FI90695B - Biospecific classification method - Google Patents

Abstract

The invention concerns a biospecific multi-parameter classification method where a combination is used of pre-manufactured microparticles, divided into different categories and representing different analytes. Microparticles in different categories contain different amounts of fluorescent dyes and they are coated with biospecific reagents which bind different analytes. The sample which is to be analysed is added and a mixture of biospecific secondary reagents which are marked with a phosphorescent molecule is added to the mixture. The fluorescent or phosphorescent dye is activated by light radiation. The strength of the fluorescence emission shows the category of the microparticles and the phosphorescence emission shows the amount of analyte which is attached to the microparticles. <IMAGE>

Description

90695

BIOSPECIFIC METHOD OF DETERMINATION - BIOSPESIFIK BESTÄMNINGSMETOD

Immunoassay is an established biospecific assay method and its various applications are widely used in routine diagnostics and research laboratories. Another group of biospecific assays, although still under development, is the DNA and RNA hybridization assay. Biospecific assays generally use two biospecific reagents, a primary reagent and a secondary reagent (e.g., antibody, DNA, or RNA probe), each of which binds to specific determinants of the analyte molecule to form a complex of three molecules (a layered structure) and a normal structure. reagents are labeled. Currently used labels include radioisotopes, enzymes, luminescent labels, and fluorescent labels.

In routine diagnostics, there is a growing need for multiparameter-15 analytics. Unfortunately, current methods do not allow the use of more than two or three labels to be measured simultaneously, as spectrometric separation of the signals from these different labels is not possible with sufficient accuracy. The emission spectra of different radioisotopes or fluoro-20 resolving labels significantly overlap and, as a result, the separation of different analytes from the same sample is poor over the required concentration range.

It is an object of the present invention to provide an improved method for multiparametric biospecific assays as well as a reagent kit for use in the method.

The features of the invention appear from claim 1.

The process according to the invention is characterized by the following steps: - Pre-preparation of microparticles divided into different classes according to the different analytes in question, so that these different classes of microparticles contain different amounts of fluorescent dye.

- These microparticles belonging to different classes are coated with biospecific primary reagents which bind the analytes in question and then the microparticles are mixed together into a suspension.

- Biospecific secondary reagents that bind different analytes are labeled with a molecule that emits phosphorescent light and these reagents are mixed together.

10 - A sample to be determined and a mixture of biospecific secondary reagents are added to the microparticle suspension, after which complexes of various analyte molecules and labeled biospecific reagents begin to bind to the reagent molecules on the surface of the microparticles due to bioaffinity.

- The binding reaction is stopped by adding a diluent and at the same time diluting the concentration of labeled biospecific reagents not bound to the microparticles.

- The fluorescent dye 20 contained in the microparticles is excited by a light beam and then the intensity of the fluorescence emission is measured, on the basis of which the microparticle class.

- The phosphorescent dye on the surface of the microparticles is excited by a light beam and then the intensity of the phosphorescence emission is measured with a photon detector whose electrical signal is proportional to that. analyte concentration.

The multiparameter assay is performed on a suspension of artificially prepared microparticles, which is a mixture of different classes of microparticles that bind different analytes. Each of the different categories

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3,90695 particles are first coated with a specific primary reagent (antibody, DNA, RNA), i.e., the microparticles serve as a solid support for these reagents and the biospecific reaction. This invention is characterized in particular by the use of a microphotometric technique to identify a class of microparticles and at the same time an analyte by means of a fluorescent dye, and a phosphorescence-based measurement to determine the concentration of an analyte on the surface of microparticles by a biospecific reaction.

It is an object of the present invention to provide a method for performing a multiparameter biospecific assay, the method being based on the use of fluorescent microparticles and a phosphorescent label, and in particular that the fluorescence signal and the phosphorescence signal can be measured over a wide dynamic range without interfering with substantially different emissions. 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 phosphorescence emission. The phosphorescent labels are excited by light pulses. The emission of fluorescent dyes and background in the microparticles has a short half-life, usually only nanoseconds, and the half-life of the phosphorescence label used herein is generally, e.g., 1 ms. The phosphorescence emission detector is activated, e.g., after a 0.5 ms delay from the excitation pulse, in which case it only measures an emission with a long half-life. In this case, neither the emission of the fluorescent dye used to detect the class of microparticles nor the background fluorescence interferes with the measurement of the biospecific label, which is an essential feature of the method of the present invention.

