WO1997001763A1 - Non-separation specific binding chemiluminescent assay - Google Patents

Abstract

The assay described herein is predicated on an observation that when acridinium ester labeled tracers are bound to their corresponding binding conjugate immobilized on a metal oxide solid phase, the measurable chemiluminescent light emission of the labeled tracer bound to the solid phase is quenched as compared to the free fraction tracer that is unattached to the solid phase. According to the invention, a non-separation specific binding assay to detect or quantify the presence of an analyte is provided where the entire reaction mixture is flashed (including unreacted tracer) and modulated signal (because of the quench effect) is associated with a reference, thus determining the amount or presence of said analyte in said sample. Disadvantages inherent in heterogeneous assays employing multiple separations may be avoided using this non-separation method.

Description

NON-SEPARATION SPECIFIC BINDING CHEMILUMINESCENT ASSAY

Field of Invention

This invention relates to a non-separation

chemiluminescent specific binding assays. Definitions

As used herein "analyte" is defined as any

substance capable of undergoing a binding reaction with

one or more specific binding partners. The term

"analyte" encompasses, but is not limited to, serum proteins, hormones, drugs, antigens, antibodies (including monoclonal, polyclonal, and fragments

thereof) , pathogens, enzymes, metabolites, coenzymes and

their binding partners, polynucleotides,

oligonucleotides, hybrids of polynucleotide or oligonucleotides, and metals and chelating agents

thereto. "Specific binding partner" is defined as a

substance capable of binding to the targeted analyte and

an analyte analogue. The term "specific binding

partner" encompasses, but is not limited to, antibodies, antigens, avidin, biotin, thyroxin, thyroxin-binding

globulin, polysaccharide, phosphorylcholine,

aminoethyldihydrogen phosphate residues, estrogen,

vitamin B-12 intrinsic factor, binding proteins, mixtures thereof, and various other proteins and

peptides including oligonucleotides and so on. "Analyte

analogue" is defined as a substance that is capable of

pairing with the selected specific binding partner of

the targeted analyte through a specific binding

reaction. "Specific binding complex" refers either to a

complex of the targeted analyte-specific binding

partner, a complex of the analyte analogue-specific

binding partner, or a complex of a first specific

binding partner-targeted analyte-second specific binding

partner "sandwich" (where the first and second binding partners may be the same or different) . The "specific binding complex" may be formed by any number of specific

binding reactions including immunological, chemical, and complementary binding. "Tracer" is defined as a labeled

specific binding partner, a labeled analyte, or a

labeled analyte analogue. "Binding conjugate" is defined as a binding moiety that may be selected from

the analyte, analyte analogue, or specific binding

partner of the analyte. "Free fraction" is defined as components of the assay that are not attached to the

solid phase.

Background of the Invention

As is well known in the art, compounds that

provide a chemiluminescent signal are currently utilized

as labels in specific binding assays. Both separation

(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 activating and measuring the chemiluminescent flash. Thus, typically, heterogeneous assay protocol

requires several steps of separation of the solid phase

reaction product from the free fraction, where decanting and resuspension in water is typically required. These separation step(s) associated with heterogeneous assays

occupy a significant amount of time and increase the chance of operator error. Homogeneous assays generally refer to assays that

typically do not require physical separation of the

solid phase reaction binding complex and free fraction

prior to activating and measuring the chemiluminescent

flash. For example, U.S. Patent No. 5,017,473 describes

a separation free solid phase immunoassay utilizing

light absorbing material (a dye) , where the material is

described as absorbing all of the chemiluminescence except that associated with the bound tracer whereby the only emission detected is due to the bound tracer.

Alternative assay systems that eliminate the need of separating the solid phase binding complex from the

free fraction are needed.

Summary of the Invention

The assay described herein is predicated on an

observation that when acridinium ester labeled tracers

are bound to their corresponding binding conjugate

immobilized on a metal oxide solid phase, the measurable

chemiluminescent light emission of the labeled tracer

bound to the solid phase is reduced (referred to herein

as "quenched") as compared to the chemiluminescent signal generated from the tracer that remains in the

free fraction unattached to the solid phase.

According to the invention, a non-separation

specific binding assay to detect or quantify the

presence of an analyte in a sample is provided, said

assay comprising: contacting said sample with a solid

phase comprising a metal oxide having attached thereto a

binding conjugate and a tracer comprising an acridinium

or benzacridinium ester labeled binding conjugate;

allowing said solid phase, tracer and sample to react to

form a reaction mixture comprising a specific binding

complex bound to said solid phase and a free fraction

unattached to said solid phase; contacting said reaction

mixture with an activating agent to flash said

acridinium or benzacridinium ester tracer and provide a

modulated chemiluminescent signal; measuring said

modulated signal; and associating said modulated signal

with a reference to determine the amount or presence of

said analyte in said sample.

Also provided with this invention is a diagnostic

kit for carrying out the above-described method. Disadvantages inherent in heterogeneous assays

employing multiple separations may be avoided using this

non-separation method. Additionally, the automation of

the assays is easier when the separation step of the

assay may be eliminated or less involved, as is

accomplished by this invention.

Description of Drawings

In the Figures included herewith, Fig. 2 is a

graphical representation of TABLE 1 of Example 2. Fig.

2 is a graphical representation of TABLE 2 of Example 3,

showing both α -Theophylline PMP and α -LH PMP. Fig. 3

is a standard curve for the measurement of theophylline,

as used as a reference in Example 4. Fig. 4 is

described in Example 5. Fig. 5 illustrates a

competition hybridization assay as described in Example

6.

Detailed Description

The present invention may be employed in detecting

and/or quantifying targeted analytes present in chemical

or biological substances. Generally any analyte

interaction that is stable under chemiluminescence activation conditions may be prepared in this assay

system.

Although various types of chemiluminescent

compounds having an acridinium, benzacridinium, or

acridan type of heterocyclic ring systems are preferred

labels, use of equivalent chemiluminescent compounds and

means for activating the labels do not depart from the

scope of this invention. Acridinium and benzacridinium

esters are currently the more preferred chemiluminescent

compounds, with preferred acridinium esters including

those compounds having heterocyclic rings or ring

systems that contain the heteroatom in a positive

oxidation state including such ring systems as

acridinium, benz [a] acridinium, benz [b] acridinium,

benz [c] acridinium, a benzimidazole cation, quinolinium,

isoquinolinium, quinolizinium, a cyclic substituted

quinolinium, phenanthridinium, and quinoxalinium, as are

well-known in the art. The tracer may be prepared by

attaching the selected binding conjugate either directly

or indirectly with a reactive functional group present

on the acridinium or benzacridinium ester, as is well known to those skilled in the art, e.g. Weeks et al. ,

Clinical Chemistry. 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 the meta position of the aryl ring.

