CA2290237A1 - Method for identifying the function of biological molecules and apparatus for carrying out the method - Google Patents
Method for identifying the function of biological molecules and apparatus for carrying out the method Download PDFInfo
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Abstract
The invention relates to a method for identifying the function of a ligand L using chromophore-assisted laser inactivation (CALI), characterized by the stages:
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b) and c).
The invention also relates to an apparatus for carrying out the method according to the invention.
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b) and c).
The invention also relates to an apparatus for carrying out the method according to the invention.
Description
_ X 29165CA BO/HW/ps Method for identifying the function of biological molecules and apparatus for carrying out. the method The invention relates to a ms~thod and an apparatus for identifying the function of biological molecules. In particular, the invention relates to a method for identifying the function of biological molecules using chromophore-assisted lase:_ inactivation (CALI) . The method is based on the specii:ic binding of a ligand to its binding partner and inactivation thereof by the CALI method.
The present invention relates tc~ the area of functional genomics and, in particular, ii, relates to a method using ligand binding partners (LBl?), preferably selected from combinatorial libraries o:~ produced by genetic manipulation, in order to modify a target ligand (L) in order to determine the function of the target ligand.
The expansion of the field of genomics has rapidly lead to the identification of complete DNA
sequences of organisms. It is assumed th~~t in the year 2003 the entire human genome will he completely sequenced and the complete structural information will be decoded. However, knowledge of the DTfA sequence on its own does not provide sufficient information for understanding how genes and diseases correlate in order to make it possible to develop more effective therapies of diseases. The plethora of information zas led to the development of new technologies in order to understand the function of these genes better. However, at present, this is not possible.
The CALI method relates to the direct inactivation of a protein. The CALI method is the most promising method for determining the i=unction of a protein. It is not always possible with gene knockouts, for example, to obtain mutations fo:~ a specific protein, and many animal systems are not readily amenable to genetic investigations. :Cn the other methods, inhibiting pharmaceuticals (ribozymes and antisenses) can be used only for a restricted number of proteins, and function-blocking antibodies and aptamers represent only a small portion of those which are produced. On the other hand, CALI can convert "binding reagents" such as antibodies or ligands into function blockers. Briefly, a probe (ligand binding partner, LBP) is labelled with the dye malachite green (MG) or another chromophore which generates free radicals after exposure to laser light of a wavelength which is not significantly absorbed by the cellular components. The MG-labelled LBP (LBP-MG) is incubated with the sample of interest. An inactivation region is selected and irradiated with a laser beam at 620 nm. The light is absorbed by MG, generating short-lived free radicals which selectively inactivate proteins bound to LBP-MG
within a radius of 15 A. The system is versatile because it can be used for in vitro and in vivo assays and for intracellular and extracellular target molecules.
CALI represents a promising tool for throwing light on the function of genes, but in the state of the art CALI is restricted to the use of whole antibodies (Jay, D.G. 1988, PNAS 85, 5454-5458) and Fab molecules (Surrey, T. et al. 1998, PNAS 95, 4293-4298) . It has been proposed that antibody molecules can be generated from the screening of monoclonal libraries or hybridomas (Wang, F. -S. & Jay, D.G. 1996, Trends in Cell Biology 6, 442-445). However, this variant is extremely time-consuming. Another restriction is that the whole antibody molecules or Fabs are relatively large and therefore CALI possibly does not inactivate the protein in cases where the distance between the MG
binding sites in the antibody molecule and the domains required for the function of the protein is large, or the sensitivity of the domain to damage by hydroxyl free radicals is low. Although it has been proposed that CALI can be used with every LBP, there are as yet no possible uses thereof.
The present invention relates tc~ the area of functional genomics and, in particular, ii, relates to a method using ligand binding partners (LBl?), preferably selected from combinatorial libraries o:~ produced by genetic manipulation, in order to modify a target ligand (L) in order to determine the function of the target ligand.
The expansion of the field of genomics has rapidly lead to the identification of complete DNA
sequences of organisms. It is assumed th~~t in the year 2003 the entire human genome will he completely sequenced and the complete structural information will be decoded. However, knowledge of the DTfA sequence on its own does not provide sufficient information for understanding how genes and diseases correlate in order to make it possible to develop more effective therapies of diseases. The plethora of information zas led to the development of new technologies in order to understand the function of these genes better. However, at present, this is not possible.
The CALI method relates to the direct inactivation of a protein. The CALI method is the most promising method for determining the i=unction of a protein. It is not always possible with gene knockouts, for example, to obtain mutations fo:~ a specific protein, and many animal systems are not readily amenable to genetic investigations. :Cn the other methods, inhibiting pharmaceuticals (ribozymes and antisenses) can be used only for a restricted number of proteins, and function-blocking antibodies and aptamers represent only a small portion of those which are produced. On the other hand, CALI can convert "binding reagents" such as antibodies or ligands into function blockers. Briefly, a probe (ligand binding partner, LBP) is labelled with the dye malachite green (MG) or another chromophore which generates free radicals after exposure to laser light of a wavelength which is not significantly absorbed by the cellular components. The MG-labelled LBP (LBP-MG) is incubated with the sample of interest. An inactivation region is selected and irradiated with a laser beam at 620 nm. The light is absorbed by MG, generating short-lived free radicals which selectively inactivate proteins bound to LBP-MG
within a radius of 15 A. The system is versatile because it can be used for in vitro and in vivo assays and for intracellular and extracellular target molecules.
CALI represents a promising tool for throwing light on the function of genes, but in the state of the art CALI is restricted to the use of whole antibodies (Jay, D.G. 1988, PNAS 85, 5454-5458) and Fab molecules (Surrey, T. et al. 1998, PNAS 95, 4293-4298) . It has been proposed that antibody molecules can be generated from the screening of monoclonal libraries or hybridomas (Wang, F. -S. & Jay, D.G. 1996, Trends in Cell Biology 6, 442-445). However, this variant is extremely time-consuming. Another restriction is that the whole antibody molecules or Fabs are relatively large and therefore CALI possibly does not inactivate the protein in cases where the distance between the MG
binding sites in the antibody molecule and the domains required for the function of the protein is large, or the sensitivity of the domain to damage by hydroxyl free radicals is low. Although it has been proposed that CALI can be used with every LBP, there are as yet no possible uses thereof.
Improvements for CALI have already been proposed (Surrey, T. et al. 1998, PNAS 95, 4293-4298).
