Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Patent
1998-02-23
2000-02-01
Ceperley, Mary E.
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
436531, 436800, 436813, 5303913, G01N 33533, C07K 1630
Patent
active
060202122
DESCRIPTION:
BRIEF SUMMARY
The invention relates to the use of a phycobiliprotein-linker peptide complex as a fluorescent tracer, notably in a fluorescent method of detecting and/or determining an analyte in a medium in which it may be present, as well as to the fluorescent conjugates constituted of said complex covalently bound to one of the elements of a ligand/receptor specific binding pair.
The use of immunological determinations in qualitative and quantitative analysis of compounds in biological fluids is currently well known.
Amongst the existing techniques, the determinations by fluorimetry have become of increasing importance.
In fact, they possess a certain number of advantages amongst which are the sensitivity, the speed of measurement, the stability and the innocuousness of the reagents labelled by fluorescent compounds, and the relatively low cost.
The phycobiliproteins are constituents of the phycobilisome of various bacteria, algae or cryptomonads and are in general of four types: the phycocyanines, the phycoerythrins, the phycoerythrocyanines and the allophycocyanines.
The proteins are made up of .alpha. and .beta. sub-units, and sometimes .gamma. sub-units, each one having one or more fluorophores, and are isolated mainly as trimers or hexamers.
Some of them are used as fluorescent labelling compounds by virtue of the many advantages they have in terms of quantum yield, absorption bands, stability and solubility (V.Oi et al, J. Cell Biology 1982, 93, 981).
Schematically, phycobilisomes are made up of two parts: and being constituted by phycoerythrins and phycocyanines.
The rods and cylinders are made up of an assembly of disks of phycobiliproteins. These disks are assembled between themselves as well as the rods to the core and the core to the thylakoid membrane via linker peptides.
These peptides are named according to their lcicalisation in accordance with the publication A. Glazer, J. Biol. Chem., 1989, 264, 1-4:
The purification of the phycobiliproteins by conventional methods makes it possible to obtain trimeric or hexameric (.alpha..beta.).sub.3 or (.alpha..beta.).sub.6 complexes which are devoid of linker peptides. The trimers have a disk shape of .apprxeq.30 .ANG. thickness and .apprxeq.120 .ANG. diameter.
The use of phycobiliproteins, notably allophycocyanine and phycoerythrin, in immunological determinations by fliiorimetry is described notably in EP 0 174 744 and 0 076 695 as well as in the patents U.S. Pat. Nos. 4,520,110 and 4,542,104.
It has been found that under certain conditions of purification of the phycobilisomes, it was possible to obtain phycobil-iprotein-linker peptide complexes (P. Fuglistaller et al., Biol. Chem. Hoppe-Seyler, 1987, 368, 353-367). However, these complexes were hitherto described in the literature as being relatively unstable and sensitive to proteases (W. Reuter and C. Nickel-Reuter, J. Photochem. Photobiol., B: Biol., 1993, 18, 51-66).
Advantageously, it has now been found that these complexes possess spectroscopic particularities with respect to the phycobiliproteins for a use as a fluorescent tracer.
In fact, such a phycobiliprotein-linker pejptide complex always possesses spectroscopic properties which are different from those of the phycobiliprotein alone, generally with: the phycobiliprotein alone.
These properties can reveal to be particularly interesting during the implementation of a detection system which uses one or more fluorescent tracers, in which, in addition to the stability of the tracers in the medium, two parameters are of utmost importance: system, determining factor of their choice.
Unexpectedly, it has now been found that: in the presence of sera of various origins containing naturally different proteases, antibodies while keeping the fluorescent properties of the complexes.
In an advantageous aspect, the phycobiliprotein used according to the invention is selected from phycoerythrin, phycoerythrocyanine, phycocyanine, allophycocyanine and allophycocyanine B.
In the rest of the description, the following abbreviations will be used to designate the prefer
REFERENCES:
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P. Fuglistaller et al, Biol. Chem. Hoppe-Seyler, vol. 368, pp. 353-367 (1987)., Apr. 1987.
Wolfgang Reuter and Claudia Mickel-Reuter; "Molecular Assembly of the Phycobilisomes from the Cyanobacterium Mastigocladus laminosus"; J. Photochem, Photobiol. B: Biol., 18 (1993) 51-66.
Vernon T. Ol, Alexander N. Glazer and Lubert Stryer; "Fluorescent Phycobiliprotein conjugates for Analyses of cells and Molecules," The Journal of Cell Biology, vol. 93, Jun. 1982, pp. 981-986, The Rockefeller University Press.
Alexander N. Glaser; "Light Guides--Directional Energy Transfer in a Photosynthetic Antenna;" Minireview; The Journal of Biological Chemistry, vol. 264, No. 1, Issue of Jan. 5, pp. 1-4, 1989.
Ceperley Mary E.
Cis Bio International
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