl was added. of pH regulator containing 0.10"14, 1013, 10-12, and 10'11 moles of normal PM979 (the oligomer that was immobilized on the PMP), to one of the tubes containing PM979-PMP and a control tube without PMP for each amount of normal added Finally, 80 μL of pH buffer containing 200 fmoles was added to all reactions.

AE-PM1076 All tubes were incubated at 56 ° C 10 μl samples were removed in duplicate of each reaction at the indicated times (0, 20, 40 and 60 minutes) were added to 100 μl of water and processed in a luminometer to determine chemiluminescent activity. The results are shown in TABLES 4 (Fig. 5) and 5. TABLE 5 shows the data derived from the 20-minute data of TABLE 4. The results shown are consistent with theoretical expectations, that is, the same maximum degree of Extinction was achieved in all hybridizations, except where the entry of PMP979 in solution exceeds the solid phase capacity.

TABLE 4 EXTINGUISHING COMPETITIVE HYBRIDIZATION TEST REACTION Input Total Signal Time PERCENTAGE normal Hybridization (RLU x lO-5) OF EXTIN- (moles) 1 (+) PMP (-) PMP CION2 -11 0 8.27 8 66 4 20 461 5 65 18 40 478 5 78 17 60 4.38 5 30 17 B 10-12 0 782 8 60 9 20 260 5 28 51 40 271 5 31 49 60 263 5 39 51 10 13 0 762 936 19 20 1 43 5 43 74 40 1 48 5 56 73 60 1 51 5 21 71 D 10 • 14 0 8 05 8 57 6 20 1 21 5 21 77 40 1 28 5 26 76 60 1 26 5 11 75 8 00 8 45 5 1 15 5 41 79 1 26 5 27 76 1 24 4 82 74 To the normal was added PM979 2 [1- (+ P P RLU) * (-PMP RLU] x 100% = Percentage of extinction TABLE 5 COMPETITIVE HYBRIDIZATION EXTINGUISHING TEST - 20 MINUTES DATA DEPT - Reaction Input (+) PMP Net (+) Normal PMP Signal signal (moles) (RLU) 1 (RLU) 2 A 10- 1 461 480 346 830 B 10- 2 259,560 144,910 C 10-13 142,860 28,210 D 10-14 120,720 6,070 E 0 114,650 0 1: Derived from the 20 minute data point of BOX 4 2 '(+ PMP RLU) * (+ PMP RLU) E i = value of "+ PMP RLU" E = value of "Reaction E" As shown by the above data, this Example demonstrates a hybridization competition assay for the synthetic DNA oligomer. Varying the amounts of the same oligomer that was immobilized in the PMP were added in solution to compete with the immobilized solid phase oligomer. for the hybridization of the AE oligomer, that is, the added oligomer that functions as a competitive standard. This resembles the competition that could arise due to the presence of an amplified zonda in the proposed assay of Example 7 EXAMPLE 7 Amplification reactions that were initiated in 0, 10"6, 10_ 8, 10_ 9 and 10-20 of the midivapant template, respectively, were determined through the addition of EDTA and placed on ice.The hybridization reactions were fixed in ice. they added 5, 20, 70 or 75 μl of pH regulator to 12 tubes, so that the final volume could be 100 μl after the addition of all the other components, as described above, then added to six tubes 50 μl of pH buffer containing 5 μg of PM979-PMP Then, 5 μl of pH buffer or one of the five replication reactions was added to the tube pairs, one with and the other without PMP. tubes 25 μl of pH buffer containing 10 fmoles of AE-MD24 All the tubes were incubated for 20 minutes at 56 ° C, then they were vaporized with reagents 1 and 2 in a luminometer to determine the chemiluminescent activity The results, shown in the CU Adro 6, do not reflect the presence of abundant replicate mid-RNA (which was estimated to be of the order of 5 fmoles per reaction). The failure of the assay to detect the presence of the midivapant sequence was due to the quality of the reagent, since the reagents were validated by titration and imitation tests with synthetic target. Additional analyzes with thermal denaturation of the target failed before addition and entry. Increased ohgomer AE. Subsequent experiments using 32 P-labeled MD24 suggest that the failure is due to inefficient hybridization of the zonda to the target, presumably due to the superior stability of a midivariant duplex. This hypothesis is supported by the results of an extinction test for the transivated individual chain structure midivariant objective, which produced a normal curve with extinction values consistent with the theoretical expectations with respect to the molar ratio of the target and zonda AE .