It has been shown that the fluorescein molecule can be used as chromophore in place of malachite green.
Fluorescein has the advantage that it is more soluble and more efficient at inactivation, and it is possible to use a more suitable light source with a continuous wavelength. It has also been shown that the Fab fragment is functional in CALI experiments. However, CALI has been modified so that one epitope (i.e.
haemagglutinin (HA)) is adapted to each target molecule of interest and subsequent CALI experiments can be carried out. Thus, only one Fab fragment, (i.e. anti-HA
Fab) serves as ligand for all target molecules which are to be inactivated by CALI.
Although this method is useful for investigations in which no non-inactivating ligands which can be used for controlled investigations are available, it has several restrictions on use in the field of functional genomics or the assessment of the target molecule. In the first place, the method is very complicated because every target molecule must be manipulated so that it includes the specific epitope.
Furthermore, the introduced epitope may break up the function of the target molecule, which will make it necessary to introduce a site which is better tolerated into the molecule. However, if this site which is better tolerated is too remote from the functional site in the molecule to be investigated and is located outside the inactivation region, CALI does not work. In the second place, although investigations have shown that Fab can be used for CALI, this cannot be generalized because antibodies are intrinsically variable. It may occur that the chromophore had a favourable attachment site in the described anti-HA Fab and this does not apply to another Fab. Hence there is a need to establish where the chromophore binds to the anti-HA Fab and to manipulate the latter for a generalization to other Fabs. Alternatively, ligands smaller than Fabs may be required to make CALI more efficient. Although the investigations further made it obvious to use smaller molecules for CALI, it was also confined to the same molecular environment as this application. It is therefore subject to the same restrictions which must be overcome for CALI to be an effective tool for functional genomics and assessment of the target molecule.
A further restriction on CALI as method for inactivating intracellular target molecules is the process of delivering the labelled ligand to the cell.
Although existing methods, such as trituration and microinjection, have functioned, this might be restricted to certain cell types. Very recent developments in electroporation may likewise be used (Rols, M.-0. et al. 1998, Nature Biotechnology 16, 168-171 ) .
The present invention is therefore based on the object of modifying or eliminating current restrictions on the CALI method. It is intended to provide novel methods which improve the efficiency and speed of carrying out CALI. It is likewise intended that the method be applicable to all binding partners and be automatable. The method is intended to permit simple determination of the function of any molecules.
The present invention therefore relates to a method for identifying the function of a ligand L using chromophore-assisted laser inactivation (CALI), characterized by the stages:
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b ) and c ) .
It has been found, surprisingly, that a combination of the CALI method with suitable methods for identifying the ligand permits rapid and simple determination of the function of a molecule. It has previously been assumed that the CALI method can be used only for known ligands with predetermined specificities such as, for example, enzyme substrates.
It was not previously possible to use CALI in cases where no known ligands were available. It was further previously impossible to combine CALI with the screening of combinatorial libraries.
CALI has now been combined according to the invention with the screening of combinatorial libraries for the first time. It has been shown according to the invention that it is possible to label a ligand at a specific site deliberately with a chromophore, which markedly increases the possibilities for the CALI
method. The method can be used to identify the function of any molecules such as, for example, proteins, peptides, carbohydrates, lipids, DNA, RNA etc.
Firstly, according to the invention an LBP with specificity for the ligand is selected.
At least one selected LBP is coupled to a marker, preferably a laser-activated marker, to form an LBP-tag. The coupling of LBP to the marker can also take place after complex formation. Then L is brought into contact with the LBP-tag to form an L/LBP-tag complex. The L/LBP-tag complex is brought into contact with an inducer, preferably irradiated with a laser beam, whereby the tag leads to the selective modification of the bound ligand (L).
The LBP is any molecule suitable for binding to a ligand. It is preferably selected from the following molecules: scFv, Fab, diabody, immunoglobulin-like molecules, peptide, RNA, DNA, PNA and small organic molecules except intact antibody molecules. In another preferred embodiment, the aforementioned LBPs are selected from a combinatorial library by one of the following methods, which are known to a skilled person:
one-stage selection, (cf. DE 19802576.9), phage display (Cwirla, S.E. et al. 1997, Science 273, 464-471), peptides on a plasmid (Stricker, N.L. et al. 1997, Nature Biotechnology 15, 336-342), SIP (Spada, S. et al. 1997, Biol. Chem. 378, 445-456), CLAP (Malmborg, A.
-C. et al. 1997, JMB 273, 544-551), ribosome/polysome display (Kawasaki, G. 1991, international patent application WO 91/05058; Hanes, J. & Pluckthun, A.
1997, PNAS 94, 4937-4942) or SELEX (Tuerk, C. &
Gold, L. 1990, Science 249, 505-510).
The binding partner is preferably derived from a combinatorial library. This can be any suitable combinatorial library, for example protein library, peptide library, cDNA library, mRNA library, library with organic molecules, scFv library with immunoglobulin superfamily, protein display library etc. The following may be presented in the libraries:
all types of proteins, for example structural proteins, enzymes, receptors, ligands, all types of peptides including modifications, DNAs, RNAs, combinations of DNAs and RNAs, hybrids of peptides and RNA or DNA, all types of organic molecules, for example steroids, alkaloids, natural products, synthetic substances etc.
The presentation can take place in various ways, for example as phage display system (for example filamentous phages such as M13, fl, fd etc., lambda phage display, viral display etc.), presentation on bacterial surfaces, ribosomes etc.
The activatable marker can be any suitable molecule which can be linked covalently or noncovalently to a binding partner and is able to produce free radicals after induction. Examples thereof are photoinducible molecules, for example peroxidase with hydrogen peroxide and laser-activatable markers.
The latter are preferably used.
In a preferred embodiment, the laser activatable marker is malachite green, fluorescein, lissamine rhodamine, tetramethylrhodamine isothio cyanate, cyanin 3.18.
Preference is furthermore given to AMCA-S, AMCA, BODIPY and variants thereof, Cascade Blue, Cl-NERF, dansyl, dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethyoxyfluorescein, DM-NERF, eosin, eosin F3S, erythrosin, hydroxycoumarin, Isosulfan Blue, lissamine rhodamine B, malachite green, methoxycoumarin, naphthofluorescein, NBD, Oregon Green 488, 500, 514, PyMPO, pyrene, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-tetrabromosulphone-fluorescein, tetramethylrhodamine, Texas Red or X-rhodamine. The irradiation takes place with laser light of a wavelength which is absorbed by the particular chromophore.