Uniform sized microparticles with a diameter of 1-200 μπι (but not limited to these sizes) can be prepared from 35 suitable polymers that are hydrophilic to such an extent that they are well suited for aqueous suspension. The surface properties of microparticles 4,90695 allow macromolecules to be bound to them by physical adsorption or covalent bonding by activating surface OH groups (L. Johansen et al., J. Immunol. Methods 59, 255-264, 1983). In the method of the present invention, a sample containing all of the analytes in question is incubated with the mixture of secondary reagents in a microparticle suspension in the smallest possible volume (e.g. 10-100 μΐ) in order to achieve the most complete reaction in a short time. Since the average distance between the analyte molecules and the labeled reagents in the microparticle suspension reaction is very small, the reaction equilibrium can be achieved quickly and resulted in the formation of complexes of reagent and analyte on the surface of the microparticles. Such a structure is commonly referred to in the literature as a layered structure. If the incubation times can be precisely adjusted, there is no need to drive the reaction to the endpoint. After incubation, the suspension is diluted sufficiently to reduce the concentration of free-labeled secondary reagent, and the amount of labeled complexes on the surface of the individual microparticles is measured phosphorometrically. In most cases, a one-fold dilution is sufficient to distinguish the signals from the bound and free-labeled reagent fraction sufficiently efficient, because the microphosphorometric detector used in this method is able to optically distinguish the phosphorescence of signal. A statistically sufficient number of microparticles are analyzed and the strength of the phosphorescence signal of each particle is recorded on a computer.

Reports describing the principles of multiparameter biospecific assays have been presented previously.

35 In most cases, however, the multiparametric method has been based on the use of solid reaction media in which biospecific reagents can be attached.

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5,90695 into distinct and optically separable regions (e.g. PCT WO 84/01031). A multiparametric biospecific assay method based on the use of artificially prepared microparticles has also been published, in which the identification of the analyte class is based on the use of microparticles of different sizes and the identification is performed by measuring the diameter of the optically analyzed particle. U.S. Patent No. 5,028,545 discloses a method of using a short-lived fluorescent dye to identify microparticles and a long-lived fluorescent dye to measure analyte concentrations. To distinguish between short-lived and long-lived fluorescence, a time-resolved fluorometer is used in the above method. In addition, a method has been developed that uses a short-lived fluorescent dye to identify microparticles and a chemiluminescent or bioluminescent molecule to measure analyte concentrations. In both of the above methods, the determination of the class of microparticles and the measurement of the binding of biospecific reagents are accomplished by recording the light emissions they emit at substantially different times, and the emission or background fluorescence of the fluorescent label used to identify the class does not interfere with biospecific reagent labeling.

The sensitivity of the measurement method of the present invention is based on a signal from a phosphorescent molecule used therein, which is measured phosphorometrically, wherein the measurement method and the measuring device are in principle completely identical to the time-resolved method and device used for measuring long-term fluorescence. Phospho-resinance is a phenomenon in which photon emission originates from the excitation states of the electron orbitals of molecules, as does fluorescence, but phosphorescence emission arises from the discharge of triplet states, whereas fluorescence is generated from the discharge of singlet states. In the literature, these phenomena and designations are clearly distinguished from each other. A large number of phosphorescent substances are known, but in general 6,90695 phosphorescent substances are solids or substances that phosphorize at low temperatures and are not commonly used to label biomolecules. However, some metalloporphyrins have been found to phosphorylate at room temperature in organic solvents (M.

P. Tsvirko & et al., Optica i Spectroscopy 34, 1094-1100, 1973). Platinopropoproporphyrin and palladium coproporphyrin have subsequently been found to phosphorize even with water as a solvent when the phosphorescent compounds are protected by the detergent 10 from the quenching effect of water and oxygen. The idea and method for using them to label biomolecules has also been proposed (A. P. Savitsky et al., Dokl. Acad. Nauk. USSR, 304, 1005, 1989). Oxygen, a potent quencher of triplet states, can be removed from the measurement solution by a variety of known oxygen scavengers added to the measurement solution. The simplest such substance is, for example, sodium sulfite (Savitsy & al. USSR patent 4201377). The advantage of porphyrins compared to e.g. lanthanide chelates is their strong light absorption and excitation at 380 to 400 nm, while lanthanide chelates have an absorption and excitation wavelength in the UV range of 280 to 360 nm, which requires a more difficult wavelength range in special construction, because -kaa. The emission wavelengths of platinumoproporphyrin and palladium coproporphyrin: 25 range from 640 to 670 nm.

The method according to the present invention is described in more detail by means of an exemplary diagram in Figure 1, which shows the apparatus with which the assay method according to the present invention can be carried out in practice. The numbers below refer to the components in Figure 1. Suitable pumps and valves, but not shown in Figure 1, are required for fluid handling. Fluorescent microparticles representing different analytes are coated with antibodies binding the different analytes and mixed into a single buffered suspension in a container 1. 10 μΐ portion of the serum sample 2 and 10 μΐ of the phosphorescent molecule-labeled secondary

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7 90695 of the mixture of substances 3 and 10 μΐ of the microparticle suspension 1 are injected into the reaction cuvette 4 and incubated, for example, at 37 ° C for a precisely controlled time of several minutes or longer. The microparticle suspension 5 is diluted or washed with phosphate-buffered saline in tank 5. During the washing, the microparticles are immobilized in the reaction cuvette either by a filter, magnetic forces or the like. The final step 10 of the wash is performed with measuring solution 6, which protects the phosphorescent molecules from quenching (e.g., a buffered aqueous solution pH 7.2 to 7.4 containing 4 mg / ml sodium sulfite and 1% Triton X-100). The microparticles in the measurement solution are then transferred as a suspension one at a time to a capillary fluorine-15 sensing detector cuvette 7, which is illuminated with a monochromatic 380-400 nm light source 8 to determine the class of microparticles based on the signal strength of the fluorescence detector 9.

