FI88545B - BESTAEMNINGSMETOD - Google Patents

Description

1,88545 Assay Method 1. Subject of the Invention

The invention relates to a solid phase bioaffinity reaction as set out in the preamble of claim 15. The bioaffinity reaction includes the following assays: immunoassays, nucleic acid hybridizations, ligand-lectin assays. The method utilizes time-resolved fluorometry in a bioaffinity reaction in which the counterpart involved in the reaction is covalently labeled with lanthanide chelate. When the bioaffinity reaction is complete, the lanthanide chelate is measured by time-resolved fluorometry.

The invention encompasses various types of biospecific and bioaffinity-based reactive counterparts, although the most common is the immunotypic reaction. The invention is described primarily as an immunochemical assay. The term refers to both competitive and non-competitive assay principles (R. Ekins et al. Pure & Appl. Chem. 57 (1985) pp. 473-482) in which one of the reacting counterparties is labeled with a measurable group. Examples of immunochemical counterparts that can be labeled with measurable moieties include antibodies, antigens, and haptens.

2. Previous Methods Related to the Invention

Over the last ten years, lanthanide chelates have become generally accepted markers in time-resolved fluorometry, e.g., in immunochemical methods (Lövgren et al .: Luminescence Immunoassays and Molecular Applications, Ed. Van Dyke, CRC-Press, 1990, pp. 233-253). From the spectroscopic and quantum chemical data, it has been concluded that Dy3 +, Sm3 +, Tb3 + and Eu3 + are the most suitable among the lanthanide ions because they emit delayed fluorescence. In view of the foreseeable practical advantages, it is generally desired that the 2 88545 lanthanide chelates used exhibit delayed fluorescence with a lifetime of more than 10 με. In addition, the lanthanide chelates used should have a sufficiently high stability constant to effectively bind the lanthanide ion during an immunochemical or other bioaffinity reaction (Lövgren, Alternative Immunoassays (1985) pp. 203-217) without losing good excitation energy absorption by the chelate and could lanthanide ion. To avoid this problem, several different alternatives have been developed, as to date no ke chelates with both good chelating ability and good energy absorption and fluorescence properties have been available.

The most widely used heterogeneous time-resolved fluoroim-munomääritys, DELFIAR assay (Wallac Oy, Turku, Finland) is Solve 15 SUT stability problem by using a covalently bound non-fluorescent lanthanide chelate the other party involved in the immunoreaction (hapten, antigen, antibody) for labeling. In the DELFIAR method, the lanthanide ion is dissociated into solution upon completion of the immunoreaction from the chelate ligand using pH 20. The ion is released from the ligand covalently attached to the solid phase-bound immunochemical counterpart.

The lanthanide ion is caused to fluoresce in an aqueous solution in a so-called developing solution containing a) a surfactant b) a chelate compound with which the lanthanide ion produces 25 fluorescence and c) a synergistic compound. The intensity and half-life of fluorescence depend not only on lanthanide but also on pH, surfactant, synergistic compound and chelating compound (Halvarson et al., J. Chem. Phys. 41 (1964) pp. 157 and 2752 , 30 and Hemmilä et al., Anal. Biochem. (1984) pp. 335-343).

In addition, only one commercial time-resolved fluorescence and lanthanide chelate immunoassay system (Cyberfluor) has been developed using 1.10-phenanthroline-2,9-dicarboxylic acid and a few new 3,88545 derivatives thereof (Khosvari et al., Organ. Chem. 33 (1988) pp. 1994-1999). These structures can be covalently attached to proteins and form fluorescent chelates in the presence of lanthanide salts. However, the stability of these chelates is relatively low so that excess lanthanide salt must be present before measuring time-resolved fluorescence from the solid phase. In addition, the measurement system is less sensitive compared to DELFlAANR.

Wieder has patented a somewhat similar system (U.S. Patent 4,058,732), although no evidence or results of system performance have been published. The reaction pair of the subject compound immunoassay is labeled with fluorescence probes of a rare earth complex such as benzoyl trifluoroacetone and the labeled compound 15 is solid phase excited and the time-resolved fluorescence is measured after a certain delay time (U.S. Patent 4,058,732, Figure 1). Also in this case, the stability of lanthanide chelate is a significant problem in practical applications.