Particularly stable acridinium and benzacridinium esters

are those having an aryl ring leaving group, with the

aryl ring having an electron donating species

(preferably C₁-C₄ alkyl or alkoxy group, most preferably

methyl) present in both ortho positions of the aryl ring

and having the reactive functional group (preferably a -

COOH which is converted to a N-succinimidyloxycarbonyl

group prior to the attachment of the binding partner) in

the meta or para position (most preferably para) , as

described in U.S. Patent No. 4,745,181 and WO 94/21823,

both of which are incorporated 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 should

be water insoluble and maintain structural integrity when exposed to water or biological fluids and may be

particulate in nature (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, microtiter plate, membrane,

film, filter paper, discs, and so on) . More preferably

the solid phase particles comprise a core of iron oxide

as described in U.S. Patent 4,554,088, issued to

Whitehead, et al., 1985 (hereby incorporated by

reference) . To assist in the attachment of the binding conjugate, preferably the metal oxide solid phase has

present a bioaffinity substance present thereon. Silane

polymeric coatings are particularly preferred

bioaffinity substances and generally may be defined as

organofunctional and silicon-functional compounds that

are characterized in that the silicon portion of the

molecule has an affinity for inorganic materials while

the organic portion of the molecule is tailored to

combine with organics. Preferably chemical reactions

to attach the binding conjugate to the solid phase

include, but are not limited to, diazotization, carbodiimide and glutaraldehyde couplings. Coupling

techniques that may be used are described in Method of

Enzy.. 2-2, p. 159-165 (1980) , and Groman, E.V., et al,

in Bio Techniques. Vol. 3, pp. 156 (1985), and U.S.

Patent 4,672,040, issued to Josephson in 1987 (each of

which are hereby incorporated by reference) .

If the present invention is practiced with gene

probes, nucleic acid hybridization may be accomplished

using the metal oxide solid phase where the

hybridizations are carried out by dispersing a nucleic

acid-coupled solid phase (most preferably a DNA

oligomer) in a reaction mixture containing molecules to

be isolated, allowing the nucleic acid-coupled solid

phase to hybridize to a complementary target sequence.

Chemiluminescent light emission of the acridinium

ester compounds may be triggered by known activating

reagents, typically a base and H₂0₂ (including H₂0₂

producing compounds) or 0₂ present in a solvent (s)

including water, ethers, esters, alcohols, and ketones,

and mixtures thereof. According to the invention, the

activating reagent (s) is contacted with the entire reaction mixture after the binding reaction has occurred

(i.e. 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 as the "first"

reagent (added first to the reaction mixture) and the

"second" reagent (added to the reaction mixture promptly

after the first reagent) . The first reagent is an

aqueous acidic hydrogen peroxide solution and the second

reagent is a basic aqueous reagent and the

chemiluminescent light is measured promptly ("promptly"

defined herein as a time period not to exceed several

minutes, more preferably less than one minute, most

preferably about 0.1 second or so after the second

reagent is added to the reaction mixture) . The acid of

the first reagent may 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 from about 0.05 H to about 0.5 N (most

preferably about 0.1 H) in an aqueous solution having from about 0.1% to about 10 % (v/v) hydrogen peroxide

present, said % based on the total volume of 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 may be used in a

concentration level of from about 0.25 N_ to about 1.25

N, (most preferably about 0.25 H) • Additionally, the

usual constituents known to those skilled in the art may

be included in the activating reagent (s) , such as,

buffer substances (including phosphate buffer, citrate

buffer, borate buffer, and so on) , various surfactants

and/or preservatives (such as described in U.S. Patent

4,927,769 and pending USSN 08/339,870, filed 11/14/94,

both commonly assigned to Ciba Corning Diagnostics

Corp.) , and proteins (including bovine serum albumin,

gelatin, casein, and so on) . The most preferred

activating reagents are: First reagen : aqueous solution

of nitric acid (preferably 0.1 N) in from about 0.1 % to

about 10 % (preferably from 0.5% to 1 %) of hydrogen peroxide; and Second reagent: aqueous solution of from

about 0.25 H to about 1.25 H of NaOH (preferably 0.25 N)

in water containing from about 0.1 to about 1 % (v/v)

surfactant (most preferably N-alkyl trimethyl ammonium

chloride) , with said % 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 quenched chemiluminescent signal provided by tracer bound to the solid phase as well as any tracer that is

left in the free fraction solution once the specific

binding reaction on the solid phase has transpired. The

percent quenching of the chemiluminescent signal is

calculated by the following equation:

Modulated Signal Counts % Quench = (1 - ) x 100%

Unquenched Signal Counts The unquenched signal counts are the counts measured

from the chemiluminescent flash of a given amount of the

unbound tracer taken in the absence of solid phase. The

given amount is defined as the same quantity of tracer as is added to the assay. The % quench calculation may

then be associated with a reference (including, for

example, a synthetic target sequence DNA and so on) to

determine the amount or presence of the analyte in the

sample. It has been observed that chemiluminescence is

quenched in proportion to the mass of solid phase

present during flashing when the solid phase is coated

with an irrelevant binding partner. The signal

modulation described here is in addition to and of

greater magnitude than quenching due to solid phase

mass, and is due to the specific binding reaction of the

acridinium ester labeled analyte and the binding partner

covalently attached to the solid phase.

The evaluation and measurement of the light

emission generated by the activation (e.g. flashing) of

the chemiluminescent label in all embodiments may be

accomplished by techniques known to those skilled in the

art. For example, luminometer instruments that may be

utilized in measuring the chemiluminescence signals are

commercially available, such as the MAGIC LITE Analyzer

(MLA I) instrument manufactured by Ciba Corning Diagnostic Corp., Medfield, Mass. The assay may also be

prepared for usage in an automated system that includes

a luminometer, with one or more photomultiplier tubes,

with multiple photomultiplier tubes described in WO

94/22002, priority filed 3/19/93, commonly assigned and

hereby incorporated by reference.

The inventive method may be practiced with various

assay systems and formats by techniques known to those

skilled in the art. Both competitive and non-

competitive (e.g. sandwich) assay formats may be practiced with immunological binding, chemical binding,

complementary binding, and combinations thereof.

According to the invention, the following are

illustrative of immunological formats that may be used.

Labeled Antigen-Competitive: In this format, the

sample containing the targeted antigen to be measured is

incubated with a solution comprising (1) a limited

amount of antibody coupled to metal oxide solid phase

and (2) a tracer comprising an antigen or antigen

analogue having attached thereto an acridinium ester

label. During an incubation of the reaction solution, the antigen in the sample competes with the labeled

antigen (or antigen analogue) for binding to the

antibody attached to the solid phase. After the

incubation period there may be up to three components

left in the reaction solution: (1) a complex comprising

the sample antigen bound with the antibody attached to

the solid phase (yielding no signal upon activation) ;

(2) a complex comprising the tracer antigen (or antigen

analogue) bound with the antibody attached to the solid

phase (yielding a quenched signal upon activation) ;

and, possibly, (3) unreacted tracer (yielding an

unquenched signal) . The amount of labeled antigen (or

antigen analogue) bound to the solid phase is inversely

proportional to the amount of antigen in the sample.

