Fluorescent detection method based on enzyme activated conversion of a fluorophore precursor substrate

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BACKGROUND OF THE INVENTION

The present invention concerns fluorescent substrates and fluorophores produced therefrom useful in fluorescent detection methods. Measurement of the fluorescence produced by these fluorophores is indicative of the presence and the amount of fluorophore in a sample.

Immunoassays employing the unique specificity of antibodies for their counterpart antigens at the molecular binding level have been demonstrated to be extremely valuable methods of analysis to determine the presence or absence of one or more components. The specificity of an antibody for its coupling partner, i.e., counterpart antigen, permits the detection of very small concentrations of specific antigens or antibodies in the presence of considerable other biomaterials (proteins, lipids, sugars, etc.) as usually found in body fluids or other biological samples.

In the past several decades, one of the more useful approaches employed to take advantage of this specificity utilizes the labeling or tagging of either the antibody or the antigen, depending upon the analyte sought in the sample. It is the label or tag which is detected in making a determination with respect to the analyte sought. Exemplary of labels that may be employed are radioactive labels which can be detected in scintillating or gamma counters, fluorochromes which can be detected in fluorometers, luminescent labels which spontaneously emit light and can be detected by light detectors, and enzymes labels which in themselves are not detectable but which, when allowed to interreact with other "substrate" molecules, produce changes in the substrate which can be detected. The advantage of the enzyme label is the fact that a single enzyme molecule can alter many tens of thousands of substrate molecules per minute, and this translates into enhanced sensitivity.

The use of enzyme labels in an assay system has been referred to as an enzyme-labelled immuno-sorbant assay, which for brevity is sometimes referred to as an ELISA technique. Such assays are the subject of a number of U.S. patents, including the following: U.S. Pat Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,016,043; RE 29,169; U.S. Pat. Nos. 4,152,411; 4,169,012; 4,228,240; 4,292,403; 4,331,761; 4,343,896; RE 31,006.

In a typical ELISA technique, a binding partner for the substance to be determined is reduced to an insolubilized form, as by adsorption of the binding partner on the wall of a plastic tube or other adsorbant, i.e., reactive surface. The substance to be determined is reacted with the insolubilized binding partner and the liquid phase of the reaction is subsequently separated. The solid phase is thereafter reacted with a determined amount of coupling partner of the substance to be determined, which coupling partner has been tagged with an enzyme. A coupled product results, and to this product there is added a liquid substrate reactable with the enzyme labels or tags of the coupling partner. The presence of resulting products are usually determined by viewing the color of the liquid phase in the final reaction mixture. Additionally, a chemical stopper is often added to the reaction mixture to inhibit further enzymatic reaction. In a chromogenic detection system, this chemical stopper often produces a change in the resulting product which causes a resultant change in color for easier visual read out. Many regard the chemical stopper, often acid or alkali, not so much as a stopper but rather as a color developer or enhancer.

To obtain greater sensitivity in an enzyme immunoassay, an end product measurable by fluorescent techniques would be preferred (see K. H. Milby in "Enzyme-Mediated Immunoassay", T. T. Ngo and H. M. Lenhoff, eds. Plenum Press, New York, pp. 325-341, 1985).

Fluorescence is a process by which a molecule that is excited by light of a given wavelength emits light at a longer wavelength. The intensity of light emission from a collection of fluorescent molecules depends on:

(1) the intensity of the excitation light source;

(2) the amount of light absorbed, which according to Beer's Law, depends directly on the concentration of the fluorescent molecules present; and

(3) the efficiency with which the fluorescent molecules convert absorbed light into emitted light (the fluorescence quantum yield of the molecule).

If a constant level of excitation light intensity is maintained, emitted light intensity is directly proportional to the number of fluorescent molecules present.

Fluorescence in general is considered to be up to 100 times more sensitive than spectrophotometric techniques or other color-change detection techniques. The use of .beta.-D-galactosidase enzyme and its fluorogenic substrate 4-methylumbelliferyl .beta.-D-galactoside has been cited in the literature as an example of this type of assay (Ishikawa E., Imagawa M., & Hashids S., "Ultrasensitive Enzyme Immunoassay Using Fluorogenic, Lumenogenic, Radioactive and Related Substrates and Factors to Limit Sensitivity", J. Biochem 73, 1319-1321, 1973).