TABLE 6 HYBRIDIZATION EXTINGUISHING TEST FOR PRODUCTS OF REPLICATION Reaction Added template + PMP RLU Percentage (moles) or Extinction medium - PMP tion1 A Regulator only - 235,545 70 pH * + 71,325 B 0 (reaction-190,240 65 mutation) 10-16 + 66,690 C 10-16. 190,055 65 + 66,570 D 10-18 - 189,950 64 + 67,820 10-19 178,570 64 + 63,780 10 • 20 164,590 63 + 60,190 -% Q = [1 - (+ PMP value) / (- PMP value)] x 100 LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) Eve H. Barlow (B) Eddie Carroll, III (C) Joseph E. Connolly (D) Michael J. Lee (E) Richard A. Martinelli (F) John T. Unger (ii) TITLE OF THE INVENTION: QUIMIOLUMINISCENT TEST OF SPECIFIC UNION WITHOUT SEPARATION (iii) NUMBER OF SEQUENCES: 7 (iv) DIRECT THE CORRESPONDENCE: (A) RECIPIENT: Ciba Corning Diagnostics Corp. (B) STREET: 63 North Street (C) CITY: Medfield (D) STATE: Massachusetts (E) COUNTRY: USA (F) CP: 02052 (v) READING FORM ON THE COMPUTER: (A) TYPE OF MEDIUM: soft disk 3.5, 1.44 Mb storage (B) COMPUTER: IBM PS / 2 PC (C) OPERATING SYSTEM: MS-DOS 6.2 (D) SOFTWARE: Word 6.0 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: Not assigned (B) DATE OF PRESENTATION: with this (C) CLASSIFICATION: not assigned (viii) INFORMATION OF THE EMPLOYEE / AGENT : (A) NAME: Judith A. Roesler (B) REGISTRATION NUMBER: 34, 237 (C) AUTHOR'S RECORD NO .: CCD-120 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (508) 359-3876 (B) TELEFAX: (508) 359-3885 (2) INFORMATION FOR THE IDENTIFICATION SEQUENCE NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 (B) TYPE: nucleic acid (C) CHAIN FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: zoned nucleic acid, PM979, having a portion complementary to the specific target sequence of Salmonella wherein bases 1-10 constitute a separating arm, bases 11-46 constitute the 5 'sequences.; fle the nanovariant template (+) and the bases 47-70 are complementary to the specific target sequence of Salmonella. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 1 1 11 21 31 (5 ') H2-CCTAGTCCAA GGGGAAATCC TGTTACCAGG ATAACGGGGT 41 51 61 TTTCTCATAA GCGCCATTGA TGTTGTCGCC (3 ') (3) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 95 (B) TYPE: nucleic acid (C) CHAIN FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acid band, MASA5, having a complementary portion to the specific target sequence of Salmonella where bases 1-10 constitute a separating arm, bases 11-71 constitute the 5 'sequences of the midivariant template (+) and bases 72-95 are complementary to the specific target sequence of Salmonella. (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) DESCRIPTION OF SEQUENCE: SEQ ID NO .: 2 1 11 21 31 (5 ') NH2-CCTAGTCCAA GGGGACCCCC CGGAAGGGGG GACGAGGTGC 41 51 61 71 GGGCACCTCG TACGGGAGTT CGACCGTGAC