Labeled Antibody-Competitive: In this format, the

sample containing the targeted antigen to be measured is

incubated with a solution comprising: (1) a limited

amount of antigen (or antigen analogue) coupled to metal

oxide solid phase and (2) a tracer comprising an

antibody having attached thereto an acridinium ester

label. During an incubation of the reaction solution, the antigen in the sample competes with the solid phase

antigen (or antigen analogue) for binding to the tracer

antibody. After the incubation period there may be up

to three components left in the reaction solution: (1)

a complex comprising the sample antigen bound with the

tracer antibody (yielding an unquenched signal upon

activation) ; (2) a complex comprising the tracer

antibody bound with the antigen (or antigen analogue)

attached to the solid phase (yielding a quenched signal

upon activation) ; and, possibly, (3) unreacted tracer antibody (yielding an unquenched signal) . The antigen in the sample and the antigen (or analogue) on the solid

phase compete for the labeled antibody. The amount of

labeled antibody that becomes bound to the solid phase

is inversely proportional to the amount of antigen there

is in the sample.

Labeled Antibody-Sandwich: In this format, which

is typically done on antigens which are large enough to

bind two antibodies (the same or different)

simultaneously, the sample is incubated with an excess

of one antibody attached to solid phase together with an excess of another tracer antibody that is labeled with an acridinium ester. The antigen becomes attached to

the solid phase via one of its antigenic determinants,

and the labeled antibody in turn becomes bound to the

antigen via a different determinant. The complex formed

on the solid phase (i.e. solid phase antibody-antigen-

tracer antibody) yields a quenched chemiluminescent

signal upon activation and the unreacted tracer yields an unquenched signal. The amount of labeled antibody that becomes bound to the solid phase is directly

proportional to the amount of antigen in the sample.

With regard to hybridization assays (e.g. gene

probe assays) , the competitive and sandwich formats may be practiced. When employed with hybridization assays, the inventive assay may take advantage of solid phase

already present in the assay from the initial

hybridization-capture step. For the hybridization

quenching phenomenon to be utilized for an assay for

specific nucleic sequences, preferably the quenching

effect is modulated by partitioning of the labeled

oligomer probe between the immobilized and solution phase sequences. In the competitive format, the

immobilized oligomer and target sequence therefore

preferably share a substantially common sequence that is

capable of hybridizing to at least a portion of the

labeled oligomer probe.

When practicing the invention in gene probe

assays, the acridinium ester may be placed on any

desired position of the oligomer, but most preferably

the label is placed at the 5' terminus of the molecule.

The gene probe assays preferably incorporate an

amplification step utilizing a DNA or RNA replicase to

generate DNA or RNA copies for each target nucleic acid

sequence in the sample by techniques well known to those

skilled in the art, such as for example, those described

in EP-A-0 481 704 (commonly assigned and incorporated

herein by reference) . After or during this

amplification step, an acridinium ester labeled oligomer

is added to the sample. The polynucleotide sequence

permits it to hybridize specifically to a portion of the

sequence which is being amplified as well as the same

sequence immobilized to a metal oxide solid phase which has been added earlier in the assay procedure. The

amount of labeled oligomer that will be captured upon

the solid phase will depend upon the relative amounts of

the complementary sequence present in solution or

immobilized on the solid phase. The amount of amplified target sequence produced from the replicase reaction is

expected to be in excess over that immobilized upon the

solid phase. Using the non-separation chemiluminescent

detection method, samples containing no or lower than a

threshold amount of target will result in a quench of

the light emission of the labeled oligomer while for

samples containing targets, the chemiluminescence of the

labeled oligomer will remain unquenched. The percent

quenching of the labeled oligomer chemiluminescence that occurs upon hybridization to an oligomer attached to the

solid phase may be calculated by the following formula:

RLU of reaction containing oligomer-solid phase X 100

RLU of minus oligomer-solid phase control

where Q represents quenching and RLU represents Relative Light Units . As shown in the calculation, the hybridization reaction and control reaction are

preferably incubated under substantially the same

conditions, as the chemiluminescence of the labeled

oligomer may generally vary with time, temperature, and

buffer conditions.

According to the invention, hybridization of the

labeled oligomer to the oligomer attached to the solid

phase will result in maximum reduction of the

chemiluminescent signal when the solid phase capacity is

in excess of the input labeled oligomer. Additionally,

an excess labeled oligomer relative to the solid phase

will result in less quenching (i.e. excess labeled

oligomer that is not hybridized will be detected with

higher efficiency by generating more chemiluminescence

relative to the control) .

The invention may be practiced using complementary

substances attached to the tracer and solid phase

conjugate. Preferred complementary substances include

biotin and avidin. Exemplary biotin compounds include,

for example, biocytin (i.e. biotinε-N-lysine) , biocytin

hydrazide, amine or sulfhydryl derivatives of 2- imminobiotin and biotinyl-ε-aminocaprioic acid hydrazide

and derivatives of biotin, including, for example,

biotin-N-hydroxysuccinimide ester, biotinyl-ε-

aminocaprioic acid-N-hydroxysuccinimide ester,

sulfosuccinimidyl 6- (biotin amido) -bromoacetylhydrazide,

p-diazobenzoyl biocytin and 3-(N-

maleimidopropoionyl)biocytin, which can be attached to

linking proteins (preferably attached to the solid phase

polymeric coating) , as is well known to those skilled in

the art. Avidin compounds that may be used include

streptavidin, succinylated avidin, monomeric avidin, and

so on. The method of attaching the avidin and biotin

(or derivative of either) either directly or indirectly

to the specified tracer or solid phase conjugate may be

accomplished through techniques known to those skilled

in the art, e.g. through reacting the amino or

sulfhydryl groups of the avidin or biotin.

With regard to immunological assays, the invention

is particularly useful for detecting or quantifying

theophylline and dinitrophenol (DNP) protein and

derivatives thereof. With regard to hybridization assays, the invention is particularly effective in

detecting or quantifying enteric pathogens, such as, for

example, Salmonella and Campylobacter species.

It is to be understood that various modifications

to the invention will be apparent to and can readily be

made by those skilled in the art, given the disclosure

herein, without departing from the scope and materials

of this invention. It is noted that the following

examples given herein are intended to illustrate and not

to limit the invention thereto.