By using fluorescent molecules as labels in immunoassays, either directly attached to an antibody or antigen (called fluorescence immunoassay, or FIA) or as a fluorogenic substrate to detect an enzyme attached to antibody or antigen (called enzyme-linked fluorescence immunoassay, EFIA, or F-ELISA, fluorescence enzyme-linked immunosorbent assay), reagent systems and instruments have been developed which allow quantitative detection of analyte levels with high efficiency and sensitivity.

FIA has the advantages of simplicity of procedure, immediate end point measurement, and excellent reproducibility (precision) of results. EFIA offers greater sensitivity than FIA, since the catalytic activity of each enzyme label can produce up to 10,000 or more fluorophores by proper choice of fluorophore precursor molecule, i.e., fluorogenic substrate. Among the fluorescent molecules most frequently used in immunochemistry are fluorescein, the rhodamines, certain coumarin (umbelliferone) derivatives, and most recently, the phycobiliproteins.

Fluorescein is currently the label of choice in FIA. Its physical properties illustrate the factors important in a fluorophore label. Fluorescein is a very efficient emitter of light, with a fluorescence quantum yield of 0.3 to 0.95 (vs. the maximum possible of 1.0) when conjugated (attached) to an antibody or antigen. It is also an efficient absorber of light, with a molar extinction coefficient [related to the probability of absorbing a photon that strikes the fluorescein] of 70,000.

Fluorescein is stable and undergoes little degradation (photobleacing or photolability) when exposed to light. Techniques for labelling antibodies or protein antigens with fluorescein are relatively simple, but are not easy to control. Fluorescein's fluorescence efficiency is affected very little by temperature, but only the dianion form of the molecule, which exists above pH 8, emits strongly, i.e., fluorescence efficiency varies considerably with pH except at pH 8 or above.

On the negative side, fluorescein emits at 525 nm, with most efficient excitation at 490 nm. Its fluorescence signal is thus subject to the following types of interference which limit its effective sensitivity: (1) overlap by endogeneous fluorescence, i.e. light emitted by sustenances such as bilirubin and hemoglobin, which are normally found in serum samples; (2) quenching, e.g., by bilirubin; and (3) inner filter effects, i.e. preferential absorption of incident light by other molecules such as hemoglobin. Its small Stoke's shift (30 nm) also leads to significant background scattering, which must be eliminated by using complex, dedicated (and thus expensive) filter systems and/or by sacrificing certain efficiencies in instrument design, e.g., off-peak excitation or emission detection. Fluorescein also self-quenches (inhibits its own fluorescence) if molecules are brought into close proximity, so multiple labeling even of large molecules like antibodies is of limited use, even though fluorescein is a small molecule.

Other fluorophores also have certain advantages and drawbacks. The rhodamines fluoresce at long wavelengths, but have poor Stoke's shifts, relatively low fluorescence quantum yields, and are extremely sensitive to pH and other environmental effects. The coumarins have relatively large Stoke's shifts and high extinction coefficients but low quantum yields, are pH sensitive, and their emission is above 500 nm, i.e., subject to endogenous sample interference.

The biliproteins have very high extinction coefficients, long wavelength emission, and in the case of R-phycoerythrin, good Stoke's shifts. They also display high quantum yields, and their emission is relatively insensitive to environment within the pH range of 5.5 to 9. These molecules are large proteins which must be isolated, e.g., from red algae. One such biliprotein, R-phycoerythrin, can provide sensitivity 10-20 times greater than that afforded by fluorescein in FIA systems. Nevertheless the large size and high molecular weight of these molecules, particularly of R-phycoerythrin, place significant limitations on applications of these molecules in immunoassays because of effects on reaction kinetics; e.g., they can't be used to replace fluorescein in fluorescence polarization or in hapten assays with competitive binding formats.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting an analyte of interest in a liquid sample. This method comprises contacting the sample with a first reagent capable of forming a complex with the analyte of interest so as to form a first reagent:analyte complex. The first reagent:analyte complex is then contacted with an oxidase-labelled reagent cable of forming a complex with the first reagent:analyte complex so as to form a first reagent:analyte:oxidase-labelled reagent complex. The first reagent:analyte: oxidase-labelled reagent complex is next treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. By exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce, and thus to emit radiation, and detecting the radiation so emitted, the analyte of interest is detected.

