CA2132708C

CA2132708C - Fluorescence immunoassays using fluorescent dyes free of aggregation and serum binding

Info

Publication number: CA2132708C
Application number: CA002132708A
Authority: CA
Inventors: Robert F. Devlin; Walter B. Dandliker; Peter O. G. Arrhenius
Original assignee: Diatron Corp
Current assignee: Diatron Corp
Priority date: 1992-03-23
Filing date: 1993-03-23
Publication date: 2005-05-10
Anticipated expiration: 2013-03-23
Also published as: CA2132708A1; EP0632893A1; EP0632893A4; WO1993019366A1

Abstract

Fluorescence immunoassays methods are provided which use fluorescent dyes which are free of aggregation and serum binding. Such immunoassay methods are thus, particularly useful for the assay of biological fluids, such as serum, plasma, whole blood and urine.

Description

DESCRIPTION
Fluorescence Immunoassays Usina Fluorescent Dyes Free of Aaareqation and Serum B:indina Field of the Inver~tion The present invention relates to methods for deter-mining the presence or amount of antigenic substances in samples. The invention is directed to fluorescence immunoassays using particular fluorescence dyes which are essentially free of aggregation and serum binding and, thus, are particularly suited for the measurement of antigenic substances in biological materials such as serum, plasma and whole blood.
:10 Background o~ tie invention The determination of the presence or amount of anti-genic substances is commonly performed by immunoassay.
Immunoassay techniques are based on the binding of the antigenic substance being assayed (the "target analyte") and a receptor for the target analyte. Either the target analyte or the receptor may be labeled to permit detec-tion. Various labels have been employed for use in immunoassays, including radioisatoDes, enzymes and ~~.~~1~~ ' , ~.
~,.

2 fluorescent compounds. Many different types of immuno-assays are known in the art, including competitive inhibition assays, sequential additian assays, direct "sandwich" assays, radioallergosorbent assays, radio-s immunosorbent assays and enzyme-linked immunosorbent , assays.
The basic reaction underlying most immunoassays is the binding of certain substance, termed the "ligand" or "analyte", by a characteristic protein (receptor) to form a macromolecular complex. These binding processes are revers~.ble reactions; and the extent of complex formation for particular analyze and receptor concentrations is regulated by an equilibrium constant according to the law of mass action. Thus, at equilibrium, some of the analyte always exists unbound (free).
In a competitive inhibition immunoassay, the unknown quantity of target anal~yte in the sample competes with a known amount, of labeled target analyze for a limited number of receptor binding sites. The reagents usually consist of a labeled target analy~te, such as an antigen, and a solid phase coupled receptor, such as an antibody.
The antigen to be assayed competes with the labeled anti-gen for binding sites on the coupled antibodies. The concentration of target axaalyte present in the sample can be determined by measuring the amount of labeled target a~aa~.yte - either "free" or .'b~und. " Th.is is an indirect assay method where the amount of labeled antigen bound to the antibodies is intrersely correlated with the amount of antigen in the test'solution. Thus, low concentrations of~
target analyte in the sample will r~sul~ in low cdncentra-t.ions of !' free" labeled taxget : analyte and high concentra-tions of "bound!' labeled target analyte, and vzce versa.
The amount of "free"° or °°b~und'! labeled target analyze is measured using a suitable detector: Quantitative determi-nations are made by comparing the measure of labeled tar-g~t a~alyte with that obtained for calibrated samples containing known quantities of the target analyte. This S lJ ~ S°~I~tJTE S H E ~T' y~ y)3/~~3~~ .. P~.'T/'US93/U2470 ~v ;

3 method has been applied to the assay of a great number of different polypeptide~ hormones, enzymes and immunoglobu-l.ins. This method may also be used as a total liquid system. .
S zt is apparent to those skilled in the art that it is not absolutely necessary that the labeled analyte be iden-tical to the un~.abeled target analyze. If there is a dif-ference between the two, for example, if the labeled ana-lyte is an analog of the target analyte, the reaction between labeled and unlabeled analytes may be considered to be competitive for the receptor binding sites; and the reaction will still provide quantitative answers, provid- , ing the difference in affinity of the analyzes is not too great . Whether or not true competition occurs in a sys'cem consisting of labeled analyze, unlabeled analyze, and receptor depends on the'nature of the labeled analyte and the specificity of the receptor.
In sequential addition assays; the reagents used are the same as in the comgetitive inhibition assay described above: However, instead of incubating them at the same time, the unlabeled antigen is first incubated with the ar~ta.body, then the labeled antigen is added.
Direct immunoassay systems are also known in the art .
Such assays,, also termed "zmmunometric'! assays, employ a labeled receptar~(antibody) rather than a labeled analyte (ant~.gen) , In these assays the amount of labeled receptor associated with the complex is proportioned to the, amount of analyze in the sample. Immunometric assays are well-suited to the detection of antigenic substances wrhich aver ~0 'able to; complex w~.th two or more antibodies at the same f.2me: In such "two-site" or "sandwich'° assays, the anti-geni:e substance has two antibodies bound to its surface at d.if f~rent l.ocatians : In a t~rpical "forward" ' sandwich assay, an antibody bound to a olid phase is f first con-tacted with the sample being; tested to form a solid phase antibody:ant~.gen complex. After incubation, the solid support is washed to remove the residual sample, including 5 ~9 ~ ST1~°LIZE S H E ET

WO 93/19366 = 1'CC/L'S93/02~70 ..

4 unreacted antigen, if any. The complex is then reacted with a solution containing a known amount of labeled anti-body. After a second incubation to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed to remove unreacted labeled antibody. The assay can be used'as a simple "yes/no" assay to determine whe-ther the antigen is present. Quantitative determinations can be made by comparing the measure of labeled antibody with that of calibrated samples containing known quanti ties of antigen. "Simultaneous" and "reverse" sandwich "' assays are also known in the art. A simultaneous assay involves a single incubatian step, both the labeled and unlabeled antibodie s being added at the same time. A
reverse assay involves the addition of labeled antibody followed by addition of unlabeled antibody bound to a suitable solid support. The sandwich technique can also be used to assay antibodies rather than antigens . Such an assay uses as a first receptor an antigen coupled to a 0 solid phase. The antibodies being tested are first bound to the solid phase-coupled antigen. The solid phase is a then washed, and then labeled anti-antibody (second receptor? is added.
The radioallergosorbent technique (R.AST) is a method for the determination of antigen-specific IgE. The method uses a solid phase coup~.ed antigen and an immunoabsorbent purified antibody labeled with a radioactive isotope . The rctethod is used to detect reaginic antibodies against vari ,, :
lous antigens which elicit allergic reactions (allergens).
The reaginic antibodies react with allergen bound to a s;;:
solid matrix. After washing of the solid phase, the ~
allergen-bound reaginic an ibodies are detected by their ability to hind labeled antibodies against IgE . A variant of RAST can be used for the determination of allergens.
The allergen to be tested is incubated with the reaginic antibody. The mixture is then tested with BAST using the same allergen coupled to the solid matrix. The allergen SE~~S "TF SHED

WO y3/~~366 - ~CT/l.'S93/02470 ~~~~i in solution reacts with the reaginic antibodies and thus inhibits the banding of these antibodies to the solid phase-coupled allergen.
Another assay method for the determination of IgE is

5 the radioimmunosorbent technique ("RIST"). In this method, the solid support is sensitized with anti-IgE and increasing amounts of labeled IgE are added to determine the maximum amount of IgE that can bind. A quantity of labeled IgE equivalent to approximately 80% of the plateau ~.0 binding is chosen. In the test experiments, this amount of labeled IgE is mixed with the serum containing the IgE
- to be tested. The test IgE competes with the labeled IgE.
The more IgE present in the test serum the less the amount of labeled IgE that binds. Thus, by producing a standard curve the amount of 2gE in a sample can be determined.
The above immunoassay methods can be applied to the assay of many different biologically active substances.
g~ohg such substances are haptex~s, hormones, gamma globu-lin, al~.ergens, viruses, virus subunits, bacteria, toxins such ds those associated with tetanus arid animal venom, and many drugs. Similar techniques can be used in non-immunologa.cal systems with, for example, specific binding proteins.
A~.th~~.gh some of the immunoassay methods described above utilize radioactive labels, those skilled in the art will appreciate that the assays can be adagted to use an alternate label, for example, a fluorophore.
If the properties of the label are not altered by 'binding, for example, as in a radioimmunoassay,~'a separa tion step is required to separate "free" from "bound"
labeled target analyte. Such assays, which require a separation step, are called "heterogeneous" assays. If the properties of the label'are altered in some way. when it is bound, no separation step is required, and the 3 S imrtlunoassay is termed '~ homogeneous . "
The measurement of target analyzes in biological fluids, such as serum, plasma and whole blood, reauires su~s°rrr~~E s~~~r

6 - PC'f/US93/02470 ., y : : ..1. .
ha n immunoassay methods which are bath specific and sensitive.
Both the specificity and sensitivity of an immunoassay depend on the characteristics of the binding interaction between the target analyte and the receptor involved. For example, the reaction must be specific for the analyte to be measured and the receptor used should not bind to any other structurally related compounds. In addition, by choosing a receptor with a high affinity for the target analyte, the sensitivity can be increased.

The label used to monitor the assay affects the sensitivity of an immunoassay. Labels currently used for immunoassay of target analytes in biological fluids include radioisotopes (radioimmt~noassay, RIA), enzymes (enzyme immunoassay, EIA); fluorescent labels (fluores-cen~e immunoassay; FIA); and chemiluminescent labels (chemiluminescent immunoassay, CzA).

RTAs are sufficiently sensitive for use in detection in lour concentrations ~f analyt~s because of their low background. They are disadvantageous in that they are heterogeneous, thus requiring a separation step before msas~,rernent of the bound and/or free portions of labelled target analyte. RIAs involve the inconvenience and haz-ards assoc~.ated with the handling and disposing of radio-isotopes. In addition, they are labor intensive and have a short shelf life due to the half-lives of radiolabels and to chemical damage produced by the emitted radiation. , EIAs have the advantage of increased signal over background, longer shelf life, lack of radiation hazards, ) They are disadvantageous in that, r and homogeneity.

because they invol~re enzyme kinetic reactions, they are t:,~:
affected by the time of the kinetic measurements, as well as by variat~.ons in temperature, gH and ionic strength.

The temperature of the enzyme incubation is particularly critical, and variations of more than 0.5C can signifi-cantly affect assay results: Thus, drifts in standard curve may result from temperature fluctuation and incon-sistencaes in sample handling. Enzyme activity may also , 58~~5T~'6JT'E SHEET

d~0 93119366 - PC'I"/fS93/02470 be affected by constituents in biological samples, such as plasma constituents. See gen:erall Strong, J.E. and Altman, R.E., "Enzyme Immunoassay: Application to Thera-peutic Drug Measurement," in P. Mover et al., Applied Therapeutic Drug Monitorinct, American Association of Clinical Chemistry (1984).
Chemiluminescent immunoassays (CTAs) offer a fairly high degree of sensitivity (picomole per liter range) but lack specificity in some instances. CIAs are disadvanta-la geous because they are heterogeneous, require expensive reagents, and are expensive to automate. See generally Boeckx, R.L., "Luminescence: A New Analytical Tool for Therapeutic Drug Monitoring," in P. Mover et al., Applied Therapeutic Druq., Monitorincc, American Association of Clin ical Chemistry (1984).
FIAs use fluorescent molecules as labels. Fluores-cent molecules (fluoraphores) are molecules which absorb light at one wavelength and emit light at another wave-length. See Burd, J.F., "Fluoroimmunoassay ~-- Application to Therapeutic Drug Measurement, " in P. Mover et al.. , Apps lied Therapeutic Druct Monitorinct, American Association of Clinical Chemistry (1984). Typically, an excitation pulse of radiation is directed onto or into a sample, followed by fluorescence of the sample, and the detection of the fluorescence radiation.
FIAs may be either heterogeneous or homogeneous. As noted above, homogeneous assays are usually sampler to perform and are thus, more amenable to automation. How-i , ~ , ~
ever, previously~known homogeneous FIAs are less sensitive than heterogeneous FIAs because high background can limit serasitivity. The heterogeneous FIA procedures can detect smaller amounts of analyte than present homogenous FIAs, but only because the separation and washing steps in the assays serve to eliminate background interference from biological substances. In solid phase fluorescent assays the solid support can limit sensitivity at the wavelengths of presently used fluors. In many cases the support S ~! ~ S°~T'~ TE S ~ E ~~' WC193f 19366 = PCT/L'S93/02470 itself will fluoresce at wavelengths of commonly used floors such as fluoresceir~ (493 nm). FIAs also offer the advantage of using stable reagents, a Another assay method uses enzyme-enhanced fluores cence technology which combines microparticle capture and antigen-antibody reaction with an enzyme rate reaction using' a fluorescent enzyme substrate. The rate reaction is monitored by steady state fluorometric measurement. Tn an enzyme-enhanced fluorescence assay, the analyte in ~0 question is "captured" by an antibody bound to a solid phase and the solid phase is washed. An enzyme is then _ bound to the captured analyte using an enzyme-anti analyte conjugate. Excess reactants are washed away and the amount of enzyme is measuxed by the addition of a i~on fluorescent substrate. As the enzymatic reaction pro ceeds, the non-fluorescent substrate is converted to the fluorescent product. For example, an alkaline phaspha-tase-labeled antibody can be used to catalyze the hydro-lysis of 4-methylumbelliferyl phosphate substrate to the fluorescent product methylumbelliferone. Thus, the rate at which the fluorescent product is generated is directly proportional to the concentration of analyte in the test solution: Enzyme-enhanced fluorescence assays, like EIAs, have the disadvantages associated with enzymes.
2S As discussed above; fluorescence is a phenomenon exhibited by certain substances, which causes them to emit light, usually in the visible range, when radiated by another light source. This is not reflection, but crea-tibn of news light ; Current commercially' available assay ' methods use fluorescein, which emits green light when radiated by a light source containing blue light.
Zn addition to fluorescing; flunrescein (and other ~' fluorophores) emit polarized light: That is, the light emitted has the same direction of polarization as the incident polarized light, if the fluorescein molecule is held ' fixed with its transition moment parallel to the electric field of the excitation. The amount of polariza S tJ ~ S'~'t~'LJTE S H E ET' ~V~ 93/19366 - P~lf/US93102~170 E
tion in the emission can be defined in terms of the inten-sity of the horizontally and~vertically polarized light, as follows:
P = ( Iv - Th) r ( Iv + Th) ( 1 ) S where Iv = intensity of vertically polarized emission Ih = intensity of horizontally polarized emission The maximum, or limiting value of polarization, for fixed, randomly oriented molecules is 0.5 (Po).
A second eguation (the Perrin equation) defines polarization in terms of physical parameters and Po:
lrp ~ ~r3 _ (~,rPo _ lr3) W + 3trry (2) where t - fluoresce:ace lifetime, a constant r = rotational relaxation time 1.5 Rotationrelaxation is further defined for spherical molecules as r = 3nV/R,T ( 3 ) where R = gas constant T = temperature, °K
n = solution, viscosity V' = volume of molecule The rotational relaxation time is a measure of the rite at vahi,ch a ' riiol~cule will rotate when free in a solo-tion. Note that the rotational relaxation time will typically be dependent primarily on the molecular volume and' shape; since solution viscosity and temperature will b~ essentially constant in a normal assay. Thus, rota-tional relaxation time; and consequently, polarization, are affected only by the hydrodynamic properties of the molecule. The smaller a molecule is, the smaller its rcatational relaxation time, and the faster it rotates StJ~S ~'~ SHEET' WO 93/1936 ' PC'f/iJS93102470 . ..
(e. g., r = 1 nsec for fluorescein, 100 nsec for large antibody complexes). For a constant, small, .fluorescence lifetime (~ nsec for fluorescein), a small molecule r retains little of the original polarization when irradi-5 aced by polarized light, because the molecule rotates rapidly and then fluoresces. an the other hand, a large molecule rotates slowly and for the same fluorescence lifetime, still retains a large degree of the original polarization when it fluoresces.
10 This dependency of polarization on molecular size can be used to determine the presence or amount of drug.
- Using a fluorescent polarizing probe in a competitive binding immunoassay provides a type of FIA called a fluor-escence polarization immunoassay (FPIA). In this type of assay, the smaller the molecule is, the smaller its rota-tional relaxation time and the faster it rotates. Typi-cally, antibody molecules are much larger than drug or drug-probe molecules. For example, r 1 nsec for fluorescein and 57 nsec for gamma globulin.
When there is a large amount of drug present, there are very few binda.ng sites available for the drug-probe. ;, As a result, most of the probe (fluorescein) is in the form of small drug-probe molecules. As these molecules rotate randomly and rapidly, a low polarization value results: When there i.s a small amount of drug present, much of the drug-probe is bound to the large antibody molecules. These molecules rotate slowly, so the emitted light will be highly polarized.
"' ' The relationship between polarization azad .drug con centration can be determined by creating a standard, or calibration; curve: 'This is done by running an assay using a range of known drug concentrations, from the lowest to highest expected concentrations, and plotting the resulting values of polarization: Thereafter, for a gi~ren ~ralue of polarization; Che drug concentration can be determined from the standard curve.
SL1~5 'TE S~EE'T