AGGTCAACTG 81 91 AACGCCCTGA GCTTT (3 ') (4) INFORMATION FOR SEQ ID NO 3 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 24 (B) TYPE nucleic acid (C) FORM OF THE CHAIN simple (D) TOPOLOGY linear (II) TYPE OF MOLECULE zonda nucleic acid , PM1076, half of the Salmonella target, complementary in PM979 and MASA5, defined in this (m) HYPOTHETICAL NO (iv) ANTI-SENSE No (v) DESCRIPTION OF THE SEQUENCE SEQ ID NO 3 1 11 21 (5") NH2- GGCGACAACA TCAATGGCGC TTAT (3 ') (5) INFORMATION FOR SEQ ID NO 4 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 48 (B) TYPE nucleic acid (C) FORM OF THE CHAIN simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: nucleic acid band PM2058, a Salmonella target, complementary to PM979 and MASA5, defined herein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) DESCRIPTION OF SEQUENCE: SEQ ID NO. : 4 1 11 21 31 (5 ') GGCGACAACA TCAATGGCGC TTATAAAGCT CAGGGCGTTC 41 AGTTGACC (3') (6) INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 48 (B) TYPE: nucleic acid (C) CHAIN FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleus of nucleic acid Sa7 , an anti-Salmonella target (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) DESCRIPTION OF SEQUENCE: SEQ ID NO .: 5 1 11 21 (5 ') GGTCAACTGA ACGCCCTGAG CTTTATAAGC GCCATTGATG 41 TTGTCGCC (3 ') (7) INFORMATION FOR SEQ ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 24 (B) TYPE: nucleic acid (C) CHAIN FORM, simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acid band, MD24 , complementary to the bases 34-57 of the midivapant template (+), that is, the bases 44-67 of MASA5, defined previously (iii) HYPOTHETICAL No (iv) ANTI-SENSE No (v) DESCRIPTION OF THE SEQUENCE SEQ ID NO 6 1 11 21 (5 ') ACGGTCGAAC TCCCGTACGA GGTG (3') (8) INFORMATION FOR SEQ ID NO. 7 (i) CHARACTERISTICS OF THE SEQUENCE "(A) LENGTH: 289 (B) TYPE nucleic acid (C) FORM OF THE CHAIN simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleus of nucleic acid, in transcription of MDV-SA2 RNA, where bases 1-61 consist of the 5 'end of the midivariant template (+), bases 62-69 and 118-123 are plasmid slug sequences, bases 70-117 are complementary to the specific target sequence of Salmonella, and bases 124-282 consist of the 3 'end of the midivariant template (+). (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 7 January 11 21 +31 (5 ') GGGGACCCCC CGGAAGGGGG GACGAGGUGC GGGCACCUCG 41 51 61 71 UACGGGAGUU CGACCGUGAC GAGCCUCGAG GCGACAACAU 81 91101111 CAAUGGCGCU UAUAAAGCUC AGGGCGUUCA GUUGACCUCG 121131141151 AGGAGUCACG GGCUGCGCUU UCGCGCAUCTJ CCCAGGUGAC 161171181191 GCCUCGAGAA GAGGCGCGAC CUUCGUGCGU UUCGGCGACG 201211221231 CACGAGAACC GCCACGCUGC UUCGCAGCGU GGCCCCUUCG 241 251 261 271 CGCAGCCCGC UGCGCGAGGU GACCCCCGAA GGGGGGU CC 288 CC (3 ')