The present invention also provides a method for detecting an analyte of interest present in a liquid sample. This method comprises contacting the sample with an oxidase-labelled reagent capable of competing with the analyte of interest for binding sites on a complementary material not normally present in the sample, and with the complementary material, the contact being effected under appropriate conditions such that the analyte of interest and the oxidase-labelled reagent competitively bind to the complementary material so as to form analyte of interest: complementary material complexes and oxidase-labelled reagent:complementary material complexes. The analyte of interest: complementary material complexes and the oxidase-labelled reagent: complementary material complexes are separated from unbound analyte of interest and unbound oxidase-labelled regent so as to obtain a mixture of analyte of interest: complementary material complexes and oxidase-labelled reagent: complementary material complexes. This mixture is then treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. By exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation, and detecting the radiation so emitted, the analyte of interest is detected.

A method for detecting a single-stranded nucleic acid sequence of interest in a liquid sample is also provided by the present invention. The method comprises contacting the liquid sample with an oxidase-labelled, single-stranded nucleic acid sequence complementary to the nucleic acid sequence of interest under hybridizing conditions so as to form an oxidase-labelled, double-stranded nucleic acid sequence. The oxidase-labelled, double-stranded nucleic acid sequence is treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrazine is then excited so as to cause it to fluoresce and thus to emit radiation, the radiation so emitted is detected, and the analyte of interest is thereby detected.

The present invention also provides a method for detecting oxidase activity in a liquid sample which comprises treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidizing agent under conditions such than an oxidase present in the liquid sample enzymatically oxidizes the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrazine is then excited so as to cause it to fluoresce and thus to emit radiation; and the radiation so emitted is detected, thereby detecting oxidase activity in the liquid sample.

Also provided by the present invention is a method for detecting an oxidizing agent in a liquid sample. This method comprises treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidase under conditions such that the nitrogen-substituted, cyclic compound is enzymatically oxidized to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrazine is then excited so as to cause it to fluoresce and thus to emit radiation, and the radiation so emitted is detected, thereby detecting the oxidizing agent.

The present invention also provides compounds having the structure

X--[(Y).sub.n --Z].sub.m

wherein:

X represents one or more nucleotides which may be the same or different, one or more amino acids which may be the same or different, an antigen, an antibody, an analyte of interest or analog of an analyte of interest;

Y represents a linker arm attaching X to Z;

Z represents an amine-substituted, ortho-fused pyrazine wherein neither pyrazine nitrogen is fused or substituted;

n represents an integer; and

m represents an integer greater than or equal to 1.

Such compounds are useful as fluorophore-labelled reagents in immunoassays, nucleic acid hybridization assays, etc.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 graphically sets forth the results obtained from the experiment described in Example 3. The plot which appears on the right of FIG. 1 illustrates the strength of fluorescent emission of different wavelengths using an excitation wavelength of 425 nm. The plot on the left of FIG. 1 is the excitation spectrum observed for an emission wavelength of 570 nm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting an analyte of interest in a liquid sample. This method comprises contacting the sample with a first reagent capable of forming a complex with the analyte of interest so as to form a first reagent:analyte complex. The first reagent:analyte complex is then contacted with an oxidase-labelled reagent capable of forming a complex with the first reagent:analyte complex so as to form a first reagent: analyte: oxidase-labelled reagent complex. The first reagent:analyte: oxidase-labelled reagent complex is next treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. Within the present application, "cyclic compound" means an alicyclic or heterocyclic compound. By exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce, and thus to emit radiation, and detecting the radiation so emitted, the analyte of interest is detected.

In one embodiment of the invention, the first reagent: analyte: oxidase-labelled reagent complex is treated with at least two structurally different nitrogen-substituted, cyclic compounds. In another embodiment of the invention, the first reagent:analyte:oxidase-labelled reagent complex is treated with the nitrogen-substituted, cyclic compound and the oxidizing agent under conditions wherein the pH is within the range from about 3.5 up to about 6.5, preferably within the range from about 4.5 to about 5.0

In still another embodiment of the invention the analyte of interest is an antigen and the first reagent and the oxidase-labelled reagent are antibodies, ligands, or haptens. In yet a further embodiment of the invention, the analyte of interest is an antibody and the first reagent and the oxidase-labelled reagent are antigens, haptens, ligands, antibodies, or anti-idiotypic antibodies.