WO 93119366 ' ~ PCf/U593102470 m One advantage of the polarization technique is the elimination of a step to separate unbound probe. Although the unbound tracer is not physically eliminated from the samples in FPIA, its contribution is readily assessed by the polarization.
Another advantage in the FPIA technique is lack of dependence on intensity. In equation (1) above for cal-culating polarization using intensity, the ratio makes the polarization value unitless, or independent of variations 1.0 in the intensity. Unlike most assays using a light mea-surement, in which it is the intensity of the light that is correlated to drug concentration (so any variations in source light intensity will directly affect the sensitiv-ity of the assay), the sensitivity of FPIAs is independent of'intensity variations. Conventional FPIAs require sepa-rate measurements of both blank and sample.
Theoretically, fluorometry is capable of being the most sensitive of all analytic tools as it is possible to detect single photon events. A problem which has plagued fluorescence immunoassays has been discriminating the ,.
fluorescent signal of interest from background radiation.
The intensity of signal from background radiation may be up to x.0,000 tames larger than the intensity of the fluorescent signal of interest.
The problem of background detection is particularly pronounced in assay of biological samples. Many of the current fluorescence assays use the fluorescent molecule, fluorescein. Fluorescein has an excitation maximum of 493 n~, and there are numerous substances in biological fluids with overlapping excitation and emission similar to fluor-escein. For example, in the analysis~of blood plasma, the presence of a naturally occurring fluorescable material, biliverd3:n, causes substantial background radiation. Such i compounds are highly fluorescent and contribute signifi-cant background signals which interfere with the label's signal, thus limiting the sensitivity of assays usin=
fluorescein labels.
S L! ~ S"1'~J'~E S ~# E E'T' CVO 93/9366 ' PCT/US93/02470 Earlier attempts to overcome the problem of back-ground radiation have met with limited success. One technique far overcoming the problem involves discrimi- ' hating against background radiation on the basis of wavelength. Filters have been used to reject detected radiation at all but a narrowly defined wavelength band.
This technique has been less than successful principally because the background radiation may also be at the same wavelength as the desired fluorescence signal, accord-ingly, still be passed through the filter and detected.
It has been recognized that for analysis of biologi-cal fluids, it would be desirable to use a dye or label which is excitable at radiations of wavelengths of greater than background radiation. However, even though the back-ground fluorescence of serum falls off at wavelengths approaching 600 nm, significant decrease does not occur until 650 nm or greater. Previous attempts to create dyes of such wavelengths have been unsuccessful. See, e:g-, Rotenberg, H. and Margarfit, R., Biochem. Journa1,229:197 0,985); and D.J.R: Lawrence, Biochem. Journal 51.:168 ( 1952 ) .
A second technique attempting to discriminate the desired fluorescent signal from the background is the so called time gating approach. Here, the fluorescent signal is observed in a short time window after the excitation.
The time window may be varied both in its length and ire ~-is starting time. Through the use of the variable time window, the detected radiation may be observed at the, ,. ;
maximal time for detection sensitivity. Hi.storically,l tk~is technique has used a fluorophore of very long decay time (such as 1,000 nanosecands) to allow the background , fluorescence to substantially decay before detection of the fluorescent signal of'interest. Generally however, long, decay time fluorophores require longer times for o~rerall analysis. i~ue to the long decay time, the light source cannot be pulsed rapidly to coiiecc data, thus requiring additional time for final analysis.
S tJ ~ S'~ITt~JTE 5 ~ E E~' Historir_ally, there have beers two excitation pulse formats for transient state fluorescent analysis. One format utilizes a single, relatively high power pulse which excites the fluorophore. The transient state is typically monitored by a high speed photomultiplier tube b~~ monitoring the analog signal representative of r_urrent as a function. of time.
Single pulse systems require sufficiently high. power to excite a large number of fluorescent molecules to make detection reliable. The other principal format f:or transient state fluorescent analysis is a digital format which utilizes repetitive excitation pulses. Ordinarily, pulses of relatively short, typically nanosecond duration, light with power in the microwatt range are repetitively supplied to the sample at rates varying from 1 to 10,600 Hz. Ordinarily, the excitation source is a lamp, such as a Xenon-lamp.
Frequently, the decay curve is measured digitally by determining the time to receipt of a photon. One commercially available system uses repetitive pulses (such as at 5,000 Hz) and pulses the photomultiplier tube at increasingly longer times after the flash in order to obtain a time dependent intensity signal. Detection in such systems has proved to be very time consuming and insensitive. A single analysis can take on the order of one hour, even at relatively high fluorescable dye concentrations (e=g. , 10-~' M) .
Recently, significant advances have been made in the area of fluorescable dyes. In one aspect, dyes being excitable by longer wavelength radiation, such as in the red and infrared wavelengths, are now available. These dyes are described in two commonly assigned Arrro.enius, U.S. Patent No.
5,403,928, entitled, "Fluorescent Marker Components and Fluorescent Probes".
Further significant advancements have been made in increasing sensitivity through data collection and analysis techniques. As disclosed in Dandliker et al., U.S. Patent No.
4,877,965, entitled "Fluorometer", time gatingf techniques are used in conjunction with data collection and analysis techniques to obtain an improved signal relative to the background. Generally, the '965 Patent considers the detected intensity as a function of time to be composed. of signals from various sources, including the desired signal source, and various undesired background sources. Optimization of the desired signal is achieved through data collection and analysis techniques.
Further significant advancements have been made in the ability to measure relevant materials in immunoassays.
For example, using the technique described in Dandliker, et al., U.S. Patent No. 5,302,349, entitled "Transient State Luminescence Assays", al.lows the bound and free form of materials in a homogeneous assay to be determined. Generally, the technique requires measurement of the time-dependent decay of the intensity of parallel and perpendicular polarization components. By measuring the time-dependent decay of various polarization states, it is possible to determine the bound and free forms of materials such as haptens, peptides, or small proteins in a homogeneous analysis format. Significantly, no separation of the bound and free materials is required.
Despite the significant and promising improvements made in the field of fluorescable dyes, and in the data analysis aspects, the actual methods and apparatus for achieving and detecting fluorescence have heretofore remained relatively unchanged. Utilizing even the most sensitive and best equipment, analysis can take an hour or 5 more, even at high concentrations of materials. When fluorometry is used far immunoassay in a clinical context, time for analysis and proper diagnosis can be absolutely critical. Patient survival can depend on accurate, timely analysis. Additionally, rapid testing would permit l0 retests of patients without having them wait significant periods of time, resulting in more rapid and accurate diagnosis. As to sensitivity, it is extremely desirable to be able to detect minute amounts of fluorescable mater-ial. However, as the amount of fluorescable material in 15 a sample decreases, the ratio of the signal increases.
Further, since the time for analysis depends on the amount of fluorescent radiation received from the detector, low concentrations generally require substantially more time to analyze.
Heretofore, the time required for analysis has been prohibitively high. Known methods and apparatus for FIAs have failed to provide rapid and accurate diagnosis and analysis of samples. This has been so despite the clear and known desirability of the use of FIAs. For example, the drug digoxin, which is used to treat congestive heart failure, has a narrow therapeutic range (,i.e., serum levels of 0.5 to 2.5 ng/ml) and is generally toxic at concentrations greater than 2.1 ng/ml. Present assays using fluorescence-based methodologies require an extract-ing process to remove interfering substances, such as proteins, in order to detect digoxin at it:a therapeutic levels. This additional extraction step increases the time, cost and equipment needed to perform the assay.
From the above discussion it can be seen that, although many different t~~pes of immunoassays currently exist, none is satisfactory for measuring small quantities of target analytes in biological ~':uids such as serum, plasma and, especially, whole blood. Accordingly, an object of the present invention is to provide improved processes far assay of antigenic substances. More speci-fically, the present invention provides fluorescence assays which allow the detection of low levels of anti-genic substances in biological samples such as serum, plasma and whale blood. The present invention also pro-vides homogeneous fluorescence assays which allow rapid and accurate determination of low levels of antigenic substances in biological samples.
Summary of the Invention The present invention is directed to methods for determining the pzesence or amount of a target analyze in a sample by using, as a label for the target analyze or a receptor which is capable of specifically recognizing the target analyte, a fluorophore moiety comprising a lumi-nescent substantially planar molecular structure coupled to two stabilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure.
By "target anal.yte" is meant the antigenic substance being assayed, for example an antigen. By "receptor" is meant a molecule or molecular component capable of specifically recognizing the target analyze, For example, an antibody may be a receptor for an antigen.
Use of such detectable labels or marker components in immunoassays is advantageous in that these: labels have substantially the same intensities of parallel and perpen-dicular components of transient state fluorescence emis-sion in the presence and absence of biological fluids such as serum. Thus, assay methods using these labels are capable of detecting low concentrations of target analyte in biological fluids.
The methods of the present invention are particularly suitable for use with the improved luorescence detection system described in commonly assigned U.S. Patent No. 5,323 1?
008 entitled "Fluorometer Detection System ".
In one aspect, the present invention is directed toward competitive inhibition assay procedures utilizing particular labels. In this aspect, the present invention is directed to a method of determining the; presence or amount of a target analyte by contacting a sample sus-pected of containing the target analyte with a known quantity of added target anaiyte or analog thereof linked to a fluorescent probe which includes a detestably labeled marker component made up of a fluorophore moiety which includes a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar mole-cular-structure; contacting the sample with a receptor capable of specifically recognizing the target ligand; and determining either the amount of fluorescent probe bound to receptor or free fluorescent probe. The amount of bound or free fluorescent probe in the unknown samples may 2.0 be compared with blank samples and samples containing known amounts of target analyte.
In a preferred embodiment, the resultant mixture of sample, fluorescent probe and receptor is diluted before measurement of the amount of bound and/or free fluorescent ~:5 probe. The dilution step allows for greater sensitivity of the assay. Particularly preferred are dilutions of 2-fold to 100-fold, preferably about 7-fold to about 50-fold, and more preferably about 35-fold.
In one aspect, the present invention provides an 30 improvement in immunoassay procedures which utilize a label for either the target analyte for analog thereof) or the receptor. The improvement is the use of a fluorophore moiety comprising a luminescent substantially planar mole cular structure coupled to two solubilizing polyoxyhydro 35 carbyl moieties, one located on either side of the planar molecular structure. Assays using this type of label are advantageous in that they are free of serum binding and aggregation and are therefore, especially suitable for testing biological samples such as serum, plasma, whole blood and urine.
In another aspect, the present invention provides a S method for performing a "sandwich" ar "two-site" immuno assay comprising the steps of:
(a) contacting a sample suspected of con-taining a target analyte with a first receptor capable of specifically recognizing the target analyte to form a complex of the target analyte and the first receptor, the first receptor being labeled with a fluorescent probe which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilized polyoxy-hydrocarbyl moieties, one located on either side of the planar molecular structure;
(b) contacting the complex with a second receptor capable of specifically recognizing the target analyte to, the second receptor being bound to a solid carrier, to farm a com-plex of the first labeled receptor, the target analyte and the second receptor bo~.xnd to the solid carrier; and (c) measuring either the amount of labeled first receptor associated with the solid car-rier or the amount of unreacted labeled first receptor.
A sandwich-type assay may be either a heterogeneous assay or a homogeneous assay. If it is heterogeneous, it may incorporate the additional step of separating the solid carrier from the unreacted labeled first receptor.
Homogeneous assays are generally preferred because they are more rapid.
In another embodiment, the assay may incorporate the additional step of relating the amount oL labeled first receptor measured in the unknown sample to the amount of labeled first receptor measured in a control sample free of the target analyse, or to the amount of labeled first receptor measured in samples containing known quantities of target analyze.
In another aspect, the present invention provides a method for a simultaneous sandwich-type ass ay comprising a method for determining the presence or amount of a target analyte in a sample comprising the steps of:
(a) simultaneously contacting a sample :LO suspected of containing a target analyze with first and second receptors capable of specifi cally recognizing the target analyte, the first receptor being labeled with a fluorescent probe which comprises a fluorophore moiety com L5 ~ prising a luminescent substantially planar mole-cular structure coupled to two solubilized poly-oxyhydrocarbyl moieties, one located an either side of the planar molecular structure, and the second receptor being bound to a solid carrier, 20 to form a complex of the first receptor, the target analyze, and the second receptor; and (b) measuring either the amount of labeled first receptor associated with the solid car-rier or the amount of unreacted labeled first 25 receptor.
In another aspect, the present invention provides a method for a simultaneous sandwich-type assay comprising a method further comprising the step of relating the amount of labeled f irsz receptor measured to the amount of 30 labeled first receptor measured for a control sample free of the target analyze, or relating to the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.
35 In another aspect, the present invention provides a sandwich-type fluorescence immunoassay method for measure-ment of a target analyze which is capable of recognizing WO 93/i9366 ' PCTIL'S93102470 two different receptors independently without mutual interference. The method utilizes two receptors, each of which is labeled with a different dye. For example, one receptor is labeled with a first dye having absorption and 5 emissian maxima of 680 nm and 690 nm, respectively, and the other receptor is labeled with a second dye having absorption and emission maxima of 695 and 70S nm, respec-tively: Detection and quantitation of the analyte can be made using either steady state or transient state measure-10 menu . In either case, for the example given, excitation would be at 680 nm and detection would be at 705 nm. This type of assay is based on energy transfer and is advan-tageous in that, it is homogeneous.
In preferred embodiments the present invention is Z5 directed to immunoassay of biological fluids, including seem, plasma, whore blood and urine. Preferably, rid blood cells in whole blood are lysed,prior to assay o~
whole blood samples: Preferred methods of lysing red blood cells include addition of tearyl-lysolecithin, 20 palma.~oyl--lysolecithin and myristoyl lysolecithin.
Depending on the type of immunoassay used, the target analyte may be an antigen. a hapten or an antibody; and the receptor may b~ an antigen or antibody. The antibody may be pr~lyclonal. or monoclonal. Preferably, the antibody is a monoclonal antibody. Monoclonal antibodies useful in the present invention may be obtained by the Kohle~ &
Milstein method reported in Nature 256:495-497 (1975). .
Alternatively, ~h.ey may be produced by recombinant i , , ~ ~ j (methods. Science 246:1275-1281 (1989) . , Tn one embodiment; the target analyte is a drug or a metabolite o~ a drug. The drug may be a steroid, hormone antiasthmatic, antineoplastic, ~n;tiarrhythmic, anticonvul-sant> antiarthra:~ic, ant~.depressant, or cardiac glycoside.
'Examples of such drugs include digoxin; digitoxin, theo phylline, Phenobarbital; thyroxine, N-;acetylproc~inamide, primidone, amikacin, gentamicin, netilmicin, tobramycin, earba.mazepine; e~hosuxima.dp, valproic acid, disopyram~.de, St~~51"E SHEET

WO 93/19366 ' . PCTlUS93/02470 s ~s ~. c~ ~ f~

lidocaine, procainamide, quinidine, methotrexate, ami- y triptyline, mortriptyline, imipramine, desipramine, van-comycin, and cyclosporine. In a preferred embodiment, the drug is digoxin.
In another embodiment, the target analyze is a pep-tide, for example, a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human choriogonado-tropin, thyroid stimulating hormone, angiotensin I, angio-tensin II, prolactin or insulin. The peptide may also be a tumor marker such as carcinoembryonic antigen. Or, the peptide may be a virus or portion thereof, for example, rubella virus or a portion thereof.
The methods Qf'the present invention provide ways of measuring target analytes in concentrations of from about I x 10'S M/L to about 1 x 10'13 M/L, and particularly in the concentration range of from about 1 ~c 10-9 M/L to about 1 x 10'~a M/L. For measurement of drugs and their metabolites, the present methods allow measurement in the range from about 5 x 10'9 M/L to about 5 x 10-1z M/L, and particularly, concentrations of from about 1 x 10'1° M/L to about 5 x M/L. For measurement of peptides; the present methods allow measurement in the range of from about 1 x 10'11 M/L
to about 1 x 1.0'lz M/L.
The measurement of amount of fluorescent probe bound or free or both -- can be determined by measuring steady~state fluorescence or by measuriztg transient state fluorescence. In a preferred embodiment, the wavelength ,of~ light measured a.s greater khan about 500 nm, preferably greater than about 650 nm, and more preferably greater than about 680 n,m or 690 nm. Because the transient.state , detection system utilises a laser diode, it is necessary for the dyes to have excitation maxima matched to the d~.ode output wavelengths. Dyes have been made available to match other commercially available laser diodes have output wavelengths of 680; '690, 720, 750, or 780 nm.
Thus, the wavelength of the light measured may be greater than about 580 nm, 690 nm, 720 nm, 750 nm ar 780 nm. The S ~ ~ ST'1?~TE S H E E°~

'VVO 93/1936t~ ,' 1PCT/L'S93/02470 ~~.~2'~U~

further into the red region of the spectrum one moves, i.e., the greater the wavelength, the greater signal ezirichment there is over background. .

In a preferred embodiment, detection and quantitation t is performed using transient state measurement. Transient state energy transfer offers improved measurements due to optimization of the wavelengths of absorption and emir-sion, as well as due to optimization of the decay times of.

the fist and second dyes. Such optimization allows removal of Rayleigh and Raman scattering; and achieving the best compromise between ef f iciency of transfer and the undesired direct excitation of the second dye by the first dye.

In one aspect, the present invention is directed to immunoassays using de~~ctably labelled marker components which comprise a fluorophore moiety which comprises a sub~tanta.ally planar macrocycl~.c multidentate ligand coordiz~ated to a central atom and two olubilizing poly-o~yhydrocarbyl moieties, one linked on either Bide of the plane of'the mul identa~e ligand to the central atom.

In one preferred aspect, the present invention is , directed to immunoassays using a marker component compris-ing a fluorophore moiety which 'comprises a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two aacial lig-ands which are coordinated to the central atom on either s~,de Qf the macrocycli.c ligand.

Marker components used in the immunoassays of the '' ; ; ;, ;
~
present invention comprise a macrocyclic multidentate.

'ligand having two solubil~,zing polyoxyhydroearbyl moieties one located'on either side of the plane-of the multiden-ate ligand exhik~it no detectable non-specific binding to serum components; and exhibit no detectable solvent sensi-tivity. These marker components also exhibit enhanced decay times which -approach their natural (fluorescent) ;

l i~et~;mes . .

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,. tw . .,...~. .f........v .. . .... n ..... .. ,. ..... , ..~.,..n. a ,......
. , v,.. .u .....>. e. ..~. .~. ~.. . .r ... ..7 n ... .... . .,..

Preferred are fluorophores which produce fluorescent light efficiently, i.e., which are characterized by high absorbitivity at the appropriate wavelength anal high fluorescence quantum yields. For certain applications, preferred fluorophores have measured fluorescence decay times cn the order of at least 2 nanoseconds and exhibit a high degree of fluorescence polarization.
Preferred solubilizing polyoxyhydrocarbyl moieties include water soluble carbohydrates such as glucose, sucrose, maltotriose, and the like; water soluble carbohydrate derivatives such as gluconic acid and mannitol and oligo saccharides and water soluble polymers such as polyvinylpyrrolidone, poly(vinylalcohol), poly(ethylenimine), polyacrylic acid, polyacrylamide, ethylene oxide copolymers such as Pluronic* (a propylene oxide copolymer, available from BASF) and Tetronic* (BASF) polyol surfactants; and polyethers, including water soluble polyoxyalkylene polymers, particularly polyethylene glycol) ("PEG") and polyethylene glycol) derivatives such as polyethylene glycol.) methyl ether, polyethylene glycol) silicon derived ethers and the like.
In one aspect, the present irxvention is directed to immunoassays using marker components comprising a fluorophore moiety which comprises a substantially planar, multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands and two polyoxyhydrocarbyl moieties which are attached as axial ligands to the central atom. Suitable central atoms are those to which may coordinate two axial ligands and which are not of high enough atomic number to cause extensive fluorescence quenching by transition to the triplet state. Preferred elements for the central atom include silicon, germanium, phosphorus, and tin, *Trade-mark 23a especially preferred are silicon and germanium.
Depending on the type of immunoassay, these marker components may be used as labels for labe7_ling an analyte, antigen, antibody or other molecule. These marker compo-nents may be optionally functionalized so as to include a linker arm which allows the marker Component to be linked to the analyte, antigen, antibody or other molecule. A
variety of linker arms which may be suited to this pur-e pose. The marker component is linked to the analyte, antigen, antibody or other molecule using conventional techniques.
The present invention is also directed to the use of divalent peptide derivatives as analogs for large mole lo cules in immunoassays. Preferably, a divalent hapten consisting of two epitopes of the same specificity con-nected by a linker about 10 nm long is used to bind to a single antibody molecule, requiring approximately 26 residues, 1~ The present invention also includes assay methods of involving cellular receptors located on the. plasma mem-brane or isolated from cytosols and synthetic ligand binders obtained by molecular imprinting.
Accordingly, it is a principal object of this inven 20 lion to provide improved FIAs with greatly enhanced sen sitivity. It is yet another object of this invention to provide FIA methods which allow rapid and accurate deter minations, often within a matter of minutes..

