FI89837C - Biospecific assay method - Google Patents

Description

8 9 8 3 7 BIOSPECIFIC METHOD OF DETERMINATION - BIOSPECIFIK BESTÄMNINGS-

Metod

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, forming a complex of three molecules (a layered structure of one) and a normal reagent. is stamped. 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 labeling labels are significantly overlapping 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 multiparameter biospecific assays as well as a reagent kit for use in the method.

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.

2 Ö9S37 - These microparticles of different classes are coated with biospecific primary reagents that bind the analytes in question and then the microparticles are mixed together into a suspension.

5 - Biospecific secondary reagents that bind different analytes are labeled with a molecule that generates or catalyzes chemical or bioluminescence, and these reagents are mixed together.

- A sample to be determined and a mixture of 10 biospecific secondary reagents are added to the microparticle suspension, followed by a binding reaction between the different analyte molecules, the microparticle-bound reagents and the labeled biospecific reagents.

- 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 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 it is possible to determine 20. microparticle class.

- When the fluorescence emission is completely extinguished, the luminescent label bound to the microparticles is activated by adding an activator to the suspension.

- The luminescence 25 generated as a result of the addition of the activator is measured by a photon detector, the electrical signal of which is proportional to the analyte concentration.

The object of the invention appears in more detail from the claims.

It is an object of the present invention to provide a method for performing a multiparameter biospecific assay, the method of which is based on the use of fluorescent microparticles and a luminescent label, and in particular that the fluorescence signal and the luminescence signal can be measured over a wide dynamic range without interference. 5 because the emissions take place at substantially different times. This multiparameter assay is performed on a suspension of artificially prepared microparticles, which is a mixture of different classes of microparticles that bind different analytes. Microparticles belonging to each of the different classes are first coated with a specific primary reagent (antibody, DNA, RNA), i.e. the microparticles act as a solid carrier 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 a fluorescent dye and a measurement based on lu-mini-sensitivity to determine the concentration of an analyte on the surface of the microparticles by a biospecific reaction.

Uniform sized microparticles with a diameter of 1-200 μιη (but not limited to these sizes) can be made from a suitable polymer that is hydrophilic to the extent that they are well suited for aqueous suspension. The surface properties of microparticles 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 a 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 possible 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 complexes of reagent and analyte labeled with reagents H 9 n / 7 4 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 run the reaction to 5 endpoints. 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 luminometrically. In most cases, a one-order dilution of 10 is sufficient to distinguish the signals from the bound and free labeled reagent fractions sufficiently efficient, because the microluminometric detector used in this method can optically distinguish the luminescence emission of the microparticle 15 at the focal point of the measurement chamber from the surrounding free labeled reagent. A sufficiently sufficient number of microparticles are analyzed and the intensity of the luminescence signal for each particle is recorded on a computer.

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

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

I; 89837 The sensitivity of the measurement method presented in this invention is based on the optimization of the luminescence reagents used therein and the concentrations of cofactors and enzymes required in connection therewith. Either core luminescence (e.g., peroxidase-luminol) or bioluminescence (e.g., luciferase-luciferin) (both referred to herein as "luminescence") can be used in this method. Both prokaryotic luciferases (e.g., Vibrio, Photobacterium, Xenorhabdus, etc.) and eukaryotic luciferases (e.g., Photinus pyralis, Pyrophorus plagiothalamus, Lampyris noctiluca, Pholas dactylus, Vargula hilgen-dorfrata together, etc.) can be combined. (luciferins), the necessary coenzymes and additional enzymes so that the light emission from the sample is a short-term strong pulse of light instead of a continuous light output. In addition, optimal concentrations of photoproteins such as aequorin, obelin, folasin or ofiophilisin can be used. If the luminescence reaction occurs on or in the vicinity of the enzyme, peroxidases or oxidases may also be used in combination with luminol or acridines (e.g., lucigenin) to generate a sufficiently strong luminescence signal for the measurement method described in this patent. These luminescent labeling methods, together with light emission amplifiers and modulators, which determine emission wavelength and kinetics, etc., are described in detail in Campbell, A. "Chemiluminescence; principles and applications in Biology and medicine", Weinheim; Deerfield Beach, FI .; VCH; Chichester: Horwood, 1988. It should also be emphasized that the above-mentioned luciferases and photoproteins can also be combined by recombinant DNA technology into IgG-binding protein A or G (Nilsson, B. & Abrahamsen, L., in Methods Enzymol., Can). 185, pp. 144-161, 1990) or directly to 35 specific recombinant antibodies such as single chain antibodies (Chiswell & McCafferty, TibTech, Vol. 10, pp. 80-84, 1992). As mentioned above, the applicability of the above-mentioned labeling methods to the 89837 6 assay method of the present invention can be further increased.