The present invention also provides a method for detecting an analyte of interest present in a liquid sample. This method comprises contacting the sample with an oxidase-labelled reagent capable of competing with the analyte of interest for binding sites on a complementary material not normally present in the sample, and with the complementary material, the contact being effected under appropriate conditions such that the analyte of interest and the oxidase-labelled reagent competitively bind to the complementary material so as to form analyte of interest: complementary material complexes and oxidase-labelled reagent:complementary material complexes. The analyte of interest: complementary material complexes and the oxidase-labelled reagent: complementary material complexes are separated from unbound analyte of interest and unbound oxidase-labelled reagent so as to obtain a mixture of analyte of interest: complementary material complexes and oxidase-labelled reagent: complementary material complexes. This mixture is then treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrzaine wherein neither nitrogen of the pyrazine ring is fused or substituted. By exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce and thus to emit radiation, and detecting the radiation so emitted, the analyte of interest is detected.

In one embodiment of the invention, the analyte of interest is an antigen, the oxidase-labelled reagent is the antigen or an analog of the antigen conjugated to an oxidase, and the complementary material is an antibody, ligand, or hapten. In another embodiment of the invention the analyte of interest is an antibody, the oxidase-labelled reagent is the same or a different antibody conjugated to an oxidase, and the complementary material is an antigen, hapten, ligand, antibody, or anti-idiotypic antibody. In yet another embodiment of the invention, the mixture is treated with at least two structurally different nitrogen-substituted, cyclic compounds.

A method for detecting a single-stranded nucleic acid sequence of interest in a liquid sample is also provided by the present invention. The method comprises contacting the liquid sample with an oxidase-labelled, single-stranded nucleic acid sequence complementary to the nucleic acid sequence of interest under hybridizing conditions so as to form an oxidase-labelled, double-stranded nucleic acid sequence. The oxidase-labelled, double-stranded nucleic acid sequence is treated with a nitrogen-substituted, cyclic compound and an oxidizing agent so as to enzymatically oxidize the nitrogen-substituted, cyclic compound to produce an amine-substituted ortho-fused pyrzaine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrazine is then excited so as to cause it to fluoresce and thus to emit radiation, and the radiation so emitted is detected, thereby detecting the analyte of interest.

In one embodiment of the invention, the oxidase-labelled, single-stranded nucleic acid sequence is treated with at least two structurally different nitrogen-substituted, cyclic compounds.

The present invention also provides a method for detecting oxidase activity in a liquid sample which comprises treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidizing agent under conditions such that an oxidase present in the liquid sample enzymatically oxidizes the nitrogen-substituted, cyclic compound to produce an amine-substituted, ortho-fused pyrazine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrazine is then excited so as to cause it to fluoresce and thus to emit radiation; and the radiation so emitted is detected, thereby detecting oxidase activity in the liquid sample.

In one embodiment of the invention, the liquid sample is treated with at least two structurally different nitrogen-substituted, cyclic compounds.

Also provided by the present invention is a method for detecting an oxidizing agent in a liquid sample. This method comprises treating the liquid sample with a nitrogen-substituted, cyclic compound and an oxidase under conditions such that the nitrogen-substituted, cyclic compound is enzymatically oxidized to produce an amine-substituted, ortho-fused pyrzaine wherein neither nitrogen of the pyrazine ring is fused or substituted. The amine-substituted, ortho-fused pyrzaine is then excited so as to cause it to fluoresce and thus to emit radiation, and the radiation so emitted is detected, thereby detecting the oxidizing agent.

In one embodiment of the invention, the liquid sample is treated with at least two structurally different nitrogen-substituted, cyclic compounds.

The methods provided by the present invention may be practiced by oxidizing the nitrogen-substituted, cyclic compound so as to form an amine-substituted, ortho-fused pyrazine which comprises an aminophenazine. Additionally, the methods provided by the present invention may be practiced by enzymatically oxidizing a nitrogen-substituted cyclic compound which comprises an amine-substituted or imine-substituted cyclic compound. Moreover, the nitrogen-substituted cyclic, compound which is oxidized may comprise an amine-substituted aromatic compound. In one embodiment of the invention the amine-substituted, aromatic compound is an ortho-substituted, diamino-aromatic compound. In another embodiment of the invention the ortho-substituted, diamino-aromatic compound is 1,2-diaminobenzene (also known as o-phenylenediamine and referred to herein as OPD), 3,4-diaminobenzoic acid, or 2,3-diaminopyridine.