In a preferred embodiment, the selected LBP or every preexisting LBP is modified so that the efficiency of coupling or binding of the marker is increased, forming a more efficient or more stable LBP-tag molecule.
For example, lysine residues can be added onto the protein, making it easier to couple dye molecules such as malachite green or fluorescein via isothiocyanate linkers. It is also possible to produce bispecific molecules by methods known per se, with one specificity being directed, for example, against a chromophore and the other against the ligand.
One variant is to produce a bispecific molecule with specificities both for the ligand and for the marker. Firstly an scFv or ligand specific for the tag is selected by means of phage display, peptides on a plasmid, SIP, CLAP, ribosome/polysome display or SELEX.
This specific ligand for the marker can then be coupled to a second domain with specificity for the target ligand (L), producing a molecule with two _ g _ specificities. This can be carried out using methods which are known to a person skilled in the field, such as, for example, by means of diabodies (Holliger, P. et al. 1993, PNAS 90, 6444-6448) through the use of a domain association such as Fos-Jun leucine zipper (0'Shea, E.K. et al. 1989, Science 245, 645-648;
Kostenly, S.A. et al. 1992, J. Immunol. 148, 1547-1553), single-chain chimeric molecules with two specificities (Gruber M. et al. 1994, J. Immunology 152, 5368-5374); Mack, M. et al. 1995, PNAS 92, 7021-7025) or chimeric SELEX. It is further possible to produce LBP by genetic manipulation so that it contains amino acids (for example Cys and Lys) which are readily amenable to chemical linkage to the chromophore (Hudson, L. & Hay, F.C. 1976, Fluorescein Conjugation technique. In Practical Immunology. Blackwell Scientific Publications, Boston, chapter 2.1.1, 14-17;
Jay, D.G. 1988, PNAS 85, 5454-5458; Surrey, T. et al.
1998, PNAS 95, 4293-4298). Compounds obtainable commercially are used in these cases.
It is possible in a preferred embodiment if an LBP is a whole antibody molecule to convert the latter into an scFv or Fab by recombinant methods known to every person skilled in the field (Clackson, T. et al.
1991, Nature 352, 624-628; Huston, J.S. et al. 1988, PNAS 85, 5879-5883).
In another embodiment, the LBP undergoes genetic manipulation so that it contains amino acid sequences which allow efficient transport of LBP into the cells in order to make CALI more widely applicable to a wide spectrum of cell types and increase the efficiency for intracellular target molecules. It has been shown that short peptide sequences (Rojas, M. et al, 1998, Nature Biotechnology 16, 370-375) and the herpesvirus VP22 protein (Phelan, A. et al. 1998, Nature Biotechnology 16, 440-443) fused to heterologous proteins lead to efficient transport into mammalian cells. Thus, for example, signal peptide sequences or homeodomains which facilitate the transport of large _ g _ protein molecules into the cells can be linked to the required binding partners by genetic engineering fusion or by a chemical coupling method. Methodological steps of this type are well known to a person skilled in this field. Methods appropriate for chemical coupling are likewise known to a skilled person, for example coupling via SH-protected cysteins.
The ligand and the LBP-tag are brought into contact under conditions under which complex formation takes place. Conditions of this type have already been investigated during research into ligand interactions, mobility tests etc. and are well known to a skilled person. These are preferably physiological conditions.
The complex formation between the ligand and the labelled binding partner takes place very specifically and is not impaired by concomitant processes or complexes.
The modified ligand is identified using a suitable chemical/physical method.
Figure 1 which is appended outlines the method according to the invention.
The invention also relates to an apparatus for carrying out the method according to the invention.
This apparatus is depicted in detail in Figure 2 which is appended. This apparatus is an automated system consisting of integrated independent units/parts for identifying the protein function. Part A relates to the preparation of LBP-tag.
An automated LBP screening machine provides specific LBPs which are directed against specific target molecules/ligands, while the chromophore synthesis apparatus produces chromophores of choice. The selected LBPs and the synthesized chromophores are linked chemically in an LBP-chromophore coupling apparatus, resulting in LBP-tag. This LBP-tag is transferred into a loading apparatus which transfers the LBP-tag into predetermined cavities which are coated with the target molecule/ligand in the assay platform. A transfer robot then moves the assay platform into the laser system in order to initiate the second part B. The samples are irradiated with the laser at the required wavelength in order to induce a modification by free radicals. An apparatus for reading the activity then passes over the cavity which has been laser-irradiated in order to record the biological or chemical activity of the irradiated samples.
All the parts of the apparatus are connected to a central computer system for monitoring and analysis.
The invention is explained in detail by the following examples.
Example 1 Selection of scFv antibodies and CALI of ~i-galactosidase i) Selection of anti-(3-galactosidase scFv antibodies by phage display.
A phage display library was produced from human single-chain antibodies (scFv) as previously described (Sheets, M.D. et al. 1998, PNAS, 95:6157-6162; Marks, J.D. et al., 1991, J. Mol. Biol. 222:581-597). ScFv antibodies specific for ~3-galactosidase were isolated from this library by phage display and biopanning using methods previously described (Sheets, M.D. et al. 1998, PNAS, 95:6157-6162; Marks, J.D. et al., 1991, J. Mol.
Biol. 222:581-597). 50 ~g of purified (3-galactosidase were coated in wells of an ELISA plate and used for selection by phage display. Three rounds of panning were carried out and the enriched phages which contained an scFv with specificity for ~i-galactosidase were subcloned into an extraction vector (Marks, J.D.
et al., 1991, J. Mol. Biol. 222:581-597) and tested for specificity for (3-galactosidase using an ELISA and by their ability to inhibit (3-galactosidase activity (Wallenfells, K., 1962, Methods Enzymol. 5:212-219). An anti-testosterone scFv was used as negative control.
