Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
2000-08-07
2004-12-21
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C436S523000, C436S524000, C436S525000, C436S073000, C436S080000, C436S169000, C156S279000, C205S169000, C427S189000, C427S191000, C427S196000, C427S201000, C427S205000, C424S278100, C252S180000, C252S182290, C252S183110
Reexamination Certificate
active
06833275
ABSTRACT:
The invention concerns compositions which comprise colloidal particles on the surface of which biomolecules are adsorbed.
Conjugates of biomolecules, such as proteins or nucleic acids, and colloidal particles are widely used for example as signal transmitters or/and as capture reagents in diagnostic and therapeutic methods. They serve for example as markers in detection procedures such as immunoassays or as microprojectiles for gene transfer. Particles that can be used are particles of metals and metal compounds such as metal oxides, metal hydroxides, metal salts and polymer cores which are coated with metals or metal compounds (cf. e.g. U.S. Pat. No. 4,313,734; Leuvering et al., J. Immunoassay 1 (1980), 77-91; Leuvering Dissertation (1984), Sol Particle Immunoassay (SPIA): The use of Antibody Coated Particles as Labelled Antibodies in Various Types of Immunoassay; Uda et al., Anal. Biochem. 218 (1994), 259-264, DE-OS 41 32 133, page 3, lines 16-18 for applications as a marker and Tang et al., Nature 356 (1992), 152-154; Eisenbraun et al., DNA and Cell Biology 12 (1993), 791-797; Williams et al., Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2730 for gene transfer applications). Furthermore it is also known that non-metallic colloidal particles such as carbon particles can be used (van Amerongen, Anabiotic '92 (1993), 193-199). At present colloidal gold particles are most frequently used.
Biomolecule-gold conjugates are prepared by firstly preparing gold sols by generally known procedures by reducing tetrachloroauric acid. Subsequently the gold sols are loaded with the biomolecule desired in each case e.g. proteins such as antibodies, protein A, protein G, streptavidin etc. The respective loading conditions (pH, buffer, concentration of the biomolecules etc) depend on the isoelectric point of the biomolecules, the MPA (minimal protecting amount) or/and the specific application of the conjugate (cf. e.g. De Mey, The Preparation and Use of Gold Probes, in: Immunocytochemistry, publisher: J. M. Polak and S.V. Noorden, pages 115-145, Wright, Bristol 1986; J. E. Beesley, Colloidal Gold: A New Perspective for Cytochemical Marking, Microscopy Handbooks 17, Oxford University Press, 1989, in particular pages 1-14; G. Frens, Nature Physical Science, 241 (1973) 20-22; J. Roth, The Colloidal Gold Marker System for Light and Electron Microscopic Cytochemistry in: Immunocyto-chemistry 2 (1983 218-284)). Explicit reference is made to the disclosure of these documents.
After loading the colloidal particles with the respectively desired biomolecule, it is necessary to stabilize the conjugates. This stabilization is intended to reduce aggregation of the particles and to saturate remaining free surfaces that are accessible to adsorption. Stabilizers used in the state of the art are inert proteins e.g. bovine serum albumin, blood substitute mixtures etc., water-soluble technical polymers such as polyethylene glycol (molecular weight 20,000 D), polyvinylpyrrolidone, polyvinylalcohol, polyvinylsulfate, dextran and gelatin (cf. e.g. De Mey, Supra; Beesley, Supra; Behnke, Eur. J. Cell Biol. 41 (1986), 326-338; DE 24 20 531 C3; and Meisel et al., J. Phys. Chem. 85 (1981), 179-187). In addition the possibility of stabilizing gold sols by phosphane complex ligands has also been described (Schmid et al., Z. Naturforsch. 45b (1994), 989-994).
When loading colloidal gold particles the procedure is usually to adjust the solution of the protein to be adsorbed to the gold as well as the gold sol to a pH close to the isoelectric point (IP) of the protein. In this connection it has been regarded as essential in the state of the art for a successful loading that the protein solution should if possible contain no additives and e.g. the ionic strength should not be above 10 mM. For the loading the protein solution is added to the gold sol while stirring or vice versa. After the protein has bound to the gold particles, a solution of a suitable stabilizer is added. Optionally the conjugate formed is subsequently purified e.g. by ultra-centrifugation or gel filtration.
The stabilizers used according to the state of the art bind adsorptively to the free surfaces of the metal particles. Longer storage or changes in the ambient conditions such as those which occur in a test by contact with the sample (blood, serum, plasma, urine), incorporation of the conjugates in test strip fleeces etc., can desorb or displace the stabilizers from the surfaces to a greater of lesser extent. This leads to a deterioration of the aggregation stability and to an increase in the unspecific reactivity. Moreover most of the stabilizers used are poorly defined products with variable quality in some cases e.g. bovine serum albumin, gelatin. This can also cause variations in the stabilizing effect.
Adsorption processes on particle surfaces are very complex and hitherto are only partially understood. It is assumed that the adsorption is due to a combination of electrostatic interactions, Van-der Waals forces and hydrophobic interactions (Beesley, Supra). Depending on the type of adsorbed biomolecule, the one or the other type of binding can dominate in this process.
Aggregates form to a certain extent in the protein-gold conjugates according to known techniques. These undesired aggregates frequently already occur before addition of the stabilizer. The reasons for the formation of aggregates can for example be that “sticky” proteins i.e. proteins with a hydrophobic surface bind together and consequently also bridge the gold particles to which they are conjugated. Therefore it has for example been described that IgG preparations should be freed of aggregates by ultracentrifugation before coupling to gold (W. D. Geoghegan, G. A. Ackerman, J. Histochem. Cytochem. 25 (1977), 1187-1200). Furthermore it is possible that hydrophobic patches that are not covered by protein on the surface of noble medal particles and in particular gold particles can interact with one another and form particle aggregates. A further cause for the occurrence of undesired aggregates can also be that hydrophobic patches on the noble metal surface that are not covered by protein interact with hydrophobic patches on proteins conjugated to neighbouring gold particles and thus cross-link the gold particles.
One frequently also observes a secondary cross-linking of protein gold conjugates which also occurs when the conjugates have already been saturated with a stabilizer. This is probably due to the fact that even after the action of conventional stabilizers, hydrophobic patches remain on the gold surface or/and on the conjugated protein by means of which an uncontrolled aggregation of protein-gold particles occurs with slow kinetics. These problems do not only occur in conjugates containing particles of gold but also in particles made of other solids, especially other metals.
Therefore the object of the present invention was to provide conjugates of colloidal particles and biomolecules in a stable form which do not have the disadvantages of the state of the art.
Surprisingly it has been found that detergents are extremely suitable for stabilizing biomolecule particle conjugates. One subject matter of the invention is therefore a composition which comprises colloidal particles on the surface of which biomolecules are adsorbed wherein the composition additionally contains a detergent.
It was found that the addition of detergents to colloidal particles, for example to a gold sol, before loading with biomolecules and/or to a solution containing biomolecules before and/or during loading of the colloidal particles, impairs binding to the particles to an extent that is considerably less than would have been expected by a person skilled in the art, and is surprisingly advantageous in several respects. The addition of detergent according to the invention prevents aggregation processes that already take place with conventional stabilizers before conjugation and before re-loading and this improves the reproducibility of the manufacturing process for the conjugates and leads to a more unif
Chin Christopher L.
Do Pensee T.
Roche Diagnostics GmbH
Rothwell Figg Ernst & Manbeck
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