The methods provided by the present invention may be performed qualitatively or quantitatively. Moreover, prior to exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce, the pH may be adjusted to greater than about 6.5. In one embodiment of the invention, the pH is adjusted to greater than about 10 prior to exciting the amine-substituted, ortho-fused pyrazine so as to cause it to fluoresce.

The oxidase utilized in the methods provided by the present invention may be a peroxidase or catalase. In one embodiment of the invention, the peroxidase may be horseradish peroxidase. Additionally, the oxidizing agent may be a peroxide, e.g. hydrogen peroxide or urea peroxide.

The present invention also provides a compound having the structure

X--[(Y).sub.n --Z].sub.m

wherein:

X represents one or more nucleotides which may be the same or different, one or more amino acids which may be the same or different, an antigen, an antibody, an analyte of interest or analog of an analyte of interest;

Y represents a linker arm attaching X to Z;

Z represents an amine-substituted, ortho-fused pyrazine wherein neither pyrazine nitrogen is fused or substituted;

n represents an integer; and

m represents an integer greater than or equal to 1.

This compound is useful as a fluorophore-labelled reagent in immunoassays, nucleic acid hybridization assays, etc.

In one embodiment of the invention, Z represents 2,3-diaminophenazine, 2,3-diamino-1,8-diazaphenazine, 6,7-diamino-1,5-diazaphenazine, or 2,3-diaminophenazine-6-carboxylic acid.

The methods and compounds provided by the present invention are based upon the fluorescent detection of a fluorophore which is produced by the enzymatic oxidation of a fluorophore precursor substrate. These novel methods and compounds will be better understood by reference to the following examples which are provided for purposes of illustration and are not to be construed as in any way limiting the scope of the present invention, which is defined by the claims appended hereto.

EXAMPLES

EXAMPLE 1

Serial dilutions were prepared of various substances, and samples of these serial dilutions were tested for fluorescence activity in a fluorimeter. The excitation wavelength bandpass in the fluorimeter was 450.+-.20 mn. The emission wavelength bandpass in the fluorimeter was 550.+-.20 nm. Readings were obtained in fluorescent signal units. The following table sets forth the reading results obtained, with the specific substance analyzed being listed under the column labelled "Substance" and with the different dilutions of the samples being indicated at the heads of the columns set forth to the right of the "Substance" column. The fluorescent signal unit reading indicated for substance #7 was in excess of 8192, 8192 being the maximum fluorescent signal unit reading available with the fluorimeter.

TABLE I ______________________________________ Dilution Substance 1:10 1:100 1:1000 ______________________________________ 1. 1,2-diaminobenzene 727 -- 528 (3.2 mM in phosphate buffered saline solution pH 7) 2. hydrogen peroxide 661 700 756 (0.01% aqueous solution) 3. horseradish 502 501 455 peroxidase-labelled avidin (1:1000 in phosphate buffered saline solution) 4. 1,2-diaminobenzene 570 537 498 with hydrogen perox- ide (3.2 mM in phosphate buffered saline solution pH 7 with 0.1% H.sub.2 O.sub.2) 5. 50 microliters 1,2- 941 769 538 diaminobenzene (3.2 mM in phosphate buf- fered saline solution pH 7) plus 50 micro- liters peroxidase- labelled avidin (1:1000 in phosphate buffered saline solution) 6. hydrogen peroxide 568 732 538 plus peroxidase- labelled avidin (1:1000 in phosphate buffered saline solution with 0.01% H.sub.2 O.sub.2) 7. 50 microliters 1,2- >8192 >8192 >8192 diaminobenzene with hydrogen peroxide (3.2 mM in phosphate buffered saline solution pH 7 with 0.01% H.sub.2 O.sub.2) plus 30 microliters peroxidase- labelled avidin (1:1000 in phosphate buffered saline solution) 8. Mixture of #7 plus 900 838 1200 100 microliters 3N HCl stopper ______________________________________

It will be seen from Table I that various ingredients employed in carrying out the method of the invention by themselves or in various subcombinations displayed only marginal fluorescence reactivity. On the other hand, the reaction product produced by the action of peroxidase-labelled avidin on the substrate 1,2-diaminobenzene in the presence of a hydrogen peroxide oxidizer resulted in a fluorescence reactivity exceeding the measurement capability of the fluorimeter even at serial dilution of 1:1000.