Examples

The solid phase in all examples consisted of

paramagnetic particles (PMP) having a silane polymeric

coating around the iron oxide core (purchased from

Advanced Magnetics Inc., Cambridge, MA) . The PMP were

activated by glutaraldehyde and conjugated with the

various specific binding partners according to the two-

step procedure as described by Groman, E.V., et al. (Bio

Techniques, 70. p. 159-165) . The acridinium ester (AE)

used in the examples was of the following structure: R, x-

wherein X is CH₃S0_4; R-_ is methyl; R₂, R₃, R₅ and R₇ are

hydrogen; R₄ and R₈ are methyl, R₆ = is an COOH converted

to a N-succinimidyloxycarbonyl group to assist in

attachment of a specific binding partner. A description

of a technique that was used in attaching acridinium

esters with binding conjugates is as found in Weeks et

al., Clinical Chemistry. 29(8), 1474-1479 (1983) and EP-

A-0 537 994 (each of which are hereby incorporated by

reference) . The assays were flashed with two reagents,

as follows: Flash Reagent 1 = about 0.3 ml of 0.1 N HN0₃ in approx. 0.5% aqueous solution of H₂0₂. Flash

Reagent 2 = about 0.3 ml of 0.25 NaOH in a approx. 0.5

% aqueous solution of ARQUAD 16-50 N-alkyl trimethyl

ammonium chloride (50% active purchased from AKZO

Chemical Inc., Chicago, IL) . Flash Reagent 1 was added

to the reaction mixture first, followed immediately with

Flash Reagent 2. Relative light units (RLU's) were

measured over a 2 second interval after injection of

®

Flash Reagents 1 and 2 using a luminometer (MAGIC Lite Analyzer "MLA I", Ciba Corning Diagnostics Corp.,

Medfield, Mass) .

The % Quench was calculated by the following

equation:

Modulated Signal Counts % Quench = (1 - ) x 100%

Unquenched Signal Counts The modulated signal counts were a measurement of the total signal provided by the reaction mixture after the

reaction between specific binding partners transpired.

The unquenched signal counts were a measurement of the light emitted from an amount of tracer added to the

assay, as flashed in the absence of solid phase. Example 1

This example demonstrates the antibody-dependent

quenching phenomenon.

Monoclonal antibodies to 2,4-dinitrophenol (DNP)

were prepared by standard methods, after immunizing mice

with a conjugate of DNP with thyroglobulin (DNP-TG) .

This conjugate was prepared by reaction of equal weights

of thyroglobulin and 2,4-dinitrobenzenesulfonic acid in 0.15 M Na₂C0₃ solution for 18 hours, followed by

exhaustive dialysis against 0.001 M sodium phosphate

buffer, pH 7.4.

The antibody (anti-DNP) was purified from ascites

fluid by precipitation of unwanted protein with caprylic

acid, followed by dialysis of the supernatant against

0.1 sodium phosphate buffer, pH 7.4.

For immobilization of the antibody, 10 mg of PMP

were activated with 6.25% glutaraldehyde in 0.1 M

phosphate, pH 7.4, for 2 hours. After washing to remove

excess glutaraldehyde, the PMP were mixed with 1 ml of a

solution of antibody (diluted to 1.5 mg/ml), and let

stand overnight. The particles were then washed several times in phosphate buffer, and finally resuspended to a

concentration of 10 mg/ml in 0.05 M sodium phosphate, pH

7.4, 0.15 M NaCl, 1 mg/ml bovine serum albumin

(PBS/BSA) .

DNP-TG (prepared as above) and a conjugate of

fluorescein isothiocyanate with bovine serum albumin

(FITC-BSA) (purchased from Sigma Chemical Co., St. Louis,

MO) were labeled with the acridinium ester (AE) as

follows: 2 mg of conjugate in 1 ml of 0.1 M sodium

phosphate, 0.15 M NaCl, pH 8.0, was mixed with 80 μl of

AE (1 mg/ml in dimethylformamide) and incubated for 1 hr

at room temp. The mixture was then added to 0.5 ml of

a 10 mg/ml solution of DL-lysine and incubated for 15

minutes. The labeled conjugate was then purified by gel

filtration on a 20 cm column of Sephadex G 25.

DNP-β-alanine was prepared as follow: One gram of

β-alanine was dissolved in 50 ml of 1 M NaHC0₃. Seven milliliters of 2,4-dinitrofluorobenzene (DNFB) was added

to 100 ml of ethanol, and this was added to the β-

alanine and stirred at room temperature for two hours.

The ethanol was removed by rotary evaporation and the remainder extracted with ether to remove excess DNFB.

Normal HCl was added to the aqueous part until a

precipitate formed. The latter was recovered by

filtration, washed with ether, and air dried. The

material was recrystallized twice: first from water

with the addition of 1 I HCl, then from NaHC0₃/Na₂C0₃,

pH=9, washed with ether, and dried.

One hundred microliters of the anti-DNP PMP

(diluted 1:30 with PBS/BSA) was incubated with either

100 μl of AE-DNP-TG (diluted with 1:15000 with PBA/BSA)

or with 100 μl of AE-FITC-BSA (diluted 1:60000 with

PBA/BSA) . After one hour, the chemiluminescent light

emission of each mixture was measured and compared to

that of the same amount of each AE-labeled conjugate in

the absence of the anti-DNP PMP. For the AE-DNP-TG

conjugate, 52% of the light output was quenched by the

particles carrying the specific anti-DNP antibody. For

the AE-FITC-BSA conjugate, which is not bound by this

antibody, only 26% of the light was quenched by the

particles. These data support that quenching is increased

upon the formation of the specific binding reaction

complex on the solid phase. These findings are contrary

to the expectation that the amount of quenching of light

emission from a given amount of labeled tracer by a

given mass of solid phase would be the same.

Example 2 The following example illustrates the effect of

addition of a DNP derivative (not labeled with

acridinium ester) to the system described in Example 1.

One hundred microliters of anti-DNP-PMP (diluted

1:30 with PBS/BSA) was incubated with 50 μl of a of AE-

DNP-TG (diluted 1:7500 with PBS/BSA) and with 50 μl of a

solution of DNP-β-alanine at concentrations of 50 to 500

ng/ml. After one hour, the chemiluminescence of each

mixture was measured. The results are shown in TABLE 1

below and graphically represented in Fig. 1.

TABLE 1

Effect of Addition of a DNP Derivative

DNP-β-Alanine (nσ/ml) Chemiluminescence (RLU)

0 545,000

50 566,000

100 802,000

250 983,000

500 1,040,000

The data show that addition of increasing concentrations

of an unlabeled DNP derivative resulted in a progressive

increase in light output. This effect is presumably due

to the unlabelled DNP competing for the immobilized

antibody's available binding sites, resulting in less of

the AE-labeled DNP binding to the particles, and thus

diminishing the quenching of chemiluminescent light

emission.