The present invention also provides particular fluor-escent probes for use in immunoassays, for instance, see Examples 3 and 11-18 below.
Definitions:
As used herein, the following terms have the follow-ing meanings unless expressly stated to the contrary:
The term "target analyte" refers to the compound or compound to be measured in an assay which may be any l0 compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor. By "analog" of a target analyte is meant a compound or com pounds capable of competing with the target. analyte for 15 binding to a receptor.
The texzn "axial ligand" refers to a substituent which, together with a macrocyclic ligand, forms a coordination complex with a central atom. The axial ligand lies normal to the plane described by the macro 20 cyclic ligand.
The term "fluorescent probe" refers to a marker component comprising a fluorophore moiety which is bonded to or coordinates either directly or via a linker arm to an analyte, antigen, hapten, antibody or other molecule 25 which is used in an assay, such as a fluoroi.mmunoassay to determine the presence of and/or quantitate a substance of interest.
The term "solvent sensitivity" refers to changes in tre fluorescence behavior of a molecule deyending on the WO 93/19365 ~ PC.'f/L'S93/0247~

solvent system in use, most notably referring to differ-ences in fluorescence behavior in aqueous solution in comparison with organic solvents (such as DMF). Many fluorophores which exhibit high fluorescence intensity in organic solvents such as DMF show substantially decreased fluorescence intensity in aqueous solution.
Fluorescence intensity is related to sample concen-tration and the intensity of the exciting radiation. The fluorescence intensity of a particular dye can be corre-laced to its characteristic light absorptivity (extinction coefficient) axed fluorescence quantum efficiency, as well - as environmental factors.
The term "specific binding pair" refers to two dif ferent molecules (or compositions) wherein one of~ the molecules has an area on the surface or in a cavity which specifically recognizes and binds to a particular spatial and polar organization of the other molecule or molecular complex involving other molecules.
The term "binding partner" refers to a molecule or molecular complex which is capable of specifically recog nizing or being recognized by a particular molecule or molecular complex.
The term "bound" refers to the condition in which a binding interaction has been formed between a molecule and its specific binding partner.
The term "decay time" is the time which must elapse in order for the concentration of excited molecules to decrease from its initial concentration to 1/e of that value .
The term °'receptor'° refers to a molecule or molecular complex which is capable of specifically recognizing or being recognized by a target analyte or analog thereof.
Brief Description of the Drawings Fig. ~. depicts an HPLC analysis of crude caged dicar-boxy silicon phthalocyanine dve preparation.
SUSTE SHEIE~"

Fig. 2 shows the absorbance of caged dicarboxy sili-con phthalocyanine dye in various so:Lverxts.
Fig. 3 describes the Jiatrori Analog Sy~~tem.
Fig. 4 depicts the decay time for caged dicarboxy silicon phthalocyanine dye.
Fig. 5 shows serum interactions of purified caged dicarboxy silicon phtha:Locyanine dye.
Fig. 6 depicts the absorbance spectrum of caged dicarboxy silicon phthalocyanine dye-C12 linker.
Fig. 7 depicts the polarization of caged dicarboxy silicon phthalocyanine dye-C12 linker at 680 nm.
Fig. 8 depicts the polarization of caged dicarboxy silicon phthalocyanine dye-C12 linker at 69() nm.
Fig. 9 depicts an HPLC Chromatograph of: caged dicar-boxy silicon phthalocyanine digoxin probe.
Fig. 10 depicts the structure of caged dicarboxy silicon phthalocyanine digoxin probe.
Fig. 11 shows the absorbance spectrum of caged dicarboxy silicon phthalocyanine digoxi.n probe in 2.0 methanol.
Fig. 12 shows the absorbance spectrum of caged dicarboxy silicon phthalocyanine digoxin probe in FPIA
buffer.
Fig. 13 shows the decay time for caged dicarboxy silicon phthalocyanine digoxin probe.
Fig. 14 shows the linearity of intensity for caged dicarboxy silicon phthalocyanine digoxin probe.
Fig. 15 shows serum/urine interactions fox caged dicarboxy silicon phthalocyanine digoxin probe.
:30 Fig. 16 depicts a comparison of TDx'~ and FAST-60 calibration curves.
Fig. 17 depicts the correlation of digoxin samples assayed by TDx° and FAST-60.
Fig. 18 depicts the effect of dilution jump on non-specific binding.
Fig. 19 depicts a digoxi:~ probe-serum calibration curve .
*Trade-mark CVO 93/y9366 PCT/L~S93/02470 28, Fig. 20 depicts a calibration curve for a high sensi-tivity digoxin assay.
Fig. 21 describes the FAST-60 digoxin assay procedure.
Fig. 22 depicts digoxin correlation - TDx~ Serum vs.
FAST-60 Whole Blood.
Fig. 23 depicts digoxin correlation - Stratus° Serum vs. FAST-60 Whole Blood.
Fig. 24 depicts digoxin correlation - FAST-60 Serum vs. FAST-60 Whole Blood:
Fig. 25 depicts digoxin correlation - TDx° Serum vs.
FAST--60 Serum.
Fig. 26 depicts digoxin correlation - Stratus° Serum vs. FAST-60 Serum.
Fig. 27 depicts the structure of caged dicarboxy silicon phthalocyanine digitoxin probe.
Fig. 28 depicts the structure of caged dicarboxy silicon phthalocyanine theoghylline probe:
Fig: 29 depicts the structure of caged dicarboxy silicon phthalocyanine phenabarbital probe.
Fig. 30 depicts the structure of caged dicarboxy silicon phthalocyanine thyroxine probe.
Fig. 3l dep~.ets the structure of caged dicarboxy silicon phthalocyanine n-acetylpr~ocainamide probe.
Fig. 32 dep~.cts the structure of caged dicarboxy silicon phthalocyanine primidone probe.
Fig. 33 depicts the structure of caged dicarboxy .
;,,, sihicon phthalocyanine phenytoin probe.
Fig. 34 dep~:cts a rubella antibody calibration curve . .
' for a sandwich assay.
Fig. 35;depicts a rubella peptide calibration curve , for an inhibition assay: ;
Fig. 36 depicts a rubella antibody calibration curve for direct polarization.
S tJ ~ 5'tITF 5 ht E ~°3' 1~V0 93/19366 . w P('T/U~93/02470 Detailed Description of the Invention The present invention provides fluorescence immuno-assay methods which have dramatic increases in sensitivity over previous methods, which can be easily performed "

because they require no separation step, and which can be used to detect and quantitate low levels of target analyte in biological samples such as serum, plasma, whole blood and urine. The FTAs of the present invention may be per-formed in small samples. For example, a digoxin assay may be performed on a 20 ~C1 sample of serum, plasma or whole blood, and the assay may be performed in about five min-utes. The ability to perform FIAs on whole blood samples is particularly significant because it allows assays t.o be .

performed at locations closer to the patient, such as ~.5 physicians' offices and emergenc~r rooms. The capability of performing FIA.s rapidly is important because, in a clinical context; patient survival can depend on accurate, timely results.

The concept of sensita.vity in fluorescence measure-menu can be usefully quantified by specifying the con-centration of tie fluoro~hore in question at which the fluorescence intensity fram the fluorophore is equal to the intensity from the background. This manner o.f expressing sensitivity emphasizes the fact that the ser~sxtivity of f7.uorescence measurements is almost always determined by the ability to discriminate between "signial"

and "background" and not by the absolute number of photons av~ilable,from the "signal:" ;

The present invention provides methods for FIAs which , solve the problem with discriminating against background ::

radiation on the basis of wavelength. The probes used in the method of the invention have excitation (and emission?

wavelengths greater than about 650'nm, preferably greater than 680 nm. This wavelength shift into the infra-red '35 range decreases background fluorescence, i.a., increases si.:~nal-to-background ratio. This decrease in background 5l! ~ S'TiTI~'TE 5 H E E'~' r ...~ :, :...; ..... ~ . ... ,.. .. . .:,.. . : . . :: ;.
v.". . , ,, . ,.. .
..,:, .:.::..;. .,..:.' '.:x;;,,:.. ,:~,::... . , ~...;;.~. :".i ;.'~.
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Y' > Fr..e .,.... .. ..,..,..:..., ....'.::1.. '...,-._._..,,:..
.,:::...,..,...,v...,.,s'.,:
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: .. ~.,... ....

Vd~ 931~936b PCT/LJS93/02470 _.
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fluorescence allows the use of fluorophore at far lower concentrations than previously used in homogeneous FIAs.
The probes used in the present assay methods have low dielectric constants, which applicants believe tend to ' S increase the Van der Waal interactions and hydrogen bond-ing, thus accelerating the antigen/antibody reaction. In, addition, applicants believe .that these probes compete for the water of hydration, thus potentiating the antigen/
antibody reaction. In other words, the probes not only 10 substantially decrease non-specific bindings to serum components, but applicants believe that they potentiate - the immunochemical reaction.
For fluorescence polarization assays, the present invention provides a further increase in sensitivity by 15 measuring transient state fluorescence rather than the steady state signal. In the steady, state mode the signal is constant over time enabling the determination of one experimental~parameter, e.a., the polarization or the anisotropy, both of which are related to molecular 20 rotational motion. In the transient state made the signals vary in a systematic way with time. This varia-tion represents a complex summation of the rates of decay arid of wolecular rotation as it changes from moment to moment in time.
25 The incxease in sensitivity from transient. state measurements stems from two sources. First, that portion of the background due to Rayleigh and Rarnan scattering disappears, in ,about 10'15 sec and, so is clean~.y removed before the transient state measurements start. This , 30 portion of background is normally an important part of the y-total in steady state measurements. , Above and beyond the removal of scattering, the tran-sient state measurements provide an additional powerful means to discriminate between the desired signal and the remaining fluorescence portion of the background. This discrimination rests upon the time dependence cf the polarized components in the fluorescence decay and makes SU~S'T'~'UTE SKEET' t-'?7Q36-13 it possible to extract the desired signal only, simultane-ously on the basis of the rate of decay of the excited state and the rate of decay of the rotational distribution imprinted by the excitation. Thus, transient state methods allow signal to be distinguished from background in ways not possible with steady state information alone.
In addition to these features, the probes exhibit a high degree of polarization, necessary for mix and read (homogeneous) fluorescence polarization assays. This l0 increase in polarization translates into increased sensitivity.
The methods of the present invention are particularly useful when used with a time-correlated transient state detection system, as described in commonly assigned Studholme, et al., U.S. Patent No, 5,323,008 entitled "Fluorometer Detection System °.
That system features transient detection along wit.z detection of the time-dependent polarization of the sample. The system uses a laser diode which can be modulated at very high frequencies, e-a., 14 MHz rats, and exhibits high output power. Typically the laser "on" time is approximately 2-3 nanoseconds. Photons from the solution are detected using a photomultiplier tube (PMT) operating in a single photon counting mode. The photon event along with the relative time of the photon event as compared with the laser pulse time is determined. By storing the individual photon event times a histogram of frequency of photons as a function of time is venerated.
Data obtained in this manner can be analyzed as described in Dandliker ~ ate", U.S. Patent No. 4,877,965, entitled "Fluorometer" or as described by Studholme, et al . , U. S . Patent 1'"10. 5,:323,008 entitled, "Fluorometer Detection System ".
The methods of the present invention also include the use of divalent peptide derivatives as analogs for large Wl'~ 93/19366 ' PCT/L'S93/02470 molecules in immunoassays. Both polyclonal and monoclonal antibody molecules are divalent. Due to the "chelate effect," the binding of a low molecular weight mimic or analog of a larger molecule will be stronger arid dissoci-ation from the antibody will be slower if both antibody .
binding sites are utilised in the bonding. Tt is within the scope of the present invention to arrange the struc-ture of the peptide analog to have twa identical sequences joined together by a linker of suitable length so as to place the two peptide sequences, in their normal config-uration in solution, in the most favorable position for reaction with the two sites on the same antibody molecule .
Preferably, several such unspecific, divalent analogs are used as a cocktail; rather than combining more~than one epitope in the same analog molecule. The latter arrangement would permit cross link~.ng of perhaps many an;t~.body molecules which might be preferable in solid phase assays,in which the formation of chains and cycles could aid in adhering to a surface. Conversely, having two identical epitop~s on the same analog molecule may "inhibit" polymerization by strongly favoring, by prox-imity factors, reaction with two sites on the same molecule.
The use of divalent peptide derivatives as analogs for large molecules in immunoassays is preferred in solution, especially in conjunction with dilution jump, due to the tighter binding afforded by the "chelate a f fect, '! resin ing in an increase, in the sens~.tivity of r , the immunoassay. , Preferably, a divalent hapten consisting of two epitoges of the same specificity connected by a linker about 20 nm long is used to bind to a single antibody molecule. Taking into account the bond distances and angles far simple peptides (L. Paining, The Nature of the Chemical Bond, Cornell University Press (1960), p. 498) and assuming a length of 0.380 nm per amino acid residue, s c~ s°rrr~~rF s ~ E ~r WO 93/ ~ 9366 ' PCT i'S93l02470 ~,~x_~~'~~~~

this would require approximately 26 residues for a ~0 nm i length.
One approach for designing a divalent hapten with , such a linker is to synthesize the epitope with a 13 resi-due linker terminating in a primary amino group. This peptide is then reacted with the bis (3-isocyanatopropyl-dimethylsilyl) derivative of dihydroxysilicon phthalocya-nine. The resulting structure has the phthalocyaninine moiety with two axial substituents, one on either side of 0 the molecular plane, each consisting of a thirteen residue peptide linker leading to the peptide epitope. The mole-cular plane of the dye moiety is perpendicular to the direction of the linker. After combination of the two peptide epitopes with the two binding sites of the 'same ~.5 antibody molecule th.e dye moiety may be located midway between the two arms of the Y-shaped antibody molecule.
The polarization changes obtainable with this type of structure may, not be as; great as yf the dye were linked through a peripheral rather than an .axial bond, and the 20 absence of PEG may result in non-sped fic binding. How-ever, if the dye moiety is held close to the antibody i-surface between the two Fab fragments after binding, it may prove to be quite protected and rotate with the long-est rotational decay 'time of the antibody (since the 2S molecular plane of the dye may lie parallel to the long .
axis of the antibody).
Alternatively, a divalent peptide hapten may be designed to utilize the PEG protected dye linked through i ,, . ; , ' a peripheral carboxyl to an amino group~on the~link~er'o 30 oz~; one of the peptide epitop~s, ~, the linkage could be the e-amino group of a lysine residue located approxi-mately midway on an interconnecting chain between the two peptide epi~opes.
Immunoassays, a class of ligand binding assays, 35 depend upon the strong and-selective binding of some analyt~ of interest to antibody specific for that analyte .
Other molecular structures that have similar strong and SL!~S T'E S~1EE°~' WQ 93/9366 -- ~CT/US93/~D2470 6 ~ i~ 6~ t''~ ~.~ , selective binding for such an analyze can serve equally well in designing an assay and such structures may have some inherent advantages over antibody. Far example, , molecules which may have desirable properties in this S context include cellular receptors located on the plasma , membrane or isolated from cytosols arid synthetic ligand binders obtained by a process known as "molecular imprinting."
The sensitivity of a ligand binding assay depends upon the binding of f inity of association constant f K) of the reaction between the analyte and the binding molecule .
For classical cytosolic steroid hormone receptors these Ks are of the order of 109 M'~. In recent work with molecular imprinting of synthetic polymers the binding constant for 1S -diazepam was found to be about 108 M'1 (Vlatakis et al . , Nature 361: 64S--647 (1993)). By contrast, the highest Ks for antibody binding are of the order of 1012 M'1 for lig-ands such as f~.uorescein and digo~in.
The magnitude of these Ks suggest that antibodies bind far more tightly than do receptors or molecular imprints. Binding processes are symmetrical and the ..
1,.
obsererad K depends upon both the "receptor" and the "ligand" and the distinction between the two is made for convenience. Applicants believe that because relatively few Ks have been measured for receptars or molecular imprinbs, there is no reason that the binding by these molecu~.es should not be potentially as tight as by anti-body,. Molecular, imprints also have the inherent advantage lof being tailored for one specific molecule and the Ks can .
be improved by he'proper placement of hydrophobic, polar hand ionic groups in the binding sites. Moreover, because these molecules are synthetic, once the optimal structure i.s.known large amounts should be readily obtainable.
Thus, the present invention includes methods of involving cellular receptors located on the plasma mem brave or isolated from cytosols and synthetic ligand binders obtained by molecular imprinting.
S LI ~ ST't°TLJTE S #~ E ET

I. Preferred Marker Components The following is a brief description of the preferred marker components to be used in the fluorescence immuno-assays of the present invention. A more complete discus-s sion is found in commonly assigned U.S. Patent No.
5,403,928.
A. P~r~~rred F,~~,uorophore Moi ties Suitable fluorophore moieties comprise a luminescent 10 substantially planar molecular structure. Preferred are fluorophore moieties in which the luminescent substan tially planar molecular structure comprises a substan tially planar macrocyclic multidentate ligand which coordinates a central atom which may coordinate with two 15 axial ligands, one on either side of the: macrocyclic ligand (~ having a trans orientation).
Preferred central atoms are elements which may form octahedral coordination complexes containing two ligands with a trans or axial orientation, on either side and 20 perpendicular to the planar macrocyclic ligand. For use as fluorescent marker components in certain applications the central atom should not have toa high atomic number (about 30 or less) so that fluorescence is not diminished through coupling interaction with orbitals of the central 25 atom.
Preferred multidentate ligands include nitrogen-containing macrocycles which have conjugated ring systems with pi-electrons. These macrocycles may be optionally substituted, including substitution on bridging carbons or 30 on nitrogens. Suitable macrocycles include derivatives of porphyrins, azaporphyrins, corrins, sapphyrins and por-phycenes and other like macrocycles which contain elec-trons which are extensively delocalized. In view of the fact that they incorporate many of the above-noted char-3~ acteristics, an especially preferred class of macrocycles comprise porphyrin derivatives, and azaporphyrin deriva-

7'7036-13 tives (porphyrin derivatives wherein at least one of the bridging carbons is replaced by a nitrogen atom). Azapor-phyrin derivatives include derivatives of mono-, di- and triazaporphyrin and porphyrazine. These rraacrocycles may S optionally have fused aromatic rings. These azaporphyrin derivatives include phthalocyanine, benzotriazaporphyrin and naphthalocyanine and their derivatives. The prepara-tion and fluorescent qualities of many of these compounds are known and some are available commercially.
For certain applications, such as fluorescence polar-ization assays, preferred are azaporphyrin derivatives which exhibit a high degree of polarization, that is, those which emit strongly polarized light. For these applications, preferred are macrocycles having lower degrees of symmetry, preferably having lower symmetry than D",. One preferred group includes macrocyc:les having at least one fused aromatic ring. Thus, preferred macro-cycles include azapvrphyrin derivatives having fused aromatic rings at positions which result in decreased symmetry. Preferred classes of azaporphyrin derivatives comprise derivatives of monoaxaporphyrin, d:iazaporphyrin, and triazaporphyrin having lower than D~" symmetry, B. P ed ub' o o a 1 of t' Preferred solubilizing polyoxyhydrocarbyl moieties include water soluble carbohydrates such as glucose, sucrose, maltotriose and the like; water soluble carbo-hydrate derivatives such as gluconic acid and mannitol, and oligosaccharides; polypeptides such as polylysin and naturally occurring proteins; and water soluble polymers such as polyvinylpyrrolidone, polyvinyl alcohol), poly (ethylenimine), polyacrylic acid, polyacrylamide, ethylene oxide copolymers such as Pluronic~'T' (a polyether) and Tetronicl" (BASF) polyol surfactants and, in particular, polyethers such as other polyoxyalkylenes including poly W~ 93/i9366 - PC.'C/US93/02470 .
,y:~YW ra (ethylene glycol), or other water soluble mixed oxyalky-lene polymers, and the like.
A particularly preferred class of solubilizing poly oxyhydrocarbyl moietie s comprises polyethylene glycol) S (PEG) and polyethylene glycol) derivatives, such as polyethylene glycol) monomethyl ether. Other suitable PEG derivatives include PEG-silicon derived ethers. Many of these polymers are commercially available in a variety of molecular weights. Others may be conveniently prepared ~.0 from commercially available materials, such as by coupling of an amino-PEG to a haloalkyl silyl ar si,lane moiety.
When linked to a fluorophore moiety, these polyoxyhydro-carbyl moieties imparf. particularly advantageous quali,ti~s of solubility in aqueous solution with improved measured 15 fluorescence decay time, and improved fluorescence inten-sity. Moxeover, the resulting marker components are water so7.uble and show decreased non-specific binding. especi-ally decreased binding tc ~exum albumin which has here-tofore been a problem with certain fluorophores, parts--20 cularly porphyxin or phtha~ocyanine dyes which have been functioa~alized with groups such as sulfonate to imP~rt increased water solubility to the molecule. Non-specifis winding of fluorophore or marker component impairs the accuracy of the resulting; immunoassay. These marker 25 components which comprise fluorophore linked to PEG may also exhibit imprcwed fluorescence intensity in aqueous solution with decreased quenching.
Suitable PEGs may vary in molecular; weight , from about art~.culari mole~_' 200 to about 20,000 or more. Choice of a p 3.0 cular weight may depend on the particular fluorophore chosen and its molecular weight'and degree of hydropho lication for which bicityas well as the particular app the f~.uorophore-PEG complex is to be used.
SUSS 'TF SHEE'T

PCT/US93/02470 . .,,n.;.. :.
,...