Several scFvs which did not inhibit (3-galactosidase activity or had a similar ~3-galactosidase activity as the negative control were used for the next step.
ii) Labelling of the scFv antibodies with fluorescein and malachite green The scFvs as obtained above (which do not inhibit (3-galactosidase activity) against (3-galactosidase were labelled with malachite green isothiocyanate or fluoroscein isothiocyanate with modifications as described (Jay, D.G. 1988, PNAS 85:
5454-5458; Surrey, T. 1998, PNAS 95:4293-4298). Stated briefly, antibodies in a concentration of 600 ~g/ml in 500 mM NaHC03 (pH 9.8) were labelled stepwise by adding malachite green isothiocyanate or fluorescein isothiocyanate (from Molecular Probes, Eugene, OR) up to a concentration of 120 ~.g/m from a stock solution (20 mg/ml or 2 mg/ml) in DMSO. After incubation with stirring at room temperature for 1 hour or incubation on ice for 4 hours, the solution was passed through a desalting column in 150 mM NaCl/50 mM NaPi, pH 7.3 (for malachite green it is necessary to centrifuge the precipitate before changing the buffer) in order to remove the marker from the labelled protein. The labelled antibodies were isolated and tested for the presence of the chromophore by W absorption.
iii) CALI of (3-galactosidase The use of the laser and the exposure to a laser beam for CALI takes place essentially as described (Beerman, A.E. and Jay, D.G. 1994, Methods Cell Biol. 44, 715-732; Surrey, T. 1998, PNAS
95:4293-4298). A 20 ~,1 sample which contained (3-galactosidase (10 ~,g/ml) and dye-labelled antibodies against ~3-galactosidase (200 ~g/ml) from ii) was placed on an ELISA plate. The total volume in the well was exposed to a laser beam after various periods of time.
The activity of the samples was measured as described above (Wallenfells, K. 1962, Methods Enzymol.
5:212-219). The negative controls consisted of an scFv chromophore/(3-Gal complex which had not been laser irradiated, and of a CALI experiment with anti-testosterone scFv.
The CALI investigation with (3-Gal-specific scFvs led to more than 90o inactivation compared with the negative controls.
The beta-galactosidase activity after CALI was followed as a function of time. The activity was measured as units which are appropriate for the test and are expressed as the ratio to the activity of the untreated beta-galactosidase.
CALI of beta-galactosidase Time (min) o active beta-galactosidase with CALI without CALI
30 <1 100 Example 2 Selection of aptamers and CALI of f3-aalactosidase i) Selection of the aptamers specific for (3-galactosidase by SELEX
DNA aptamers specific for (3-galactosidase were isolated from a random DNA library by SELEX using methods previously described (Morris, K.N. et al. 1998, PNAS 95:2902-2907). Various concentrations (0.1 to 2 mg/ml) of (3-galactosidase were used for the selection.
After 25 selection rounds, the enriched DNA aptamers were sequenced and tested singly for specificity against (3-galactosidase and the ability to inhibit ~3-galactosidase activity (Wallenfells, K. 1962, Methods Enzymol. 5:212-219). Aptamers specific for the UlA
protein were used as negative control in the (3-galactosidase inhibition tests.
Several aptamers which did not inhibit galactosidase or had a similar (3-galactosidase activity as the negative control were selected for the next step.
ii) Labelling of nucleotide aptamers with fluorescein and malachite green The selected DNA aptamers which did not inhibit (3-galactosidase activity were used for the labelling with fluorescein and malachite green. Several methods, including both enzymatic and chemical synthesis, which are known to a person skilled in the field were used in order to insert dye-labelled nucleotides as part of the aptamer (Igloi, G.L. 1996, Anal. Biochem. 233:124-129;
Meyer, K.1: & Hanna, M.M. 1996, Bioconjug. Chem. 4:401-412; Nelson, P.S. et al. 1992, Nucleic Acids Res.
20:6253-6259; Thiesen, P. et al. 1992, Nucleic Acids Symp. Ser 27:99-100; Proudnikov, D. & Mizabekov, A.
1996, Nucleic Acids Res. 24:4535-4542; Rosemeyer, V. et al. 1995, Anal. Biochem. 224:446-449; Richardson, R.W.
& Gumport, R.I. 1983, Nucleic Acids Res. 11:6167-6184;
Kinoshita, Y. et al. 1997, Nucleic Acids Res. 25:3747-3748).
The labelled aptamers were isolated by polyacrylamide gel electrophoresis and tested for the presence of the chromophore by W absorption.
iii) CALI of ~i-galactosidase The use of the laser and the exposure to a laser beam for CALI takes place essentially as described (Beerman, A.E. and Jay, D.G. 1994, Methods Cell Biol. 44, 715-732; Surrey, T. 1998, PNAS
95:4293-4298). A 20 ~1 sample which contained (3-galactosidase (10 ~.g/ml) and dye-labelled antibodies against (3-galactosidase (200 ~,g/ml) from ii) was placed on an ELISA plate. The total volume in the well was exposed to a laser beam after various periods of time.
The activity of the samples was measured as described above (Wallenfells, K. 1962, Methods Enzymol.
5:212-219) .
The beta-galactosidase activity after CALI was followed as a function of time. The activity was measured as units which are appropriate for the test and are expressed as the ratio to the activity of the untreated beta-galactosidase.
CALI of beta-galactosidase Time (min) % active beta-galactosidase with CALI without CALI
30 <1 100 Example 3 Production of scFv by genetic manipulation for selective labelling with fluorescein and malachite green and use thereof in CALI
In order to avoid the restriction of unpredicatable labelling of scFv with the chromophore, a sequence of Lys residues was introduced by genetic manipulation at the following sites in the scFv or Fabs: (1) N terminus, (2) C terminus, (3) linker region of the scFv. Standard gene synthesis methods known to a person skilled in the field are used for this (Prodromou, C. & Pearl, L.H. 1992, Prot. Eng. 5: 827-829). After the labelling with the chromophore as described in Example 1 ii., the presence of the chromophore was detected and determined quantitatively by W absorption as described above. In addition, the recombinant scFvs labelled with the chromophore were tested for binding to (3-Gal by ELISA as described above. These scFvs were then tested by CALI as described in Example 1 iii).
The scFvs produced by genetic manipulation and labelled with the chromophore retained their ability to bind to (3-Gal and showed in the CALI experiments more than 950 inactivation of (3-galactosidase activity.
It has been shown that the fluorescein molecule can be used as chromophore in place of malachite green.
Fluorescein has the advantage that it is more soluble and more efficient at inactivation, and it is possible to use a more suitable light source with a continuous wavelength. It has also been shown that the Fab fragment is functional in CALI experiments. However, CALI has been modified so that one epitope (i.e.
haemagglutinin (HA)) is adapted to each target molecule of interest and subsequent CALI experiments can be carried out. Thus, only one Fab fragment, (i.e. anti-HA
Fab) serves as ligand for all target molecules which are to be inactivated by CALI.