Several subsequent alternative lanthanide chelates have good fluorescence properties and a sufficiently high stability constant to chelate and maintain stability of lanthanide ion under most bioaffinity assay conditions. All of these chelates or compounds can be covalently linked to a single compound involved in the bioaffinity assay. For example, in a solid phase, competitive or non-competitive immunoassay, time-resolved fluorescence is measured at the end of the immunoassay directly from the immobilized labeled reaction pair. Alternatively, various homogeneous (no separation) assays can be developed to measure by time-resolved fluorescence either a change in the solution concentration of the labeled reaction pair (U.S. Patent 4,920,195 and European Patent 0203047) or a change in the fluorescence intensity and / or lag time of the lanthanide chelate used. a bioaffinity reaction between labeled and 4,885,45 unlabeled reaction pairs (U.S. Patent 4,587,223; European Patent Application 88850314.1).

The separation step can be avoided by selectively measuring the amount of labeled compound bound to the solid phase (European Patent Application 86300588.0).

In most of the methods described, time-resolved fluorescence is measured either directly from the labeled compound on the solid phase or by using the dissociation-assisted lanthanide difluoroimmunoassay (DELFIAR). In this method, the lanthanide ion as such is rapidly dissociated at low pH, whereby a new, highly fluorescent chelate is formed with the components of the so-called developing solution. Both options work, although they have 15 certain downsides.

If time-resolved fluorescence is measured directly from a labeled bioaffinity compound on a solid phase, a highly fluorescent permanent lanthanide chelate is required. The optical requirements of the measuring device are high, as the excitation light must be directed at a limited surface used as a solid phase in the determination. In addition, many solid surfaces emit significant background fluorescence directly upon excitation, which has a long half-life that limits the sensitivity of the assay.

25 DELFIAR technology does not have e.m. limitations, as lanananide is measured from a solution in which it has formed a new highly fluorescent chelate. But the dissociation of the lanthanide ion from the solid phase, the labeled compound, and the subsequent subsequent formation of a new chelate in solution make the development step very sensitive to external lanthanide contamination. This results in an immediate increase in the background of the "background" fluoro 5,88545 (Diamandis, Clin. Biochem. 21 (1988) pp. 139-150). The possibility of contamination has been ruled out with the use of lanthanide chelates, which are both persistent and fluorescent.

In addition, International Patent Application WO 87/04523 discloses how lanthanide chelate can be released from the solid phase by eluting the immobilized antibody-antigen-antibody fluorophore complex in an acidic solution before measuring the fluorescence.

10 3. The invention

The invention relates to time-resolved fluorometry and measurement of fluorescence from a lanthanide chelate label after a solid phase bioaffinity reaction. The bioaffinity reaction includes the following methods: immunoassays, nucleic acid hybridizations, ligand-lectin assays, and ligand-receptor assays. After the solid phase bioaffinity reaction, the lanthanide chelate is released from the solid phase bound bioaffinity complex into the solution and can be measured directly from the solution by a time-resolved fluorometer. Lanthanide chelate is released from the solid phase by breaking the bond between the bioaffinity compounds on the solid phase, for example, by one or more of the following factors: pH, temperature, detergent, chaotropic salt, organic solvent and ionic strength.

4. Detailed Description of the Invention

The invention is applicable to many different types of biospecific affinity reactions, although the most common is an immunochemical reaction. For this reason, the invention is described primarily as an immunochemical assay. The various biospecific affinity reactions in question are illustrated in Figures 1-5. Competitive (competitive) reaction options 1-3 are depicted in Figures 1-3 and non-competitive (88545 (non-competitive) reaction options 1-2 in Figures 4 and 5. Both the competitive and non-competitive immunoassay principle is described above, assuming that the reaction the immunoassay section has been completed.

5 The following reference numerals are used in Figures 1-5: solid phase 1, carrier protein kp, antibody 2, antigen or hapten 3, antigen 4, permanent and fluorescent lanthanide chelate Kel: Eu, bioaffinity bond arrow bio, covalent bond hard and lanthanide chelate and bioaffinity com-10 A breakable covalent bond between pieks kkov.