C. Absorbance and Polarization Behavior of Preferred Marker Components These marker components which comprise a central atom , (for example, Silicon) coupled to two PEG moieties may be characterized by measurements of transient state fluores- , cence. ~n such measurements the intensity of the two components polarized either parallel or perpendicular to the direction of polarization of the exciting pulse is monitored over a time periAd equal to about 3 times the ZO decay time of the marker component. Such curves reflect extinction coefficient, quantum yield, decay time and state of polarization and supply sensitive indications on the chemical and physical condition of the marker component.
~.5 For example, if the excited state is being deacti-vated or converted to the triplet state the overall intensities are lowered and the decay times shortened. If the rotary brownian' ~rotian of the molecule is being a~aered by an increase in viscosity or by being bound to 20 a large molecule; the ratio of the intensity of the parcel-lel to the perpendicular component is increased.
Some marker components according to the present invention show, within experimental error of about 5%, the same intensities, decay time and polarization in DMF (an 25 organic solvent) as in ~.Ap (saline azide phosphate, an aqueous neutral buffer). To'some extent these properties are shared by other marker component preparations. A
distinctive and important property of the marker comps= r' vents' of the'' present invention is ainsensitivity to (and 30 lack of binding to) the components in serum which is evi-de~ced by a lack of shy measured effect of serum on the intensities, decay time ar relative magnitudes of the polarized components of the fluorescence. This property is crucial for the marker components to be useful for 35 applications such as assays using biological materials.
S Ll ~ S~tT'LITE S H E E?' ' WO 93/19366 ' PCT/LrS93/02470 f .. ., ents D, Pre aration of Preferred Marker Cornnon According to one method of preparing the 'preferred marker components of the present invention, the appropri-ate fluorophore moiety having hydroxy or halide groups as axial ligands is reacted with a reactive form of the solu-bilizing polyoxyhydrocarbyl moiety in a ligand exchange reaction according to the general reaction scheme:
Mcl-CA- (X) 2 + 2 (SM) ~ Mcl-CA- (SM) 2 + 2X
whexein Mcl denotes the macrocyclic ligand, CA the central 1C1 atom, X the displaced ligand and SM the solubilizing moiety. This reaction may be carried out neat or, if desired, in solvent. Suitable solvents include quinoline, THF, DMF, imidazole and the like. Suitable reaction tem peratures may vary, depending on the nature of the macro ~,5 cyclic starting material and the solubilizing group. The reaction is generally complete in about 2 minutes to about z~ hours: The r~actian mixture can be conveniently heated under reflex or by mans 'such as a sand bath. For eon venience, the reaction may be carried out at ambient 20 pressure:
It is believed that this reaction takes place in two steps, with one polyoxyhydrocarbyl group coordinating as an axial ligand at a tine.
y~hen used as fluorescent labels in fluorescence 2~ immunoassays, these marker components may be linked to one member of a specific -Mnding pair ( "labelled binding part ner") or an anal~g of such a member. The marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
30 II. Caged DicarbeY_y Silicon Phthalocyanine Dye Example 1 Preparation of a Caned Dicarboxy Sil~.co~
_'-' phthalocyanine D~~e unless otherwise stated, all chemicals used in the 3~ synthesis of ghthalocyanine derivatives were purchased from Aldrich Chemical Co., Milwaukee, WI. Amino SUSTE S~iE~T

;'7036-13 terminated polyethylene glycol and phthaloc:yanine deriva-tives were synthesized according to published procedures.
See, e.a., Reference 18 of U.S. Patent No. 5,403,928.
5 A. Preparation of Diimino~so~.nd ine In a three-neck, 100 ml round-bottom flask fitted with a reflux condenser and a gas inlet tube was placed phthalonitrile (12.8 g), and methanol (50 ml), and the mixture was stirred while ammonia gas Was slowly intro-l0 duced. In order to prevent the possible flow of the reaction mixture into the ammonia source, and in-line trap was employed. After the reaction mixture appeared to be saturated with ammonia, 0.33 g of dry potassium tertbut-oxide was added with stirring.
15 Stirring was continued and the reaction mixture was heated to reflux for three hours with continued introduc-tion of ammonia. Care was taken to avoid fouling of the gas inlet with the crystalline product. At the end of the reflux period a pale green solid had farmed. The solid 20 was collected by filtration and washed with a small volume of cold (4°C) methanol. (This compound is appreciably soluble in methanol.) This material was dried and used for the next step without further purification. Yield was '7 g (about 50%) .
25 B. re a ati i 1,2,4,5-Tetracyanobenzene (Pfaltz & Bauer, 0.5 g, 2,8 mMol ) was suspended in methanol ( 10 ml ) in a three-neck round-bottom flask fitted with a reflux condenser and a gas inlet tube. The mixture was stirred at 25°C without 30 external cooling while ammonia gas was rapidly introduced.
During the first two minutes of ammonia introduction the temperature of the reaction mixture rose to greater than 50°C and the suspended solid dissolved r_ompl.etely. Within 5 minutes a precip.itate~began to form. Stirring at 40-35 50°C with slow introduction of ammonia was continued for 1~0 93/19366 ' P~ I'/i;'S93/02470 r . . ~., ~s~s~~~ o~

1 hour. The precipitated solid was collected by filtra-Lion, washed with methanol, and dried. This product exhibited a very low solubility in methanol.
C. Pre aration of Dic anosilicon hthaloc arsine Dichloride (Compound I) In a dzy 50 ml round-bottom f lack was placed dicyano-diiminoisoindoline (350 mg, 1.8 mMol) along with diimino-isoindoline (1.0 g, 5.9 mMol) and quinoline (Fluka, 20 ml) . The miaeture was stirred at 25°C while silicon tetra-~.O chloride (Aldrich, 2.0 ml, 18 mMo1) was added dropwise over a period of 1 minute. The f Task was then f fitted with a reflux condenser (using teflon tape) and a calcium chloride drying tube and stirred for one minute at 25°C.
At this time the reaction flask was lowered into a large oil bath maintained at 180--185°C and efficient mag netic stirring was continued for 30 minutes. The oil bath was then remcwed and the contents of the flask were allowed to cool to room temperature.
The dark reaction mixture was carefully treated with water (5 m1) and then diluted with 45 ml of a 30% HC1 solution. The resulting dark precipitate was collected by filtration on a ~.0 cm Buchner funnel. 'Washing with water and then acetone left a blue solid (1 gram) which was air dried and used without further purification for the next 2~ 'reaction step.
D ~ H drol sis of Dic anosilicon hthaloc arsine Dichloride ,. , , , , ,~Compo~:nd I I ) The crude dicyanophthalocyanine from step (C) ( 1 grim) was placed in a f bask with a stir bar and 6 ml of concentrated sulfuric acid and stirred at 50°C overnight.
The mixture was then carefully diluted with 4 ml water and heated to 100°C for an additional 20 hours. Cooling and dilution with water (20 ml) gave a blue precipitate which was collected by ~ilt~ration and washed with water . The soi.id was then transferred to a flask along with a stir StI~S "~F S~IEF'T

bar and 20 ml of a 1.0 M potassium carbonate solution and stirred and heated at reflex for one hour. The suspension was then slowly and carefully acidified with concentrated HC1 and then filtered and the resulting solid was washed with water and acetone and dried in a desiccator. This material (0.7 g) was used without further purification in the next step.
E. Preparation of 2~,3-Dicarboxvc~htha~5~ryani~ato-bis- (3-~F~f-imidazol-1-Ylparbonyl? am~,n,opropvl-~dime,~hvlsil-anQlatol silicon (Comgound IT,~~, The crude dicarboxy silicon phthalocyani.ne dihydrox-ide from step (D) (85 mg) was placed in a vial along with a stir bar and imidazole (160 mg, ~2.3 mMol) and 1 ml of dry DMF. The mixture was stirred for 5 minutes at 25°C
and then 3-isocyanatopropyldimethylchlorosilane (Petrarch, 110 ~cl, 0.68 mMol) was added to the stirred mixture over a period of 0.5 minutes. The vial was capped in order to exclude moisture and stirring at 25°C was continued for 20-40 hours. (A 40 hour reaction time appeared to result in an improved yield.) The vial was then opened and the dark blue mixture was diluted with methanol (4 ml) and filtered through #545 Celite*to remove solids. The fil-trate was concentrated on a rotovap using high vacuum and a water bath maintained at 40°C. The dark residue was 2S then slurried with silica gel (1-3 g) and methanol (5 ml) and the methanol was removed on a rotovap under aspirator pressure. The blue residue was then suspended in toluene and transferred to a silica gel column prepared from 15 ml 23-400 mesh silica gel (EM Science) and toluene. This column had been washed with 50~r methanol in toluene.
Increasing the solvent polarity by increasing the methanol content of the solvent to io% brought about the migration of a distinct band which was collected. This material was saved but not used for further transformations.
*Trade-mark PCTlUS93102470 ...: i1r~ 93! 19366 ~' ~~'~i~~
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a3 t Increasing the solvent polarity by slowly increasing the methanol content of the eluant to 30% brought about .
the migration of a second blue band which. was collected within a 20 ml volume of 30% methanol. This material was transferred to a round bottom flask. Removal of solvent on a rotovap under high vacuum at 25°C left a residue which appeared to include an appreciable quantity of imi dazole along with the blue dye. This material was used without further purif ication for the next step . The yield of compound III was approximately 3 mg.
g, Pre aration of Amine-Terminated Pol eth lane G1 col Polyethylene glycol) monomethyl ether (Aldrich, average M.W. 2000, 10 g, 5 mMol) was placed in a 100 ml round-bottom flask along with a stir bar and 55 ml toluene. The flask was fitted with a short-path distil lation apparatus and immersed in a heating bath. Toluene was slowly distilled at 760 mm Hg until the distillate was no longer cloudy. This required the removal of about 15 ml of toluene.
The relatively water-free PEG solution was allowed to coal to 40°C: When this temperature had been attained, carbonyldiimidazole (Aldrich, 1.2 g, 7.5 mMol) was added to the stirred solution in one portion. Stirring at 30 40°C was continued overnight with protection from atmos ph~ric moistuz°e.
Water (100 ~sl, 3.75 mMol) was then added to the rear-,. tion mixture and efficient magnetic stirring was, continued, until the evolution of COZ gas could no longer be observed (about 15 minutes). .
Most of the toluene was removed on the rotovap at a 30°C under high vacuum leaving a viscous, colorless oil.
This material was diluted with isopropanol (20 ml) and added to a stirred solution of 1,2 ethylenediamine (Fluka, 6.7 ml, 100 mNi~l) in isopropanol (15 ml) over a period of five minutes. After completion of the addition the clear solution was maintained at 40pC for four hours.
S9J~ST'd't'LJTE SiiE

WtD 93/19366 ' PCT/L'S93/02470 :,~'::.

At this time isopropanol (150 ml) was added to the reaction mixture. The diluted solution was allowed to stand at 4°C overnight, resulting in the formation of a voluminous mass of white crystals. This solid was col-S lected on a 10 cm Buchner funnel, and subsequently recrystallized from isopropanol.
Drying under high vacuum over sulfuric acid afforded 7 grams of the crude amine, suitable for use as a reagent.
Stricture of the product was confirmed by IR.
ZO The amine content of polyethyleneglycol amine, pre pared as outlined above, was determined to be > 70 moleo - by the following method:
25 ml of ~.Oo solution of the amine in methanol was allowed to react with an equal volume of a 6% solution of 1S malefic anh~rdride in THF: The reaction mixture was allowed to stand for 0.5 hours at 25°C and was then diluted to 1..0 ml, with methanol. A S ~l aliquot of this final solution was injected;on to an analytical RP18 reverse phase HPLC
column using 30% methanol in water as the initial mobile ~0 phase. Using n-propylamine as an internal standard allowed for accurate-quantification of the UV-absorbing ac~rl-PEG derivative; which was eluted in 80% methanol and was detected at 254 nm:
Analysis of the infrared spectrum of amine-terminated 25 PEG can also provide a-convenient means of estimating the product yield.
G. Reaction of Compound III with Amine-Terminated Poly-;, eth~rl~ne Glycol ( Compound IZT) The product of step (E) (Compound IIT) (3 mg, 5 x 10'3 30 mi~iol) , which had been obtained in partially purified form by chromatography on silica gel, was dissolved in methanol (1 ml): The mzxture was stirred while amine-terminated PEG (product of step (F) . 100 mg, 5x10-'2 mMol) was added.
Th:e resulting deep blue solution was heated to reylux for 35 one hour.
S ~J ~ S'Ti°CCJT'E S H E E~'°

Removal of methanol under aspirator pressure at 25°C
left a viscous blue ail which was taken up in water (0.5 ml) and applied to a small (10 ml wet volume) DEAE Sepha-dex'~anion ion exchange column (Pharmacia, 3.5 meq/g, 40-'~ 120 micron, basic form < 1M K~CO~) . The water-soluble blue dye was retained quantitatively by the column. The column was washed with water (15 ml) and the blue dye was then eluted in greater than 70% yield with :10-20 ml of a 15% aqueous acetic acid solution.
10 Water and acetic acid were rernaved under high vacuum and the blue residue was taken up in a small volume of methanol and applied to a C18 reverse phase semi-prepara-tive HPLC column. The major product., detected at 675 nm as a single peak, eluted with 80% aqueous methanol (con-15 taining 0.6% acetic acid) and comprised about 50% of the sum of the material which was recovered from the column.
Fractions containing the major product were combined and solvent was removed under high vacuum leavinc3 a blue resi-due ( app rox . 0 . 5 mg , 10 ~' mMo 1 ) .
20 NMR (DCCl~: 8 -2.85 (5, 12H), b -2.29 (m, 4H), b -1.30 (m, 4H), b 1.80 (m, 4H), ~ 3.6 (br.s, 300-400H), b 8.39 (m, 6H), b 9.68 (m, 6H), b 10.56 (S, 2H). dote: Because the sample had been previously dissolved in DzO, the acidic protons, RCOOH, were not observed.
25 Fig. 1 is a typical HPLC chromatogram of this prepar-ation (compound IV). The fraction containing compound IV, with retention time of approximately 25-26 minutes, was designated "B" fraction. The yields for a typical dye preparation range from 25-65% of this fraction. Fractions 30 from several chromatographic runs were pooled, dried in vacuo and analyzed. The absorbance of the "B" fraction was measured in a Perkin-Elmer* spectrophotometer using various solvents. As shown in Fig. 2, vex-y little "sol-vent sensitivity" can be seen between methanol, dimethyl-35 formamide and FPIA buffer (100 nM phosphate pH 7.5 with 0.01% gamma globulin).
*Trade-mark WO 93!1'9366 ' PCT/US93/02470 J.

The fluorescence decay time for the "B" fraction was determined to be 4.3 nanoseconds. The measurements were made on the "Diatron Analog System". In the Diatron ' Analog System, transient-state fluorescence was detected using a high speed, "gateable" photo-multiplier tube (PMT). The combination of being able to rapidly change the PMT gain and the use of high power laser pulses enabled the viewing of the fluorescence decay of dyes with a single excitation pulse. In practice, many pulses were averaged to obtain improved data. These analog signals coming from the PMT were captured by a digitizer which took the analog signal and cut it into 512 time bins for analysis. r The Diatr~n Analog System is diagramed in Fig. 3.

The tunable dye laser used was a PTI model PL2300 nitrogen laser with a dye laser module. By changing the ,laser dye and adjusting the dye laser grating, 600 picosecond pulses with peak power of near 40 KWatts could be generated at wavelengths from 340 to 900 nm.

A beam splitter was used to send a portion of each.

pulse to a pulse detector which consists of a high speed Hamamatsu photo diode. The resulting output of the photo-diode was fed into a pulse shaper which converted the resulting 800 picosecond (ps) pulse into a 100 nanosecond 2S (ns) purse. This 100 ns pulse was then used as a gate for the Hamamatsu microchannel plate PMT whose gain was changed by 10,000 within a 2 ns time period. The PMT

stayed at the high gain until the 100 ns was over. ;

The dye laser module, reaction cell and pulse , detector was positioned and connected such that the PMT

was dated to its high ~ensitivit~r state approximately 2 ns , after the laser pulse passed thrc~ixgh the reaction cell.