The microparticles are then transferred one at a time to 20 capillary phosphorescence detector cuvettes 10. The photon emission is measured by a photon detector 11 operating in the range of 640 to 670 nm, which is optically connected to the detector cuvette. The photon counter 12 readings obtained for the different microparticles are proportional to the different readings in the serum sample; : 25 for analyte concentrations.

The method according to the present invention and the equipment required for it can be implemented in practice in many different ways. However, it is essential in the invention that each individual microparticle representing a different class of analyte is identified and measured separately and thus a multi-parameter biospecific assay can be performed on the same sample. Identification is based on fluorescence measurement and measurement of the biospecific reagent label on phosphorescence, as described above. With a time-resolved measurement system, the relatively short-lived fluorescence emission from the detection dye and the background can be distinguished from the phosphorescence emission because their light emissions occur at different times and the fluorescence emission is completely stopped before the phosphorescence emission through. The essential content of the invention is here.

Fluorescent microparticles can be prepared by combining a suitable fluorescent compound with a polymeric material. Organic fluorescent compounds, e.g., POPOP, bisMSB, fluorescein, rhodamine, etc., can be added to the monomers in the preparation of microparticles ("Theory and Practice of Scintillation Counting", Ed. JB Birks, Pergamon Press, 1967, pp. 321-353) and thus the polymerization forms a solid fluorescent material. The material is processed into microparticles in the same process. The compound used to identify the microparticles is added to the different batches in different amounts so as to form microparticles whose fluorescence intensity differs substantially and by a factor of two.

The analysis of the microparticles can be performed in a capillary measuring chamber either by stopping the flow or while the flow is running. In the measuring instrument, microphotometric observation of the microparticles takes place in a thin measuring cuvette into which the diluted microparticle suspension is introduced. The microparticle suspension is first measured with a fluorescence detector and the analyte class of each microparticle is determined by the intensity of the fluorescence. Next, the concentration of the analyte is determined by measuring the phosphorescence intensity. The aperture of the Mit-30 probe cuvette is optimally selected according to the size of the microparticle used, and the phosphorescence emission is measured with a photomultiplier tube or other sensitive photon detector or a suitable image detector. A microchannel image amplifier and a half-wire wired image detector (CCD) can also be used as a detector. A sufficient number of microparticles are identified and analyzed statistically to obtain an accurate result for each analyte. The measurement results are related to the measurements performed on standard samples, on the basis of which the final results are calculated.

It is also characteristic of the present invention that the microphotometric measurement system disclosed herein is capable of optically distinguishing light emission from the surface of microparticles from light emission from a solution in the vicinity of the microparticles. This resolution is based on the fact that when the suspension is diluted considerably, only one microparticle fits into the focus area of the microphosphorometer at a time and the background signal caused by the free labeled reagents in the solution is negligible. This method achieves sufficient resolution of the free and bound fraction, but if necessary, the separation can be enhanced by any known separation method such as washing, filtration or centrifugation.

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

Claims (5)

1. Biospecific multiparametric assay method, using pre-made, divided into different categories and different analytes representing microparticles, where the microparticles of different categories contain different amounts of fluorescent dye and where the various categories of the microparticles are coated with biospecific reagents, such as biospecific reagents. various analytes, wherein - the various microparticles belonging to different categories are mixed into a suspension and the sample to be analyzed is added to this suspension, - to the suspension is added a mixture of biospecific secondary reagents, whereby a bioaffinity-based binding reaction starts between the analyte molecules, the labeled - the suspension is diluted to decrease the concentration of free labeled reagents, - the fluorescent dye is activated by light irradiation and the intensity of the fluorescence emission is measured for each The fluorescence signal is converted into an electrical signal to identify the category of the microparticle on the basis of the strength of the electrical signal derived from the fluorescence signal, characterized in that the mixture of the secondary reagents is labeled with a phosphorescent molecule and that phosphorus each microparticle is measured with a time-resolving photon detector and on the basis of the strength of the electrical signal received from the detector, the amount of analyte attached to each microparticle is analyzed. Method according to Claim 1, characterized in that the fluorescent substance on the microparticles is a compound which achieves especially short-term fluorescence emission. 3. A method according to claim 1 or 2, characterized in that a derivative of platinum acoproporphyrin or palladium coproporphyrin is used as a label for the secondary reagent. 4. Reagent packaging for biospecific multiparametric assay containing pre-made, divided into different categories and different analytes representing microparticles, where the microparticles of different categories contain different amounts of fluorescent dyes and where the various categories of microparticles belonging to them are coated with biospecific reagents, different analytes, and biospecific secondary reagents, representing different analytes characterized in that the biospecific secondary reagents are labeled with a phosphorescent molecule. 5. Reagent packaging according to claim 4, characterized in that the biospecific secondary reagents are labeled with a platinum acoproporphyrin or palladium coproporphyrin derivative.