According to the invention, the lanthanide chelate is released into solution by breaking one of the described bioaffinity bonds. In this connection, the covalent bond can also be broken. When using a method in which bioaffinity bonds are broken, no cleavable covalent bond is required between the fluorescent lanthanide chelate and the labeled bioaffinity compound. During each release process, the chelate complex between the lanthanide ion and the chelate ligand must be kept intact. Thus, the fluorescent lanthanide chelate is released into the solution as such, from which it can be efficiently measured by a time-resolved fluorometer. In this case, neither the background caused by the solid phase material nor any external lanthanide contamination can affect the result.

The bioaffinity bond between the reactants (antibodies, haptens, antigens, nucleic acids, receptors, lectins, carbohydrates, hormones, etc.) consists mainly of hydrophobic interactions, hydrogen bonds, Van der Waals forces, and ionic interactions. Thus, these bonds must be broken to release the fluorescent lanthanide chelate into solution. Several factors are known for breaking bioaffinity bonds, such as pH, ionic strength, chaotropic salts, detergents, organic solvents, etc. These factors can be used to break the bioaffinity bond to release the fluorescent lanthanide chelate into solution.

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The use of cleavable bifunctional molecules and bonds is known to identify biomacromolecule contact sites and has also been used successfully in affinity chromatography to elute highly tightly bound ligands (Jayabaskaran et al. Prep. Biochem. 1987, 17, p.

121-141, Montan et al. Arch. Biochem. Biophys. 1982, 218, pp. 101-108, Singh et al., Arch Biochem. Biophys. 1979, 193, pp. 284-293, Herman et al., Anal. Biochem. 1986, 156, pp. 48-55), in the purification of macromolecular reagents (Schwarzberg, 10 U.S. Patent 4,272,506), in the formation of bidirectional synthetic vesicles (Chang et al., Chem. Lett. 1987, pp. 1385-1388), in bidirectional enzyme immobilization (Carlsen, Hind. Antibiot. Bull. 1978, pp. 105-108) and determination of ligand titers and solid state chemical status (Marburg et al., Anal. Biochem. 1989, 181, p. 242). -249). There has also previously been a patented reagent (British Patent 1,597,758) used in assays based on bioSpecific affinity reactions. The reagent comprises a labeled immunochemical component consisting of a raultin conjugate having a plurality of analytically detectable moieties joined together by cleavable covalent bonds. This multiconjugate, in turn, is linked to the immunochemical component by multiple cleavable bonds.

To the best of our knowledge, no cleavage of bioaf-25 affinity bonds has been used in the past to introduce a label into solution after a solid-phase bioaffinity reaction. It is also not known that cleavage of a single covalent bond to release a single label was used concurrently with cleavage of bioaffinity bonds.

* * 8 88545

The following is a list of the most commonly known covalent bonds and the methods used to break the bond:

Bond to be broken Chemical cutting method

Disulfide Reduction 5 Visinal Glycols Periodate

Phenyl ester Base / hydroxylamine 1-oxidized sulfone Base

Azo Dithionite Bis-Glycol Ester Hydroxylamine 10 Thioester Hydroxylamine

In addition to chemical methods, exposure can also be used to break photosensitive bonds (Chow, The Chemistry of Amino, Nitraso and Nitro Compounds and Their Derivatives, Part 1, Ed. Patai, J. Wiley & Sons, N.Y. 1982, pp. 181-287). O-nitrophenyl groups are typical examples of light-cleavable bonds: D.H. Rich & s.K. Gurwara, J. Am.Chem.Soc. 97 (1975) 1575:

Z-NHCO - CH2OCOR hv Z-NHCO - {CHO + HOCOR

NO2 Ν <ζ "^ 20 D. H. Rich & s. K. Gurwara, Tetrahedron Lett. 1975 301:

Z-NHCO - CH2NHCO0 hv> Z-NHCO - <0> -CHO + H, NCOR

m2 NOT "^ B. Amit & Patchornik, Tetrahedron Lett. 1973 2205: / r3 -h ^ - ^ R1COOH + other lighting products 25 R2 no2 * β 88545

Several alternative cleavable covalent bonds and chemical cleavage methods, as well as light, can be used to cleave the covalent bond so that the fluorescent lanthanide chelate is released into solution.