A filter was positioned in, front of the PMT to guard against high scatter signals when required. A lens was used to image the fluorescence onto the PMT microchannel plate: Also, ~ rotatable polarizer was positioned in the S l! ~ 5°~i'~°L!?'E ~' ~ ~

i'7036-13 output optical path to measure the time dependent polari-zation of the fluorescence, High voltage from 1000 to 3400 volts wa;a supplied to the PMT, The output of the PMT was connected to a Tek tronix 7912AD Programmable Digitizer.
A computer was used to trigger the laser. The laser output was detected by the digitizer via a connection to the pulse detector (not shown). A programmable sweep on the digitizer set up the time spread to be measured after the laser pulse from 10 ns to as high as several seconds.
Typically, the system was operated such that 512 data points were generated over a 20 ns time period.
The natural log Eln) (intensity) of the dye prepara tions was plotted versus time and subjected to least square linear regression analysis. These data are shown in Fig. 4.
The dye preparations were analyzed for their inter-action with serum protein. The dye preparations were adjusted to 5 x l0'' M/L in FPIA buffer. These dye prep-arations were added to the following solutions to a final dye concentration of S x 10'1' M/L: FPIA buffer, 0.5% bovine gamma globulin, 5.0% bovine serum, 5,0% normal human serum, 5.0% pooled human serum and 5.0% whole blood lysate. These data are shown in Fig. 5. Typically, when a dye binds to a protein non-specifically (as can be seen with the "C" fraction), a significant increase in fluor-escence polarization occurs. This makes it impossible to distinguish the specific polarization due to antibody binding from the non-specific due to protein-dye inter-action. The "B" fraction showed only minimal interaction aver buffer as determined by measurement of transient state polarization.
Example 2 Linl~~rs In certain polarization assays, it is advantageous to use a spacer or linker. These .inker cr spacer arms are *Trade-mark !VO 93/d936b P~CT/(JS93/OZ~t70 useful when different ligands are terminated by either a !
carboxyl or amino group. Tn addition, such compounds are e.
important when the probe needs to be separated (stood off) from the molecule with the antibody binding epitope. This may be necessary to reduce the potential of non-radiated transfer of energy when antibody binds the specific epi tope and/or to eliminate stearic hinderance. These linker/spacer arms are generally the same in both the ligand-probe and ligand-protein immunogen used to raise antibodies to the ligand; in order to create a specific binding pair. In polarization immunoassays, it is desir-able that the spacer create a relatively inflexible linker moiety.
Because the caged diearboxy silicon phthalocyanine is y5 ~a .c~rboxy-terminated dye, it is advantageous to have an amino terminated dye coupled to a carboxy terminated lig and. Various linkers (spacer arms) have been evaluated.
Such compounds include giperazine, ethylenediamine, hex anediamine, 6-amixao hexanaic acid, 5-aminobutanoic acid, 2d I.2-amizaododecanoic said, alan~.ne and other amino acids .
The folloc~aing ' methodology for preparation of the phthalocyanine-12 amino dodecanoic acid compounds is an example of the genoral reaction for such linkers: To 1.o mg of caged phthalocyanine dye (the "B" fraction of step 25 G of eacample 2) in 200 ~1 of 500 DMF in water was added 2 mg of 1-hydroxybenzotriazole (HOBT) and 1 mg 12-amino dodecanoic acid. The suspension was gently warmed until al.l ingredients were dissolved. This took from I-2 hours .
At this'time 3.0 mg of 3-dimethylaminopropyl carbodiimide~ , 3d way added and mixed thoroughly. The reaction mixture was allowed to react overnight at 4°C. The reaction became slightly turbid and was clarified by centrifugation. The new abducts were purified on reverse phase C-18 columns by HPLC.
35 ~nthen 12-aminododecanaic acid was used as a linker, the ~aolarization ira glyce~'ol of the dye increased from p =
0:280 to p 0.340. Concomitantly, a 10 nm shift :.0 890 SLJ~S'T~1J~'E SHEET' VV~ 93/193bb ' PCT/US93/02470 s Y

nm occurred, which matches commercially available 690 nm laser diodes. The change increased the dynamic range of the assay from 0.03 to greater than 0.30 millipolarization in buff er when bound to an antibody molecule. In addi-S tion, the 10 nm shift increased the signal-to-background ratio by moving away from the excitation maximum of f luor . escing background molecules found in biological fluids.
The absorbance spectrum in methanol for the purified caged dicarboxy silicon phthalocyanine dye-linker is shown 10' in Fig. 6. There is a 10 nm shift from 680 for the frac tion "a" dye (Fig. 2); to 690 nm for the dye-linker. In addition, transient state fluorescence polarization was measured on the Diatron Analog System described in Exam-ple I, in FPIA buffer and glycerol at 680 nm and in 15 glycerol at 690 nm. These data are shown in Figs. 7 and 8.
IIx. Synthesis of Caged Dicarboxy Silicon Phthalocyanine Dioxin Probe Digoxin is a glycosylated steroid which, when used in 20 patients with, congestive heart failure, increases cardiac r.
output, decreases heart size, venous pressure and blood volume, and relieves edema. As noted above, digoxin has a very marrow therapeutic range (serum levels of 0.5 to 2.S ng/ml) and is generally toxic at concentrations 25 greater than 2.l ng/ml. Accordingly, there is a need for a digoxin assay which can accurately and precisely deter-mine digoxin concentrations at these levels.
~~ , , , Example 3 Diexoxin Probe Preparation~ Caced Dicarboxy 30 Silicon Phthalocvanine-Dic~oxicxenin A ~ Preparation of 3 -~Cetodictoxictenin A mixture of 488 mg digoxigenin, 7.S ml toluene, 3.75 ml cyclohexanone; and SSO mg aluminum isopropoxide was hewed under reflux for 2.3 hours and then concentrated 5~~5 TE S~dEET

W~ 93/19366 ' . P~I"/US93/02470 ,~~ , t~
,, in_ vacuo to half of its original volume. Two hundred gel water was added and the mixture was evaporated in vacuo to dryness. The powdery solid was dried in vacuo over over-night. The dry residue was stirred in 25 ml methanol and 5 the resulting mixture was filtered. The residue on the , funnel was washed with 25 ml methanol. The filtrate and washing were combined and evaporated in vacuo affording 920 mg white solid.
B. Preparation of 3-Aminodiqoxigenin 10 A mixture of 92O mg 3-ketodigoxigenin, 693 mg ammo-nium acetate, and 730 mg, NaBH2CN was stirred in 48 ml methanol at room temperature overnight. Concentrated HC1 (35 ml) in 20 ml methanol and 5 ml water were added.
after gas evolution had_subsided, the sol~,rent was removed 15 in vacuo. The residue was stirred in 15 ml water and then extracted with 2 x 20 ml methylene chloride. The water phase was gummy materiel was evaporated in vacuo and the residue was dried leaving a granular solid, This dry solid was extracted'with 2 x 20 ml dimethylformamide (DMF) 20 and the solution centrifuged. The clear DMF solution was ,.
evaporated in vacuo affording 684 mg white solid. The entire amount was dissolved in 25 ml methanol and the solution was stared at -20°C overnight. A white crystal-lino material; which had deposited, was removed by fil-25 txation and washed with 300 ~1 cold methanol. The fil-trate was applied to two washed EM 576 silica TLC plates .
~l,f~er being developed ~:n 90 ml chloroform + 25 ml methanol the chrnmatdgram shcawed 7 bands visible under 254 nmi uv. ~, .
Band Number 1, Rf 0.10, was -rempved from the plates, 30 extracted with 4 x 40 ml methanol and the SiOz was cen-trifuged out . The supernates were combined and evaporated in ~racuo affording 70 mg white solid.
C. Synthesis of Probe The digoxin probe was prepared as follows : 4 . 2 mg of 35 3-aminodigoxigenin was placed in a 3.0 ml reaction vial S~~s~~ S~F

WO 93J~9366 ' PCI"/i;S93/02470 and dissolved with 100 ~.1 DMF . In a separate vial , 1 . 0 mg of caged dicarboxy silicon phthalocyanine (Compound IV
from Example 1 (G) ) was dissolved in 400 ml DMF and then transferred to the reaction vial along with 200 ul of wash DMF for a total of 600 ~Cl. 4.2 mg of 1-hydroxybenzotria-zale (HOBT) was added to the reaction vial, which was then dissolved and mixed well. To make the final reaction mix-tuts, 10.5 mg of 1-ethyl-3-(3-dimethylaminopropyl/carbodi-imide)-HC1 (EDAC) was added and mixed thoroughly. The reaction mixture containing digoxin-phthalocyanine probe was stored at 4.0-8.0°C overnight.
D. Purification of Probe The digoxin-phthalocyanine probe was purified as fol lows: a slurry of S gm C-18 was made in acetone and poured into a 1x15 cm glass column. The acetone was removed by the application of light pressure, and the column was equilibrated by the addition of 4 column vol-umes of 70% methanol/30% water. The reaction mixture cor~taining digaxin-phthalocyanine probe was applied to the column and flushed with 70% methanol/30% water. The probe was eluted with 80a methanol/20o water, concentrated by vacuum and further purified by two subsequent passes on H~~,C. After the second chromatograph an HPLC, the probe was brought to dryness in vacuo. A portion was dissolved in methanol and a portion was dissolved in 100 mM lVaPO~
buffer containing 0.1°~ sodium azide and l.Oa bovine gamma globulin (pH 7.5). Fig. 9 depicts a chromatograph of the HPDC method' semi-p~'eP C-18 column with a mobile phase and gradient elution of meth~nol/wa~er. Fig. 10 depiets the 30- structure of the digoxin-phthalocyanine probe.
A~alysis of Probe The probe was'analy~ed in a Perkin-Elmer spectro-photometer (Lambda 4 c) in two solvents, methanol and 100 mM phosphate, pH 7.5: Figs. 11 and 12 are repre-~~ntative spectra. Fluorescence decay time was determined ~ l! ~ 5°T1Z'L,'TE S I~ E ~'1' W~ 93/19366 ' P~C'T/L'S93/02470 ~, t:..:. .
,~

to be.4.7 ns using the Diatron Analog System described in Example 1, (Fig. 13).
F. Linearity in Buffer and 1.0% Bovine Serum Albumin To determine the sensitivity and linearity of the .
transient sate measurement system and development of a digoxin assay, it wa's necessary to determine the concen-tration of the phthalocyanine-digoxin probe. The probe was purified by one additional pass through HPLC for a total of three (3) passes through HPLC chromatography using reverse phase C18 columns. The probe was dried under vacuum and dissolved in a 100 mM phosphate buffex, pH 7.5 containing 0.01% bovine gamma globulin. The absorbance maximum was determined and the absorbance of the probe solution was measured. Concentration was l5 determined using the following formula: A = cxBC, where A
Absorbance, a = extinction coefficient; B = path length, and C = concentration. Thus; C ~ A In this example:
aB
of = 1' . 6 X 105, B _ 1 cm, and A = 0 . 227 . A,ccordingly; the concezztration of the stock phthalocyanine-probe solution was ' = 1: 45 X 10'6 M/L. a Based upon this value of 1:45 X 10'~ M/L, dilutions of appropriate concentrations were prepared in FPIA buffer 'and in l.0% bov~:ne serum albumin. The results are shown in Fig. 14:
The lxnear~,ty of intensity of probe from 6.5 X 10-9 ~
to 1 X 10°x3 M digoxin in~ both FPI:A. buffer and FPIA buffer with. 1% BSS demonstrates the ab~.lity of the probej'to:'~~unG- ;
tion in a protein solutionwithout interaction with-bind-ing-components.
As can be seen from'the data presented in Table 2;
the polarizations of free dye ("B°' fraction) and 'free prolae in various sera are similar, with the polarizations being slightly higher gor the digoxin prcabe: This is 'cons-intent with the increase in molecular size and asym-;:metry o~ the probe. Tn addi.tion, the changes observed in 5 U ~ 5'°~'i~'L1°f F S ~l a.
W4~ 93/~1~3E6 ' P~'/LJ593/02470 ,.,:

buffer vs the serum solutions is consistent with a change s in viscosity as defined by the following equation:
r = 3 nV
RT
where: R - gas constant; T - temperature (in °R). n -solution viscosity, and ~l = volume of molecules.
Table 1 Phthalocyanine Digoxin Probe: Serum Interactions Comparison of milli-Polarization (mP) O.So 5.Oo S.Oo FPIA Gamma Bovine Normal Buffer Globulin Serum ~-Iuman Serum ~~~n Fraction 8.0 20.0 22.0 30.0 Digoxin Probe 26.1 S1~~ 38.5 40.5 G. Serum Ura.ne Interactions A comparison of a fluorescein-digoxin probe analyzed on the Abbott TDx~ Fluorescence Polarization .Analyzer and the phthalocyanine digoa~in probe analyzed with the Diatron FAST-60 Analyzer is presented in Fig. 15. In this ex~m-ple, both probes were tested at the normal (workizag) concentration used when performing a digoxin assay with the TDxT''' and, FA.~T--60 a~ialyzers ( i . a . , 2 . 5 ~ l0~lo and 5 . 5 X 15°11 M/~) . See' Example 4 for a description of the p~,atron FAST-60' Analyzer. In this figv.re, the intensity le~re~,s are plotted as background equivalents.
AS can' be se~era from Fig, i5, the fluorescein probe is' only slightly detectable above background in 5o serum and completely non-detectable in 10o urine. In contrast, the phthalocyanine digoxin Pgobe is detectable at a very sig-nificant level above background in both the same serum and urine samples.
S~~S °~~ S~IEE't°

