Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms

Definitions

• the invention relates to a bioaffinity reaction performed on a solid phase.
• the bioaffinity reaction comprises the following assays: immunoassays, nucleic acid hybridizations, ligand-lectin assays.
• the method utilizes time-resolved fluorometry in connection with the bioaffinity reaction, the other partner taking part in the reaction being covalently labelled with a lanthanide chelate.
• the lanthanide chelate is assayed by time- resolved fluorometry.
• the invention covers different biospecific and bio- affinity-based reactive partners, although an immuno- reaction is the most common type.
• the invention is thus primarily described as an immunochemical assay.
• the term refers to both competitive and non-competitive assay principles (R. Ekins et al. Pure & Appl. Che 57 (1985) pp. 473-482) , in which one of the reactive partners is labelled with a measurable group.
• Such immunochemical reagents that can be labelled with measurable groups include antibodies, antigens and haptens.
• lanthanide chelates have become generally accepted labelling agents in connection with time-resolved fluorometry e.g. in immunochemical methods (L ⁇ vgren et al.. Luminescence Immunoassays and Molecular Applications, Ed. van Dyke, CRC-Press, 1990, pp. 233-253). From spectroscopic and quantum chemical data it has been inferred that in the lan ⁇ thanide series, Dy + , Sm + , Tb and Eu + are the most suitable, because they emit delayed fluorescence. Taking into account the foreseeable advantages in practice, a finding that the lanthanide chelates show delayed fluorescence with a life time longer than 10 ⁇ s has generally been hoped for.
• the lanthanide chelates used should have a sufficiently high stability coefficient for being capable of binding a lanthanide ion efficiently during an immunochemical or some other bioaffinity reacion (L ⁇ vgren, Alternative Immunoassays (1985) , pp. 203-217) without losing the good absorption of excitation energy by the chelate and for being capable of transferring excitation energy to the chelated lanthanide ion.
• Several different alternatives have been developed to avoid this problem, because chelates having both good chelating ability and good energy absorption and fluorescence properties have not been available.
• the DELFIA Assay (Wallac Oy, Turku, Finland) , the stability problem has been solved by using a covalently bound non-fluorescent lanthanide chelate for labelling the partner taking part in the immunoreaction (hapten, antigen, antibody) .
• the lanthanide ion is dissociated from the chelate ligand into the solution at pH 4 after the immunoreaction has been completed. The ion is separated from the ligand which is covalently bound to the immunochemical partner bound in the solid phase.
• a developer solution containing (a) a surface- active agent, (b) a chelate compound, with which the lanthanide ion exhibits fluorescence, and (c) a synergistic compound.
• the intensity of the fluorescence and its half-life are dependent not only on the lanthanide but also on the pH value, on the surface- active agent, on the synergistic compound and on the chelate compound (Halvarson et al., J. Chem. Phys. 41 (1964), pp. 157 and 2752, and Hemmila et al.. Anal. Biochem. (1984) , pp. 335-343) .
• lanthanide chelates developed later have good fluorescence properties and a suffi ⁇ ciently high stability coefficient for chelating a lanthanide ion and for retaining stability under most conditions for bioaffinity assay. All these chelates or compounds can be covalently bound together with one compound taking part in the bioaffinity assay. For example in a competitive or non-competitive immunoassay on a solid phase, the time-resolved fluorescence is measured after the completion of the immunoassay directly from the immobilized labelled reaction partner. Alternatively, different homogeneous assays (no dissociation) can be developed for measuring by time-resolved fluorescence of either the change in the solution concentration of the labelled reaction partner (U.S.
• the dissociation stage can be avoided by measuring the content of the compound bound in the solid phase selectively (European Patent Application 86300588.0).
• the time-resolved fluorescence is measured either directly from the labelled compound on a solid phase or by using the principle of the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA R ).
• DELFIA R dissociation-enhanced lanthanide fluoroimmunoassay
• the DELPHIA assay has none of the limitations men ⁇ tioned above, because the lanthanide is measured from a solution in which it has formed a new intensively fluorescent chelate.
• the developing stage is very sensitive to external lan- thanide contaminations. This will result in the immediate growth of "background” fluorescence (Diaman- dis, Clin. Biochem 21 (1988) , pp. 139-150) .
