FI111758B - Use of lyoluminescence for analytical purposes - Google Patents

Description

Use of Lyoluminescence for Analytical Purposes 111758

Lyoluminescence (LL) is the process by which the dissolution of a substance produces light (G. Reynolds, Journal of Luminescence, 54, 43 (1992)). The present invention relates to the utilization of the 5 loluminescence phenomenon in the detection of labels in biochemical and analytical and clinical chemistry assays.

Luminescent (fluorescent or phosphorescent) labels are often used as aids in various analytical methods. By means of these, another substance 10 (X) is labeled or labeled and (X) can be identified and its amount analyzed by a label using various known techniques. Such labels, organic compounds and metal chelates, may be excited by light or electrically and thus produce a specific luminescence for the labels (I. Hemmilä, "John Wiley & Sons, New York, 1991; E. 15 Diamandis. , Clinical Biochemistry, 23, 437 (1990), L. Faulkner and A. Bard, in "Electroanalytical Chemistry", Vol. 10, A. Bard, (Ed.), Marcel Dekker, New York, 1977, p. 228). The mechanism of lyoluminescence, which is only partially known in mechanism, has previously been applied only to dose measurement of radioactive radiation such that the radiation forms 20 holes and electrons trapped in solid state alkali halides or sugars in proportion to the duration and intensity of the radiation. The dose of radioactive radiation is obtained by dissolving the crystals in the luminophor solution and measuring the intensity of the light produced (G. Reynolds, Journal of Luminescence, 54-992) 43).

It is known from radiation dosimetric studies that certain hydrated metal ions: ·· and organic luminophores can be excited by LL-1 processes in aqueous solutions (G. Reynolds, Journal of Luminescence, 54 (1992) 43). However, information on the concentrations of various rare metal ions or complex fluorophores is generally of no practical importance. In addition, in previous studies; v, 30 LL intensity dependence on the concentration of luminophores has produced narrow. · * ·. in the concentration ranges, bell curves where two different concentrations of luminophore produce the same signal (G. Reynolds, Journal of Luminescence, 54 (1992), 43, '' Figures 24 and 25). Such a system has no use in analytical chemistry and for these reasons has not been able to apply LL to practical analytics.

: T: 35 By the experimental arrangements of the present invention, the clock curves are avoided and the techniques disclosed exhibit excellent responses of the labels, the graphs of which are linear over a range of 10 orders of magnitude. In contrast to the radiation dosimetric methods, these methods work in 2 111758, that is, the salt is irradiated at a constant dose, and the amount of molecules labeled with a luminophore is usually measured on the basis of the light produced in the solution. This invention mainly relates to cases where the analyte itself cannot be made luminescent by the LL process, but the amount of analyte is indirectly measured using luminescent labels (L). They are generally covalently bound to either analyte (X) or to agent (B) selectively binding to the analyte, such as an antibody. The amount of analyte (X) can be measured indirectly either by a competitive reaction with (X) and labeled (X), (XL), or by allowing the labeled (B), (BL) to react selectively with (X): with 10 to form a complex (XBL). In all cases, the parameter to be measured in the last step is the label, which in the present invention is usually measured by LL after suitable separation or washing steps. According to the present invention, LL-based measurement provides a significant advantage over a portion of bioaffinity-based assay methods.

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Typically, analyte (X) is a biomolecule that is measured by an antibody that is extremely selective in binding to it based on bioaffinity. It may also be selective binding reactions between nucleic acids or any other process capable of separating the labeled bound analyte (X-L, X-B-L or the like) from free (X-L) or (B-L). It is essential that a selective binding and / or purification and separation process is achieved between the free and the lemma. . equivalence that gives the original amount of (X) in the sample directly or »·! inversely proportional to the LL signal. Unlike the stamp itself; measurement, measurement of non-luminescent analytes is remarkably economical '; 25 in clinical diagnostics and environmental and agrochemical applications.

• ·

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• '* In measurements based on the binding properties of biological molecules, such as

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In immunoassays, the analyte (X) to be measured can be selectively fixed from a mixture of different molecules to a solid-phase antibody and adhered to it: 30 molecules can be measured by means of another antibody, also labeled or labeled as (X). with a suitable marker. Tracers may be ·: ·· radioactive isotopes, enzymes, light-absorbing, fluorescent or phosphorescent molecules, certain metal chelates, etc., which are attached by a solid bond to an antibody. Alternatively, the purified (X) can be labeled and determined with the help of • i i! . The amount of 35 unlabeled (X) unknown samples in a competitive reaction. Many measurement methods for DNA and RNA are based on selective bioaffinity and can thus be measured analogously. Other chemical and biochemical analyzes can also be performed. Various measurement techniques and strategies that can be used in Immunodiagnostics 3,117,558 are described in The Immunoassay Handbook, edited by David Wild, Stockton Press Ltd., New York, 1994, pages 1-618.

The two main lines of development in biochemical, clinical chemistry, and environmental chemistry, each with their own advantages, are, on the one hand, accurate, rapid and standardized analyzes performed on large complex automated devices in research centers and large hospital laboratories, and on the other.

The present invention relates in particular to the latter processes. They can be used, for example, to monitor a patient's own illness at home, for quick diagnoses at a doctor's office, or for agrochemical or environmental analyzes in the wild. In all of these, the speed at which the results are obtained and the freshness of the sample as well as the ease with which the analytical result is obtained are the 15 most important factors. However, in this case, the measuring device must be as cheap and easy to use as possible, so that its acquisition or learning to use it is not an obstacle. On the other hand, since the number of assays to be measured is often relatively small, the cost of chemicals needed for one assay can be much higher than for large lab automators. Optimally, the assay method is a sophisticated, low-skill, preferably commercially made, test series of pre-dosed chemicals. After the sample is added; v the analytical result is obtained with a simple, easily transferable device with

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! broken or serviceable parts.

'; The use of radioactive labels in the methods of the present invention is excluded due to radiation legislation, although the equipment required may be simple and otherwise appropriate. Photometers are available that can measure the light absorption of colored solutions, but methods based on this are often too insensitive. In addition, the need for optical · '· * 30 components in photometers makes the device expensive. For special purposes small '··. cheap portable light meters, luminometers. These are equipped with ·; · ', * photomultiplier tube or photodiode capable of measuring very low luminous intensities. Luminometers are mainly used in biochemical applications. |. for measuring light from luciferase reactions. Basically luciferase enzyme '. 35 can be used as a label. In this case, the luciferase reaction must always be coupled with biochemical ATP or NADH formation or removal reactions, which makes the method difficult and, in the case of highly degradable reaction components, poorly preserves the test system. Methods based on fluorescence and phosphorescence (luminescence) 4111758 are generally very sensitive. Such devices require special excitation by electrical or pulsed light. This makes the measuring device complicated and expensive.

The object of the invention is a simple method and apparatus for the detection of label compounds which can be utilized in particular in bioaffinity based assay methods, such as immunoassay and DNA probe methods used by non-experts or in specific conditions at home, in doctors' offices, on farms or in environmental chemistry.