Although this method is useful for investigations in which no non-inactivating ligands which can be used for controlled investigations are available, it has several restrictions on use in the field of functional genomics or the assessment of the target molecule. In the first place, the method is very complicated because every target molecule must be manipulated so that it includes the specific epitope.
Furthermore, the introduced epitope may break up the function of the target molecule, which will make it necessary to introduce a site which is better tolerated into the molecule. However, if this site which is better tolerated is too remote from the functional site in the molecule to be investigated and is located outside the inactivation region, CALI does not work. In the second place, although investigations have shown that Fab can be used for CALI, this cannot be generalized because antibodies are intrinsically variable. It may occur that the chromophore had a favourable attachment site in the described anti-HA Fab and this does not apply to another Fab. Hence there is a need to establish where the chromophore binds to the anti-HA Fab and to manipulate the latter for a generalization to other Fabs. Alternatively, ligands smaller than Fabs may be required to make CALI more efficient. Although the investigations further made it obvious to use smaller molecules for CALI, it was also confined to the same molecular environment as this application. It is therefore subject to the same restrictions which must be overcome for CALI to be an effective tool for functional genomics and assessment of the target molecule.
A further restriction on CALI as method for inactivating intracellular target molecules is the process of delivering the labelled ligand to the cell.
Although existing methods, such as trituration and microinjection, have functioned, this might be restricted to certain cell types. Very recent developments in electroporation may likewise be used (Rols, M.-0. et al. 1998, Nature Biotechnology 16, 168-171 ) .
The present invention is therefore based on the object of modifying or eliminating current restrictions on the CALI method. It is intended to provide novel methods which improve the efficiency and speed of carrying out CALI. It is likewise intended that the method be applicable to all binding partners and be automatable. The method is intended to permit simple determination of the function of any molecules.
The present invention therefore relates to a method for identifying the function of a ligand L using chromophore-assisted laser inactivation (CALI), characterized by the stages:
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b ) and c ) .
It has been found, surprisingly, that a combination of the CALI method with suitable methods for identifying the ligand permits rapid and simple determination of the function of a molecule. It has previously been assumed that the CALI method can be used only for known ligands with predetermined specificities such as, for example, enzyme substrates.
It was not previously possible to use CALI in cases where no known ligands were available. It was further previously impossible to combine CALI with the screening of combinatorial libraries.
CALI has now been combined according to the invention with the screening of combinatorial libraries for the first time. It has been shown according to the invention that it is possible to label a ligand at a specific site deliberately with a chromophore, which markedly increases the possibilities for the CALI
method. The method can be used to identify the function of any molecules such as, for example, proteins, peptides, carbohydrates, lipids, DNA, RNA etc.
Firstly, according to the invention an LBP with specificity for the ligand is selected.
At least one selected LBP is coupled to a marker, preferably a laser-activated marker, to form an LBP-tag. The coupling of LBP to the marker can also take place after complex formation. Then L is brought into contact with the LBP-tag to form an L/LBP-tag complex. The L/LBP-tag complex is brought into contact with an inducer, preferably irradiated with a laser beam, whereby the tag leads to the selective modification of the bound ligand (L).
The LBP is any molecule suitable for binding to a ligand. It is preferably selected from the following molecules: scFv, Fab, diabody, immunoglobulin-like molecules, peptide, RNA, DNA, PNA and small organic molecules except intact antibody molecules. In another preferred embodiment, the aforementioned LBPs are selected from a combinatorial library by one of the following methods, which are known to a skilled person:
one-stage selection, (cf. DE 19802576.9), phage display (Cwirla, S.E. et al. 1997, Science 273, 464-471), peptides on a plasmid (Stricker, N.L. et al. 1997, Nature Biotechnology 15, 336-342), SIP (Spada, S. et al. 1997, Biol. Chem. 378, 445-456), CLAP (Malmborg, A.
-C. et al. 1997, JMB 273, 544-551), ribosome/polysome display (Kawasaki, G. 1991, international patent application WO 91/05058; Hanes, J. & Pluckthun, A.
1997, PNAS 94, 4937-4942) or SELEX (Tuerk, C. &
Gold, L. 1990, Science 249, 505-510).
The binding partner is preferably derived from a combinatorial library. This can be any suitable combinatorial library, for example protein library, peptide library, cDNA library, mRNA library, library with organic molecules, scFv library with immunoglobulin superfamily, protein display library etc. The following may be presented in the libraries:
all types of proteins, for example structural proteins, enzymes, receptors, ligands, all types of peptides including modifications, DNAs, RNAs, combinations of DNAs and RNAs, hybrids of peptides and RNA or DNA, all types of organic molecules, for example steroids, alkaloids, natural products, synthetic substances etc.
The presentation can take place in various ways, for example as phage display system (for example filamentous phages such as M13, fl, fd etc., lambda phage display, viral display etc.), presentation on bacterial surfaces, ribosomes etc.
The activatable marker can be any suitable molecule which can be linked covalently or noncovalently to a binding partner and is able to produce free radicals after induction. Examples thereof are photoinducible molecules, for example peroxidase with hydrogen peroxide and laser-activatable markers.
The latter are preferably used.
In a preferred embodiment, the laser activatable marker is malachite green, fluorescein, lissamine rhodamine, tetramethylrhodamine isothio cyanate, cyanin 3.18.
Preference is furthermore given to AMCA-S, AMCA, BODIPY and variants thereof, Cascade Blue, Cl-NERF, dansyl, dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethyoxyfluorescein, DM-NERF, eosin, eosin F3S, erythrosin, hydroxycoumarin, Isosulfan Blue, lissamine rhodamine B, malachite green, methoxycoumarin, naphthofluorescein, NBD, Oregon Green 488, 500, 514, PyMPO, pyrene, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-tetrabromosulphone-fluorescein, tetramethylrhodamine, Texas Red or X-rhodamine. The irradiation takes place with laser light of a wavelength which is absorbed by the particular chromophore.
In a preferred embodiment, the selected LBP or every preexisting LBP is modified so that the efficiency of coupling or binding of the marker is increased, forming a more efficient or more stable LBP-tag molecule.