• the possibility of contamination is excluded by the use of lanthanide chelates which are both stable and fluorescent.
• the invention relates to time-resolved fluorometry and a fluorescence measurement from a lanthanide chelate label after a bioaffinity reaction taken place on a solid phase.
• the bioaffinity reaction comprises the following methods: immunoassays, nucleic acid hybridizations, ligand-lectin assays and ligand- receptor assays.
• the lanthanide chelate is released into the solution from the bioaffinity complex bound to the solid phase, and it can be measured directly from the solution by time-resolved fluoro ⁇ metry.
• the invention can be applied in several biospecific affinity reacions of different types, although the immunochemical reaction is the most common type. For this reason, the invention is primarily described as an immunochemical assay.
• the different biospecific affinity reactions that are feasible are illustrated in Figs, l to 5.
• the competitive reaction alterna- tives 1 to 3 are described in Figs. 1 to 3 and the non-competitive reaction alternatives 1 to 2 in Figs. 4 and 5. Both the competitive and the non-competitive principle of immunoassay are described above presuming that the immunoassay part of the reaction has been completed.
• Figs. 1 to 5 the following reference signs are used: solid phase 1, carrier protein kp, antibody 2, antigen or hapten 3, antigen 4, stable and fluorescent lanthanide chelate Kel:Eu, bioaffinity bond arrow bio, covalent bond kov, and cleavable covalent bond between the lanthanide chelate and the bioaffinity compound kkov.
• the lanthanide chelate must be released in the solution. This can be performed either by cleaving one of the bioaffinity bonds described or by breaking the covalent bond or by combining these two alternatives. Using a method in which only the bioaffinity bonds are broken, no cleavable covalent bond is needed between the fluores ⁇ cent lanthanide chelate and the labelled bioaffinity compound. During each releasing process, the chelate complex between the lanthanide ion and the chelate ligand must remain intact. Thus only the fluorescent lanthanide chelate is released into the solution, in which it can be measured effectively by time-resolved fluorometry. Therefore, no background induced by the material of the solid phase nor any external lanthanide contamination can interfere with the result.
• the bioaffinity bond between the reagents is mainly composed of hydrophobic interactions, hydrogen bonds, Van der Waals forces and ionic interaction. Therefore, these bonds must be broken for releasing the fluorescent lanthanide chelate into the solution.
• reagents antibodies, haptens, antigens, nucleic acids, receptors, lectins, carbohydrates, hormones, etc.
• hydrophobic interactions hydrogen bonds
• Van der Waals forces and ionic interaction Therefore, these bonds must be broken for releasing the fluorescent lanthanide chelate into the solution.
• Several factors are known for breaking bioaffinity bonds, such as the pH value, ion strength, caotropic salts, detergents, organic solvents, etc. These factors can be used for breaking the bioaffinity bond to release the fluores- cent lanthanide chelate into the solution.
• breakable bifunctional molecules and bonds is known for detecting the contact sites of biomacro- molecules, and they have also been successfully used in affinity chromatography for elution of very strongly bound ligands (Jayabaskaran et al., Prep. Biochem. 1987, 17, pp. 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), for purification of macromolecular reagents (Schwarzberg, U.S.
• Patent 4,272,506 for formation of bidirectional synthetic vesicles (Chang et al., Chem. Lett. 1987, pp. 1385-1388) , for bidirectional immobilization of enzymes (Carlsen, Hind. Antibiot. Bull. 1978, pp. 105-108) , and for determination of the chemical state of ligand titres and the solid phase (Marburg et al., Anal. Biochem. 1989, 181, pp. 242-249).
• the prior art includes also a patented reagent (British Patent 1,597,758) that is used in assays based on biospecific affinity reactions.
• the reagent comprises a labelled immunochemical component composed of a multiconjugate with several analytically detectable groups connected to each other by breakable covalent bonds.
• the said multiconjugate is bound by several breakable bonds to the immunochemical com ⁇ ponent.
• the fluorescent lan ⁇ thanide chelate can be released into the solution either by breaking one of the visible bioaffinity bonds or by breaking a covalent bond or by combining these two alternatives. During the releasing process, the chelate complex between the lanthanide ion and the chelate ligand is kept intact.