This object is achieved by using luminescent labels under properly selected conditions, such that the labels are chemically excited with LL and the resulting light is measured by a simple luminometer or the amount of light produced can be analyzed by a device having no electrical components by a photosensitive film. as described. A particular advantage of lyoluminescence tuning is that the equipment does not require expensive light sources, optics, potentiostats, expensive electrode materials such as gold or platinum, or chemicals hazardous to the environment and users. The cost of such a device is low and the use is simple or in some cases the actual device is not needed at all. The immunological methods themselves may be highly sophisticated, making analysis easy.

i 't The method is based on storing radiation energy in solid state reagents! UV irradiation or coagulation of energetic reducing metals; and

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! 25 release of stored energy by the dissolution process. An amount commensurate with the amount of analyte is allowed to excite during the dissolution process,, as a result of redox reactions * I, · 'generated by radicals moving from the solid state to the solution, or energy transfer from excited ions or molecules formed by these radicals. Also, some metals, such as aluminum and magnesium, when dissolved, can produce highly reactive intermediates, which often function as efficiently in LL- * systems as the intermediates produced by irradiated salts. The following illustrates the physical and chemical mechanisms involved in LL.

, ·, I 35 Energy storage during gamma or X-ray irradiation is based on the formation of trapped electrons and gaps in the solid reagent (reaction 1). However, such an irradiated product is poorly suited for practical analysis due to the fact that both light and temperature elevation 111758 cause recombination of electron-hole pairs, which problem can be avoided by the following staining methods. Energy can also be stored in solid state in the so-called. additive coloring methods (Reactions 2a-2b) and electrolytic coloring methods (J. Schulman and W. Compton, "Color Centers in 5 Solids", Pergamon Press, New York, 1962) (Reactions 3a-3b). The F-center is an electron trapped in the halide ion vacancy and the V-center is an opening analog of the F-center.

hv> 10 eV

(1) alkali halides -> ^ centers + ^ centers 10 alkali metal vapors at high pressure and temperature (2a) alkali halides -> F centers 15 halogen gas at high pressure and temperature (2b) alkali halides V centers 20 point cathode at high temperature (3a) alkali halides At high temperature:. (3b) Alkaline halides -> Centers t * · · • · · · · · · · · · · · · · · · · · · · · Energy storage can also be based on fragmentation of molecules, eg ... homolytic cleavage of peroxodisulfate during UV irradiation (reaction 4) or isomerization (reaction 6).

»· * Hv> 4eV

C: 35 (4) S2082- - 2 SO4-

• h v> 4eV

(5) P2082- -> 2 P042-

; ; ; h v> 4.8 eV

. ·. : 4 0 (6) KN03 -> potassium peroxonitrite 6 111758

The energy stored during the above reactions (1-6) can be released by dissolving the treated solid in solution. The radicals thus formed may produce luminescence by redox mechanisms (e.g., reactions 7a-7b). In reactions 7c-7e, A represents an organic redox excitable molecule (e.g., luminol 5 or fluorescein) or a metal chelate (e.g., Ru (bpy) 32+ or Tb chelates having an aromatic moiety in the ligand).

h2o (7a) F-center eaq- (hydrated electron) 10 H2O (7b) V-center - + X (X = F, Cl or Br) (7o) A + eaq- (or + X :) -> Ared (or Aox + X) -) 15 (7d) Ared (or Aox) + X (or eaq-) A * (7e) A * -> A + hp (visible light) 20

The excitation of the label-specific luminescence can also be based on energy transfer (reactions 7f-7h).

* ·; (7f) eaq- + X · -> (X -) *: 25 * * · •; - | (7g) (X -) * + A ^ X- + A * (7h) A * -> A + hv (visible light) 30 I ·, ·· *. If the solvent itself or its additives can function energetically as a suitable oxidant or reducing agent, excitation can be accomplished by the addition of a solid 'LL' which, upon dissolution, produces only an oxidizing or solely reducing radical. For example, by addition of additively colored alkali halide: 35 to a solution containing peroxodisulfate ions are obtained in aqueous solution; at the same time, extremely oxidizing and reducing conditions whereby a maximum of 6.3 eV (8a-8b) of energy can thus be introduced into the luminescent reactions.

7 111758 h2o

(8a) Fkeskus -> eaq. E ° (eaq-) = -2.9 V vs. SHE 5 (8b) eaq- + S2082- -> S04- + S042-. E ° (S04 / S042 ') = 3.4 V vs. SHE

(8c) eaq- + P2084- P04 · 2- + P043- · E ° (P04-2- / P043-) = 2.5 V vs. SHE

(8d) eaq- + H 2 O 2 - »OH + OH-, E ° (OH / OH ') = 2.7 V vs. SHE

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Similarly, the luminescence process can be initiated by dissolving the oxidizing hydrated radical generator. In the exemplary case (Reactions 9a-9c), solvent 15 acts as an excitatory reducing agent. Instead of a solvent, a radical former which produces a strongly reducing radical (e.g., formate ion) as a result of single-electron oxidation is often more suitable as a reducing agent.

20 h 20 (9) UV-treated K 2 S 2 O 8 -> 2 SO 4 '+ 2 K +; (9a) Tb (III) + S04 - »Tb (IV) + S042 '- i 25 (9b) Tb (IV) + water -> Tb (III) *: (9c) Tb (III) * -> Tb ( lll) + hv t * 30; V Some labels, such as luminol and isoluminol derivatives, can be excited by producing strong oxidants such as sulfate, phosphate, hydroxyl, halogen or dihalo radicals in the immediate vicinity of the labels without the presence of a strong reducing agent being necessary. In this case, the luminophores can be excited according to the following reaction scheme '; * *: (10a) Luminophor + n Ox- - intermediates + n Οχ- (10b) intermediates -> product + hp 8 111758

Ox above is one of the strong single-electron oxidants mentioned above and Luminophore is, for example, a derivative of luminol or isoluminol. For example, luminol produces, when oxidized, radical intermediates and, as the final intermediate, 3-5 aminophthalates in the excited state, which ultimately emits light.

Highly reducing metals can be used to produce lyoluminescence, for example by contacting an exposed metal surface with an aqueous solution by etching the protective oxide layer with an acid or base, after which either the active metal surface itself acts as a radical-generating reductant, or dissolved al , Al (I) or Al (II) also act as a reducing agent in the solution (i.e., at a distance greater than the tunneling distance of the electron from the metal / solution interface). The reducing agent should generally produce secondary high oxidizing radicals (e.g., hydroxyl, sulfate, or phosphate) from suitable starting materials such as, for example, dissolved oxygen, hydrogen peroxide, peroxodisulfate or peroxodiphosphate ions in accordance with the above reactions (8b), that in the reactions, instead of the hydrated electron, the reducing agent may be a reducing metal, a metal ion, or an atomic hydrogen.

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The energy released in the above leaching process is converted into light. , by means of a label compound based on the production of redox reactions (or energy transfer) »; ; luminescence of the label compound. The analytical method itself is based; ; for measuring the number of label molecules. Between label molecules and analyte »*; Equivalence is achieved in known ways, such as based on bioaffinity (see Figs.