For example, lysine residues can be added onto the protein, making it easier to couple dye molecules such as malachite green or fluorescein via isothiocyanate linkers. It is also possible to produce bispecific molecules by methods known per se, with one specificity being directed, for example, against a chromophore and the other against the ligand.
One variant is to produce a bispecific molecule with specificities both for the ligand and for the marker. Firstly an scFv or ligand specific for the tag is selected by means of phage display, peptides on a plasmid, SIP, CLAP, ribosome/polysome display or SELEX.
This specific ligand for the marker can then be coupled to a second domain with specificity for the target ligand (L), producing a molecule with two _ g _ specificities. This can be carried out using methods which are known to a person skilled in the field, such as, for example, by means of diabodies (Holliger, P. et al. 1993, PNAS 90, 6444-6448) through the use of a domain association such as Fos-Jun leucine zipper (0'Shea, E.K. et al. 1989, Science 245, 645-648;
Kostenly, S.A. et al. 1992, J. Immunol. 148, 1547-1553), single-chain chimeric molecules with two specificities (Gruber M. et al. 1994, J. Immunology 152, 5368-5374); Mack, M. et al. 1995, PNAS 92, 7021-7025) or chimeric SELEX. It is further possible to produce LBP by genetic manipulation so that it contains amino acids (for example Cys and Lys) which are readily amenable to chemical linkage to the chromophore (Hudson, L. & Hay, F.C. 1976, Fluorescein Conjugation technique. In Practical Immunology. Blackwell Scientific Publications, Boston, chapter 2.1.1, 14-17;
Jay, D.G. 1988, PNAS 85, 5454-5458; Surrey, T. et al.
1998, PNAS 95, 4293-4298). Compounds obtainable commercially are used in these cases.
It is possible in a preferred embodiment if an LBP is a whole antibody molecule to convert the latter into an scFv or Fab by recombinant methods known to every person skilled in the field (Clackson, T. et al.
1991, Nature 352, 624-628; Huston, J.S. et al. 1988, PNAS 85, 5879-5883).
In another embodiment, the LBP undergoes genetic manipulation so that it contains amino acid sequences which allow efficient transport of LBP into the cells in order to make CALI more widely applicable to a wide spectrum of cell types and increase the efficiency for intracellular target molecules. It has been shown that short peptide sequences (Rojas, M. et al, 1998, Nature Biotechnology 16, 370-375) and the herpesvirus VP22 protein (Phelan, A. et al. 1998, Nature Biotechnology 16, 440-443) fused to heterologous proteins lead to efficient transport into mammalian cells. Thus, for example, signal peptide sequences or homeodomains which facilitate the transport of large _ g _ protein molecules into the cells can be linked to the required binding partners by genetic engineering fusion or by a chemical coupling method. Methodological steps of this type are well known to a person skilled in this field. Methods appropriate for chemical coupling are likewise known to a skilled person, for example coupling via SH-protected cysteins.
The ligand and the LBP-tag are brought into contact under conditions under which complex formation takes place. Conditions of this type have already been investigated during research into ligand interactions, mobility tests etc. and are well known to a skilled person. These are preferably physiological conditions.
The complex formation between the ligand and the labelled binding partner takes place very specifically and is not impaired by concomitant processes or complexes.
The modified ligand is identified using a suitable chemical/physical method.
Figure 1 which is appended outlines the method according to the invention.
The invention also relates to an apparatus for carrying out the method according to the invention.
This apparatus is depicted in detail in Figure 2 which is appended. This apparatus is an automated system consisting of integrated independent units/parts for identifying the protein function. Part A relates to the preparation of LBP-tag.
An automated LBP screening machine provides specific LBPs which are directed against specific target molecules/ligands, while the chromophore synthesis apparatus produces chromophores of choice. The selected LBPs and the synthesized chromophores are linked chemically in an LBP-chromophore coupling apparatus, resulting in LBP-tag. This LBP-tag is transferred into a loading apparatus which transfers the LBP-tag into predetermined cavities which are coated with the target molecule/ligand in the assay platform. A transfer robot then moves the assay platform into the laser system in order to initiate the second part B. The samples are irradiated with the laser at the required wavelength in order to induce a modification by free radicals. An apparatus for reading the activity then passes over the cavity which has been laser-irradiated in order to record the biological or chemical activity of the irradiated samples.
All the parts of the apparatus are connected to a central computer system for monitoring and analysis.
The invention is explained in detail by the following examples.
Example 1 Selection of scFv antibodies and CALI of ~i-galactosidase i) Selection of anti-(3-galactosidase scFv antibodies by phage display.
A phage display library was produced from human single-chain antibodies (scFv) as previously described (Sheets, M.D. et al. 1998, PNAS, 95:6157-6162; Marks, J.D. et al., 1991, J. Mol. Biol. 222:581-597). ScFv antibodies specific for ~3-galactosidase were isolated from this library by phage display and biopanning using methods previously described (Sheets, M.D. et al. 1998, PNAS, 95:6157-6162; Marks, J.D. et al., 1991, J. Mol.
Biol. 222:581-597). 50 ~g of purified (3-galactosidase were coated in wells of an ELISA plate and used for selection by phage display. Three rounds of panning were carried out and the enriched phages which contained an scFv with specificity for ~i-galactosidase were subcloned into an extraction vector (Marks, J.D.
et al., 1991, J. Mol. Biol. 222:581-597) and tested for specificity for (3-galactosidase using an ELISA and by their ability to inhibit (3-galactosidase activity (Wallenfells, K., 1962, Methods Enzymol. 5:212-219). An anti-testosterone scFv was used as negative control.
Several scFvs which did not inhibit (3-galactosidase activity or had a similar ~3-galactosidase activity as the negative control were used for the next step.
ii) Labelling of the scFv antibodies with fluorescein and malachite green The scFvs as obtained above (which do not inhibit (3-galactosidase activity) against (3-galactosidase were labelled with malachite green isothiocyanate or fluoroscein isothiocyanate with modifications as described (Jay, D.G. 1988, PNAS 85:
5454-5458; Surrey, T. 1998, PNAS 95:4293-4298). Stated briefly, antibodies in a concentration of 600 ~g/ml in 500 mM NaHC03 (pH 9.8) were labelled stepwise by adding malachite green isothiocyanate or fluorescein isothiocyanate (from Molecular Probes, Eugene, OR) up to a concentration of 120 ~.g/m from a stock solution (20 mg/ml or 2 mg/ml) in DMSO. After incubation with stirring at room temperature for 1 hour or incubation on ice for 4 hours, the solution was passed through a desalting column in 150 mM NaCl/50 mM NaPi, pH 7.3 (for malachite green it is necessary to centrifuge the precipitate before changing the buffer) in order to remove the marker from the labelled protein. The labelled antibodies were isolated and tested for the presence of the chromophore by W absorption.
iii) CALI of (3-galactosidase The use of the laser and the exposure to a laser beam for CALI takes place essentially as described (Beerman, A.E. and Jay, D.G. 1994, Methods Cell Biol. 44, 715-732; Surrey, T. 1998, PNAS
95:4293-4298). A 20 ~,1 sample which contained (3-galactosidase (10 ~,g/ml) and dye-labelled antibodies against ~3-galactosidase (200 ~g/ml) from ii) was placed on an ELISA plate. The total volume in the well was exposed to a laser beam after various periods of time.