'17036-13 IV . Digoxin Assays Example 4 Competitive Serum Assay for Diaoxin:
Seduential HindincLProcedure Digoxin reacts with serum albumin and other serum proteins at many reaction sites, Prabes made with a fluorescent dye and digoxin will also react. "Nonspeci-fic" binding or serum protein interactions were minimized in this procedure by the action of the cyclodextrin, which 1G has an affinity for digoxin which exceeds digoxin's affi-nity for constituents in serum. Thus the cyclodextrin interferes with the binding of digoxin with serum consti-tuents, but allows far binding of digoxin with digoxin antibody. Thus, the assay was designed to allow both the serum digoxin and the digoxin probe to react with the digoxin antibody.
100 uL of serum sample was mixed with 25 ~L of rabbit antidigoxin and 500 ~L Buffers (100 millimolar phosphate buffer, pH - 7.6 with 0.01 bovine gamma-globulin, 0.5°c gamma-cyclodextrin and 0.1~ sodium azide). The mixture was incubated for 5 minutes. 25 ~.L of digoxin probe (as prepared according to Example 31 and 200 ~L of Buffer2 (100 millimolar phosphate buffer, pH - 7.6 with O.Ol~c bovine gamma-globulin and 0.1°-. sodium azide~ were added and the mixture was incubated for 20 minutes.
In a study of 20 random human serum samples it was found that the serum-digoxin probe interaction would vary from sample to sample, and that the variation may be as much as l0-15 millipolarization units. The buffers in the present example were formulated to eliminate this varia-tion to a relatively constant millipolarization of 70.
Transient state polarization was measured as described in Studholme, et al . , U.S. Patent No. 5,323,008 entitled "Fluorometer Detection System ".
The transient state optical system was installed in the Diatron "FAST-60 Analyzer," which contains a laser diode operating at 685 nm was pulsed at ' PCT/US93/02470 i .":_:.
~~~~ , s a 10 MHz rate. Typically, the laser "on" time was approx-imately 3 nanoseconds. Photons from the solution were detected using a photomultiplier tube (PMT) operating in a single photon counting mode. The photon event along 5 with the relative time of the photon event as compared with the laser pulse time was determined. By storing the individual photon event times a histogram of frequency of photons as a function of time was generated.
The Diatron FAST-60 Analyser includes a transient 10 state optical system installed in an automated fluores cence reader designed ~.o measure fluorescence from immuno - assay reactions: The reader contains the optical system, motor control for position reaction cuvettes in front of the optical system, thermal control to hold the system at 7,5 ~5°C and a computer link to control the reader, analyze and display results and print those results. For immuno assay use, the results were formatted into transient-state polarization units,or, by using a calibration curve, the results were transformed into concentration units of the 0 analyte being measured.
Commercial serum calibrators for digoxin determina-tion (obtained from Abbott Laboratories) were analyzed to obtain a standard curve. Sample blanks were prepared for each sample or calibrator by performing the same steps 25 with the exception that buffer was added in place of the digoxin probe. The sample blank was measured and sub-tracted from the measurements for all reaction mixtures.
The procedures were performed at 25°C.
' ' Fig. 16 ' hows a comparison of digoxin calibr~atiori 30 curves by a standard fluorescence polarization procedure (Abbott's TDx° Fluorescence Polarization Analyzer) and the homogeneous sequential binding assay procedure described in this Example. Fig. 17 displays a correlation plot of 37 serum samples assayed by a commercial digoxin test 35 system manufactured by: Abbott Laboratories (TDx° Digoxin II In Vitro Test, Product #9511-60) and assayed by the digoxin assay procedure described in this example. A
SU~STt~°LJTE SH~~'T' p~./US93102470 d t:
_.
~~ 93/19366 ~ ~ ~.
c of 0.96 and a slope of 0.98 were determined.
correlation a and y-intercept indicate no systematic bias.
The slop F_xample 5 Competiti~re-.Serum_Assay f~ lgaxi~n'..
~ uf~ion Jur~~ Procedure_ tion jump Procedure described in this Example The dilu erformed in the presence of high allows the assay to be p s~~ri,; and was designed to reduce "non-coxxcentrations of ,ions vahich compete with the antibody for specif~.c" interac ' n and digoxin probe : While not wish° .
binding to the digoxi b any particular theorya aPPlicants ~; ing to be bound y when sample ~ antibody and d~.goxin probe are believe that reaction volume,, the "nonspecific"
incubated in a small 'initially compete with the antibody for interacts~ns in a.nd digoxin probe. then the solution bixrding to digox ~~ bns ecif is" pxotein interactions tend' to is diluted, the n P .
and only the specific antibody reaction disappear rapidly remains.
'ution jump procedure, 20Q ~L of ' To perf ~rm the dil rotor (Abbott Laboratories) or serum sample was 2p serum calib of antibody (rabbit anti-digoxin anti-mixed with 250 ~.L
oxin probe and 1000 ~L, FPLA buffer.
body)', 250 ~,~, of d~.g ated for 30 minutes at 35°C~ A
fihe mi~cture was incub of the reaction mixture was removed and variable volume buf f er . For example 17 0 ~L of ~5 added to; 900 ~.L 'ot FPIA
in 900 ~L of buffer provided a fW al probe reacted mixture conc~ntraaion. of 5. x 10-11 M
r ~'' ure Bias diluted, the degr~e,of As the reaction maxt in decreased while the amount of specifl-nonspecif is bind 9 shed nearly constant. As shown by 30 ca.lly bound Probe rema de',icted graphically in fig. 18, for non-the reault~s p re (probe concentrat~.on of 3.5 x diluted reaction mixtu olarization was ~-89 mP : As the IO-to M) , the resulting P
ted; the polarization decreased reaction. mixture was dzlu d alimit (near 152'mP) at a 7-fold dilu-35 until it ruche tion of the reaction mixture.
SU~ST~ 5'~~~'T

i Wd 93/19356 P(.°T/L'~93/02470 An alternate dilution jump procedure was also done in which 20 ~1 of commercial serum calibrator or serum sample was mixed with 80 ~.1 of lysing/buffer (5 x 10'' M/L
stearyl-lysolecithin in .001 M/L Tris HC1 buffer at pH 8) S diluent, and 10 ~l of rabbit anti-digoxin was added and mixed. This mixture was allowed to incubate at 35°C for minutes, At this time, 25 ~.1 of digoxin probe was added a:nd incubated an additional 15 minutes. To this reaction mixture 1.0 ml of FPIA buffer was added (dilution jump y vortexed and the transient state polarization measurements were made in the Diatron FAST-60 Analyzer described in Example 4.
The calibration curve using the dilution jump pro-cedure is shown in Fig. 19.
is Examt~le 6 Gomt~etitive Serum Assay for Diaoxin:
Signal-to-Background Comt~arisons fox Transient-State and Steady-State Measurements The signal~to-background ratios for steady state and trahsient state measurement were determined as follows: ' .. The steady state fluorescence intensity measure-ments were made on the Abbott TDx~ Fluorescence Polariza-tion Analyzer using the '° Photo Check Mode , '° Both back-ground and floor measurements wire taken by removing the S reference solutions' from the calibration carousel and substituting varying dilution of fluorescein from 1.7 X
10,'e M/L to 1 . 7 , X, 10'12 M/L in 1 . 0% bovine ,serum albumin . -2. The steady state fluorescence of the caged sili con ghthalocyanine-digoxin probe was measured in a modi-3Q fled TDx~ Fluorescence Polarization Analyzer. . These modifications were made by replacement of the input filter with a' 680, 10 mm 1/2 bandwidth filter and the output with a RG715 color Mass filter. The concentrations of the probe solutions were determined in a similar manner as 35 those determined ~.n Example 3. Dilutions were prepared in l.Oa bovine serum albumin in concentrations of 1.4 X 10'~
S UI ~ 5°f ~'i~'f F S H E ~~' W(~ 93li936fi PCT/L'S93/02470 _ .
I
0 1.
M/L to 1.4 X 10-lz M/L. The measurements were made as in 1 above. ' 3. The transient state measurements were made in the .
i Diatron FAST-60 System using.the same solutions as in 2 , above, but at concentration of 1.4 X 10'g M/L to 1.4 10'1' _ M/L.
The data are summarized in Table 2. The signal-to-background data is represented as a ratio (signal counts/
background count).
Table 2 Signal-ta-Background Ratio Comparisons Serum Calibrator 680 nm Probe Intensity Blank Intensity Probe/

Fluorophore Seounts/15 sec) ~counts/15 sec) Blank S teady-State 437,2 466,848 6.5 Transient-State 264,404 4,048 65.3 Thus; applicants have shown that steady state assays can be configured with acceptable sa.gnal-to-background ratios using the caged dicarboky silicon phthalacyanine digoxin px'obe which is measured at 680 nm. There was an approxi-mately l0-fold enhancement in this ratio When transient state techniques were used to time-discriminate against fast fluorescexs within the background and scattering bands zn immunoa~~ays wh~.ch measure analytes at very low concentrations; for example, digoxin at 5 x 10-1 to 60 x ~~J,o' ~/L~ 'the concentrati;on of fluorescez~~ probe in' the ~ , fluorescein steady assay system is 2.5 x 10'1 M/L. These assays require an extraction step to remove the digoxin bound to serum proteins (approximately 25-40%) and fluor-escers bound to proteins that interfere in the measurement of fluorescence polarization. In serum, the background fludroscenee is higk~ly polarized due to this protein bind-ing, which can mimic specific polarization due to antibody binding. These must ,be removed before the assay can be nerfarmed. Many of these fluorescers are excited by 493 S tJ ~ S'~'~~t JT'E S ~ ~ ~'T' . r ~:.. , , ;
r ~ r ..r,-.. :
.. , . ,. , ". .. . u~ ..
..u..._x:,.".. . r, , .".. ., ...... . , . .. .:r. ~ ,.. t . ~ . ... .. . _ ,_ ,_....
..,. , ., .. _. ._ :~i:.. ..,... . , .. .. , . , .. ...-~.1. :.;-.
.

r W~ 93/193fi~b ' PC'f/US93/02470 .. .
:': ,.~ a ~p ~~~~~~~ ~
59 , nm light, which corresponds to the excitation maximum of these fluorescein based assay systems. In this example, the signal to background is at best 2.5:1. However, when unextracted serum is added as in a homogeneous assay using the fluorescein steady state measurement technique, the probe fluorescznce is not detectable at the concentration 'of serum needed to run the assay (Fig. 15).
This principle can be illustrated by the data in Table 3. For a steady state fluorescein-based assay, the fluorophore concentration at which fluoxophore signal equals background is 1.6 x 10'9 M/L. This is far above the concentration of fluorophore needed to perform an accept-able digoxin assay, i . e-. , an assay which can detect and quantitate digoxin at therapeutic levels. In other words, the fluor cannot be measured over fluorescence of background.
As can be seen from the data in Tables 2 and 3 , there is approx~.mately an S-fold improvement in steady. state caged dicarboXy si~.icon phthaZocyanine measurement over steady state fluorescein measurement. This improvement increases an additional 10-fold when transient state measurements are made Casing the caged dicarboxy silicon phtYa.alocyanine probe : Additionally, there is a dear 100--fold imgx~ovement of the transient stake measurements over the currently used fluorescein steady state measurements.
Table 3 Signal-to-aackaround Comparison: , .~~ , Fluorophore Caneentration Where Fluor~hore Sicrnal Ernaals Background ' _Technolcaay Fluorophore Concentration Steady'State Fluorescein 1:6 X 10'9 M/L
Steady State Phthalocyanine Probe 2.4 X 10'1° M/L y Transient State Phthalocyanine Probe 1.4 X 10'11 M/L
5~U~5'~'tl°1.1°~°E SHEE'I°

P~.T/U593/02d70 WC~ 93/19366 ' 60 Example 7 Competitive Serum Assay for DlCIOXIT1:
High Sensitivity Assay , In this assay, the sensitivity of the digoxin assay described in Example 5 is increased by a factor of 10.
The concentration of caged dicarboxy silicon phthalocya-nine digoxin probe was determined by the procedure out-lined in Example 3 to be 4.2 X 10'lz M/L. In this assay the total reaction valume was reduced by 50 percent and all reactants were reduced 10-fold. To increase the polarize tion values, the incubation times were increased to 5 and _ l5 minutes for the sequential addition, competitive bind ing format.
The procedure is as' follows: 50 ~C1 of lysir~g/buffer (see Example 5 above) diluent was mixed with 2.0 ~.1 serum calibrator or serum, plasma or whole blood sample and 5.0 ~1 of rabbit anti-digoxin antibody. This mixture was y incubated for 5 minutes and 2.5 ~1 of digoxin proY~e was added and incubated an additional 15 minutes. After this ineubation, 1.0 ml ,FPI~1. buffer was added as a dilution jump. The transient stake polarization measurements were G:
made on the Diatron FAST-60 Analyzer described in Exam-Ple 4.
Sample blanks were prepared for each sample or calibrator by performing the same steps, with the exeep tion that buffer was added ~.n place of the probe. The sample blank'was then measured and subtracted from the measurements for the entire reaction mixtures.
;. ; F,ig ; 20 ~ displays a calibration curve for ccinmerdial serum calibrators containing known cancentration of digoxin, which were assayed using the high sensitivity procedure.
Example 6 Pre~ai-ationo~ Whole Blood Calibrators Whole bloocz was obtained from two donors by drawing ' blood into Vacutainer~' (Bector Dickinson) tubes containing 5~~5'~°1T~J~'E SHEF"f WO 93/19366 ~'C.'T/U593102470 x~'a ~~. .s~! .;~ ;
;~ :~ .due 8 EDTA anticoagulant. The tubes were mixed thoroughly on a standard laboratory sample rotator. Based on the average specific gravity of blood being 1.056, a series o~ six 2 ml volumes of whole blood were weighed using standard gravimetric technique. These samples were then spiked using a USP grade digoxin (200 ng/ml) to final concen-trations of 0, 0.5, 1.0; 2.0, 3.0, and 5.0 ng/ml whole bland. 'fhe whole blood calibrators were stored at 4.0 -

8.0 °C and were used within two weeks.
ZO Example 9 W_ hole Blood Dictoxin Assay- Sictnal-to-Background Ratio Serum Versus Whole Blood Applicants determined the signal-to-background ratio , of the whole blood preparations which were pregared as described in Example 8 and compared the whole blood (i.e., blank) intensity to the probe intensity. The resulting values comparing the whole blood and sexwm signal-to-background ratios are shown in Table 4. These measure-ments were made at working digoxin probe concentration of 5 X 10'1'' M/L in the transient state system. It can be seen that the net probe intensities remained constant even when the background intensities fluctuated. In a typical steady state fluorescein digoxin assay where the digoxin is extracted by precipitation of proteins, the average sa.gnal-to-noise ratio i s 2 to 1 at probe concentrations of 2.5 X 10'1 M/L, as c~ntrasted with those found by homoge-neous transient state fluorescence for serum and whole , .. ~ ~ i . . ~ ' ~
,' . f blood of 77.6:1 and 26.7:1, respectively.

Table Signal-to-Background Ratio Comparisons a for Serum and Whole Blood z Probe xn,tensity Blank Intensity counts 15 sec? (counts/15 sec Probe/.Blank Serum 113>2gc 1,466 77.6 Whole Blood 100,254 3,757 20.7 Sl3~STE SHEET

. . . ~.. .. : ..._.. ~ .... :.. .... . . ~..,.. ._... . . . . . . . .
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.. . . . ,. .. .. . . , .. ,... , . . .. , , ... . .. , . , .. ., . .

f dV~ 93/19366 F°CT/US93/0247a , Examrle 10 Homo eneous Whole Blood Diqoxin Assav - Clinical Study Previous whole blood immunoassays have been limited by many factors. For example, separation steps are S required in many assay systems, enzymes and other sub stances released from red blood cells cause interference in the assays, and the instrumentation is incapable of measuring analyzes or reaction products through whole blood hemolysates. Applicants have developed a homoge ° 10 neous whole blood assay system which offers the clinical laboratory and other testing facilities significant advan-tages over currently used methods, including decreased J.abor cost, and decreased sample manipulation. In addi-tion, with a homogeneous 5 to 1C minute assay, the pro-15 cedure can be brought much closer to the patient, for example, to the bedside, emergency care facilities clinics and satellite testing facilities.
Digoxin is widely distributed in body tissues. Serum and plasma have been the accepted samples for the assay of 20 digoxin using the current commercially available test kits . Studies have shown a relative constant relationship between heart muscle and serum digoxin levels, thus vali-dating the use of digoxin serum levels in monitoring patients receiving the drug (Doherty, J.E., et al., 1978, 2S "Clinical Pharmacokinetics of Digitalis Glycosides." Pro gress in Cardiovascular Diseases, Vol. XXT, No. 2 (Sept./
Oct.)). Because whole blood has not been routinely used a,s ~a medium fox assay in digoxin therapy, the following ,, ; , study was undertaken to determine: (1) the distribution y 3c? of digoxin in seaum, plasma and red cell components of y blood; t2).the percentage discrepancy, if any, in digoxin y levels of serum, pl~.sma and whole blood assays; and (3) correlation among two currently commercially available assays - the Abbott TDx° serum assay and the Dade Stratus°
3S serum assay - and the assay of the present invention. The clinical study was conducted using 43 patient samples, collecting 1 EDTA tube for whole blood or plasma levels 5th~S?'F SHE

_ W~ 93/99366 P'CT/US93/02~d70 r and 1 tube for serum levels. Each sample pair was ana-lyzed for serum digoxin levels determined by the Abbott TDx~, Dade Stratus~ and Diatron FAST-60 Systems. Plasma levels were analyzed by Abbott TDx~ and Diatran FAST-60 Systems. Whole blood levels were analyzed by Abbott TDx and Diatron FAST-60 Systems. The Abbott TDx~ System used the TDx~ Digoxin II In Vitro Test, Product #9511-60 (Abbott Laboratories): The Dade Stratus System used the Dade Stratus~ Digoxin Fluorometri.c Enzyc~e Immunoassay (Dude Diagnostics Division of Baxter Healthcare Division, Miami, Flora.da). The Diatron FAST-60 System used the methods described ?z~ Example 5 and the apparatus described in Examr~le 4 and Studholme, et al.; Lyon & Lyon Docket No. 195/129.

In this study, the primary concern was whether the whole blood digoxin values were similar to the serum values. Thus, to reduce the number of variables, the whale blood hysates were clarified by centrifugation befare assay.

The study subjects were randomly selected patients currently on active dig~xin therapy. The following sam- r Ales wexe taken from each patient: (1) Red stopper Vacu-tainerT''' tube (no EDTA) for serum collection (a minimum of 2 ml required); and (2) Purple stopper Vacutainerz~' tube (EDTA) fear whole bload assay and for plasma preparation (a ma.nimum of 4 ml required). Both tubes drawn at the same time. All blood; serum and plasma was stored at 4C until as~a~ed.: A1,1 assays xun within 24 ; hours aft=er , draw~.ng . , , .

The Diatron FAST-60 Digoxin Assay System consisted of '(1) caged dicarboxy,silic~n phthalocyanine digoxin probe ir_ FPIA buffer w~.th 1% bovine gamma globulin; (2) rabbit anti-digoxin in FPIA buffer with 0.1% bovine gamma globu- -i.

lin; (3) iysing/buffer diluent; and (4) FPIA buffer (100 mM phosphate buffer with 1% sodium azide end .O1%

3S bovine gamma globulin).

~ ~! ~ S'i't I'%'E '~ ~i E E'T

. . . ... . , ; .- , , , ; .; . . ...,.; , ,;; . : , ~; , . , ~ : ;~ ;
;:
r.. ~ .:.:.-.~ ..
. ~.:.: ~...... .....,.w. .... ,.:., ,.,. ... .. , ,.. ..... .,....
.....:....
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.... ,.... .:, .. . . .. .. ........ .... ...

~'~ y3/a936~6 F'~T/US93/02470 .
~: _ Assay procedures were performed as follows:
1. Serum A. Abbott TDx~ Digoxin II In Vitro Test - .
Performed according to manufacturer's instructions.
B. Dade Stratus~ - Performed according to manufacturer's instructions. Serum values performed by Pathology Medical Laborator ies, 11160 Roselle Street, San Diego, California 92121.
C. Diatron FAST-60 - See Fig. 21.
2. Plasma A. Abbott TDxa Digoxin II In Vitro Test Performed according to manufacturer's instructions:
B. Diatron FAST-60 - See Fag. 21.
3. Whole Blood A. vAbbott TDx~ Digoxin II In Vitro Test Performed according to manufacturer's instructions, except for the precipitation step. Digoxin extraction from whole blood ~