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 are required for fluid handling, but are not shown in Figure 1. Fluorescent microparticles representing different analytes are coated with antibodies that bind different analytes and mixed together into a single buffered suspension in a container (1). 10 μΐ of the serum sample (2) and 10 μ 10 of the mixture of biotin-labeled secondary antibodies (3) and 10 μ ja of the microparticle suspension (1) are injected into the reaction cuvette (4) and incubated at 37 ° C lasting several minutes or longer. The microparticle suspension is diluted or washed with phosphate-buffered saline in tank 5 additionally containing bovine serum albumin (100 mM Na-K-phosphate, pH 7.4, 0.9% NaCl, BSA 1 mg / ml) for 20 min. During the washing, the microparticles are immobilized in the reaction cuvette either by a filter, magnetic forces or the like. A buffered solution (6) of streptavidin covalently labeled with firefly luciferase is injected into the reaction cuvette (4) to obtain biotin in a form suitable for the luminescence assay. The microparticle suspension is then introduced into a capillary fluorescence detector cuvette (7), which is illuminated with a monochromatic light source (8) to determine the class of microparticles based on the signal strength provided by the fluorescence detector (9). The same suspension is then introduced into a capillary luminescence detector cuvette (10) together with an activated activating solution (11) containing 0.1 M TRIS-C1, pH 7.75, 0.1 mM D-Luciferin, 0.1 mM ATP, 1 μΜ pyrophosphate, 1 mM MgCl2 and 1 μΜ coenzyme A. This solution activates the luminescence reaction and the emitted photons are counted with a photon detector (12)

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% 9 8 3 7 7 connected to the detector cuvette. The photon counter readings obtained for the different microparticles are proportional to the concentrations of the different analytes in the serum sample.

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 analytes is identified and measured separately and thus a multi-parameter biospecific assay can be performed on the same 10 samples. Identification is based on fluorescence measurement as described above. With a suitable measurement arrangement, relatively short-lived fluorescence emission and luminescence can be distinguished because their light emissions occur at different times and the fluorescence emission is completely stopped before the luminescence reaction is activated and does not significantly interfere with the luminescence determination of the analyte concentration. 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, etc., fluorescent lanthanide chelates, and fluorescent inorganic microcrystals can be added to monomers in the preparation of microparticles ("Theory and Practice of Scintillation Counting", Ed. JB Birks, Perga-Mon Press, 1967, p. Press). -353) and thus a solid fluorescent material is formed in the polymerization. An important aspect in the selection of a fluorescent compound is that its light absorption region does not fall in the same region as the luminescence emission. 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 with a fluorescence intensity that differs substantially by a factor of 35 and e.g. by a factor of two.

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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, micropropometric detection 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 suspension is activated by syringes-10 by introducing a luminescence activator. The analyte concentration is determined by measuring the luminescence intensity. The aperture of the measuring cuvette is optimally selected according to the size of the microparticle used and the luminescence emission is measured with a photomultiplier tube or other sensitive photon detector or a suitable image detector. A microchannel image amplifier and a semiconductor image detector (CCD) can also be used as a detector. A sufficient number of microparticles are identified and analyzed to obtain a statistically 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 microluminometric measurement system disclosed herein is capable of optically distinguishing light emission from the surface of microparticles from light emission from a solution. This resolution is based on the fact that when the suspension is diluted considerably, only one microparticle fits into the focus area of the microluminometer at a time and the background level signal from the free labeled reagents in solution 30 is low. 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.

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Claims (4)

9 H 9 o X 7 1. A biospecific multiparameter assay method using prefabricated microparticles of different classes and representative of different analytes, in which different classes of microparticles contain different amounts of 5 fluorescent dyes and in which different classes of microparticles are coated with different analytes-binding biospecifiers the microparticles are mixed together into a suspension and the sample to be analyzed is added to this suspension, to reduce the concentration of labeled reagents, - the fluorescent dye is excited by light radiation and the fluorescence emission intensity is measured for each particle - the fluorescence signal is converted into an electrical signal for the identification of a class of microparticles on the basis of the strength of the electrical signal obtained from the fluorescence signal, characterized in that - the mixture of secondary reagents is labeled with a luminescence which generates or catalyzes luminescence on the surface of the microparticles and produces luminescence emission and luminescence emission is measured by an optically resolving photodetector and the amount of analyte molecule associated with each microparticle is analyzed based on the strength of the electrical signal obtained from the detector. Process according to Claim 1, characterized in that the fluorescent agent for the microparticles is a compound which causes very short-lived fluorescence emission, such as POPOP or bisMSB. Process according to Claim 1, characterized in that the microparticle fluorescent substance is a compound which causes prolonged fluorescence emission, such as lanthanide dicelate or a fluorescent inorganic microcrystal. 4. A reagent kit for a biospecific multiparameter assay, characterized in that it comprises, in separate packages, 20. prefabricated microparticles of different classes and representative of different analytes, wherein the microparticles of different classes contain different amounts of fluorescent dye and the microparticles of different classes are analyte-binding biospecific reagents, - a mixture of secondary reagents labeled with a biospecific luminescence-generating or catalyzing molecule, and - an activator that generates or catalyzes luminescence on the surface of microparticles and provides luminescence emission. I: 1X 8 9837