1> · V »e.g., The Immunoassay Handbook, Edited by David Wild, Stockton Press Ltd., New; "York, 1994, pages 1-618). The label compound may be a luminescent metal chelate or

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'·' 'Is an organic molecule or a polymeric particle containing luminophores attached through a suitable moiety to a bioactive molecule. According to the invention, I t Ί! When the dissolution process is stabilized, the same number of label molecules yields the same I t »· ... · light signal. The amount of light can be integrated over the whole or only part of the dissolution *; ··.

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With a Lyoluminometer, tuning is usually done with a disposable test tube! , 35 contains a constant amount of LL-producing solid material, and the label removed from the solid carrier I * »': or, when the latex particles act as a solid carrier, a particle suspension is injected onto the salt while light is measured. Paramagnetic latex particles are particularly advantageous because they make the washing 9111758 simple and quick to perform. Other alternative procedures may also be used. For example, peroxonitrite in UV-irradiated potassium nitrate is very stable in basic solution (0.1 M NaOH), whereby this solubilization product can be treated like any liquid chemilunescent reagent and the luminescence process can be triggered by acid addition. Also, the LL-producing material can be dried into a film in a desired reaction vessel prior to irradiation, whereby a hundred LL-producing thin films or LL-producing powder can be dispensed into a liquid stream from a turret-type dispenser.

Lyoluminescence can be measured, depending on the size of the analyte assay range, either using a cheap semiconductor detector or, in the case of analytes requiring extreme sensitivity, using a photomultiplier tube. In difficult non-electric conditions, battery powered devices may be used, or the device may be based on the use of photosensitive film. Due to differences in film development processes, a standard light source may be needed for comparison, which may function as a so-called. Tritium lamp. It is comprised of a tritium-containing glass capsule, the inside of which is coated with a luminescent layer that is excited by the action of tritium to produce a standard light output. The glass capsule prevents β-radiation from entering the outside of the lamp, so this standard light source is completely safe in use and is used for example.

20 as components of the night guns of Finnish weapons. The light output from the tritium lamp to the film can be standardized to the desired level with optical filters and distance control. When using a film as a detector, the exact result can be read t * from the film with special densitometers, but with the sensitivity of Polaroid films'. ! it is sufficient that the analytical result be obtained immediately without the assistance of a photographic laboratory '; 25 comparing the brightness of the specimen spot with the brightness of the lower and upper standard spot produced by two differently calibrated standard light sources. The highest sensitivity when using a photosensitive film is achieved with x-ray films or other ultra-sensitive films (such as the Kodak 3200 ASA), but then film development in a photographic laboratory is essential. The advantage is, however, that a fresh sample can be used and only the film is sent for development. In this case, the result of the analysis is reliable, although the result is not: ··; immediately after the analysis. As is known, especially '' ': biological samples deteriorate to non-representative quite rapidly and cause large errors in the results J -. A photographic film can also be replaced by a battery-powered CCD or; 35 digital cameras, making the test result easy to use in a computer-readable format.

10 111758

Useful labels for use in the invention include, for example, derivatives of the following organic luminophores: 9-fluorenylmethyl chloroformate (emission 309 nm), luminol (emission 420 nm), fluorescein (emission 516 nm), salicylate derivatives (emission in the range 400-450 nm), aminon emission in the range of 400-500 nm) and coumarins (emission in the range of 450 nm to 550 nm), derivatives of aromatic lanthanide (III) chelates, such as suitable derivatives of terbium (III) complexes with 1 12 3 3 of the following ligands: N - (4-aminobenzyl) ) diethylenetriamine-N, N, N, N-tetraacetate (abbreviation Tb (III) -1), 4- (phenylethyl) (1-hydroxybenzene) -2,6-diyl) bis-methylenenitrile) tetrakis (acetate) (Tb (III) -2); 4-Benzoyl (1-hydroxybenzene-1) -2,6-diyl) -bis (methylenenitrile) tetrakis (acetate) (Tb (III) -3), N - (4-Aminobenzyl) -diethylenetriamine-N1, N1, N3, N3, -tetraacetate (Tb (III) -4); 4-methyl (1-hydroxybenzene) -2,6-diyl) bis (methylenenitrile) tetrakis (acetate) (Tb (III) -5) (strongest emission line for all Tb (III) chelates at 545 nm), derivatives of some of the transition metal chelates such as e.g., ruthenium (II) and osmium (II) trisbipyridyl and derivatives of trispyrazyl complexes (emission in the range 550-650 nm).

In the description of the invention some possible chemical reaction mechanisms for the production of lyoluminescence are described, but the process according to the invention may also be based on some other solid process of solubilization which results in excitation of the label. Similarly ^ the structure of the labels may vary so that they can emit light under UV light! range to infrared range and accordingly the measuring device is wide-ranging * · ·! 25 limits. In practice, the LL tuning itself can be performed in many different ways, depending on the device used. The invention is characterized by the features set forth in claims 1 and 2 *.

* 1 »* · ·

In the following, the invention will be further illustrated by diagrams and non-limiting thereof

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•; '30 with examples and related illustrations.

• »• 1 •• Il • ·: ': Figure 1. Block diagram of a Lyoluminometer. (1) A test tube or cell containing a solid material producing lyoluminescence. (2) Tube through which the solution or suspension of the label: 35 is injected into the lyoluminometer cell. (3) Optical filter selected on the basis of the emission spectrum of the label.

11 111758

Figure 2. Schematic of a lyoluminometer without electrical components. (1) Light-sealed housing. (2) Light-sensitive film. (3) A test tube or cell containing a solid material producing lyoluminescence. (4) The tube through which the solution or latex particle suspension is injected into the lyoluminometer cell. (5) 5 For a tritium lamp. (6) Tritium lamp2. (7) Tritium LampTrn Distance Adjusting Screw. (8) Tritium Lamp2 Distance Adjusting Screw. (9) Gray filter,. (10) Gray Filter2.

Figure 3. Detection of N- (6-aminobutyl) -N-ethyl isoluminol (ABEI) by various lyoluminescence methods. (a) Additively colored KCl, pH 11.5, 3x101M K2S20810 (open circle, dotted line), (b) Electrolytically colored KCl, pH 11.5, 3x10'4M K2S208 (open square, dotted line), (c) ) UV-irradiated K2S2C> 8, pH 12 (circle), (d) UV-irradiated IC2Og, (tip square), (e) UV-irradiated KNO3, pH 11 (square), (f) Aluminum, pH12, 0.03 M K4P208.

Figure 4. Lyoluminometric immunoassay of p2-microglobulin on latex particles based on a label detection step for UV-irradiated L2 produced by K2S208 or K4P208 or KN03 with AHEI.

Figure 5. Lyoluminometric immunoassay of p2-microglobulin on (a) the inner surface of the aluminum cup, (b) on the surface of the aluminum particles, or (c) on the surface of the latex particles based on a label detection step under aluminum conditions to produce LL with AHE.

