FI96367C - Biospecific assay method - Google Patents

Description

96367 BIOSPHERIC DETERMINATION METHOD

Immunoassay is an established biospecific assay method and its various applications are widely used in routine diagnostics and research. Another group of biospecific assays, although still under development, is the DNA and RNA hybridization assay. Most commonly, these methods are based on tracer technology, which means that a label is attached to a specific reagent. Currently used labels include radioisotopes, enzymes, luminescent labels, and fluorescent labels.

There is a growing need for multiparameter analytics in routine diagnostics. Unfortunately, current methods do not allow the use of more than two or three labels of bioaffinity reagents to be measured simultaneously, because spectrometric separation of the signals from these different labels is not possible with sufficient accuracy. The emission bands of different radioisotopes or fluorescent labels are significantly overlapping and, as a result, the separation of different analytes from the same sample is poor over the required concentration range.

Multi-parameter determination can be performed e.g. so that the specific bioaffinity reagents corresponding to each analyte are attached to different microparticles which act as solid reaction media in a reaction solution containing the necessary reagents and to which a sample is added. One or more fluorescent dyes D2 / D3, ... D * are incorporated inside or on the surface of the microparticles. (hereinafter D *.) for the identification of the microparticle in question and at the same time the analyte by means of the signals given by these fluorescent dyes. The biospecific reaction on the surface of each particle measures the on the basis of the signal F of the analyte specific for the particle and the photoluminescence label attached to the reagents.

Concentration of 35 analytes in the reaction solution. The fluorescent dyes Dk and the photoluminescent label F used in these known methods 2 96367 are selected so that the signals they give can be distinguished from each other by means of different signal timings.

5 In the following text, the dyes Dk used to identify the class of microparticles are referred to as fluorescent dyes and the biospecific reaction substance F is referred to as a photoluminescent label, and the latter may be either a fluorescent, phosphorescent, or corn or bioluminescent label.

In the method described above, the determination of the class of microparticles may be based on measuring the concentration of one short-lived dye contained in the microparticle so that the concentration of the dye in question varies in microparticles of different classes, e.g. 1, 2, 4, 8, etc. based on the dye combination. To measure the bioaffinity reaction, a fluorescent lanthanide chelate (U.S. Pat. No. 5,028,545), a bio- or core-luminescent compound (FI-Pat. No. 89837) or a phosphorescent compound (FI-Pat. No. 90695) can be used as a photoluminescent label in these methods.

The methods described in the other patents mentioned above are based on the simultaneous use of fluorescent and photoluminescent labels with different emission discharge times. These methods are based in particular on the fact that the signal of the photoluminescent label F can be measured over a wide dynamic range without the signals of the fluorescent labels Dk interfering with the signal F, since the emissions occur at substantially different times. In practice, this means that the measurement is performed by a time-resolved measurement method that effectively separates short-lived fluorescence emission from long-lived emission. Dyes Dk and Stamp F

3 963 67 tuning occurs with a light pulse and the release time of the dyes Dk is generally at least two orders of magnitude shorter than that of the label F. If a photoluminescent label is used with a release time of 10-1000 5 microseconds (e.g. lanthanide chelates or metalloporphyrins), the dyes Dk may be dyes with a fluorescence discharge time of less than 10 ns. If a bioluminescent label is a bio- or chemiluminescent label having an emission discharge time of at least several seconds and also with a chemical activator Transparent, the dyes Dk may be not only conventional fluorescent dyes but also, for example, lanthanide chelates or metalloporphyrins or other fluorescent or phosphorescent compounds. not less than 10 microseconds and not more than 10 milliseconds.

It is clear that the measuring device required in this method becomes simpler if all the fluorescent dyes Dk have the same excitation wavelength and the emission wavelengths differ so much that they can be distinguished by simple spectrometric methods.

Examples of such fluorescent dyes are, for example, the commercially available and commonly used fluorescent dyes fluorescein, RBE (rhodamine), RBE + Texas Red and RBE + CY, the latter two being the so-called "tandem" dyes, i.e., two-color conjugates. In tandem dyes, an efficient energy transfer from one dye to another occurs and tuning is performed at the excitation wavelength of the first dye and emission is obtained at the emission wavelength of the second dye. The combination of these four dyes can be excited at one and the same wavelength of 488 nm and the emissions occur at 520, 35,560, 613 and 680 nm. Corresponding combinations of several 4,936,67 dyes and tandem dyes are known.