EXAMPLE 2

To determine the structure of the fluorophore produced by oxidation of OPD with HRP and hydrogen peroxide, a reaction mixture containing the fluorophore was concentrated to give a crop of dark brown needles, which were then isolated from the concentrated solution by filtration. The resulting dark brown needles were dissolved in butyl alcohol, applied to a KBr plate and allowed to dry. An IR spectrum of the sample was recorded using an IBM FTIR spectrometer.

The recorded spectral data indicated the presence of unsubstituted amino groups, as well as an aromatic heterocyclic compound which contains nitrogen. This data indicated that the reaction mixture contained 2,3-diaminophenazine (DAP).

Authentic 2,3-diaminophenazine and 2,2'-diaminoazobenzene were chemically synthesized by standard literature methods (see Gupta and Srivastava, Indian J. Chem. 9:1303-1304, 1971 and Omote, et al., Chemistry and Industry, p. 996, 1971, respectively).

Product formation from the enzymatic oxidation of OPD using 0.1 M sodium citrate, pH 5, 0.005 M hydrogen peroxide, 100 ng/ml HRP and 0.015 M OPD was monitored by thin layer chromotography on Kieselgel.RTM. 60F.sub.254 silica gel plates (E. M. Scientific, N.J.) using the solvent system benzene:ethanol:acentone:0.25% ammonium hydroxide (100:30:5:2). Additionally, authentic DAP, 2,2'-diaminoazobenzene and the starting material OPD were run on the plates. Only one product was observed from the enzymatically oxidized reaction mixture of OPD. This product had an R.sub.f value identical to that of authentic DAP and fluoresced strongly under a black light.

The reaction mixture described above was diluted 1/50 in 0.1 M sodium citrate, pH 5, and its emission spectrum was obtained and compared with that of authentic DAP (1.3.times.10.sup.-5 M) in 0.1 M sodium citrate, pH 5. Using an excitation wavelength of 440 nm on a McPherson Model FL750 spectrofluorimeter, the maximum wavelength of emission for authentic DAP was 565 nm compared with 562 nm for the OPD reaction mixture.

These data indicate that the product of enzymatic oxidation of OPD is DAP and, further, that the DAP product is responsible for the observed fluorescence.

EXAMPLE 3

A reacted mixture of 1,2-diaminobenzene, hydrogen peroxide, and peroxidase-labelled avidin (substance #7 in Table I) was analyzed in an Aminco-Bowman scanning spectrofluorimeter to determine fluorescence reactivity in response to a given excitation wavelength, and to determine the excitation spectrum for a fluorescent emission of a given wavelength. As may be seen with reference to FIG. 1, peak emission was observed at the wavelength 565 nm with an optimum excitation wavelength of 415 nm. A Stoke's shift of 150 nm in the sample analyzed was demonstrated.

In Table II below, the Stoke's shifts experienced with some of the more commonly known fluorophores is set forth and compared with the Stoke's shift demonstrated with diaminophenazine.

TABLE II ______________________________________ Stoke's Fluorophore Excitation (nm) Emission (nm) Shift (nm) ______________________________________ Fluorescein 495 525 30 Tetramethyl- 550 585 35 rhodamine 4-methyl 375 450 75 umbelliferone Fluorescamine 394 475 81 Lucifer 430 540 110 yellow VS R-phycoerythrin 495 575 80 Diaminophenazine 415 565 150 ______________________________________

EXAMPLE 4

Serial dilutions (in phosphate buffered saline solution) of anti-feline IgG antibody labelled with horseradish peroxidase were reacted with 1,2-diaminobenzene and hydrogen peroxide (3.2 mM with 0.01% H.sub.2 O.sub.2) in a series of glass test tubes. After a five minutes reaction, 20 microliters of each tube'2 contents were transferred to wells in a plastic tray. The contents of these wells were then read in a fluorimeter for fluorescence quantitation. Table III below sets forth the respective dilutions of the antibody and the fluorimeter reading obtained.