* n * ", l; Figure 6. Lyoluminometric immunoassay of p2-microglobulin on the inner surface of Al cups, based on a label detection step on LL produced by an electrolytically alkalized aluminum electrode, the label compound being a Tb (III) chelate isothiocyanate derivative.

1 hr

Figure 7. Immunoassay immunoassay for thyroid stimulating hormone (TSH) with latex particles based on a label detection step based on '*; · * magnesium under acidic conditions to produce LL (a), aluminum under alkaline conditions to produce LL (b) or additively staining. .iin (c) where the label compound is a derivative of Ru (bpy) 32+ chelate.

Fig. 8. Lyoluminometric immunoassay of phospholipase A2 with latex particles based on a label detection step on LL produced by additively stained KCI, the label compound being an isothiocyanate derivative of Tb (III) chelate.

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Figure 9. Lyoluminometric immunoassay of phospholipase A2 with latex particles based on a label detection step on LL produced by additively stained KCl with the label compound being AHEI.

5 Figure 10. Lyoluminometric immunoassay of phospholipase A2.n latex particles based on a label detection step in the presence of aluminum P2084 " complex with Tb (III) -EDTA chelate prior to detection.

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Figure 11. Lyoluminometric immunoassay of li2-microglobulin on microtiter plates by first detaching the Tb (III) ion from the antibody and measuring the released Tb (III) ion with a new complex using a staining detection step based on UV irradiated K2S208 or to 15 LL with the label compound being an isothiocyanate derivative of Tb (III) chelate.

Figure 12. Standard plot of l $ 2 microglobulin lyoluminometric immunoassay with label containing liposomes containing luminophores and label detection based on LL produced by electrolytically stained KCl in the presence of S2082 'ions.

, Figure 13. Lyoluminometric immunoassay for ft2-microglobulin; ; * standard graph with UV-removable luminophor and stamp • · * '; ; the detection step being based on LL produced by additively stained KCI.

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Figure 14. Lyoluminometric immunoassay of β2-microglobulin based on AHEI-labeled antibody and label removal from solid support prior to production of LL with UV-irradiated potassium peroxodisulfate.

: 30 Figure 15. Lyoluminometric detection of the Philadelphia chromosome by DNA · ... 'hybridization with the label being Tb (III) chelate; · · · Isothiocyanate derivative.

! 35 **: Example 1. Preparation of energy salts and calibration curves of N- (6-aminobutyl) -N-ethyl-isoluminol (ABEI, Wallac Oy) by various LL methods.

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Additive staining of potassium chloride. 500 mg of microcrystalline KCl (Merck, Suprapur) was additively stained with a potassium vapor pressure of 15 torr and 630 ° C for 12 h.

5 Electrolytic staining of potassium chloride. KCl (Merck, Suprapur) was initially dried by heating at 500 ° C in vacuo, and then the single crystal was grown by the Czochralski method under argon atmosphere. A 12 x 12 x 20 mm piece of crystal was electrolytically stained by the matrix cathode method at 700 ° C with a current of 1 mA.

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UV irradiation of potassium peroxodisulfate, potassium peroxodiphosphate and potassium nitrate. Each salt was applied in turn to the glass plate surface as a thin layer (approximately a grain size layer) and irradiated for 4 h with a Philips TUV / 15W, G15T8 UV lamp at a distance of about 6 cm. After irradiation, the salt was collected in a test tube and homogenized by shaking.

Convective injection. When the diameter of the injection tube and the geometry of the tube head and the shape of the LL cell tube are correctly selected, standard precision pipettes used in laboratories 20 can perform standard solution injection so that convection in solution in the cell is reproducibly similar from one injection to another. Most preferably, the injection is battery-powered; ; * a stepper motor pipette with adjustable injection speed.

I · I »- 25 ABEI Calibration Curves. (a) 15.0 * mg mg of additively stained KCl was weighed into a 1.2 mL disposable tube and 1.00 mL was injected into the tube. ABEI solution in 3 µM NaOH in 3x10 ”4 mol / L potassium peroxodisulfate (open v’ circle, dashed line), (b) 15.0 mg was weighed in a 1.2 mL disposable test tube

electrolytically stained KCl, which after milling was finely ground to a powder of agate mortar, and 1.00 mL of ABEI solution was injected into the test tube.

In a NaOH solution containing 3 x 10 4 mol / L potassium peroxodisulfate (open square, dashed line), (c) 10.0 mg of UV-irradiated K2S208 was weighed in a 1.2 mL disposable tube and 1.00 mL of ABEI was injected into the tube. solution in 0.01 M NaOH solution (circle), (d) 10.0 mg of 35 UV irradiated K4P208 were weighed into a 1.2 mL disposable test tube and 1.00 mL of 0.01 M ABEI solution was injected into the tube. NaOH in solution (vertical), (e) 10.0 mg of UV-irradiated KNO3 was weighed in a 1.2 mL disposable test tube and 1.00 mL of ABEI in 1 mM M NaOH (square) was injected into the tube (f) 1.0 ml of luminol solution in 0.01 M NaOH solution, 14111758 containing 0.03 mol / L K4P208 was injected into an aluminum cup. Measurements were made with a photon-counted lyoluminometer (S. Kulmala and K. Haapakka, Analytica Chimica Acta, 294 (1994) 13), whose injection tube structure and geometry, and thus the hydrodynamics of the injection, were redesigned and the 450 nm interference filter was changed to 450 nm filter. . The measured calibration curves are shown in Figure 3.

Example 2. Lyoluminometric immunoassay of & 2-Microglobulin using 10 N- (6-aminohexyl) -N-ethyl isoluminol as a label). LL was produced with UV-irradiated potassium peroxodisulfate.

Antibody coating of latex particles. The latex particle stock suspension (Sigma, LB-8) was diluted 1: 100 in TSA buffer (0.05 mol / L Tris-HCl, pH 7.75, 0.9% 15 NaCl, 0.05% NaN 3). 100 µL of this dilution was taken, to which was added 100 µL of a solution containing 6 mg / mL of mouse anti-IJ2 microglobulin antibody (clone 6G12, Labmaster Oy, Turku) in TSA buffer and incubated overnight. The particles were separated from the supernatant by centrifugation and washed with wash solution (0.01 M Tris-HCl, pH 7.75, 0.9% NaCl and 0.02% Tween 20). Thereafter, the latex particles were saturated overnight in TSA buffer supplemented with 0.1% bovine serum albumin.

t

Preparation of labeled antibody. Another mouse anti-ft2 microglobulin antibody; (clone 1F10, Labmaster Oy, Turku) was labeled with N- (6-aminohexyl) -N-ethyl-? 25 isoluminol (AHEI) Schroeder et al. (Methods in Enzymology, Vol. 57, M. DeLuca (Ed.), Academic Press, N.Y., 1978).

# · V * Bz-Microglobulin standard. Standards (0.4, 1.6, i. · 30, 4.0, 8.0 and 16 mg / L) were prepared from β2-microglobulin (75.5 mg / mL, Labmaster Oy, Turku) purified from human ascites fluid. buffer supplemented with 7.5% bovine serum albumin.