Example 3

Monoclonal antibodies to theophylline and

lutenizing hormone (LH) were generated by standard

techniques after immunization of mice with 8- carboxypropyltheophylline-thyroglobulin and LH

respectively. Polyclonal antibody to theophylline was

produced by immunization of rabbits with 8-

carboxypropyltheophylline-thyroglobulin. Monoclonal and

polyclonal antibody (anti-LH) were purified as described

above for Examples 1 and 2 except that activation and

coupling were done in 0.01 M sodium acetate buffer (pH

5.5) . Particles were finally resuspended at 25 mg/ml in

PBS/BSA buffer. Theophylline standards were prepared by

diluting a stock solution of theophylline (Sigma) into

PBS/BSA for buffer based standards or drug-free human

serum for serum standards. Standards from a

Theophylline RIA kit obtained from Clinical Assays were

also used. PMP immobilized with either theophylline

monoclonal antibody (THEO) or LH monoclonal antibody was

serially diluted from 2.5 mg/ml to 0.156 mg/ml in

PBS/BSA buffer. Five hundred microliters of PMP was

dispensed to test tubes, the buffer was removed after

magnetic separation of the particles and 100 μl of water

was added to resuspend the particles. AE-THEO (0.4 million RLU/tube) diluted in PBS/BSA was added to each

tube. The chemiluminescent light emission was

immediately measured using the MLA I.

The RLU observed for the solution of AE-THEO with

the anti-theophylline PMP and the anti-LH PMP (modulated

signal total counts) were compared to the RLU measured

in the absence of PMP (unquenched total counts) . The

results are summarized in TABLE 2 as follows.

TABLE 2

Quenching of AE-Theophylline in the Presence of Anti-

Theophylline Antibody PMP (Anti-THEO) and

Anti-Luteotropic Hormone Antibody PMP (Anti-LH)

Antibody (PMP) mg/tube RLU Quench % on PMP

0 335937 (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-THEO 0.156 30915 92

Anti-THEO 0.312 24900 94

Anti-THEO 0.625 19635 95

Anti-THEO 1.25 13255 97 TABLE 2 is graphically represented in Fig. 2, where

separate lines represent α -Theophylline PMP and α -LM

PMP, as labeled. The data shown in TABLE 2 and Fig. 2

illustrate that the Quench % observed when the anti-

THEO PMP was present seemed to be relatively independent

of the concentration of PMP mass over this range. In

contrast, quenching of counts in relation to the mass of

particles was observed when the particles were coupled

with an "irrelevant" antibody (anti-LH) was obtained.

Example 4

Patient samples were obtained from an clinical

laboratory (Metpath, Inc.) . Theophylline levels for

patient samples were obtained using a commercially

available assay, TDX Theophylline Assay (Abbott

Laboratories, Abbott Park, 111.) .

Non-separation theophylline assays were run as

follows. Standards, controls or patient samples were

incubated with AE-theophylline and monoclonal anti-

theophylline PMP at room temperature for 10 minutes.

Chemiluminescence was measured in the MLA I without

further manipulation of the tubes. At low analyte concentration, most of the tracer was bound to the solid

phase and did not flash, thus giving low counts. As the analyte concentration increased, more tracer remained

unbound, thus giving a higher signal. A sample standard

curve is shown in Fig. 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 the control, with the

control data shown in TABLE 4.

TABLE 3 NON-SEPARATION THEOPHYLLINE ASSAY

Theophylline, μg/ml

PATIENT CONTROL NON-SEPARATION ASSAY

TDX INVENTION

(μg/ml) (μg/ml)

1 5.2 8.3

2 16.0 13.0

3 7.1 6.8

4 11.1 12.9

5 13.2 9.3

6 4.6 4.8

7 23.2 19.8

8 14.6 14.6

9 5.0 6.4

10 15.9 10.5

11 8.7 6.8

12 5.6 6.1

13 11.4 7.7

14 14.4 9.0

Correlation: Non Separation Assay = 2.42 + 0.656 TDX R 0.874 EXAMPLES 5- 7

Hybridization Assays

The nucleic acid sequences used in the

hybridization assays described in Examples 5-7 are

provided in sequence listing herewith, where the

following abbreviations are used in presenting the

hybridization assays: adenine (A); thymine (T) ; uracil

(U) ; guanine (G) ; cytosine (C) .

PM979: shown as SEQ. NO. 1, where bases 1-10

constitute a spacer arm, bases 11-46 consist of the 5'

sequence of the nanovariant (+) template, and bases 47-

70 are complementary to a Salmonella specific target

sequence.

MASA5 : shown as SEQ. NO. 2, where bases 1-10

constitute a spacer arm, bases 11-71 consist of the 5'

sequence of the midivariant (+) template, and bases 72-

95 are complementary to a Salmonella specific target

sequence.

PM1076 : shown as SEQ. NO. 3, where half of the

Salmonella target, complementary to PM979 and MASA5 was

employed. PM2058 : shown as SEQ. NO. 4, where the Salmonella

target, complementary to PM979 and MASA5 was employed.

Some versions of this sequence have a 5' -amino group for

conjugation with acridinium ester, as described herein.

SA7 : anti-Salmonella target, shown as SEQ. No. 5.

MD24 : shown as SEQ. NO. 6, where a probe

complementary to bases 34-57 of the midivariant (+)

template; i.e. bases 44-67 of MASA5 was used. Some

versions of this sequence have a 5'amino group for

conjugation with acridinium ester, as described herein.

MDV-SA2 RNA Transcript : shown as SEQ. NO. 7,

where bases 1-61 consist of the 5' end of the

midivariant (+) template, bases 62-69 and 118-123 are

plasmid linker sequences, bases 70-117 are complementary

to a salmonella specific target sequence, and bases 124-

282 consist of the 3' end of the midivariant (+)

template.

The solid phases consisted of PMP to which one of

the amplification oligomer probes was covalently

attached (via a 5' -terminal amino group using a hetero-

bifunctional coupling reagent for PM979, or by glutaraldehyde activation of the PMP for MASA5) . The AE-

PM1076 and 5' ³²P-PM2058 sequences are complementary to

the anti-target portion of the immobilized probes

(therefore hybridization and capture occur

simultaneously) . The PM979-PMP was prepared using PM

979 oligomer (obtained from Promega Corp., Madison, WI)

and a thio-terminated PMP (purchased from Advanced

Magnetics, Cambridge, MA) . The AE labeled PM1076 (AE-

oligomer) was prepared using the dimethyl acridinium

ester label and PM1076 oligomer obtained from Promega

Corp. The MASA5-PMP was prepared using MASA5 oligomer

(obtained from Promega Corp.) and a thio-terminated PMP

purchased from Advanced Magnetics. The PM 2058 oligomer

was also obtained from Promega Corp. The sodium citrate

was purchased from Mallinckrodt, Inc., St. Louis, MO.

The NaCl, Tris EDTA and Tween-20 were all purchased from

Sigma Chemical, Co., St. Louis, MO) and the BSA

(Fraction V) from Miles, Inc.