• the experiment is arranged in a way similar to a non- competitive assay (Fig. 4) except that the covalent bond is excluded.
• Polystyrene microtitre wells were coated with a monoclonal anti-hTSH antibody and the surface was impregnated with BSA.
• Another monoclonal anti-hTSH antibody was labelled with fluorescent europium chelate, namely with an isothiocyanate derivative of 4-aminophenyl-ethynyl-2,6-bis(N,N-bis(carboxymethyl)- aminomethyl)pyridine-Eu.
• This assay was performed in a single stage in the coated microtitre wells contain ⁇ ing 50 ⁇ l of determination buffer, hTSH standard, and 50 ⁇ l of the labelled antibody (50 ng) . Incubation was carried out at room temperature (30 min) during continuous shaking of the microtitre plate.
• the wells were sucked dry and washed six times, after which the bioaffinity bond was broken by adding 200 ⁇ l various breaking solutions.
• the wells were shaken for 2 min, whereafter the time-resolved fluorescence was measured from each breaking solution at intervals of 2, 7, 32, 68 min.
• the composition of the breaking solutions was the following: each contained 50 mM carbonate buffer with a pH value of 10.0 and 0.1% BSA and either
• Fig. 11 The signal level of the highest standard (500 ⁇ lU/ml) and its stability are shown in Fig. 11. In each case, a complete standard diagram was the aim. Fig. 8 shows the standard curve on the use of 20% ethanol.
• Fig. 12 shows the fluores ⁇ cence measured from the solution in the conditions described above after a breaking time for 2 and 53 minutes.
• test was carried out in a manner analogous to the non-competitive assay presented in alternative 1.
• Polystyrene microtitre wells were coated with a monoclonal anti-hTSH antibody, and the surface was impregnated with BSA.
• Another monoclonal anti-hTSH antibody was labelled with a fluorescent europium chelate which is an isothiocyanate disulphide deriva ⁇ tive of 4-aminophenylethynyl-2,6-bis-(N,N-bis(carboxy- methyl)-aminomethyl)pyridine-Eu.
• the wells contained 50 ⁇ l of hTSH standard (0, 1, 10, 50, 250 and 500 ⁇ lU/ml) and assay buffer (50 ⁇ l) containing the labelled antibody (50 ng) . After incubation, the wells were sucked dry and washed six times before adding the breaking solution (200 ⁇ l) .
• the solution contained 20% EtOH, 2 mM dithiotreitol and 0.1% BSA in 50 mM carbonate buffer, pH 10. After shaking for 2 minutes, the time-resolved fluorescence of the europium chelate in the solution was measured. The results are given in Fig. 9.
• test procedure is described in connection with the competitive assay in Fig. 3.
• Polystyrene microtitre wells are coated with polyclonal anti-rabbit antibody.
• a polyclonal rabbit anti-17- ⁇ -OH-progesterone antibody was diluted in the assay buffer (1:50,000) , and 100 ⁇ l of the dilution was added into each well. Thereafter, 25 ⁇ l of standard was added (7, 16, 28, 57, 102 and 261 nM) .
• stage 3 a 17- ⁇ -OH-progesterone derivative, labelled with an isothiocyanate disulphide derivative of 4-aminophenyl- ethynyl-2 ,6-bis(N,N-bis(carboxymethyl)-aminomethyl)- pyridine-Eu, was added (100 ⁇ l, 4.8 nM) . Incubation was performed for 3 hours at room temperature by continuous shaking. Next, the wells were sucked dry and washed six times, whereafter 200 ⁇ l of the breaking solution was added (as in Example 3) and the time- resolved fluorescence of the europium chelate in the solution was measured after 2 minutes had elapsed from shaking.
• Fig. 10 shows the standard diagram of 17- ⁇ -OH-progesterone assay. Themaximum signal obtained for the 0-standard was 55,000 cps.

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• 1991
  • 1991-03-12 FI FI911200A patent/FI88545C/fi not_active IP Right Cessation
• 1992
  • 1992-03-12 WO PCT/FI1992/000068 patent/WO1992016839A1/en active Application Filing

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