Claims (26)

1 A specific binding assay without separation for detecting or quantifying the presence of an analyte, said assay comprising (a) contacting a sample with a solid phase, comprising a metal oxide having a conjugate attached thereto, and a tracer, comprising a binding conjugate labeled with acpdinium or benzacpdinium ester, (b) allowing the solid phase, the tracer and the sample to react to form a reaction mixture comprising a specific binding complex attached to said phase. solid and a free fraction not bound to the solid phase, (c) contacting the reaction mixture with an activating agent to vaporize the acpdmium ester tracer and provide a modulated chemiluminescent signal, (d) measuring the modulated signal, and (e) associating the modulated signal with a reference to determine the amount or presence of the analyte in said sample.

2 An assay according to claim 2 wherein the specific binding complex is formed through an immunological reaction

3. An assay according to claim 2, wherein the solid phase of metal oxide comprises iron oxide

4. An assay according to claim 3, wherein the activating agent comprises a first reagent comprising an aqueous solution of acid hydrogen peroxide and a second reagent comprising a basic aqueous solution, the first reagent being in contact with the mixture of reaction before the second reagent does.

5. An assay according to claim 4, wherein the first reagent has an acid component selected from the group consisting of nitric acid, hydrochloric acid, sulfuric acid, and mixtures thereof, and the second reagent has a base selected from the group consists of sodium hydroxide, potassium hydroxide, lithium hydroxide, and mixtures thereof

An assay according to claim 5, wherein the first reagent of said acid is present in an amount ranging from 0 1 N_ to about 0 5 N_, in an aqueous solution of hydrogen peroxide present in an amount varying from about 0.1% to about 10% (v / v) based on the total volume of the reaction of the first aqueous reagent, and in the second reagent, said base is present in the aqueous solution in a concentration ranging from about 025 N to about 1 25 N

An assay according to claim 6, wherein the first reagent comprises an aqueous solution of 0 1 N of nitric acid from 025% to 1% hydrogen peroxide and the second reagent comprises an aqueous solution of 025 N of hydroxide of sodium and a surfactant present in an amount ranging from about 0 1% to about 1% (v / v) based on the total volume of the second aqueous reagent

An assay according to claim 7, further characterized in that the analyte is selected from the group consisting of theophylline or a dinitrophenol protein

An assay according to claim 1, further characterized in that the assay is a hybridization assay and the specific binding complex is formed through complementary binding

An assay according to claim 9, wherein the solid phase of metal oxide comprises iron oxide.

An assay according to claim 10, wherein the activating agent comprises a first reagent comprising an aqueous solution of acid hydrogen peroxide and a second reagent comprising a basic aqueous solution, the first reagent is contacted with the reaction mixture before the second reagent does.

12. An assay according to claim 1, wherein the first reagent has an acid component selected from the group consisting of nitric acid, hydrochloric acid, sulfuric acid, and mixtures thereof and the second reagent has a base selected from the group consisting of of sodium hydroxide, potassium hydroxide, lithium hydroxide and mixtures thereof

13. An assay according to claim 12 further characterized in that the first reagent of said acid is present in an amount ranging from about 0 1 N to about 0.25 J in an aqueous solution of hydrogen peroxide present in an amount ranging from about 0. 1 5 to about 10% (v / v), based on the total volume of the solution of the first aqueous reagent, and in the second reagent, said base is present in the aqueous solution in a concentration ranging from about 025 t ± a approximately 1.25 N ..

An assay according to claim 13, wherein the first reagent comprises an aqueous solution of 0 1 N of nitric acid from 0.5 to 1% (v / v) of hydrogen peroxide, and the second reagent comprises an aqueous solution of 025 N, of sodium hydroxide and a surfactant present in an amount ranging from about 0.1% to about 1% (v / v) based on the total volume of the second aqueous reagent

15. An assay according to claim 1, further characterized in that the binding reaction is immunological and the analyte is selected from the group consisting of ti of i lina and a dinitrophenol protein.

16. An assay according to claim 1, further characterized in that the binding reaction is complementary to the binding and the analyte is an enteric pathogen

17. A specific binding assay without separation according to claim 1, for detecting or quantifying an analyte in a sample, the assay comprising, in the established order: a) incubating a solution comprising a sample, a solid phase comprising the oxide of metal coupled to a specific binding pattern to the analyte; and a tracer comprising said analyte or analyte analog having an acpdinium or benzacridinium ester attached thereto, b) allowing the solution to react to form a reaction mixture comprising a specific binding complex comprising the tracer linked to the solid phase and a free fraction comprising an unreacted tracer; c) contacting the reaction mixture with a first activating reagent comprising an acid hydrogen peroxide solution; d) contacting the mixture of step C with a second activation reagent comprising a basic aqueous solution to provide a modulated chemiluminescent signal, e) measuring the modulated chemiluminescent signal, f) associating the modulated signal with a reference to determine the amount or presence of the analyte in said sample

An assay according to claim 17 wherein the amount of tracer from step A is separately activated to emit a chemiluminescent light emission which is measured in the absence of the solid phase resulting in a non-extinct signal count measurement, a percentage of extinction effect is calculated using the modulated signal of step e) and the signal count not extinguished through the equation Modulated signal accounts extinction% = (1-) x 100% Signals not extinguished signal and said percentage of extinction is associated with the reference of step (f) to provide a determination of the presence of or the amount of the analyte.