Example 5

This example illustrates the quenching of AE-

oligomer chemiluminescence by hybridization to oligomer-

PMP

Hybridization reactions containing PMP979

immobilized on PMP (PM979-PMP) and AE-PM1076 (AE-

oligomer) were set up on ice by adding 40μl of 60 mM

sodium citrate, 600 mM sodium chloride, 10 mM tris-

(hydroxymethyl) aminomethane (Tris) HCl, 1 mM ethylene

diaminetetraacetic acid (EDTA) , 0.1 % (w/v) BSA, and

0.02 % (v/v) polyoxyethylene (20) sorbitan monolaurate

(TWEEN-20) at pH 7.5 (hereinafter "buffer") to ten

tubes. Secondly, 5 μl of buffer containing 50 μg of

PM979-PMP was added to five of the ten tubes, and 5 μl

of buffer alone was added to the remaining five tubes

for control reactions. Finally, 5 μl of buffer

containing 10, 32, 100, 320, and 1000 femtomoles (1 fmol

1 x IO^"15 moles) of AE-PM1076 was added to pairs of

tubes, one group containing PMP and one control group

without PMP (control for AE decomposition during

hybridization) . All reactions were incubated at 56° C and 5 μl samples were removed at 0, 75, 120, and 180

minutes, added to 100 μl of water, flashed with Reagents

1 and 2 in a luminometer to determine the

chemiluminescent activity.

The % Quench was calculated from the signals

measured from the various tubes and are presented in

Fig. 4 . As shown in Fig. 4 , the chemiluminescence of

all reactions containing oligomer-PMP decreased

dramatically relative to the corresponding control

without oligomer-PMP, up to a maximum of 74%, upon

hybridization. Also the data demonstrate conditions

where the relative input of AE-oligomer and oligomer-PMP

generated significant quenching. The conditions of the

reaction having 100 fmol input of the AE-oligomer, where

the solid phase capacity is five-times the input of AE-

oligomer, were chosen to test the feasibility of the

competitive assay of Example 6.

Example 6

Hybridization reactions were set up on ice by

adding 10 μl of buffer (described in Example 5) alone or

10 μl of buffer containing 100 μg of PM979-PMP to each of five tubes. Next, 10 μl of buffer containing 0, 10^"

¹⁴, IO^"13, IO^"12, and 10^"11 moles of standard, PM979 (the

oligomer that was immobilized on the PMP) , was added to

one of the tubes containing PM979-PMP and one control

tube without PMP for each amount of added standard.

Finally, 80 μL of buffer containing 200 fmol of AE-

PM1076 was added to all reactions. All tubes were

incubated at 56° C. Duplicate 10 μl samples were

removed from each reaction at the indicated times (0,

20, 40, and 60 minutes) , added to 100 μl of water and

processed in a luminometer to determine chemiluminescent

activity. Results are shown in TABLES 4 [Fig. 5) and 5.

TABLE 5 shows data derived from the 20 minute data from

TABLE 4. The results shown are consistent with

theoretical expectations, i.e. the same maximum extent

of quenching was achieved in all hybridizations except

where the input of PMP979 in solution exceeds the solid

phase capacity.

TABLE 4 COMPETITIVE HYBRIDIZATION QUENCHING ASSAY

REACTION Standard Hybrid. Total Signal

Input Time (RLU x IO^"5) Quench %^2"

(moles) (min) (+)PMP (-) PMP

20 4.61 5.65 18

40 4.78 5.78 17

60 4.38 5.30 17

B io^"12 0 7.82 8.60 9

20 2.60 5.28 51

40 2.71 5.31 49

60 2.63 5.39 51

20 1.43 5.43 74

40 1.48 5.56 73

60 1.51 5.21 71

20 1.21 5.21 77

40 1.28 5.26 76

60 1.26 5.11 75

E 0 8.00 8.45 5

1.15 5.41 79

1.26 5.27 76

1.24 4.82 74

^1- Standard was added PM979.

²- [1- (+PMP RLU) ÷ (-PMP RLU)] x 100 % = % Quenching TABLE 5

COMPETITIVE HYBRIDIZATION QUENCHING ASSAY

-20 minute data point-

Reaction Standard (+)PMP Net (+)PMP Input Signal Signal (moles) (RLU)^1' (RLU)^2'

0 114,650 0

1: Derived from the 20 minute data point of TABLE 4 2: (+PMP RLU)i ÷ (+PMP RLU) _E. i = "+PMP RLU" value

E = "Reaction E" value

As shown by the data above, this example demonstrates a

hybridization competition assay for synthetic DNA

oligomer. Varying amounts of the same oligomer that had

been immobilized on the PMP were added in solution to

compete with the solid phase immobilized oligomer for hybridization of the AE-oligomer, i.e. the added

oligomer functioned as a competitive standard. This

mimics the competition that would take place due to the

presence of amplified probe in the proposed assay of

Example 7.

Example 7

Amplification reactions that had started with 0,

IO^"16, IO^"18, 10^"19, and IO^"20 moles of midivariant

template, respectively, were terminated by addition of

EDTA and put on ice.

Hybridization reactions were set up on ice by

adding 5, 20, 70, or 75 μl of buffer to 12 tubes, so

that the final volume would be 100 μl after addition of

all other components, as described above. Next 50 μl of

buffer containing 5 μg of PM979-PMP was added to six

tubes. Then 5 μl of buffer or one of the five

replication reactions was added to pairs of tubes, one

with and one without PMP. Finally, 25 μl of buffer

containing 10 fmol of AE-MD24 was added to all tubes.

All tubes were incubated for 20 minutes at 56°C then flashed with Reagents 1 and 2 in a luminometer to

determine the chemiluminescent activity.

The results, shown in Table 6, do not reflect the

presence of abundant replicated midivariant RNA

(estimated to be on the order of 5 pmol per reaction) . The failure of the assay to detect the presence of

midivariant sequence was not due to reagent quality,

since reagents had been validated by titration and mock

assays with synthetic target. Additional trials, with

heat denaturation of the target prior to addition and

increased input of AE-oligomer, also failed. Subsequent experiments using ³²P-labeled MD24 suggest that the

failure is due to inefficient hybridization of the probe to the target, presumably due to higher stability of a midivariant duplex. This hypothesis is supported by the

results of a quench assay for transcribed single

stranded midivariant target, which produced a standard

curve with quench values consistent with theoretical

expectations with respect to the molar ratio of target

and AE-probe. TABLE 6 HYBRIDIZATION QUENCH ASSAY FOR REPLICATION PRODUCTS

Reaction Added Template + PMP

(mol) or Mean RLU % Quench^1'

- PMP

A b >uuffffeerr oonnllyy - - 2 23355,,554455 70

+ 71,325

B 0 - 1 19900,,224400 65

(mock + 66,690 reaction)

+ 66,570

D i 1o0^"18 — 1 18899,,995500 64

+ 67,820

+ 63,780

+ 60,190

1. .Q = [1- (+PMP value) /(-PMP value)] x 100

SEQUENCE LISTING

fl) GENERAL INFORMATION

(i) APPLICANTS:

(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 INVENTION:

NON-SEPARATION SPECIFIC BINDING CHEMILUMINESCENT ASSAY

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Ciba Corning Diagnostics

Corp.