Another example of a suitable combination for the dyes D * is the fluorescent chelates of the lanthanide ions Tb, Dy, Eu and Sm (FI-Pat. No. 931198). Since the ligands of all these chelates have an absorption in the range of 300-360 nm, their fluorescence can be excited at one and the same wavelength. The emission wavelengths of these chelates are 545, 575, 613 and 643 nm, respectively.

A third example of a suitable combination for the dyes Dk are inorganic microcrystals of yttrium oxide sulfide and zinc sulfide and the like, which are commonly used as colored fluorescents, e.g. in television tubes. These substances use, for example, lanthanides or other metals as fluorescence activators, which determine the fluorescence emission wavelength of the crystal.

This invention relates to the selection of fluorescent dyes suitable for the multiparameter biospecific assay methods described above. These substances 20 should be required to have the following characteristics: 1) they should have a common fluorescence excitation wavelength and preferably in the excitation band of the bioaffinity label, 2) have separate narrow fluorescence emission bands that are easily spectrometrically separable from each other, 3) have a short discharge time i.e., the nanosecond order if, for example, lanthanide chelates or metalloporphyrins are used as the photoluminescent label. Furthermore, it is advantageous for the functionality of the invention if 5) they do not have a small number of long-lasting fluorescent or phosphorescent excitation states and if 6) these compounds are chemically stable and highly fluorescent in the microparticle polymer and if there is a large difference between their excitation wavelengths and emission wavelengths.

The features of the invention appear in claim 1. The invention is characterized in that the fluorescent dyes Dk are selected from one or more derivatives of the following classes of tetrapyrrole compounds: porphyrins, chlorines, bacteriochlorins, chlorophylls, purpurines, phthalosins fluorescence excitation wavelength, and 10 - separate narrow spectrometrically separated fluorescence emission bands, and - the discharge time of their fluorescence excitation states is short compared to the discharge time of the photoluminescent label F.

Since the excitation bands of the fluorescent or phosphorescent bioaffinity labels in the present invention are in the range of 300nm to 420nm, the above dyes (fluorescein, RBE, RBE + Texas Red and RBE + CY) 20, like many other commonly used conventional fluorescents, do not the dyes used to dye the microparticles satisfy the above-mentioned condition 1. The emission spectra of these substances are relatively broad, and therefore also do not satisfy the above-mentioned condition 2 well enough. EN-pat. in application no. The lanthanide chelates used in the process described in 931198 do not meet conditions 4 and 5. The same applies to the use of fluorescent microcrystals.

This invention relates to the selection of dyes for use in the identification of microparticles, and in particular to compounds of the above conditions 1-6 are found among the following tetrapyrrole compounds: porphyrins, chlorines, bacteriochlorines, chlorophylls, purpurines, phthalosynes and phthalosides, 96367 6. These compounds generally have broad overlapping absorption bands in the near-ultraviolet range (320nm-450nm) and narrow distinct fluorescence emission bands in the red and 5 near-infrared regions (600nm-800nm). These compounds can be produced synthetically or microbiologically (Porphyrins, D. Dolphin, Ed., Elsevier,

Amsterdam-N.-Y.-London, 1980, Vl-3) and have been used in various analytical applications (DB Papkovsky, 10 Appl. Fluor. Technology 3 (1991) 16-23; EP 0127797; EP 0071991; Russian patent SU 1,659,477).

Examples of tetrapyrrole compounds include:

1) deuteroporphyrin IX 2) mesoporphyrin IX

3) dimethyl ester of protoporphyrin (IX) 4) octaethyl porphine 5) tetraphenyl porphine 6) tetra- (2-methoxy) -phenyl porphine 7) chlorine of coproporphyrin dimethyl ester 8) coproporphyrin dimethyl ester bacteriochlorine 9) aluminum phthalocyanine 10) aluminum phthalocyanine 10)

Table 1 shows the excitation wavelengths (Xexc) and emission wavelengths (Aem) of these tetrapyrrole compounds. Figures 3a-3j show the excitation and emission spectra of these compounds. It can be stated that the spectra of the measured compounds satisfy conditions 1-3.

These tetrapyrrole compounds can be supplemented in a multiparameter assay method with other known organic fluorescent compounds having the same excitation wavelength and having an emission band on the shorter side of the emission range of the tetrapyrrole compounds. As an example of such a compound, coumarin 120 is shown in Table 1.

