Maija-Liisa Mäkinen - Academia.edu (original) (raw)
Papers by Maija-Liisa Mäkinen
Analytical Chemistry, 2004
We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-res... more We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energytransfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z′ factors.
Analytical Biochemistry, 2004
Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparamet... more Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.
"Protein Engineering, Design and Selection", 1995
Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for... more Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.
Analytical Chemistry, 2004
We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-res... more We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energytransfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z′ factors.
Analytical Biochemistry, 2004
Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparamet... more Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.
"Protein Engineering, Design and Selection", 1995
Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for... more Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.