* · *

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• <»•: · j Immunochemical Assay. 20 μΙ of a 1:20 diluted suspension of the coated latex particle (approximately 180 million latex particles) was added to 1.5 mL polypropylene centrifuge tubes saturated with bovine serum

I · I

! , 35 with albumin. The standards were diluted 1:50 in assay buffer (0.05 mol / L Tris-HCl, pH

'' 7.75 containing 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin and 0.01%

Tween 20) and added to the bottom of the cuvette (40 µL). Next, 120 µL of labeled antibody in assay buffer was added and incubated for 1 hour with shaking at room temperature, after which the particles were separated by centrifugation and washed 3 times with wash solution and once with 0.02% TWEEN 40 (p / p). The particles were then suspended in LL measuring solution (0.01 M NaOH) and injected into a measuring cuvette containing 10.0 mg of UV-irradiated K2S208. LL was measured as in Example 1e 5. The standard curve is shown in Figure 4.

Example 3. Lyoluminometric immunoassay of R2 microglobulin using AHEL as a label. LL was produced by dissolving aluminum in NaOH in the presence of P2084 'ions.

Antibody coating of the aluminum cup. Aluminum sheet (0.3 mm thick) was pressed into aluminum cups with a volume of 500 μί. The cups were coated with a mouse anti-IJ2 microglobulin antibody (clone 6G12, Labmaster Oy, Turku) by standing in cup for 500 overnight with 500 μl of antibody (10 pg / mL) in 0.2 M NaH 2 PO 4. The next day, the electrode was washed six times with wash solution and equilibrated overnight in saturation solution (0.05 M Tris-HCl, pH 7.75 containing 1 g bovine serum albumin, 60 g sorbitol, and 1 mmol CaCl 2). The aluminum cup was aspirated and stored dry.

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Antibody coating of aluminum filings powder. Aluminum filament powder was coated with a mouse anti-IJ2 microglobulin antibody (clone 6G12, Labmaster Oy, Turku) in a manner similar to that of Example 1 for coating latex particles.

'! 25 Preparation of labeled antibody. Another mouse anti-1332 microglobulin antibody (clone 1F10, Labmaster Oy, Turku) was labeled with AHEU as in Example 2.

£ 2 m / 'Krog / obu // "/> 7 / -stendard /. Standards were prepared from B2 microglobulin as in Example 2.

30

Immunochemical assay on aluminum cups. The standards were diluted 1:50 in assay buffer (0.05 mol / L Tris-HCl, pH 7.75 containing 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin, and 0.01% Tween 20) and added to the bottom of the Al-cup (40 μl). Next, 120 μl of labeled antibody in assay buffer was added and incubated for 1 35 hours with shaking at room temperature, after which the cups were washed 4 times with washing solution and once with distilled water. The cups were then transferred one at a time to the lyoluminometer and 400 µL of 0.01 M NaOH was injected into each Al cup.

16 111758 solution containing 0.03 mol / L <4P208. The light was integrated for 300 s and the measured calibration curve is shown in Fig. 5, curve (a).

Immunochemical assay using aluminum powder. 30 μΙ of a 1:20 diluted suspension of coated aluminum 5 powder was added to 1.5 mL polypropylene centrifuge tubes saturated with bovine serum albumin. The standards were diluted 1:50 in assay buffer (0.05 mol / L Tris-HCl, pH 7.75 containing 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin, and 0.01% Tween 20). ) and added to the bottom of the cuvette (40 μί). Next, 120 μl of labeled 10 antibodies in assay buffer was added and incubated for 1 hour with shaking at room temperature, after which the aluminum powder was separated by centrifugation and washed 3 times with washing solution and once with 0.02% TWEEN 40 (w / v). The test tube was aspirated dry and transferred to an LL measuring cell, after which 400 μl of 0.01 M NaOH solution containing 0.03 mol / L K4P208 was injected into the tube. The light was integrated for 300 s and the measured calibration curve is shown in Figure 5, curve (b).

Immunochemical Assay Using Latex Particles. The assay was performed as in Example 2 except that the particles were finally washed with distilled water supplemented with 0.02% TWEEN 40 (w / w), after which the particles were suspended in 400 µL of 0.01 M 2 O NaOH , 03 mol / L K4P208, and the suspension was injected into aluminum cups. The light was integrated for 300 s and the measured calibration curve is shown in Fig. 5, curve (c).

: Example 4. Lyoluminometric immunoassay of B2 microglobulin on the inner surface of A1 cups using Tb (III) chelate isothiocyanate as a label. LL was produced by electrolytic cathodization of aluminum in the presence of S2082 'ions.

The aluminum cups were coated with antibody as in Example 3.

* · 30 Preparation of labeled antibody. Another mouse anti-β-microglobulin antibody (clone 1F10, Labmaster Oy, Turku) was labeled with the Tb (III) -4 chelate isothiocyanate derivative [Tb3 + - N2- (4-isothiocyanatobecyl) -diethylenetriamine-N1, N1, N3, N3 - 'tetraacetate] (Wallac Oy, Turku) by reacting the antibody with chelate in a 1:60 molar ratio at pH 9.5. The pH is adjusted with 1 M Na 2 CO 3 solution. The labeled 35 antibody was purified from excess unreacted chelate by gel filtration (Sepharose 6B 1 x 50 cm, Sephadex G-50 1 x 5 cm) using TSA buffer (0.05 mol / L Tris-HCl, pH 7.75, 0.9 % NaCl, 0.05% NaN 3) as eluate. Typically, this 17111758 yields 5-10 chelate molecules bound to one antibody molecule. To improve shelf life, 0.1% bovine serum albumin was added to the labeled antibody.

Immunoassay with aluminum cups was performed as in Example 3. Subsequently, a 0.5 M solution of Na 2 SO 4 containing 0.01 mol / L K 2 S 2 O 8 was pipetted into the Al cup. A platinum wire was placed in the cell as a counter electrode and electrolysis was initiated at 10 V dc with the Al electrode as the cathode. The light was measured for 300 s with the interference filter being a 546 nm filter. The measured calibration curve is shown in Figure 10.

Example. 5. Lyoluminometric immunoassay for TSH. LL was produced by dissolving magnesium in hydrochloric acid, aluminum in sodium hydroxide solution or additively stained KCl in the presence of S2082 'ions.

15

The streptavidin-coated paramagnetic latex particles, the Ru (bpy) 32+ -labeled anti-TSH monoclonal antibody, and the biotin-labeled anti-TSH monoclonal antibody were made by Boehringer Mannheim (Elecsys TSH Immunoassay kit).

20

Production of standards. Standards were diluted from TSH stock solution (52300 pU / mL, Wallac Oy) in dilution buffer (Labmaster Oy, Turku). Standards! * concentrations were 9.0, 54.0 and 324 pU / mL.