was accomplished as follows: to 360 ~cl whole blood, an equal volume of Abbott -Precipitation Reagent (Digoxin II? was added with .immediate vortexing for 30 sec onds:' The conical tubes were centrifuged at 10,000 RPMs for two minutes. The ,, ,; , slightly brownish supernate was; removed ; .
very careful~.y with a Pasteur pipette and , transferred to the sample cup, to avoid the transfer of small particles. , B' Diatron FAST-60 - See Fig. 21.
Red blood cells were lysed prior to assay by addition of lysing buffer (0:001 M/L Tris buffer, pY~ 8.0 containing 3~ 5 x Z0'; M/L stearyl-lysolecithin). Palmitoyl-lys~lecithin and myristoyl-lysolecithin in Tris buffer are equally ~ l.6 ~ 5°t~t.lTE S ~d .. :.. ~ ~ .r ..: ....., . , , ~ .; . .:-. . . . . ..,~ . ". : :.: , , ,,: . .
:.. , ,,., .
c: .,... . r :.:.: t -c., ..v;:,' .'".'.~. , . .av.::~ - , ~ ::~. :.~. ~'., . ;...,:.. , ..~.:':..n r.~:~:. ~..., ,: ~ '.;...~.:r :=:, ._,.., ...-,:.. ......~.n~'.. ..
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WO 93/19366 ' P~CTlLjS93/02470 ' ~~~~~~~
effective. These reagents are preferred in that they do not interfere with the ~_mmunoassay at this concentration, and that red cell ghost particle size is reduced in 30 to 60 seconds, thus reducimg ar~y effect of light scatter dur-5 ing the fluorescence measurements resulting in a homoge-neous, non-separation whale blood assay.
The results of the testing are tabulated in Table 5.
The values from the TDx° and Stratus~ are the result of single point testing. Because the FAST-60 procedure used 10 "manual gipetting" and automated instrumental analysis, the samples were run in duplicate and the raw values averaged.
The correlation data are found in Figs. 22 through 26. These include TDx~ serum versus FAST-60 whole blood 15 (Fig. 22), Stratus° serum ~rersus FAST-60 whole blood (Fig. 23) FAST-60 serum versus FAST-60 whole blood ( F 'ig , 24 ) , TI7x~ serum versus FAST- 6 0 serum ( Fig . 2 5 ) and Stratus~ serum versus FAST-6O serum (Fig. 26).
As can be (seen in Figs: 22 and 24, a high degree of 20 correlation exists between TDx~ serum versus FAST-60 whole blood, with R -- 0.96 and 0.97, respectively. When the correlation data in Fig. 24 is compared to Fig. 23, it can be seen that the data is system consistent within the FAST-60 System. Figs. 25 and 26 show a comparison of TDx~
25 serum versus FAST-60 s~:rum and Stratusa serum versus FAST-60 serum assay data with co~r~elations of R - 0.96 and 0 . g7 , respectively . '.l-lgain., it could be interpreted that alslight system bias exists:
The' raw' digoxin values f eir the assays aye found' in 30 Table 5. A comparison ,of the FAST-60 serum and FAST-60 whole blood systems with tie TDx° and Stratusp Systems reveals only small differences between the mean values.
This is surprising and remarkable w~.en considering the differences inherent in the various methods used,: A
35 composite CV of 12:7% was found when comparing data for all assays.
5 ~ ~ 5~'i'tJ'TF ~ ~i E

W~ 93/19366 - PCT/U~93/02~70 ' TABLE S

Abbott Diatron PML

TDx FAST-60 StratusMean %C.V

ng/mL ng/mL ng/mL

S Serum Plasma WB Serum PlasmaWB Serum All All 1 0.87 0.80 1.00 0.72 0.82 0.92 0.80 0.85 10.0 2 2.51 2.08 2.75 2.78 2.02 2.08 2.50 2.39 12.6 3 2.16 2.21 2.46 2:14 1.92 2.OS 2.10 2.15 7.2 4 1.11 0.92 l.ll 1.28 1.07 1.24 0.70 1.06 17.3 0 S 0.85 1.03 1.41 1.25 1.24 1.46 0.80 1.15 21.0 6 1.85 1.96 1.83 1.85 1.87 1.95 1.80 1.87 3.0 7 1.0B 1.35 0.90 0.82 1.19 0.77 1.30 1.06 20.4 8 0.82 0.8? 1.04 0.80 0.76 0.88 0.70 0.84 12.0

9 2.79 3.13 3.23 3.93 4.04 4.34 3.40 3.55 14.6 1 5 10 0.96 0.82 0.98 0.97 0.73 0.75 1.00 0.89 12.2 - 11 0.78 0.88 1.21 0.92 0.96 1.00 1.00 0.96 12.8 12 1.80 1.74 2.43 2.26 1.85 1.90 2.00 2.00 11.9 13 0.60 0.61 0.6? 0.49 0.46 0.53 0.70 0.58 14.4 14 7..70 1.50 1.91 1.77 1.35 1.68 1.70 1.66 20.2 2 0 15 1.06 0.96 1.27 1.20 0.96 0.95 1.10 1.07 11.0 16 0.53 0.53 0.60 0.68 0.45 0.43 0.60 0.55 15.0 17 0.08 0.02 0.00 0.22 0.27 0.00 0.00 0.08 7,80.77 0.74 1.08 1.09 0.95 0.83 0.90 0.91 14.3 19 1:66 l.S2v 2.Z0 1:86 1.68 1.75 1.70 1.75 9.7 2 S 20 2.41 2.23 3.24 3.04 2.32 2.60 2.60 2.63 13.2 21 1.19 1.32 1.62 1.53 1.39 0.98 1.20 1.32 15.3 22 1. 1.13 1.51 0.53 1.12 0.94 ~ 0.90 1.03 26.7 OS

23 1.64 1.58 2.01 1.28 1. 1.55 1.40 1.55 14.2 24 1.65 1,62 2.35 1.83 1.62 1.77 1.50 1.76 14.7 3 0 25 2.95 2.87 3.59 3.82 2:93 3.66 3.00 3.26 11.6 26 3.41 3.13 3.65 4.20 3.80 4.06 3.30 3.65 10.1 27 0.52 0.55 0:71 0.82 0:74 0.42 O.SO 0.61 22.5 28 2.54 2.48 2.91 2.92 2.56 3.00 2.60 2.72 7.4 29 1.93 1.84 2:31 2.07 2.02 2.20 1.70 2.01 9.6 ~ ~ 30 2.64 3.02 3:44 3.45 4.64 3.55 2.60 3.33 19.3 31 3.64 3.49 4.34 3.51 3.83 4.21 3.60 3.80 8.4 32'!1:24 1.06 ~ 1:13 1.12 1.23 0.86 1.10 1:11 '10;~

33 0..68 0.55 0:53 0.61 0.44 0.81 0.70 0.62 18.5 3q 1.26 1.23 1.28 1,36 p,97 1.38 1.20 1.24 10.1 ~ Q 3S 1.'74 1.67 2.05- 2.00 2.04 1.98 1.80 1.90 7.6 , 36 1.16 0:95 1.17 1.17 0.87 0.99 1.00 1.04 10.8 37 0.85 0.98 1.12 0.93 0.96 0.86 0.90 0.94 9.0 38 2.65 2.77 3:06 3.26 3.30 2.93 2.90 2.98 7.5 39 0.95 0.96 1:22 1.19 1. 0.90 1.00 1.04 11.0 OS

4 S --".00.66 0.63 0:67 0.76 0.73 0.82 0.80 0.72 9.4 .~1'0.84 0.89 1.03 1.02 0.93 1.01 1.00 0.96 7.1 S U E3 STT~dT'E S H E El PCT/US ;/0270 ~

W~ 93/19366 ,. i ~,~~~ ~~ ~ ' 42 0.9:9 0,67 0.90 0.70 0.64 0.72 0.?0 0.69 16.3 43 1.20 1.16 1.54 1.43 1.16 1.06 1.30 1.26 12.4 Meanl.47 1.45 1.75 1.66 1.56 1.60 1.49 1.57 12.7 oCV57.3 57.4 57:1 62.5 66,6 67.6 58.5 60.0 P.:

In addition, the digoxin values for whole blood, plasma and serum obtained on the FAST--.60 system were extrapolated from a single composite calibration curve.
Thus, it would appear that satisfactory digoxin values could be obtained using either specimen and extrapolation from only one calibration curve: In a clinical setting thin is important a.n that less sample needs to b~ drawn from the patient, and also, any one of the blood specimen can be used in the measurement of digoxin. This saves the patient from a needless extra venipuncture and saves'the 1S laboratory time; and additional cost for time and mater~:als .
The above examples involve the preparation end use of a Caged dicarboxy silicon phthalocyarine digoxin probe.
Those skilled in 'the art will appreciate that other types of fluorescent dic~oacin probes which Comprise a detectably labeled marker component which COmpri.ses a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxy-hydrocarbyl moieties; one located on either side of the planar mol~CUlar structure;- can b~ prepared: Those skilled in the art will also readily appreciate the' fact ~ha~ Caked diCarl~oxy silicon phthalocyanine probes can be prepared for other analyzes, as well. For, example, small anal~rtes such as amikacin, gentamicin, netilmicin, tobra-;mycin, carbamaz~pine, ethosuxima.de, valproiC acid, diso-PY~'amad~, lidocaine,' Procainamide, quinidine, meth~trex-ate, amitriptyline, mortripyline, imipramin~, desipramine, vanCOmycin and cyc3osporine arc part~.cularly suited for the assays described here~.n due to their size.
3S For exempla, as described in Examples 11--1°T below, sucn probes were prepared for digitoxin, theophylline;
phenobarbital, thyroxine, N-acetylnrocainamide, primidone S~J~S TE SHEE?"

WO 93/19366 " PCT/US93/02~70 6 , and phenytain, Those skilled in the art will alsa recog nize that caged dicarboxy silicon phthalocyanine probes can be prepared for peptides. For example, as described in Examples 18 and lg below, such probes were prepared for rubella virus peptide.
Example 11 Synthesis of Caked Dicarboxy Silicon Phthalocr~anine-Diqitoxin A. Preparation of 3-Aminodic,~itoxicxenin 3-aminadigitoxigenin was prepared by procedures - similar to those described in Example 3 above for the digoxin probe.
E. Preparation of Probe The digitoxin probe was pxepared as follows: 0.8 mg 2S of 3-aminodigitoxigenin was placed in a 3,0 ml reaction vial and dissolved zn Z00 ~cl DMF. Caged dicarboxy silicon phtha~.ocyanine ~ (1. 0 mg) was added to the reaction vial .
Also added to the reaction vial were 0.5 mg HOBT and 2.0 mg EDAC and the resulting mixture was thoroughly mixed.
The reaction mixture was stored overnight at about 4 to 8°C.
C. Purification of Probe The digitoxin probe was purified by procedures similar to those described in Example 3 for the digoxin probe. The structure of the caged dicarboxy silicon p'h~h~'~.ocyariine-digitoxi,n probe' is shown in Fig . 27 .
Example 12 Synthesis of Caged Dica~boxv Silicon Phthalocvanine-Theophvlline A: P~epara~ionof Theoph~rlline 8-Butyric Acid ,~ mixture of 3I.3 g glutaric anhydride, 25 g 5.5-diamino-1,3-dimethyl uracil, and 300 ml N,N-dimethyl-an~,line was heated under reflux for 4 hours. Upon SIJ~STE SHEET' l~Gl 93! 19366 ' PCT/ US93102470 W , j i i cooling, the product crystallized from the dark, clear reactian mixture. The crystals were collected by fil-tration, washed with benzene, then crith methanol, and .
dried affording 187 g light yellow solid. , B. Preparation of Theo~hylline-8-(N-2-Aminoethvl~
Bu_tyramide To a starred mixture of 453 mg theophylline-8-butryic acid, 6 ml DMF and 4 ml (THF) was added 240 ~C1 triethyl-amine. The resultant solution was cooled in ice and 220 ~:0 ~.1 isrsbutylchloroformate was added. After 1 hour the slurry was added-to 2 ml ethylenediamine cooled in ice:
The reaction mixture was maintained at 0°C for 6 hours and then concentrated to dryness. The residue, upon frac-~icinal crystallization from chloroform + ethanol provided 369 mg pure theoghylline-8-(~T-2-aminoethyl)butyramido.
~. Preparation of Probe The theophylline probe was prepared as follows:
~:.2 mg 'theophylline-8-(N-2-aminoethyl)butyramide was placed in a 3.0 ml reac ion vial and dissolved in 100 ~zl DMF.' In a separate vial, caged dicarboxy silicon phthalo-cyanine (1:0 mg) way dissolved in 400 ~cl'DMF and then transferred to the reaction vial along with 200 u,l of wash DMF (for a total of 600,1 DMF). To the r~aGtion vial wad added 6.1 nng of IHt~BT; dissolved and mixed well. To make the ffinal reaction mixture, 7.0 mg EDAC was added and the resu~.ting' mixture,mix~d thoroughly., The reaction mixture j ~ ; , ' ( i' Y
was stored~overnight at about 4 to 8°C.
D. Purification of Probe The theophylline probe was purified using procedures simihar to hose descra.bed in Example 3 for tha digoxin xarolae. The structure of the caged dicarboxy silicon ohtk~alocyanzne-digoxigenin probe is shown in Fig: 28.
Examb l ~ 1: 3 SIJ ~S ~°F S H EE°T

WO 93/19366 ' PCT/L1593/02470 ~:~. ,1 1Ya ':~ ' ~ ~~ ..
'f~ ~ r Synthesis of Caned Dicarboxy Silicon Phthalacyanine-Phenobarbital A. Preparation of Nitra~henobarbital Phenobarbital, 663 mg, was dissolved in 2.7 ml con- .
5 centrated sulfuric acid cooled in ice. With stirring, a cold solution of 0.16 ml concentrated nitric acid in 0.65 ml concentrated sulfuric acid was added dropwise over a period of ~ minutes. After 1/2 hour in the cold, the reaction mixture was poured into ice water. The precipi-

10 tote was collected, washed with water, and dried in vacuo affording 0.03 g white solid.
B. Preparation of Aminaphenobarbital Nitrophenobarbital (225 mg) was stirred in a mixture of 3 cnl concentrated HC1, 2 'ml acetic acid and 2 . 5 ml THF .
15 To the slurrr~r mixture was added a solution of 370 mg SnCl2 in 1 ml concentrated HCl and l ml acetic acid. After stirring at room temperature fnr 2 hours, the reaction mixture was concentrated to give an oily residue. To this residue, 1~?aHCO~ solution was added until the pH was about 20 7. The precipitate was collected, washed with water and .
dried in vacuo leaving 449 mg white solid. This solid was stirred in 10 ml THF ~.z~d centrifuged to remove the inor ganic material. The supernatant lic~ua.d was evaporated and the residue was dried in vacuo affordirxg 138 mg light yel 25 low solid.
C., Preparation of Probe ~' The ph~nobarb~.tal probe was prepared as follows:~l.2 ~ .
mg of 5-ethyl-5-(aminophenyl)barbituric acid (P-amino- .;
phenobarbi~al) was placed in a 3.0 ml reaction vial and , 30 dissolved with -200 ~1 DMA':-'Tn a separate vial the caged dicarboxy silicon phthalocyanine (1.0 mg) was dissolved in 200 ~1 DMF and then transferred to the reaction vial. To the reaction vial was added 2.~ mg HOBT, dissolved and mixed well. To make'~he final reaction mixture, 2.8 mg oz ~ L~ ~ STt~IJ'TF S ~i E ~' WO 93/19366 ' F'C°f/U593102470 i EDAC was added and mixed thoroughly. The reaction mixture , was stored overnight at about 4 to 8°C.
D. Purification of Probe The phenobarbital probe was purified using procedures similar to those described in Example 3 for the digoxin probe. The structure of the caged dicarboxy silicon phthalocyanine probe is shown in Fig. 29.
Examtale 14 Synthesis of Caged Dicarboxy_ Silicon Phthalocvanine-Thvxoxine A. Preparation of Th~rroacetic Acid Ethvlenediamine ;
To a stirred mixture of 100 mg thyroacetic acid in ml pyridine was added 16 mg of N-hydroxysuccinimide and 27.6 mg N,N'-dicyclohexylcarbodiimide. The mixture was 1,5 stirred for 2 hours at, room temperature and transferred to 4°C for 18 hours. The crystals were removed by filtration and 8.03 mg of ethylen~diamirie was added to the filtrate J
w~.th stirring. This xeaction was allowed to proceed an additional 24 hours at 4°C and was dried i~ va~uo result ing in whitish-gray powder. The material was stored at -20°C in a desiccator.
B. Preparation of Probe The thyroxi:ne probe was prepared as fral~.ows: 1.0 mg of tetraiodothyroacetic acid-ethylenediamine (Tetras-EDA) was placed ixa a 3 . 0 ml "reaction vial and ,dissolved in 100 , r ~,1 D~~, In a separate vial, caged dicarboxy silicon phthalocyanine (1.0 mg) was dissolved in 400 ~.1 DMF and then transferred to the reaction vial along with X00 ~.1 of wash DMF for a total'of 600 ~,1. To the reaction vial was added ~:. 8 mg Ht,~B"~, dissolved and -mixed well . To the final reaotioz~'mixture; 1.5 mg EDAC was added and the result~.ng mixture mixed thoroughly. The reaction mixture was stored cavernighc at about 4 to 8°C.
S U STI~tJ?°~ S ~i ~ ET

!~O 93/~936g ' PCT/~.JS93/02470 .

C. _Purification of Probe The thyroxine probe was purified using procedures similar to those described in Example 3 for the digoxin probe. The structure of the caged dicarboxy silicon phthalocyanine-thyroxine probe is shown Fig. 30.
Example 15 Svnthesis of Caqed Dicarbo Silicon ~Phthaloc anine-N-Acet 1 rocainamide A. Preparation of Desethyl-N-Acet~lprocainamide Desethyl-N-acetylprocainamide was prepared by dis-solving 1.0 g of p-acetamidobenzoic acid and 0.7 g N-hydroxysuccinimide in 20 ml pyridine. To this solution was added l.4 g of N,N'-dicyelohexylcarbodiimide. The reaction mixture was placed at 4°C for 18 hours, at which ~.5 time the~crystals were removed by filtration. The filtra-tion was brought to room temperature and with stirring, 0.51 g N-ethyl,ethylenediamine was added. Stirring con-tinued for 3 hours, the solution was cooled to 4°C and allowed to react for an addition 24 hours at 4°C. The second crop of crystals was removEd by ~~.ltration, dis-solved in 25 ml of distilled water. The pH was adjusted to 10'with sodium hydroxide to form a white precipitate of desethyl-N-aoetylprocainamide. The resultant precipitate was dried in vacuo and .stored at -20°C in a desiccator.
B. Preparation of Probe The N-Acetylprocainamide probe was prepared as fol-~i , ; ~ i lows: I.O mg desethyl-N--Acetylprocainamide was placed in a 3.0 ml reaction vial and dissolved with 100 ~.1 DMF. In a separa a 'vial caged dicarboxy silicon phthalocyanine (1.O mg) was dissolved irk 400 ~C1 DMF and then transferred to the reaction vial along with 200 ~.l of wash DMF for a - total of 600 ~.1. To the reaction vial was added 4.2 mg HOBT, dissolved and mixed well. To make the final reac-tion mixture, 10:5 mg EDAC was added and mixed thoroughly.
S!!~S 'fF SHED?' WO 93/19366 ' PCTlUS93/02470 The reaction mixture was stirred overnight at about 4 to 8°C.
C. Purification of Probe The N-Acetylprocainamide probe was purified using procedures similar to those described in Example 4 for the digoxin probe. The structure of the caged dicarboxy sili con phthalocyanine-N-acetylprocainamide probe is shown in Fig. 31.
Example l6 Synthesis of Caaed Dicarboxy Silicon Phthalocyanine-Primidone A. Preparation of Nitr~oprimidone Primidone, 1.60 g; was dissolved with stirring in 8 ml concezztrated sulfuric acid and cooled in ice . A cold solution of 4~5 gel concentrated nitric acid in 2 ml con centrated sulfuric acid was added over a period of 10 min-ut~s. After 2 hours at 0°C the reaction mixture was poured into ice water neutralized with cold sodium hydrox-ide: The precipitate was collected; washed with water and 2p dried in vacuo affording 1:79 g white solid.
~: partition of Aminoprimidone Ni.troprimidone 1.79 g was dissolved with heating in 15 ml concentrated, HC1 and 3S ml THF. The salution was allowed to cool to room temperature. A solution of 4.86 Sz~Cl2 in 3 ml concentrated HC1 and 3, ml THF was added over a period of'10 minutes. After stixring at room~tempera-tur~ overnight, the reaction mixture was made basic wi h ~~
ammonium hydroxide: The THF layer was separated and evaporated to dryne~s> The residue was dried in vaeuo, stirred a.n In ml THF and centrifuged to remove inorganic material. The clean THF solution was evaporated in vacuo to provide a residue which upon fractional crystallization from THF and petroleum either yielded 519 mg pure amino nrimiczane . .
S L~ ~ S°l'f F S H ~ E°

V!'O 93119366 ' P~CT/US93/0247U . , C. Preparation of Probe The primidone prabe was prepared as follows: 0.8 mg 5-ethyl-5-(4-aminophenyl)hexahydropyrimidine-4,6-dione (p-aminoprimidone) was placed in 100 ~.1 DMF in a 3.0 ul reaction vial. To the reaction vial was added 1.0 mg .
caged dicarboxy silicon phthalocyanine (1.0 mg) and 3.1 mg HOBT. To make the final reaction mixture, 3.9 mg EDAC was added along with 150 ml DMF and the resulting mixture mixed thoroughly. The reaction mixture was stored over 10. night at about 4-8°C.
D. Purification of Probe The primidone: probe was purified using procedures similar to those described in Example 3 for the digaxin probe. The structure of the caged dicarboxy silicon phthalocyanine-primid~ne probe is shown in Fig. 37.
Examt~la 17 S~nthes~.s of Cacxed Da.carboxvSilicon Phthalocvanine-Phenytoin 1~, Preparation of Probe The phenytoin probe was prepared as follows: 1.2 mg of diphenylglycine was placed in a 3.O m1 reaction vial and dissolved with 100 ~cl DMF . In a separate vial , dicar-bo~yphthalocyana.ne (3.0 mg) was dissolved in 400 ml DMF
and then transferred to the reaction vial along with 200 ~,l of wash DMF for a total of 600 ~1. To the reaction vial was added 6.1 mg of ~iOBT, dissolved arid mixed well.
,, , i , To,make the final reaction, mixture,' 7.0 mg of EDAC was , added and mixed thoroughly. The reaction was stored at 4.0-$.0°C overnight.
B. Purification of Probe The phenytoin-ph~halocyanine probe was purified using procedure similar to those described in Example 3 for the digaxin probe. The structure of the caged dicarboxy sili-con phthalocyanine-phenytoin probe is shown in Fig. 33.
S~~S TE SHEET

Exam~cl a Rubella Anti-Ig~~robe A. Labeling of Goat Anti-Human IaG
Caged dicarboxy silicon phthalocyanine dye (12 5 ,moles) prepared according to Example 1 and purified by DEAE Sephadex* chromatography was mixed with 1 ml of pyridine-pyridinium chloride buffer made by mixing 5 ml 1 M HCl with 0.5 ml pyridine. The solution was taken to dryness in a sublimation apparatus and the excess pyridine 10 and pyridinium chloride was removed, thus assuring that all acetate ion present would be removed. The dry resi-dual dye Was dissolved in anhydrous dichloromethane to make a 3.5 mM solution.
The carboxylic acid groups of the dye were converted 15 to the imidazolide by mixing 1 ml of 3.5 mM dye with '760 ~1 of 0.46 M carbonyl diimidazole and allowing 1.5 hour at room temperature for reaction, after which the solvent was removed i~r v a DMF was scavenged free of water and reactive amines 20 by adding carbonyl diimidazole to a final concentration of 0.1 M.
To 100 ~1 of scavenged DMF was added 10 ~ul of HZO and 100 ul of this mixture was added in the cold to the dry activated dye. After 1 minute this mixture was added to 25 a mixture of 600 ~cl IgG solution containing 6 mg of goat anti-human IgG and 100 ~l of 100 mM phosphate, pH 7.6.