5 Similar methods can be applied to other bioaffinity assays in addition to immunochemical assays. For example, in nucleic acid hybridization assays with a solid phase, the label can be released into solution upon completion of the hybridization assay. Figures 6 10 and 7 show typical such assay options. Only the release of fluorescent lanthanide chelate into solution is missing from the presentations. In addition to the reference numerals shown in Figures 1-5, the following notations are used: immobilized probe 5, target DNA 6, labeled probe 7, biotinylated probe 8, biotin bt, and immobilized streptavidin 9.

Also in hybridization assays, the fluorescent lanthanide chelate can be released into solution either by breaking one of the visible bioaffinity bonds or by cleaving the covalent bond or by combining the two alternatives. During the release process, the chelate complex between the lanthanide ion and the chelate ligand is kept intact.

The invention can be further illustrated by the following non-limiting examples: Example 1:

Cleavage of the immunoaffinity bond in a non-competitive hTSH assay

The experiment is performed in the same way as the non-competitive assay (Figure 4) except that the covalent bond is omitted.

10 88545 Specification

Polystyrene microtiter wells were coated with a monoclonal antibody to hTSH and the surface was impregnated with BSA. Another monoclonal antibody to hTSH was labeled with a fluorescent europium chelate, an isothiocyanate derivative of 4-aminophenylethynyl-2,6-bis (imet, Ν-bis (carboxymethyl) aminomethyl) pyridine-Eu. This assay was performed in a single step in coated microtiter wells containing 50 μΐ of assay buffer, hTSH-10 standard, and 50 μΐ of labeled antibody (50 ng). Incubation was performed at room temperature (30 min) with constant shaking of the microtiter plate. The wells were aspirated dry and washed six times, after which the bioaffinity bond was broken by adding 200 μΐ of different cleavage solutions. Wells 15 were shaken for 2 min, after which time-resolved fluorescence was measured from each cleavage solution at 2, 7, 32 every 68 min. The composition of the cleavage solutions was as follows: all containing 50 mM carbonate buffer, pH 10.0 and 0.1% BSA and either 20 0.2 Triton X-100 or 20% Ethanol or 10% Glycerol or 1.0% Acetone or 10% Propanol or 25 5 mH NaSCN

The signal level of the highest standard (500 μΐυ / ml) and its stability are shown in Figure 11. In each case, a complete standard plot was sought. Figure 8 shows a standard curve using 20% ethanol.

I: 11 88545

Example 2;

Breaking of a breakable covalent bond

In a model study, the fluorescence signal alone was dependent on cleavage of the covalent bond, as no bioaffinity bond was involved.

Configuration process

Polystyrene microtiter wells were coated with 1 Mg of BSA (200 μΐ) labeled with a europium chelate derivative of isothiocyanate disulfide (SCN-S-S) (Example 1).

10 Cleavage solution (200 μΐ) was added to the coated wells and 2 min. after shaking, the time-resolved fluorescence of the europium chelate in solution was measured. Various cleavage solutions made in 50 mM carbonate buffer were tested.

15 Dithiothreitol EtOH BSA

Truncation solution pH mM%% 1 9 2 - 2 9 5 5 0.1 3 9 10 10 0.5 20 4 10 2 5 0.5 .5 10 5 10 6 10 10 - 0.1 7 11 2 10 0.1 8 11 5 - 0.5 25 It is essential that BSA was not released from the solid phase during cleavage. Figure 12 shows the fluorescence measured from the solution under the conditions described above after a cleavage time of 2 and 53 minutes.

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Example 3:

Simultaneous disruption of immunoaffinity bonds and a cleavable covalent bond in a non-competitive hTSH assay 5 The performance of the assay is consistent with the non-competitive assay described in Option 1.