( (BB)) S STTRREEEETT:: 63 North Street

(C) CITY: Medfield

(D) STATE: Massachusetts

(E) COUNTRY: USA

(F) ZIP: 02052

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette 3.5 inch, 1.44

Mb storage

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS 6.2 (D) SOFTWARE: Word 6.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Not Assigned

(B) FILING DATE: Herewith (C) CLASSIFICATION: Not Assigned

(viii) ATTORNEY INFORMATION:

(A) NAME: Judith A. Roesler

(B) REGISTRATION NUMBER: 34,237 (C) DOCKET NUMBER: CCD-120 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (508) 359-3876

(B) TELEFAX: (508) 359-3885

(2) INFORMATION FOR SEQ ID NO. 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, PM979, having a portion complementary to a Salmonella specific target sequence, where bases 1-10 constitute a spacer arm, bases 11-46 consist of the 5' sequence of the nanovariant (+) template, and bases 47-70 are complementary to the Salmonella specific target sequence.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: SEQ ID NO. : 1:

1 11 21 31

(5') NH₂-CCTAGTCCAA GGGGAAATCC TGTTACCAGG ATAACGGGGT

41 51 61

TTTCTCATAA GCGCCATTGA TGTTGTCGCC (3 ' )

(3) INFORMATION FOR SEQ ID NO. 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 95

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Nucleic acid probe, MASA5, having a portion complementary to a Salmonella specific target sequence, where bases 1-10 constitute a spacer arm, bases 11-71 consist of the 5' sequence of the midivariant (+) template, and bases 72-95 are complementary to the Salmonella specific target sequence.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: 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) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, PM1076, half of the Salmonella target, complementary to PM979 and MASA5, defined herein.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: SEQ ID NO. : 3: 1 11 21

(5') NH₂-GGCGACAACA TCAATGGCGC TTAT (3')

(5) INFORMATION FOR SEQ ID NO. 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, PM2058, a Salmonella target, complementary to PM979 and MASA5, defined herein.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: SEQ ID NO. : 4:

1 11 21 31

(5' ) GGCGACAACA TCAATGGCGC TTATAAAGCT CAGGGCGTTC

41

AGTTGACC (3' )

(6) INFORMATION FOR SEQ ID NO. 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, SA7, an anti- Salmonella target.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No (v) SEQUENCE DESCRIPTION: SEQ ID NO. : 5:

1 11 21 31 (5' ) GGTCAACTGA ACGCCCTGAG CTTTATAAGC GCCATTGATG

41 TTGTCGCC (3')

(7) INFORMATION FOR SEQ ID NO. 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, MD24, complementary to bases 34-57 of the midivariant (+) template; i.e. bases 44-67 of MASA5, defined herein.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: SEQ ID NO. : 6:

1 11 21

(5') ACGGTCGAAC TCCCGTACGA GGTG (3')

(8) INFORMATION FOR SEQ ID NO. 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 289

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Nucleic acid probe, MDV-SA2 RNA Transcript, where bases 1-61 consist of the 5' end of the midivariant (+) template, bases 62-69 and 118-123 are plasmid linker sequences, bases 70-117 are complementary to a Salmonella specific target sequence, and bases 124-282 consist of the 3' end of the midivariant (+) template.

(iii) HYPOTHETICAL: No

(iv) ANTISENSE: No

(v) SEQUENCE DESCRIPTION: SEQ ID NO. : 7:

1 11 21 31 (5') GGGGACCCCC CGGAAGGGGG GACGAGGUGC GGGCACCUCG

41 51 61 71

UACGGGAGUU CGACCGUGAC GAGCCUCGAG GCGACAACAU

81 91 101 111

CAAUGGCGCU UAUAAAGCUC AGGGCGUUCA GUUGACCUCG

121 131 141 151

AGGAGUCACG GGCUGCGCUU UCGCGCAUCU CCCAGGUGAC

161 171 181 191

GCCUCGAGAA GAGGCGCGAC CUUCGUGCGU UUCGGCGACG

201 211 221 231 CACGAGAACC GCCACGCUGC UUCGCAGCGU GGCCCCUUCG

241 251 261 271

CGCAGCCCGC UGCGCGAGGU GACCCCCGAA GGGGGGUUCC

288

CC (3')

Claims

THAT WHICH IS CLAIMED IS:

1. A non-separation specific binding assay to

detect or quantify the presence of an analyte, said

assay comprising:

(a) contacting a sample with a solid phase,

comprising a metal oxide having attached thereto a

binding conjugate, and a tracer, comprising an

acridinium or benzacridinium ester labeled binding

conjugate;

(b) allowing said solid phase, tracer and sample

to react to form a reaction mixture comprising a

specific binding complex attached to said solid phase

and a free fraction unattached to said solid phase;

(c) contacting said reaction mixture with an

activating agent to flash said acridinium ester tracer

and provide a modulated chemiluminescent signal;

(d) measuring said modulated signal; and

(e) associating said modulated signal with a

reference to determine the amount or presence of said

analyte in said sample. 2. An assay according to Claim 1 wherein said

specific binding complex is formed by an immunological

reaction.

3. An assay according to Claim 2 wherein said

metal oxide solid phase comprises iron oxide.

4. An assay according to Claim 3 wherein said

activating agent comprises a first reagent comprising an

aqueous solution of acidic hydrogen peroxide and a

second reagent comprising a basic aqueous solution, with

said first reagent contacted with said reaction mixture

prior to said second reagent.

5. An assay according to Claim 4 wherein said

first reagent has an acid component selected from the

group consisting of nitric acid, hydrochloric acid,

sulfuric acid, and mixtures thereof and said second

reagent has a base selected from the group consisting of

sodium hydroxide, potassium hydroxide, lithium hydroxide

and mixtures thereof.

6. An assay according to Claim 5 wherein in said

first reagent said acid is present in an amount ranging

from about 0.1 __I to about 0.5 N in an aqueous solution of hydrogen peroxide present in an amount ranging from

about 0.1% to about 10 % (v/v) , based on the total

volume of the aqueous first reagent solution, and in

said second reagent said base is present in the aqueous

solution in a concentration ranging from about 0.25 E to

about 1.25 E-

1 . An assay according to Claim 6 wherein said

first reagent comprises an agueous solution of 0.1 E

nitric acid in from 0.5% to 1% hydrogen peroxide and

said second reagent comprises an aqueous solution of

0.25 M 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 aqueous second reagent.

8. An assay according to Claim 7 wherein said

analyte is selected from the group consisting of

theophylline or a dinitrophenol protein.

9. An assay according to Claim 1 wherein said

assay is a hybridization assay and said specific binding

complex is formed by complementary binding.

10. An assay according to Claim 9 wherein said

metal oxide solid phase comprises iron oxide. 11. An assay according to Claim 10 wherein said

activating agent comprises a first reagent comprising an aqueous solution of acidic hydrogen peroxide and a

second reagent comprising a basic aqueous solution, with

said first reagent contacted with said reaction mixture

prior to said second reagent.

12. An assay according to Claim 11 wherein said

first reagent has an acid component selected from the

group consisting of nitric acid, hydrochloric acid,

sulfuric acid, and mixtures thereof and said second

reagent has a base selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide and mixtures thereof.