The activity of the samples was measured as described above (Wallenfells, K. 1962, Methods Enzymol.
5:212-219). The negative controls consisted of an scFv chromophore/(3-Gal complex which had not been laser irradiated, and of a CALI experiment with anti-testosterone scFv.
The CALI investigation with (3-Gal-specific scFvs led to more than 90o inactivation compared with the negative controls.
The beta-galactosidase activity after CALI was followed as a function of time. The activity was measured as units which are appropriate for the test and are expressed as the ratio to the activity of the untreated beta-galactosidase.
CALI of beta-galactosidase Time (min) o active beta-galactosidase with CALI without CALI
30 <1 100 Example 2 Selection of aptamers and CALI of f3-aalactosidase i) Selection of the aptamers specific for (3-galactosidase by SELEX
DNA aptamers specific for (3-galactosidase were isolated from a random DNA library by SELEX using methods previously described (Morris, K.N. et al. 1998, PNAS 95:2902-2907). Various concentrations (0.1 to 2 mg/ml) of (3-galactosidase were used for the selection.
After 25 selection rounds, the enriched DNA aptamers were sequenced and tested singly for specificity against (3-galactosidase and the ability to inhibit ~3-galactosidase activity (Wallenfells, K. 1962, Methods Enzymol. 5:212-219). Aptamers specific for the UlA
protein were used as negative control in the (3-galactosidase inhibition tests.
Several aptamers which did not inhibit galactosidase or had a similar (3-galactosidase activity as the negative control were selected for the next step.
ii) Labelling of nucleotide aptamers with fluorescein and malachite green The selected DNA aptamers which did not inhibit (3-galactosidase activity were used for the labelling with fluorescein and malachite green. Several methods, including both enzymatic and chemical synthesis, which are known to a person skilled in the field were used in order to insert dye-labelled nucleotides as part of the aptamer (Igloi, G.L. 1996, Anal. Biochem. 233:124-129;
Meyer, K.1: & Hanna, M.M. 1996, Bioconjug. Chem. 4:401-412; Nelson, P.S. et al. 1992, Nucleic Acids Res.
20:6253-6259; Thiesen, P. et al. 1992, Nucleic Acids Symp. Ser 27:99-100; Proudnikov, D. & Mizabekov, A.
1996, Nucleic Acids Res. 24:4535-4542; Rosemeyer, V. et al. 1995, Anal. Biochem. 224:446-449; Richardson, R.W.
& Gumport, R.I. 1983, Nucleic Acids Res. 11:6167-6184;
Kinoshita, Y. et al. 1997, Nucleic Acids Res. 25:3747-3748).
The labelled aptamers were isolated by polyacrylamide gel electrophoresis and tested for the presence of the chromophore by W absorption.
iii) CALI of ~i-galactosidase The use of the laser and the exposure to a laser beam for CALI takes place essentially as described (Beerman, A.E. and Jay, D.G. 1994, Methods Cell Biol. 44, 715-732; Surrey, T. 1998, PNAS
95:4293-4298). A 20 ~1 sample which contained (3-galactosidase (10 ~.g/ml) and dye-labelled antibodies against (3-galactosidase (200 ~,g/ml) from ii) was placed on an ELISA plate. The total volume in the well was exposed to a laser beam after various periods of time.
The activity of the samples was measured as described above (Wallenfells, K. 1962, Methods Enzymol.
5:212-219) .
The beta-galactosidase activity after CALI was followed as a function of time. The activity was measured as units which are appropriate for the test and are expressed as the ratio to the activity of the untreated beta-galactosidase.
CALI of beta-galactosidase Time (min) % active beta-galactosidase with CALI without CALI
30 <1 100 Example 3 Production of scFv by genetic manipulation for selective labelling with fluorescein and malachite green and use thereof in CALI
In order to avoid the restriction of unpredicatable labelling of scFv with the chromophore, a sequence of Lys residues was introduced by genetic manipulation at the following sites in the scFv or Fabs: (1) N terminus, (2) C terminus, (3) linker region of the scFv. Standard gene synthesis methods known to a person skilled in the field are used for this (Prodromou, C. & Pearl, L.H. 1992, Prot. Eng. 5: 827-829). After the labelling with the chromophore as described in Example 1 ii., the presence of the chromophore was detected and determined quantitatively by W absorption as described above. In addition, the recombinant scFvs labelled with the chromophore were tested for binding to (3-Gal by ELISA as described above. These scFvs were then tested by CALI as described in Example 1 iii).
The scFvs produced by genetic manipulation and labelled with the chromophore retained their ability to bind to (3-Gal and showed in the CALI experiments more than 950 inactivation of (3-galactosidase activity.
Claims (9)
1) Method for identifying the function of a ligand L using chromophore-assisted laser inactivation (CALI), characterized by the stages:
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b) and c).
a) selecting a ligand binding partner (LBP) with specificity for the ligand L, b) coupling the LBP to a laser-activatable marker (tag) to form LBP-tag, where appropriate after previous modification of the LBP with the aim of more efficient binding to the marker, c) bringing the ligand L into contact with at least one LBP-tag to form an L/LBP-tag complex, and d) irradiating the L/LBP-tag complex with a laser beam, whereupon the irradiated LBP-tag selectively modifies the bound ligand, it being possible to interchange the sequence of stages b) and c).
2) Method according to Claim 1, characterized in that the LBP is selected from dsFv, scFv, Fab, diabody, immunoglobulin-like molecules, peptides, RNA, DNA, PNA, and small organic molecules, except intact antibody molecules.