• · = »» '; 25 Lyoluminometric immunoassay. The immunochemical assay itself was performed according to the instructions of Boehringer Mannheim, except that the standards were pipetted with 50 pL, »»

A solution of Ru (bpy) 32+ -labeled anti-TSH in 100 µL and a solution of biotin-labeled anti-TSH in 100 µL in a test tube where the first 25 min incubation was performed with shaking. After incubation, 200 µL of freshly: · * 30 mixed suspension of streptavidin-coated magnetic latex particles were pipetted into the mixture and shaken for 10 min. After washing according to the instructions, the particles were washed once more with distilled water supplemented with 0.02% TWEEN 40 (w / w). The particles were suspended: "*: finally in the measuring solution according to each method.

. In LL produced by magnesium, the particles were suspended in 1.00 mL of 0.1 M HCl

solution containing 0.005% TWEEN 40. The suspension was injected in a lyoluminometer into a test tube with a 1.0 cm piece of Mg tape (Merck art.

18 111758 no. 5812) and LL were integrated for 400 s with a lyoluminometer interference filter of 620 nm. The standard curve is shown in Figure 7, curve (a).

In aluminum-produced LL, the particles were suspended in 1.00 mL of 0.1 M NaOH solution containing 0.005% TWEEN 40 and 0.01 mol / L K 2 S 2 O 8. The suspension was injected into the Al cup on the lyoluminometer and LL was integrated for 600 s. The standard curve is shown in Figure 7, curve (b).

In LL produced by additively stained KCl, the particles were suspended in 1.00 mL of 10 0.2 M borate buffer containing 0.005% TWEEN 20 and 3x10 4 mol / L K 2 S 2 O 8.

The suspension was injected in a lyoluminometer into a test tube containing 25.0 mg of additively stained KCl powder. LL was integrated 10.0 s from the time of injection. The standard curve is shown in Figure 7, curve (c).

Example 6. Lyoluminometric immunoassay of phospholipase A2 (PLA2) with latex particles. LL was produced by additively stained KCl, the label being an isothiocyanate derivative of Tb (III) chelate.

Antibody coating of latex particles. 20 stock suspension of latex particles (Sigma, LB-8) were diluted 1: 100 in TSA buffer (0.05 mol / L Tris-HCl, pH 7.75, 0.9% NaCl, 0.05% NaN 3). 100 pL of this dilution was taken, to which was added 100 pL of a solution containing 5.7 mg / mL of anti-PLA2 antibody (clone 2E1, Labmaster, Turku) in TSA buffer and incubated overnight. Particles 7! was separated from the supernatant by centrifugation and washed with the wash solution. After this ! The latex particles were saturated overnight in TSA buffer supplemented with 0.1% bovine serum albumin.

Preparation of a Labeled Antibody. A polyclonal sheep antibody to human pancreatic phospholipase A2 (affinity purified, Labmaster Oy, Turku) * »* 30 was labeled with the Tb (III) -1 chelate isothiocyanate derivative [Tb3 + - N- (p- • * '···' isothiocyanatobecyl) -diethylenetriamine-N1, N2, N3, -tetraacetate], (Wallac Oy,

Turku) by reacting the antibody with chelate in a 1:60 molar ratio at pH 9.5. The pH was adjusted with 1 M Na 2 CO 3 solution. The labeled antibody was purified from excess unreacted chelate by gel filtration (Sepharose 6B 1 x 50 cm,; 35 Sephadex G-50 1 x 5 cm) using TSA buffer as mobile phase. Typically, I1 * thus obtains 5-10 chelate molecules bound to one antibody molecule. To improve shelf life, 0.1% bovine serum albumin was added to the labeled antibody.

i9 111758 5

Production of standards. Human pancreas PLA2 (Labmaster Oy, Turku) was prepared in TSA buffer supplemented with 7% bovine serum albumin, standard series 0, 1.5, 9, 54, 324 ng / mL.

Immunoassay. 20 µl of a diluted suspension of the coated latex particle 1.20 (approximately 180 million latex particles) was taken into 1.5 mL centrifuge tubes saturated with bovine serum albumin. 10 µl of standard and 20 µg of Tb (11) -1-labeled antibody (500 ng) was added to the suspension and incubated for 20 min at room temperature, after which the particles were separated by centrifugation and washed twice with wash solution and once with LL measurement buffer (0, 2 mol / L borate buffer, pH 7.3 supplemented with 0.02% TWEEN 40 (w / w)). The particles were then suspended in LL measurement buffer and LL was measured as in Example 1 (a) but with a 546 nm interference filter on the lyoluminometer. The LL assay buffer in this case was a 0.2 M borate buffer (pH 7.3) containing 3 x 10 4 mol / L K 2 S 2 O 8 and 0.02% TWEEN 40. The suspension was injected in a lyoluminometer into a test tube containing 20.0 mg of additively stained KCI.a. The standard curve is shown in Figure 8.

20

Example 7. Lyoluminometric Immunoassay for Phospholipase A2; latex particles based on the detection step of the label. LL was produced electrolytically; 25 stained with potassium chloride.

The method used the same antibody-coated latex particles and standards as in Example 6.

»I I

: · '30 Preparation of labeled antibody. Polyclonal sheep antibody '···' to human pancreatic phospholipase A2 (affinity purified, Labmaster Oy, Turku) •: was labeled with AHEU and purified as in Example 2.

,. Immunoassay was performed as in Example 6 except that

. The latex particles were finally suspended in a solution of 0.01 M NaOH containing 1 mmol / L

K4P208. The suspension was immediately injected in a lyoluminometer (450 nm interference filter) into a test tube containing 20.0 mg of powdered electrolytically stained KCl. The standard curve is shown in Figure 9.

20 111758

Example 8. Lyoluminometric Immunoassay for Phospholipase A2 Using Latex Particles as a Solid Carrier in an Assay Using Enzymatic Amplification. LL was produced by additively stained KCl or UV-5 irradiated K2S208.

The latex particles were coated with antibody as in Example 6.

Labeling of antibiotics with alkaline phosphatase. Polyclonal sheep anti-PLA? 10 was labeled by the alkaline phosphatase (ALF) maleimide method (E. Ishikawa, M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamuguchi and T. Ueno, J. Immunoassay, 1983, 4, 209).

Immunoassay. 20 µL of a 1:20 diluted suspension of the coated latex particle (about 180 million latex particles) was taken into 1.5 mL centrifuge tubes made of polypropylene saturated with bovine serum albumin. 20 µL of standard and 20 µl of ALF-labeled antibiotic (600 ng) were added to the suspension and incubated for 15 min at room temperature, after which the particles were separated by centrifugation and washed twice with the washing solution. The particles were suspended in 200 µL of a solution containing 1 mmol / L of substrate (phosphate ester of 5-fluorosalicylic acid, FSAF, Kronem

Systems Inc. Mississauga, Ontario, Canada). The supernatant was separated for 15 min ·. after incubation and 100 µL of supernatant was transferred to a test tube containing; ·. 0.900 mL of 0.01 mol / L K2S208 in 0.5 M NaOH. The solution was injected into an Al cup and: LL was measured through a 420 nm interference filter to give a standard curve (a),

, ,, | 25, shown in Figure 10. An alternative detection method was as follows: 150 L

:. supernatant was added to 850 µL of a 0.5 mM Tb (III) -EDTA solution in 0.2 M NaOH

, In solution, and the solution was mixed with a pipette and incubated for 15 min. 25 µL was added to the solution

* * · 0.01 M potassium peroxodisulfate solution and the mixed solution was injected into the Al cup. The LL signal was measured for 300 s at 545 nm to give PLA2 ·,,,, the 30 standard curves shown in Figure 10 (b).