13. An assay according to Claim 12 wherein in

said first reagent said acid is present in an amount

ranging from about 0.1 E to about 0.5 E in an aqueous solution of hydrogen peroxide present in an amount

ranging from about 0.1% to about 10 % (v/v), based on

the total volume of the aqueous first reagent solution, and in said second reagent said base is present in the aqueous solution in a concentration ranging from about

0.25 E to about 1.25 E-

14. An assay according to Claim 13 wherein said

first reagent comprises an aqueous solution of 0.1 E

nitric acid in from 0.5% to 1% (v/v) hydrogen peroxide

and said second reagent comprises an aqueous solution of

0.25 IS 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 aqueous second reagent .

15. An assay according to claim 1 wherein said

binding reaction is immunological and said analyte is

selected from the group consisting of theophylline and a

dinitrophenol protein.

16. An assay according to claim 1 wherein said

binding reaction is complementary binding and said

analyte is an enteric pathogen.

17. A non-separation specific binding assay to

detect or quantify an analyte in a sample, said assay

comprising, in the order stated:

(a) incubating a solution comprising: a

sample; a solid phase comprising a metal oxide coupled to a binding partner specific to said analyte; and a

tracer comprising said analyte or analyte analogue

having attached thereto an acridinium or benzacridinium

ester;

(b) allowing said solution to react to form a

reaction mixture comprising a specific binding complex

comprising said tracer bound to said solid phase and a

free fraction comprising unreacted tracer;

(c) contacting said reaction mixture with a

first activating reagent comprising an acidic hydrogen

peroxide solution;

(d) contacting said mixture of step (c) with

a second activating reagent comprising a basic aqueous

solution to provide a modulated chemiluminescent signal;

(e) measuring said modulated chemiluminescent

signal;

(f) associating said modulated signal with a

reference to determine the amount or presence of said

analyte in said sample.

18. An assay according to claim 17 wherein the

amount of tracer of said step (a) is separately activated to emit a chemiluminescent light emission that

is measured in the absence of said solid phase resulting

in a unquenched signal count measurement; a % quench

effect is calculated using said modulated signal of

said step (e) and the unquenched signal count by an

equation:

Modulated Signal Counts % Quench = (1 - ) x 100% ;

Unquenched Signal Counts and said % quench effect is associated with said

reference of said step (f) to provide a determination of

the presence of or the amount of said analyte.

19. An assay according to claim 18 wherein said

first reagent comprises an aqueous solution of 0.1 N

nitric acid in from 0.5% to 1% hydrogen peroxide and

said second reagent comprises an aqueous solution of

0.25 N 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 aqueous second reagent.

20. A non-separation specific binding assay to

detect or quantify an analyte in a sample, said assay

comprising, in the order stated: (a) incubating a solution comprising a

sample; a solid phase comprising a metal oxide coupled

to an analyte or analyte analogue specific to said

analyte; and a tracer comprising a specific binding

partner to said analyte or analyte analogue having

attached thereto an acridinium or benzacridinium ester;

(b) allowing said solution to react to form a

reaction mixture comprising a specific binding complex

comprising said tracer bound to said solid phase and a

free fraction comprising unreacted tracer;

(c) contacting said reaction mixture with a

first activating reagent comprising an acidic hydrogen

peroxide solution;

(d) contacting said mixture of step (c) with

a second activating reagent comprising a basic aqueous

solution to provide a modulated chemiluminescent signal;

(e) measuring said modulated chemiluminescent

signal;

(f) associating said modulated signal with a

reference to determine the amount or presence of said

analyte in said sample. 21. An assay according to claim 20 wherein the

amount of tracer of said step (a) is separately

activated to emit a chemiluminescent light emission that

is measured in the absence of said solid phase resulting

in a unquenched signal count measurement; a % quench

effect is calculated using said modulated signal of said

step (e) and the unquenched signal count by an equation:

Modulated Signal Counts % Quench = (1 - ) x 100%

Unquenched Signal Counts and said % quench effect is associated with said reference of step (f) to provide a determination of the presence of or the amount of said analyte.

22. An assay according to claim 21 wherein said

first reagent comprises an aqueous solution of 0.1 N

nitric acid in from 0.5% to 1% hydrogen peroxide and

said second reagent comprises an aqueous solution of

0.25 S 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 aqueous second reagent. 23. A non-separation specific binding assay to

detect or quantify an analyte in a sample, said assay

comprising, in the order stated:

(a) incubating a solution comprising a

sample; a solid phase comprising a metal oxide coupled

to a first binding partner specific to said analyte; and

a tracer comprising a second binding partner to said

analyte having attached thereto an acridinium or

benzacridinium ester, wherein said first and second

binding partner may be the same or different;

(b) allowing said solution to react to form a

reaction mixture comprising a specific binding complex

comprising said tracer bound to said analyte which is

bound to said to said solid phase and a free fraction

comprising unreacted tracer;

(c) contacting said reaction mixture with a

first activating reagent comprising an acidic hydrogen

peroxide solution;

(d) contacting said mixture of step (c) with

a second activating reagent comprising a basic aqueous

solution to provide a modulated chemiluminescent signal; (e) measuring said modulated chemiluminescent

signal;

(f) associating said modulated signal with a

reference to determine the amount or present of said

analyte in said sample.

24. An assay according to claim 23 wherein the

amount of tracer of said step (a) is separately

activated to emit a chemiluminescent light emission that

is measured in the absence of said solid phase resulting

in a unquenched signal count measurement; a % quench

effect is calculated using said modulated signal of

said step (e) and the unquenched signal count by an

equation:

Modulated Signal Counts

% Quench = (1 - ) x 100% ;

Unquenched Signal Counts and said % quench effect is associated with said

reference of said step (f) to provide a determination of

the presence of or the amount of said analyte.

25. An assay according to claim 24 wherein said

first reagent comprises an aqueous solution of 0.1 N

nitric acid in from 0.5% to 1% hydrogen peroxide and said second reagent comprises an aqueous solution of

0.25 -SI 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 aqueous second reagent.

26. A diagnostic kit for use in non-separation

assay to detect or quantify the presence of an analyte

in a sample, said assay comprising a non-separation

specific binding assay to detect or quantify the

presence of an analyte, said assay comprising:

(a) contacting a sample with a solid phase

comprising a metal oxide having attached thereto a

binding conjugate and a tracer comprising an acridinium

or benzacridinium ester labeled binding conjugate;

(b) allowing said solid phase, tracer and sample

to react to form a reaction mixture comprising a

specific binding complex attached to said solid phase

and a free fraction unattached to said solid phase;

(c) contacting said reaction mixture with an

activating agent to flash said acridinium ester tracer

and provide a modulated chemiluminescent signal;

(d) measuring said modulated signal; (e) and associating modulated signal with a

reference to determine the amount or presence of said

analyte in said sample.