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Figure 1 shows curves illustrating the discharge rates of the emissions of these tetrapyrrole compounds 1-10. The logarithmic vertical axis of the graph indicates the signal strength and the horizontal axis indicates 5 times in milliseconds. For measurement, the compounds were mixed into a solid polymer matrix (Merckoglas) and the curves were measured with a time-resolved fluorometer. Curve 11 is the result obtained with a pure matrix, the emission discharge time of which is known to be short. Curve 12 is the result from 10 empty cuvettes. It can be concluded from the results that compounds 1-10 have a short emission discharge time and meet condition 4.

Figure 2 shows the long-term emission intensities of these same samples in a time window starting at 100 microseconds and ending at 1000 microseconds after the excitation pulse. The results show that the signal intensities do not differ significantly from the signal intensities given by coumarin (11), pure matrix (12) and empty cuvette (13), with the exception of compound 4, which has twice the long-term emission component of the other compounds. However, the double difference is not significant in this application. It can be concluded from the results that the compounds do not exhibit long - lasting fluorescent or phosphorescent excitation states and meet condition 5.

Figures 3a, 3b, .... 3j show the excitation and emission spectra of tetrapyrrole compounds 1-10 and Figure 3k shows the excitation and emission spectra of coumarin. From these spectra it can be easily stated that it is easy to select from them at least three different compounds, the emission spectra of which are clearly different from each other.

As an example, the emission spectra of three compounds 1,6 and 10 together with the emission spectra of coumarin are shown in Figure 4b. The emission of each compound can be measured over a dynamic range of at least one order without interference from the other 35 compounds, with concentrations of the other compounds varying by one in the corresponding amount of 8,936,367. The excitation spectra of the same compounds are shown in Figure 4a. All compounds are excited in the 350-400 nm wavelength band. The compounds were in a solid matrix (Merckoglas). The solid line represents 5 excitation spectra and the dashed line the emission spectrum.

Microparticles having fluorescent dyes such as tetrapyrrole compounds inside, on the surface layer or on the surface can be prepared in many different known ways. Dyes can be covalently attached to the surface of the particles if there are chemically active groups on the surface of the particles (Molday, R.S. et al., J. Cell. Biol., 1975, 64, 75-88). The dyes can be absorbed into the surface layer of the microparticles by first swelling the microparticles in an organic solvent and after absorbing the dye, the organic solvent is washed and evaporated. These methods are described e.g. in the following microparticle manufacturers' publications: Dyeing Large Particles, published by The Dow Chemical Co., 1972, and Uniform Latex Particles, published by Seragen Diagnostics Inc. Naturally, dyes can be added during the preparation of microparticles prior to polymerization.

It will be apparent to those skilled in the art that various embodiments of the invention may vary within the scope of the claims set forth below.

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Claims (2)

1. Biospecific multiparametric assay method, using a mixture of pre-made, microparticles divided into different categories as solid supports representing different analytes, where microparticles belonging to different categories are coated with different biospecific reagents, which bind different analytes and where the microparticles contain one or more different fluorescent dyes I in one or more concentrations depending on the microparticle category, in which method one mixes the various microparticles belonging to the different categories and adds the sample to be analyzed to the mixture and adds biospecific reagents labeled with a photoluminescent marker. and - activates the fluorescent dyes Dk and the photoluminescent markers F, and - with a time-delayed fluorimeter, measures the fluorescence fluorescence of the microparticle dye Dk to determine the microparticle category and the photoluminescence The marker of marker F for determining the amount of analyte, characterized in that, as fluorescent compounds Dk, one or more of the following tetrapyrrole compound derivatives were selected: porphyrins, chlorines, bacteriochlorines, chlorophylls, purpurines, pheophorbids, phthalosyanines and n-phthalosyanines. Dk - have common excitation path length for the fluorescence, and - have distinct narrow from each other spectrometrically distinguishable emission bands, and - the dissolution time of the fluorescence excitation state of them is short compared to the dissolution time of emission of the photoluminescent marker F. Method according to Claim 1, characterized in that one or more of the following tetrapyrrole compounds' derivatives are used as fluorescent compounds: 1) deuteroporphyrin IX, 2) mesoporphyrin IX, 3) protoporphyrin (IX) dimethyl ester, 4) octaethyl porphine, 5) tetrafenyl pore. , 6. tetra- (2-methoxy) -phenylporphine, 7) coproporphyrin 5 dimethyl ester chlorine 8) coproporphyrin dimethyl ester bacteriochlorine, 9) aluminum phthalosyanine, 10) zinc phthalosyanine.