Example 9. Lyoluminometric Immunoassay of B2 Microglobulin on Microtiter Plates by first detecting Tb ion after immunochemical reaction from 35 antibodies and measuring the released Tb ion with a new complex by labeling step based on UV-irradiated K2S208 or additively stained L2. being an isothiocyanate derivative of Tb (III) chelate.

2i 111758

Coating of microtiter plate with antibody. 200 μΙ (10 mg / mL) mouse anti-B2 microglobulin antibody (clone 6G12, Labmaster Oy, Turku) in 0.2 M NaH 2 PO 4 was added to the wells of the microtiter plate. The next day, the wells were washed six times with 5 washes (0.01 M Tris-HCl, pH 7.75, 0.9% NaCl, and 0.02% Tween 20) and equilibrated with a solution (200 µL / well) of 0.05 M Tris-HCl, pH 7.75, and 1 g bovine serum albumin, 60 g sorbitol and 1 mmol CaCl 2 per liter of solution. Next, the wells were sucked dry and stored moist.

The antibody was labeled with an isothiocyanate derivative of Tb (III) -1 chelate, as in Example 4.

Immunochemical assay. The standards were diluted 1:50 in assay buffer (0.05 mol / L Tris-HCl, pH 7.75 containing 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin, and 0.01% Tween 20) and added to the well (40 µL). Next, 160 µL of labeled antibody (500 ng clone 1F10, Labmaster) was added in assay buffer.

Oy, Turku). The immunochemical reaction was allowed to occur and after one hour the wells were washed six times with the washing solution and 200 µL of 0.1 M glycine (SO 2 buffer, pH 2.5) was added to the well and incubated for 15 min. Subsequently, the previous solution was taken into a 150 μ -4 20 L measuring cuvette and 50 µL of a 0.5 mol / L Na 2 SO 4 solution containing 5 × 10 mol / L ligand 5 was added and 800 µL of 0.2 Na 2 B 4 O 7 buffer was added. The formed Tb (III) -5 chelate was quantitated by injecting the solution in a lyoluminometer (545 nm: interference filter) into a test tube containing 10.0 mg of UV-irradiated K2S208.

I ·,: The standard curve is shown in Figure 11.

• *: ··; Example 10. Lyoluminometric Immunoassay of Microglobulin B2 Using Liposomes for Binding Tb-5 Chelate to Antibody. LL was produced electrolytically! ... with stained KCI and S2082 ions.

30

Liposomes containing the terbium-5 complex were prepared and then bound to an antibody (anti-U2 microglobulin, clone 6G12, Labmaster Oy, Turku) (GP.

Vonk, B. Wagner, Clinical Chemistry, 37, 1519 (1991). The immunochemical reaction was carried out as in Example 9 but in a buffer which did not contain surfactants. After washing, 230 µL of 0.1% Triton X-100 in 0.1 M glycine buffer (pH 3.2) was added to the well and incubated for 10 min. 200 pL of the solution was taken into a test tube and 60 pL of a 0.5 mol / L solution of Na 2 CO 3 containing 1 x 10 3 mol / L ligand 5 was added.

22 111758

Finally, 0.800 mL of 0.05 M Na2B407 buffer was added to the test tube, mixed and injected in a lyoluminometer (545 nm interference filter) into a test tube containing 20.0 mg of finely ground electrolytically stained KCl. The standard curve is shown in Figure 12.

5

Example 11. Immunometric determination of S2-microglobulin by UV-removable label. LL was produced by additively stained KCl in the presence of S2082 'ions.

The wells of the microtiter plate were coated in a similar manner as in Example 9.

Antibody labeling. The antibody (clone 1F10, Labmaster Oy, Turku) was labeled with UV-removable label (Rhodamine Green sulfosuccinimidyl Ester, Molecular Probes, R-7091, Eugene, USA) according to the manufacturer's instructions.

15

Immunoassay was performed in a well of a microtiter plate as in Example 9 except that 200 μΐ of 0.05 M Na2B407 solution was pipetted into the wells of the strip.

2 0 Removal of the stamp by UV light and EL measurement. The microtiter plate strip was exposed from above with a UV lamp (Philips HPLR) for 4.0 min. From each well, 180 µl of solution was taken .. into a test tube. To this was added 820 μί 0.05 M Na2B407 solution containing 5x1 O'4 mol / L

I; K2S2Oe and the mixed solution were injected in a lyoluminometer into a test tube containing; 30.0 mg of additively stained KCl. The Lyoluminometer had an optical filter of 500 µm at a 25 nm high pass filter. The standard curve is shown in Figure 14.

»» \ Example 12. Lyoluminometric immunoassay of B2 microglobulin using AHEL as a label. The label was removed from the solid support by chaotropic reagent and LL was produced with UV-irradiated potassium peroxodisulfate.

30

Coating of microtiter plate with antibody. To the wells of the microtiter plate was added 200 µL of pL (10 mg / mL) mouse anti-IJ2 microglobulin antibody (clone 6G12, Labmaster Oy, Turku) in 0.2 M NaH2PO4 solution. The next day, the wells were washed six times with washing solution (0.01 M Tris-HCl, pH 7.75, 0.9% NaCl and 0.02% Tween 20) and! , Was equilibrated with a solution (200 μΙ / well) of 0.05 M Tris-HCl, pH 7.75 containing 1 g of bovine serum albumin, 60 g of sorbitol and 1 mmol of CaCl 2 per liter. Next, the wells were sucked dry and stored moist.

23 111758

Preparation of labeled antibody. Another mouse anti-R2 microglobulin antibody (clone 1F10, Labmaster Oy, Turku) was labeled with N- (6-aminohexyl) -N-ethyl isoluminol (AHEI) according to Schroeder et al. (Methods in Enzymology, Vol. 57, 5 M. DeLuca (Ed.), Academic Press, N.Y., 1978).

βζ-microglobulin standard. Standards (0.4, 1.6, 4.0, 8.0, and 16 mg / L) of β2 microglobulin purified from human ascites fluid (75.5 mg / mL, Labmaster Oy, Turku) were prepared in TSA buffer supplemented with 7.5% bovine serum albumin.

10

Immunochemical assay. The standards were diluted 1:50 in assay buffer (0.05 mol / L Tris-HCl, pH 7.75 containing 0.9% NaCl, 0.05% NaN3, 0.5% bovine serum albumin, and 0.01% Tween 20). ) and added to the well (40 µL). Next, 160 µL of labeled antibody (500 ng, clone 1F10, Labmaster 15 Oy, Turku) was added in assay buffer. The immunochemical reaction was allowed to occur and after one hour the wells were washed six times with the washing solution. Next, the labeled antibody adhering to the wells of the immunocomplex was chaotropically removed with 0.2 mol / L borate buffer, pH

9.2 to which was added 1.75 mol / L NaSCN (250 mL). Finally, 200 µL of the solution was transferred to a test tube to which 800 µL of 0.2 mol / L borate buffer, pH 9.2, was added. The mixed solution was injected in a lyoluminometer (450 nm interference filter) into a test tube containing 10.0 mg of UV-irradiated K2S208. LL was measured as in Example 2. The standard curve is shown in Figure 14. Instead of the chaotropic reagent used in this example; * the labeled antibody or labeled antigen may also be cleaved from the solid support; ; as in Finnish Patent 88545.