The reaction was allowed to proceed for 4.5 hours at room temperature and overnight at 4°C. A pardon of the reac-tion mixture was equilibrated with 10 mM phosphate, pH 7.6 30 by two treatments in a Minicori concentrator iAmicon Cor-poration, Danvers, MA, USA) and passed through a hydroxy-lapatite column (Bio Rad Laboratories, Richmond, CA, USA) equilibrated with 10 mM phosphate, pH 7.6. Free dye eluted at this stage and the labeled antibody was recov-3=~ ered by elution with 100 mM phosphate, pH 7.6.
B. Analysis of Probe *Trade-mark 1W0 93/~93G6 PCT/US93/02470 ~~~~ ~~

The labeled antibody was found. by absorbance measure-menu at 280 nrn and 682 nm to contain an average of 1.3 moles of dye/mole of IgG. It was Shawn to react specifi-cally in a solid phase sandwich assay in which adsorbed rubella virus antigen was coated with human anti-rubella which enabled reaction with the labeled antibody. Tran-sient-state fluorescence intensity measurements in the 680 nm region were used to quantify the bound labeled anti-body. Specificity was further tested by correlation with a standard method. A series of 40 patient samples were , _ run; 4 were negative by bath methods, 35 were positive by both while one was positive by the standard method and negative transient state fluorescence.
In clinical pathology and medical screening, speci ficity is defined as the proportion of individuals with negative test results for the disease that the test is intended to revel, i.~e., true negative results as a proportion of the total. number of true negative and false positive results. In this example, by this definition, this assay demonstrated 1000 specificity. In addition, sensa.tivity of a procedure can be defined as that pro-portion of inda.viduals wa.th a paaitive test result for the disease that the test intended to reveal, i.a., true pasi-tive results as a proportion of the total true positive and false negative results. These data indicate a 97.2%
sensitivity for this assay system. Although we report a relatively small number of samples the performance of the assay, demons;trat~s the use of an antibody labeled; with: the , marker components in a sandwich assay. See F'ig. 34.
Example l9 S~rnthesis of Caged Dicarboxy Silicon Phthalocyanine Synthetic Rubella Peptide A. pre~arat~.on of Probe A synthetic rubella peptide, for example, a portion of the El protein of the rubella virus (Therien strain), SII~ST'E S~i~ET

can be synthesized by standard peptide synthesis proce-aure. The coupling of caged dicarboxy silicon phthalo-cyanine (prepared according to Example ly to the synthetic rubella peptide was a four step process:
1. Dye activat~.gn_ - Sufficient caged dicarboxy silicon phthalocyanine in dimethylformamide to give a molar ratio of caged dicarboxy silicon phthalocyanine dye to peptide of 1.3 was activated by adding 50 moles of carbonyldiimidazole per mole of dye in dichloromethane to l0 form an imidazole.
2. Decomggsition o_~ excess ca,,~bo~,vldiimidazole -The dichloromethane was evaporated from the activation mixture and water was added to decompose the excess carbonyldiimidazole.
3. Cougling~ ~o Peptide - The solution was buffered by adding for each ~cmole of carbonyldiimidazole used 1 ~1 of a mixture of 100 ~cl of pyridine, 2.38 ml water and 620 ul of 1 M HC1. The resulting solution was added to the dry peptide. Alternatively, the carbonyldiimidazolide was reacted with the peptide in DMF.
B. ~urifi~"tion cl~~ Probe Purification of caged dicarboxy silieon phthalo-cyanine labelled peptide from the reaction mixture was carried out by high performance liquid chromatography on a reversed phase C" column using a water-methanol gradient.
Ex~mp 1 a 2 0 Immunoloaical Evaluation of Phthalocvanine-Rubella Probe Two assay procedures were performed in order to waluate the phthalocyanine-Rubella probe. The phthalo-rvanine-Rubella Probe was diluted in a 0.01 Tris buffer pH 8.0 containing 0.1 o bovine serum and 0.025°s Tweeri 20.
*Trade-mark WO 93119366 FC,"T/CJS9310~470 :. , -.
a ~a .

The probe concentration was determined to be 1.1 x 10-1' M/L. Rubella peptide calibrators were made by diluting in the 'iris buffer to the following concentrations: 0.0, 1.0 .
x 10'12, 2.7 x 10y~2, and 5.4 x 10'2, 2.7 x 10'11, and 5.4 x 10'11. The antibody was made by hyper-immunizing a rabbit .
with the Rubella peptide. Dilutions were made in the Tris buffer described above.
A. Com etitive Bindin Assa Se ential Format To a series of small conical test tubes was added 25 ' ~,l Tris buffer, 20 ~.1 antibody solution and 10 ~1 antibody solution and l0 ~,l of each peptide calibrator. The tubes were incubated at 3S°C for 10 minutes. At this time, ~.1 of probe was added to each tube and incubated an addita:onal 20 minutes a~ 35°C. After incubation 1.0 ml of 15 iris buffer was'. added and transient Mate polarization measurements were made.
The typical inhibition curve is shown in Fig 35. The 'data obtained clearly demons~ra~ed a sensitivity of l..o x 10'kx N1/L of peptide in a homogeneous fluorescence polariza t~.on assay.
D, Antibody Titration Curve To~a series o~ small conical test tubes, varying di.lut~.ons ~f rubella 'antibody (20 ~cl) was incubated with 20 ~.1 Tris buffer and l5 ~1. probe for 20 minutes a~ 35°C.
After incubation 1.0 ml of Tris buffer was added ~:o each tube; ,and the tra~.sient state, polarization was . measured.
~ ~
The data obtained are shown in Fig. 36. .
A typical antibody was obtained at a probe concentra-tion of 2.0 x 10°11 MIL. As can be seen from the data, the probe when depolarized in buffer has a polari.zata:on of S1 millipolarization units (mP) and when bound to antibody ~,,j' exhibits a polarization of X15 mP, with a dynamic range of 164 mP. Thus, indicating the ability to use a homogeneous polarization assay for detection of rubella virus anti-bodies zn human serum samples.
5 ~ ~ 5'TTTLIT~ S H ~ ~'I°

yyp g3/ig3~,~ ' PCT/US93/02470 ~~~f~ f ~~

Those skilled in the art will recognize that the methods used in the above examples relating to rubella peptide are applicable to other peptides. For example, probes can be made for peptide hormones such as luteiniz-S ing hormone, follicular stimulating hormone, human chorio-gonadotropin, thyroid stimulating hormone. Angiotensin I, Angiotensin II, prolactin and insulin. Probes can be made for peptides such as tumor markers (for example, carcino-embryonic antigen) as well.
20 To assist in understanding the invention, the results of a series of experiments have been provided. The above examples relating to the present invention should not, of course, be construed as limiting the scope of the inven-tion. Such variations of the invention, now known or ~.5 later developed, which would fall within the purview of those skilled in the art are to be considered as falling within the scope of the invention as hereinafter claimed.
SIJESTE SHEE'1°°

Claims

CLAIMS:

1. A method for determining the presence or amount of a target analyte in a sample, comprising the steps:
(a) contacting a sample suspected of containing a target analyte with a known quantity of an added target analyte or analog thereof linked to a fluorescent probe wherein the fluorescent probe comprises a detestably labeled marker component, wherein the detestably labeled marker component comprises a fluorescent porphyrin or azaporphyrin that is both: (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of a planar molecular structure;
(b) contacting the sample with a receptor capable of specifically recognizing the target analyte;
(c) determining the amount of the fluorescent probe linked to the added target analyte or analog bound to the receptor or the amount of fluorescent probe linked t:o the added target analyte or analog which is nor bound to the receptor; and (d) comparing the amount of the bound or unbound fluorescent probe with the amount of fluorescent probe in a standard sample free of the target analyte or containing the target analyte in a known amount.

2. The method of claim 1, which further comprises diluting the resultant mixture from step (b) by an amount of from 2-fold to 100-fold.

3. The method of claim 2, wherein the diluting is by an amount of from about 7-fold to about 50-fold.

4. The method of claim 3, wherein the diluting is by an amount of from about 35-fold.

5. A method for determining the presence or amount of a target analyte in a sample, comprising the steps:
(a) contacting a sample suspected of containing a target analyte with a first receptor capable of specifically recognizing the target analyte to form a complex of the target analyte and the first receptor, the first receptor being labeled with a fluorescent probe which comprises a fluorescent porphyrin or azaporphyrin that is both; (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of a planar molecular structure;
(b) contacting the complex with a second receptor capable of specifically recognizing the target analyte, the second receptor being bound to a solid carrier, to form a complex of the first labeled receptor, the target analyte and the second receptor bound to the solid carrier;
(c) determining the amount of the fluorescent probe associated with the solid carrier or with the first receptor present unreacted, thereby measuring either the amount of the labeled first receptor associated with the solid carrier or the amount of the unreacted labeled first receptor; and (d) relating the amount of the labeled first receptor measured to the amount of the labeled first receptor measured for a control sample free of the target analyte, or relating the amount of the labeled first receptor measured with the amount of the labeled first receptor measured in samples containing known amounts of the target analyte.

6. The method of claim 5, further comprising the step of separating the solid carrier from the sample and the unreacted labeled first receptor.

82 A method far determining the presence or amount of a target analyte in a sample, comprising the steps of:
(a) simultaneously contacting a sample suspected of containing a target analyte with first and second receptors capable of specifically recognizing the target analyte, the first receptor being labeled with a fluorescent probe which comprises a fluorescent porphyrin or azaporphyrin that is both: (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of a planar molecular structure and the second receptor being bound to a solid carrier, to form a complex of the first receptor, the target analyte, and the second receptor;
(b) determining the amount of the fluorescent probe associated with the solid carrier or with the first receptor present unreacted, thereby measuring either the amount of the labeled first receptor associated with the solid carrier or the amount of the unreacted labeled first receptor; and (c) relating the amount of the labeled first receptor measured to the amount of the labeled first receptor measured for a control sample free of the target analyte, or relating the amount of the labeled first receptor measured with the amount of the labeled first receptor measured in samples containing known amounts of the target analyte.

8. The method of claim 7, further comprising the step of separating the solid carrier from the sample and the unreacted labeled first receptor.

9. The method of claim 5 or 7, wherein the first receptor is labeled with a first fluorescent probe and the second receptor is labeled with a second fluorescent probe, the first fluorescent probe having a different absorption and emission than the second fluorescent probe, and wherein absorption of one of the fluorescent probes overlaps with emission of the other fluorescent probe.

10. The method of any one of claims 1 to 9, wherein the sample is a biological fluid.

11. The method of claim 10, wherein the biological fluid is plasma.

12. The method of claim 10, wherein the biological fluid is whole blood.

13. The method of claim 12, wherein red blood cells in the whole blood have been lysed.

14. The method of claim 13, wherein the red blood cells have been caused to lyse by use of a compound selected from the group consisting of stearyl-lysolecithin, palmitoyl-lysolecithin and myristoyl-lysolecithin.

15. The method of claim 10, wherein the biological fluid is urine.

16. The method of any one of claims 1 to 15, wherein said target analyte i.s selected from the group consisting of an antigen, a hapten, and an antibody, and the receptor is selected from the group consisting of an antigen and an antibody.

17. The method of claim 16, wherein the antibody is a monoclonal antibody.

18. The method of any one of claims 1 to 15, wherein the target analyte is a drug or a metabolite of a drug.

19. The method of claim 18, wherein the drug is a steroid, hormone, antiasthmatic, antineoplastic, anti-arrhythmic, anticonvulsant, antiarthritic, antidepressant, or cardiac glycoside.

20. The method of claim 18, wherein the drug is digoxin.

21. The method of claim 18, wherein the drug is N-acetylprocainamide.

22. The method of claim 18, wherein the drug is phenobarbital.

23. The method of claim 18, wherein the drug is primidone.

24. The method of claim 18, wherein the drug is theophylline.

25. The method of claim 18, wherein the drug is thyroxine.

26. The method of claim 18, wherein the drug is digitoxin.

27. The method of any one of claims 1 to 15, wherein the target analyte is a peptide.

28. The method of claim 27, wherein the peptide is rubella virus peptide.

29. The method of claim 2l, wherein the peptide is a portion of the E1 viral protein of rubella virus peptide.

30. The method of any one of claims 1 to 29, wherein the method is capable of detecting the target analyte in a concentration of from about 1 × 10-5 M/L to about 1 × 10-3 M/L.

31. The method of any one of claims 1 to 29, wherein the method is capable of detecting the target analyte in a concentration of from about 1 × 10-10 M,/L to about 1 × 10-12 M/L .

32. The method of claim 18, wherein the method is capable of detecting the drug or metabolite in a concentration of from about 5 × 10-9 M/L to about 5 × 10-12 M/L.

33. The method of claim 32, wherein the method is capable of detecting the drug or metabolite in a concentration of from about 1 × 10 1° M/L to about 5 x 10-10 M/L.

34. The method of claim 32, wherein the drug is digoxin.

35. The method of claim 33, wherein the drug is digoxin.

36. The method of claim 27, wherein the method is capable of detecting peptide at a concentration of from about 1 × 10-10 M/L to about 1 × 10-12 M/L .

37. The method of claim 36, wherein the peptide is rubella virus peptide or a portion thereof.

38. The method of any one of claims 1 to 37, wherein the determination of the amount of the fluorescent probe is conducted by steady-state fluorescence measurement.

39. The method of any one of claims 1 to 37, wherein the determination of the amount of the fluorescent probe is conducted by transient state fluorescence measurement.

40. The method of any one of claims 1 to 39, wherein the determination of the amount o.f the fluorescent probe is made by measurement of light at a wavelength of greater than 500 nm.

41. The method of claim 40, wherein the determination of the amount of the fluorescent probe is made by measurement of light at a wavelength of greater than 650 nm.

42. The method of claim 40, wherein the determination of the amount of the fluorescent probe is made by measurement of light at a wavelength of 680 nm.

43. The method of claim 40, wherein the determination of the amount of the fluorescent probe is made by measurement of light at a wavelength of 690 nm.

44. The method of claim 40, wherein the determination of the amount of the fluorescent probe is made by measurement of light at a wavelength of greater than about 700 nm.

45. The method of any one of claims 1 to 44, wherein the two solubilizing polyoxyhydrocarbyl moieties comprise axial ligands which coordinate to the central atom.

46. The method of claim 45, wherein the solubilizing polyoxyhydrocarbyl moieties are selected from the group consisting of polyethers, polyols, water soluble carbohydrates, and water soluble carbohydrate derivatives.

47. The method of claim 45 or 46, wherein the central atom is capable of forming octahedral coordination complexes.

48. The method of claim 47, wherein the central atom is selected from the group consisting of silicon, germanium, phosphorus and tin.

49. The method of claim 45, wherein the solubilizing polyoxyhydrocarbyl moieties comprise polyethylene glycol or a water soluble polyethylene glycol derivative thereof.

50. The method of claim 49, wherein each of the polyoxyhydrocarbyl. moieties has a molecular weight of about 200 to about 20,000.

51. The method of anyone of claims 1 to 50, wherein the fluorescent probe comprises a fluorescent tetrabenzotriazaporphyrin derivative.

52. The method of any one of claims 1 to 50, wherein the fluorescent probe is selected from the group consisting of tetrabenzotriazaporphyrin, 27-phenyltetrabenzotriazaporphyrin, a.nd 27-(p-methylphenyl)tetrabenzotriazaporphyrin.

53. The method of any one of claims 1 to 52, wherein the central atom is silicon.

54. The method of any one of claims 1 to 47, wherein the fluorescent porphyrin or azaporphyrin has a low degree of symmetry so as to enhance the polarization of emission parallel to polarization of absorption.

55. The method of claim 54, wherein the central atom is silicon or germanium.

56. The method of claim 55, wherein the porphyrin or azaporphyrin has a lower symmetry than D4h.

57. The method of claim 56, wherein the porphyrin or azaporphyrin has at least one fused aromatic ring.

58. The method of claim 56, wherein the fluorescent probe comprises a fluorescent porphyrin derivative wherein 1 to 3 bridging carbon atoms are replaced by nitrogen.

59. The method of claim 57, wherein the porphyrin or azaporphyrin comprises a fluorescent phthalocyanine derivative.

60. The method of claim 58, wherein the fluorescent porphyrin derivative is a fluorescent tetrabenzotriazaporphyrin derivative.

61. The method of any one of claims 1 to 4, wherein the detestably labelled marker component, in the presence of serum components in aqueous solution, is characterized by transient state fluorescence emission having parallel and perpendicular components of substantially the same intensities as without serum.

62. The method of claim 61, wherein the detestably labelled marker component comprises a fluorescent tetrabenzotriazaporphyrin derivative.

63. The method of claim 61, wherein the detestably labelled marker component comprises a fluorescent phthalocyanine derivative.

64. The method of any one of claims 1 to 47, wherein the fluorescent probe has a decay time in the range of from about 1 nanosecond to about 50 nanoseconds.

65. The method of claim 64, wherein the decay time is in the range of from about 5 nanoseconds to about 20 nanoseconds.

66. A method for determining the presence or amount of a target analyte in serum comprising the steps:
(a) contacting serum suspected of containing a target analyte with a known quantity of an added target analyte or analog thereof linked to a fluorescent probe, wherein the fluorescent probe comprises a detectable labelled marker component, wherein the detectable labelled marker component comprises a fluorescent porphyrin or azaporphyrin that is both: (i) coordinated to a central metal atom and (ii) coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure;
(b) contacting the serum with a receptor capable of specifically recognizing the target analyte or analog;

(c) determining the amount of the fluorescent probe linked to the added target analyte or analog bound to the receptor or the amount of the fluorescent probe linked to the added target analyte or analog which is not bound to the receptor; and (d) comparing the amount of the bound or unbound fluorescent probe with the amount of the fluorescent probe in a standard serum sample free of the target analyte or containing the target analyte in a known amount.

67. The method of any one of claims 1 to 18 or claim 66, wherein the fluorescent probe comprises a caged dicarboxy silicon phthalocyanine probe of the formula:
in which:
n is such an integer that HOCH2CH2(OCH2CH2)n OH has a molecular weight of from about 200 to about 20,000;
R2 is -COOH; and Q is such a group that H2N-Q is 3-aminodigoxigenin, 3-aminodigitoxigenin, theophylline-8-(N-2-aminoethyl)butyramide, p-aminophenobarbital, tetraiodothyroacetic acid-ethylene diamine, desethyl-N-acetylprocainamide, p-aminoprimidone, diphenylglycine, or an antibody.