TABLE III ______________________________________ Visual Subjective Dilution of Fluorescence Under Anti-Feline Fluorimeter Black Lamp (Wave- IgG Antibody Reading length of 365 nm) ______________________________________ 1. 1:800 8192+ 4+ 2. 1:3200 5195 2+ 3. 1:12800 4400 2+ 4. 1:51000 3705 2+ 5. 1:204000 2743 2+ 6. 1:816000 1525 0 7. Buffer 611 0 ______________________________________

Also indicated in Table III is the visual subjective fluorescence observed under a black lamp, with grading being done on a zero to four basis, zero being no observed fluorescence and four being indicative of clearly observed and distinct fluorescence of high intensity.

EXAMPLE 5

Three-fold serial dilutions were prepared from D.immitis (Heartworm) antigen (2 mg per ml) beginning with a 1:10 dilution in 0.01 M carbonate-bicarbonate buffer, pH 9. Five microliters of each antigen dilution were applied as spaced droplet deposits along the surface of a nitrocellulose strip. The strips were then incubated at 37.degree. C. for thirty minutes and washed with phosphate buffered saline solution pH 7.0 containing 0.3% ovalbumin and 0.05% Tween (detergent).

Dilutions were prepared of dog anti-D.immitis IgG (1:50, 1:250, 1:1250, and 1:6250) in the phosphate buffered saline solution described. Each antibody dilution was used in the soaking of a strip containing each antigen dilution. The strips were then incubated for one hour at room temperature.

Biotin-labelled goat anti-dog antibody IgG diluted 1:100 in the above-described phosphate buffered saline solution was then applied to the deposits on the various strips and the strips incubated for one hour at room temperature. The strips were then washed for fifteen minutes with phosphate buffered saline solution.

The assay was then developed by overlaying the deposits with horseradish peroxidase-labelled avidin diluted 1:3000 in phosphate buffered saline solution and the strips incubated for one hour. The strips were then washed with phosphate buffered saline solution.

Each of the deposits was then punched out of the nitrocellulose strips using a paper punch, and the punched deposits placed in respective wells of a carrier such as a Track XI.RTM. microtiter strip (Microbiological Associates, Inc., Bethesda, MD) described in U.S. Pat. No. 4,468,371 and DES 279,817. Twenty microliter volumes of 1,2-diaminobenzene and hydrogen peroxide solution (3.2 mM with 0.01% H.sub.2 O.sub.2) were placed in the wells and the reaction allowed to proceed for five minutes. The contents of the various wells were then washed with water.

The discs of cellulose then analyzed in a fluorimeter utilizing an excitation wavelength of 450.+-.20 nm and an emission wavelength bandpass of 550.+-.20 nm. The discs were also usually analyzed to determine subjectively fluorescence under a black light (360 nm), and to determine fluorescence with a fluorescence microscope with blue excitation and green filter in the eye piece. Table IV sets forth the fluorimeter readings obtained in signal units for the various deposits, and the subjective fluorescence noted.

TABLE IV ______________________________________ Black Light Microscope- Fluorimeter Subjective Subjective Well Reading Fluorescence Fluorescence ______________________________________ 1 5301 4+ 4+ 2 2718 - .+-. 3 4623 4+ 5+ 4 2429 1+ .+-. 5 2786 - .+-. 6 5718 3+ 4+ 7 6026 2+ 3+ 8 5862 3+ 2+ 9 2508 - .+-. ______________________________________

EXAMPLE 6

Clear polypropylene capillary tubes were precoated with a 5% polycarbonate polymer (Lexan) in methylene dichloride solvent. After evaporation of the solvent, a uniformly thin polymeric transparent film of the polycarbonate was left as a residual on the inner surfaces of the tubes.

A dilution of unlabeled antibody directed against herpes simplex virus (diluted in 0.01 M carbonate-bicarbonate buffer pH 9) was then adsorbed on the coated inner surfaces of the capillary tubes with insolubilizing of the antibody.

After adsorption of the capture antibody and washing with phosphate buffered saline solution pH 7, serial dilutions of herpes virus antigen extracted from tissue culture and suspended in phosphate buffered saline solution were drawn up into the interior of the tubes and the tubes and their contents incubated for thirty minutes. The tubes were then emptied and flushed with phosphate buffered saline solution.

A dilution of a second antibody to