Example 13. DNA hybridization method of the Philadelphia chromosome with latex fragments. Lyoluminescence was produced with UV irradiated K2S208.

: 30 Labeling the probe with Tb chelate. The oligonucleotide supplemented with amino groups (TTCGGGAAGTCGCCGGTCATCGTAGA- (C-NH2) 25-5 ', Wallac, Turku) was labeled with *: ··· an isothiocyanate derivative of Tb chelate except that the labeled:' ': Nucleotide was purified NAP-10 columns (Pharmacia, Uppsala, Sweden).

! , 35 Labeling the probe with biotin. Another was also prepared for the method

'T I

A probe (C- (NH 2 -C) -GTCGTAAGGCGACTGGTAGTTATTCCTT-5 ', Wallac, Turku) labeled with biotin. The N-hydroxysuccinimide ester derivative of biotin (3.7 µL Ν, Ν-dimethylformamide) was reacted with the probe overnight (5 nmol at 24 111758 in 50 µL, 50: 1 molar) at pH 9.5, +4 ° C. The pH was adjusted by adding Na 2 CO 3 to a final molarity of 50 mmol / L. The probe was purified as a Tb-chelated probe.

5 Coating of latex particles with streptavidin. The latex particles were coated with streptavidin, as in Example 2, and the latex particles were coated with the antibody.

Hybridization. A 10,170 bp fraction amplified by the Ph1 chromosome of the human cell line K562 cells (Turku University Hospital, Turku) was used.

as a positive sample and distilled water served as a negative control. Hybridization was performed in 1.5 mL polypropylene centrifuge tubes saturated with bovine serum albumin. 20 µL of a suspension of coated latex particles (approximately 180 million latex particles) was added to the tubes. The positive sample and the negative control were kept at 100 ° C for 10 min and cooled in an ice bath, then centrifuged in a microcentrifuge for 1 min at 12,000 rpm. The sample was pipetted onto 50 μΙ of latex particles followed by 150 μΙ of assay buffer containing 2 ng of biotinylated probe and 2 ng of Tb-chelated probe. The assay buffer contained 33.72 g of NaCl, 0.25 g of NaN3, 2.5 g of bovine serum albumin, 0.25 g of bovine serum gamma globulin and 0.05 mL of Tween 40 per liter of 25 mM

Tris-HCl buffer, pH 7.75. The reaction was allowed to proceed for 2 h at 50 ° C. Next, the particles were separated by centrifugation and washed as in Example 5. Finally, the particles! was suspended in 0.05 M Na2B407 solution and injected in a lyoluminometer (545 nm * 1 'interference filter) into a test tube containing 10.0 mg of UV-irradiated K2S2O8.

; A bar graph of the experiment is shown in Figure 15.

Ml • · I 1 I ·

* I

'·: 30 <· 1 I »» · ». 35 • »♦

Claims (4)

1. A method based on lyoluminescence to be used in the detection phase of embossed compounds in biochemical definition processes based on bioafifinity, as well as in definitive methods for clinical chemistry, characterized in that energy is stored in solid material in such form that it produces lyoluminescence according to additive dye production. Reactions: High Pressure and High Temperature Alkali Metallic Alkali High ^ Center of High Pressure and High Temperature Halogen Gas Alkali ibalides * Center or by electrolytic staining procedures according to the following general reactions, high temperature point alkaline cathode alkali halides alkali halides - * Vcentmm 'or by UV radiation according to the following general reactions hv> 4eV (4) S2082' - 2 SO4 · hv> 4eV (5) P2Og2 '- 2 PO4 2' 'hv> 4.8 eV (6) KNO3 - > potassium peroxonitrite, or the energy can be stored in the form of strongly reducing metals, such as aluminum or magnesium, and that in the methods imprints detected by means of lyoluminescence are used, and the excitation of the embossed compounds is based on the energy produced or formed in connection with the solution process, and has continued until the solution is converted to light emitted by the embossed compounds, the amount of the actual analyte object being measured by measuring the luminescence formed. 2. A method according to claim 1, characterized in that the luminescence of the embossed compound is formed either by redox reaction mechanisms according to the following general reactions: solute solid - * · solute + intermediate, intermediate + dissolved additive -> the secondary intermediates produced by the additive. which reactions the intermediates formed in the solid solution dissolution are either reducing units such as undervalent metal ions, solvated electrons, hydrogen atoms or oxidizing units, such as sulfate, hydroxyl or phosphate radicals, or radicals containing halogens or pseudohalogens; and that the additive is a peroxo compound, an amine, an oxalate or a formate, and where the excitation of the stamp is based on reactions belonging to the following reaction series: A + reducing intermediate - A + d + product of the reducing intermediate A + oxidizing intermediate - »AoX + product of the oxidizing intermediate, Ared + oxidizing intermediate —► A * + product of the oxidizing intermediate, Αοχ + reducing intermediate - * A * + product of the reducing intermediate, Ared Aox * A * + A, A * - A + hv (visible light), in which reactions A is an excitable embossed compound, Ared an electronically reduced form of the embossed compound, A <, x is a single electronically oxidized form of the embossed compound, and the intermediate is rather a primary intermediate which has formed in the solution of a solid, yes 111758 or a secondary intermediate produced by additives in the solution; or according to the following general equations: Luminophore + oxidizing intermediate -> luminophore intermediate Luminofor intermediate - »products + light or by energy transfer according to the following general equations: (X -) * + A -» X '+ A *, A * -> A + hv (visible light), where A is an excitable marker and X is an oxidizing radical such as sulfate, phosphate or hydroxyl radical, or another radical formed from halides or pseudohaloids produced by said radicals. Method according to claims 1 and 2, characterized in that the measurement of the light emitted from the marker, and thus the quantitation of the stamp and indirectly the actual analysis object can be carried out with a lyoluminometer or with another corresponding device, such that the marker to be measured , is introduced into the measuring tank of the device, and in the measuring tank the conditions according to claims 1 and 2 are formed, after which the amount of the marker is measured either instantaneously or for a longer period. Method according to claims 1 and 2, characterized in that the bioaffinity-based determination process is carried out on a solid phase and the luminophore that is excited by lyoluminescence, or the antibody or antigen which is embossed with luminophore, releases the solid phase after the bioaffinity reaction and washing by breaking the bond (s) between the bioaffinity compounds that are on the solid phase under the influence of one or more of the following factors: pH, temperature, UV light, ultrasound, detergent, chaotropic site, strongly reducing or oxidizing conditions, organic solubility and / or high ionic strength.