19. An assay according to claim 18, wherein the first reagent comprises an aqueous solution of 0 1 N, of nitric acid of 0 5% to 1% of hydrogen peroxide and the second reagent comprises an aqueous solution of 025 N of hydroxide of sodium and a surfactant present in an amount ranging from about 0 1% to about 1% (v / v), based on the total volume of the second aqueous reagent

A specific binding assay without separation according to claim 1, for detecting or quantifying an analyte in a sample, the assay comprising, in the established order a) incubating a solution comprising a sample a solid phase comprising an oxide of metal coupled to an analyte or analogue of anahto specific to anahto, and a tracer comprising a specific binding pattern for the analyte or analyte analog having an acpdinium or benzacpdinium ester attached thereto, b) allowing the solution to react to form a reaction mixture comprising a specific binding complex. comprising the tracker bound to the solid phase and a free fraction comprising an unreacted tracer, c) contacting the reaction mixture with a first activating reagent comprising an acid hydrogen peroxide solution, d) contacting mixing step C with a second activation reagent comprising a basic aqueous solution to provide a modulated chemiluminescent signal e) measuring the modulated chemiluminescent signal, f) associating the modulated signal with a reference to determine the amount or presence of the analyte in said sample

An assay according to claim 20 wherein the amount of tracer from step A is separately activated to emit a chemiluminescent light emission which is measured in the absence of the solid phase resulting in a non-extinct signal count measurement a percentage of extinction effect is calculated using the modulated signal of step e) and the signal count not extinguished through the equation Modulated signal accounts% extinction = (1 -) x 100% Signals not extinguished signal and said percentage of extinction is associated with the reference of step (f) to provide a determination of the presence of or the amount of the analyte

22. An assay according to claim 21, wherein the first reagent comprises an aqueous solution of 0 1 N of nitric acid from 0.5% to 1% hydrogen peroxide and the second reagent comprises an aqueous solution of 0.25 N hydroxide. sodium and a surfactant present in an amount ranging from about 0.1% to about 1% (v / v), based on the total volume of the second aqueous reagent.

23. A specific binding assay without separation according to claim 1, for detecting or quantifying an analyte in a sample, the assay comprising in the established order: a) incubating a solution comprising a sample, a solid phase comprising an oxide of metal coupled to an analyte analogous analogue or analyte; and a tracer comprising a second analyte-binding pattern having an acpdinium or benzacpdinium ester attached thereto, wherein the first and second binding patterns can be the same or different, b) allowing said solution to react to form a mixture of reaction comprising a specific binding complex comprising the tracer bound to the analyte, which is bound to said solid phase and a free fraction comprising an unreacted tracer; c) contacting the reaction mixture with a first activating reagent comprising an acid hydrogen peroxide solution, d) contacting the mixture of step C with a second activating reagent comprising a basic aqueous solution to provide a modulated chemiluminescent signal, e) measuring the modulated chemiluminescent signal f) associating the modulated signal with a reference to determine the amount or presence of the analyte in said sample

An assay according to claim 23 wherein the amount of tracer from step A is separately activated to emit a chemiluminescent light emission which is measured in the absence of the solid phase resulting in a non-extinct signal count measurement, a percentage of extinction effect is calculated using the modulated signal of step e) and the signal count not extinguished through the equation Modulated signal accounts extinction% = (1-) x 100% Signals not extinguished signal and said percentage of extinction is associated with the reference of step (f) to provide a determination of the presence of or the amount of the analyte

25. An assay according to claim 24, wherein the first reagent comprises an aqueous solution of 0.1 N nitric acid from 0.5% to 1% hydrogen peroxide and the second reagent comprises an aqueous solution of 0.25 N hydroxide sodium and a surfactant present in an amount ranging from about 0.1% to about 1% (v / v), based on the total volume of the second aqueous reagent.

26. A diagnostic kit for use in an assay without separation to detect or quantify the presence of an analyte in a sample, said assay being a specific binding assay without separation to detect or quantify the presence of an analyte, the equipment comprises: (a) ) a solid phase, comprising a metal oxide having attached thereto a binding conjugate, and a tracer, comprising a labeled binding conjugate with acridinium or benzacridinium ester; b) an activating agent to vaporize the acridinium ester tracer and c) optionally normal or controls or both.