3) Method according to either of Claims 1 or 2, characterized in that the LBP is derived from a combinatorial bank, excepting hybridomas.
4) Method according to any of Claims 1 to 3, characterized in that the laser-activatable marker is malachite green, fluorescein, lissamine rhodamine, tetramethylrhodamine isothiocyanate, cyanin 3.18, AMCA-S, AMCA, BODIPY and variants thereof, Cascade Blue, Cl-NERF, dansyl, dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethyoxyfluorescein, DM-NERF, eosin, eosin F3S, erythrosin, hydroxycoumarin, Isosulfan Blue, lissamine rhodamine B, malachite green, methoxycoumarin, naphthofluorescein, NBD, Oregon Green 488, 500, 514, PyMPO, pyrene, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-tetra-bromosulphonefluorescein, tetramethylrhodamine, Texas Red or X-rhodamine.
5) Method according to any of Claims 1 to 4, characterized in that the LBP-tag is modified by attaching lysine residues.
6) Biological molecule obtained by a method according to any of Claims 1 to 5.
7) Apparatus for carrying out a method according to any of Claims 1 to 5, characterized in that it is an automated system consisting of integrated independent units/parts for identifying the protein function and comprises the following constituents:
- an automated LBP screening machine for producing specific LBPs which are directed against specific target molecules/ligands, - a chromophore synthesis apparatus for producing chromophores, - an LBP-chromophore coupling apparatus for linking the selected LBPs and the synthesized chromophores, - a loading apparatus for transferring the LBP-tag into predetermined cavities which are coated with the target molecule/ligand in the assay platform, - a transfer robot arm for moving the assay platform into the laser system, - an apparatus for reading the activity, - a database, - a central computer system.
- an automated LBP screening machine for producing specific LBPs which are directed against specific target molecules/ligands, - a chromophore synthesis apparatus for producing chromophores, - an LBP-chromophore coupling apparatus for linking the selected LBPs and the synthesized chromophores, - a loading apparatus for transferring the LBP-tag into predetermined cavities which are coated with the target molecule/ligand in the assay platform, - a transfer robot arm for moving the assay platform into the laser system, - an apparatus for reading the activity, - a database, - a central computer system.
8) Use of an LBP-tag for carrying out a method for identifying the function of a ligand L using chromophore-assisted laser inactivation.
9) Use of a ligand L, the function of which having been identified by the method of claim 1, for the development of a drug for the treatment of a disease.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19854195.3 | 1998-11-24 | ||
| DE19854195A DE19854195C2 (en) | 1998-11-24 | 1998-11-24 | Method for identifying the function of biological molecules and device for carrying out the method |
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| Publication Number | Publication Date |
|---|---|
| CA2290237A1 true CA2290237A1 (en) | 2000-05-24 |
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ID=7888852
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002351419A Abandoned CA2351419A1 (en) | 1998-11-24 | 1999-09-24 | Method and device for automated chromophore-assisted laser/light inactivation (cali) |
| CA002290237A Abandoned CA2290237A1 (en) | 1998-11-24 | 1999-11-23 | Method for identifying the function of biological molecules and apparatus for carrying out the method |
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| CA002351419A Abandoned CA2351419A1 (en) | 1998-11-24 | 1999-09-24 | Method and device for automated chromophore-assisted laser/light inactivation (cali) |
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| EP (2) | EP1149280B1 (en) |
| JP (2) | JP2002530670A (en) |
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| DK (1) | DK1149280T3 (en) |
| ES (1) | ES2186412T3 (en) |
| WO (1) | WO2000031517A1 (en) |
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| JP4288571B2 (en) * | 2003-05-21 | 2009-07-01 | 独立行政法人科学技術振興機構 | Method for analyzing physiological function of target substance |
| DE102005046583A1 (en) * | 2005-09-28 | 2007-03-29 | Eppendorf Ag | Real-time polymerase chain reaction device, has monitoring device with electrical device for examining electrical function of light emitting diodes, where monitoring device generates signals when determining malfunction of diodes |
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| DE3818654A1 (en) * | 1988-06-01 | 1989-12-14 | Benedikt Dr Med Jean | Selective, laser-induced inactivation of proteins by fluorochrome labelling |
| US5784152A (en) * | 1995-03-16 | 1998-07-21 | Bio-Rad Laboratories | Tunable excitation and/or tunable detection microplate reader |
| US5780857A (en) * | 1996-10-04 | 1998-07-14 | Wallac Oy | Apparatus for imaging biochemical samples on substrates |
| DE69839501D1 (en) * | 1997-02-27 | 2008-06-26 | Cellomics Inc | System for screening biological cells |
-
1998
- 1998-11-24 DE DE19854195A patent/DE19854195C2/en not_active Expired - Fee Related
-
1999
- 1999-09-24 CA CA002351419A patent/CA2351419A1/en not_active Abandoned
- 1999-09-24 AU AU61966/99A patent/AU761573B2/en not_active Ceased
- 1999-09-24 WO PCT/EP1999/007126 patent/WO2000031517A1/en active IP Right Grant
- 1999-09-24 EP EP99948861A patent/EP1149280B1/en not_active Expired - Lifetime
- 1999-09-24 DE DE59903278T patent/DE59903278D1/en not_active Expired - Fee Related
- 1999-09-24 JP JP2000584281A patent/JP2002530670A/en active Pending
- 1999-09-24 ES ES99948861T patent/ES2186412T3/en not_active Expired - Lifetime
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- 1999-11-23 CA CA002290237A patent/CA2290237A1/en not_active Abandoned
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| JP2002530670A (en) | 2002-09-17 |
| DE19854195A1 (en) | 2000-06-29 |
| EP1004873A1 (en) | 2000-05-31 |
| EP1149280B1 (en) | 2002-10-30 |
| ES2186412T3 (en) | 2003-05-01 |
| JP2000206116A (en) | 2000-07-28 |
| AU6196699A (en) | 2000-06-13 |
| AU761573B2 (en) | 2003-06-05 |
| EP1149280A1 (en) | 2001-10-31 |
| WO2000031517A1 (en) | 2000-06-02 |
| DE59903278D1 (en) | 2002-12-05 |
| ATE227021T1 (en) | 2002-11-15 |
| DK1149280T3 (en) | 2003-03-03 |
| DE19854195C2 (en) | 2001-02-01 |
| CA2351419A1 (en) | 2000-06-02 |
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