Procedure flow;

Polystyrene microtiter wells were coated with monoclonal antibody to hTSH and the surface was impregnated with 10 BSA. Another monoclonal antibody to hTSH was labeled with a fluorescent europium chelate, an isothiocyanate disulfide derivative of 4-aminophenylethynyl-2,6-bis- (N, N-bis (carboxymethyl) aminomethyl) pyridine-Eu. During the immunoassay (30 min at room temperature, continuous shaking)), the wells contained 50 μΐ hTSH standard (0, 1, 10, 50, 250 and 500 μΐυ / ml) and assay buffer containing labeled antibody (50 ng) (50 μΐ ). After incubation, the wells were aspirated dry and washed six times before adding the digestion solution (200 μΐ). The solution contained 20% EtOH, 2 mM dithiothreitol and 0.1% BSA in 50 mM carbonate buffer, pH 10. After shaking for two minutes, the time-resolved fluorescence of the europium chelate in the solution was measured. The results are shown in Figure 9.

Example 4: Simultaneous disruption of immunoaffinity bonds and a cleavable covalent bond in a competitive assay for 17-alpha-hydroxyproesterone

The experimental setup is described in connection with the competing assay in Figure 3.

13 88 545 Configuration progress

Polystyrene microtiter wells are coated with a polyclonal anti-rabbit antibody. Polyclonal rabbit anti-17-alpha-OH-progesterone antibody was diluted in 5 assay buffers (1: 50,000) and 100 μΐ of the dilution was added to each well. 25 μΐ of standard (7, 16, 28, 57, 102 and 261 nM) was then added. Finally, in step 3, a 17-alpha-OH-progesterone derivative labeled with a 10 isothiocyanate disulfide derivative of 4-aminophenethynyl-2,6-bis (Ν, Ν-bis (carboxymethyl) aminomethyl) pyridine-Eu was finally added (100 μΐ, 4.8 nM). The three hour incubation was performed at room temperature with constant shaking. The wells were then aspirated dry and washed six times, followed by the addition of 200 μ 200 of digestion solution (as in Example 15) and 2 min. after shaking, the time-resolved fluorescence of the europium chelate solution was measured. Figure 10 shows a standard plot of the 17-alpha-OH-progesterone assay. The maximum signal reached for the 0 standard was 55,000 cps.

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Claims (7)

14,88545 Claims; A solid phase bioaffinity assay method, wherein measurement 5 is performed using fluorometry, in particular time-resolved fluorometry, with fluorescent lanthanide chelates, wherein a single lanthanide chelate is covalently attached to one bioaffinity compound of the bioaffinity reaction. at the end of the bioaffinity reaction, wherein after the bioaffinity reaction and washing the lanthanide chelate, containing both the chelating structure and the lanthanide ion in chelated form, is released from the solid phase, characterized in that the lanthanide chelate is released from the solid phase factor: pH, temperature, 20 detergent, chaotropic salt, organic solvent and ionic hvuus. Process according to claim 1, characterized in that when the lanthanide chelate is released from the solid phase, it is still covalently bound to one bioaffinity compound of said bioaffinity reaction. Process according to Claim 2, characterized in that, after release, the same lanthanide chelate is measured from solution using fluorometry, in particular time-resolved fluorometry, with the chelate still bound to said bioaffinity compound. 35 Process according to Claim 1, characterized in that the lanthanide chelate, containing both the chelating structure and the lanthanide ion, is released from the solid-phase-bound, labeled bio-affinity compound by breaking the cleavable covalent bond between the labeled bioaffinity compound and a single chelating agent, for example. .5 by oxidative hydrolysis or light or some combination thereof. Process according to Claim 4, characterized in that, after release, the same lanthanide chelate is measured from solution by means of fluorometry, in particular time-resolved fluorometry. Method according to one of the preceding claims 1 to 5, characterized in that the bioaffinity reaction is an antigen-antibody reaction, a nucleic acid hybridization reaction, a ligand-receptor reaction or a ligand-lectin reaction. Process according to one of the preceding claims 1 to 6, characterized in that the chelated lanthanide ion is either europium, terbium, samarium or dysprosium. 16 88545 Patentkrav;