Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-11-12
2001-01-30
Houtteman, Scott W. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023400, C536S023510
Reexamination Certificate
active
06180769
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The subject invention is directed at a method for preparing strongly negatively charged non-proteinaceous macrobiomolecules which are linked to plastic.
DESCRIPTION OF RELATED ART INCLUDING INFORMATION DISCLOSED UNDER 37 C.F.R. 1.97 and 1.98.
Not applicable
Strongly negatively charged biological macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and glycosaminoglycans (GAG) play a crucial part in cell functioning. In principal strongly negatively charged implies at least 1 negative charge per pentasaccharide of the biomolecule in so far as this is composed of pentasaccharides. DNA and RNA contain 1 negative charge per nucleotide. Glycosaminoglycans are heterogenous in this respect and can contain 1 to 4 negative charges per disaccharide. Compounds with such charge structure can be applied in the invention from which proteins, polypeptides, oligopeptides and peptides, the so-called proteinaceous molecules, are excluded. It is generally known that such proteinaceous molecules can coat polystyrene in physiological salt solutions (for example 0.15 M NaCl) however for non-proteinaceous molecules until now preferably spacer molecules have been applied.
DNA encodes all proteins, RNA is an intermediate in protein synthesis and glycosaminoglycans (such as heparin) play a part during fundamental processes such as growth, differentiation and blood coagulation. By development of molecular biological techniques nucleic acids have begun to play an increasingly large part in analysis of biological material. Fragments of DNA that are specific for example for causative organisms of disease (bacteria, viruses etc.) can be used for detection of these organisms and diagnosis of hereditary diseases also occurs at DNA level. Besides the strongly developing diagnostic importance of DNA/RNA, nucleic acids are also often applied for synthesis (also at industrial level) of specific proteins via so-called recombinant DNA techniques. Antibodies directed against nucleic acids play a part in diseases of connective tissue and autoimmune diseases such as Systemic Lupus Erythematosis. From this it is clear that good analysis possibilities of in particular DNA and RNA are of crucial importance. Microtitre plates are often used for analysis of a large number of samples. In diagnostic routine assays microbeads, tubes and strips are also used a lot. Microtitre plates are plastic plates (usually of polystyrene) which contain 96 wells in which the sample is analysed. A known example of use of such plates is for ELISA's (Enzyme Linked Immuno Sorbent Assays). A large disadvantage of these plates is that for assays wherein biological micromolecules with a strong negative charge have to be immobilized, such molecules hardly bind to the plastic plates. Immobilized macromolecule is however required for many tests. At the moment spacer molecules are used to bind negatively charged molecules to plastic. The spacer molecules bind both to the plastic and the macromolecule. Examples of such spacer molecules are protamin and polylysine, which are both positively charged proteins. Another option is the modification of DNA/RNA via biotinylation whereby it binds to avidin which can be bound in turn to plastic. Both methods require additional steps, are costly and also sensitive to disturbances. Such a disturbance is the provision of false positive results which cannot be acceptable in particular for diagnostic tests.
In Journal of Colloid and Interface Science (1990) 136: 519-526 for example Yamaoka et al describe the linkage of deoxyribonucleic acid and poly(citydyl acid.poly(inosinic acid) to poly(styrene)latex. In this procedure Yamaoka et al first dialyse the nucleic acid against the salt with which linkage is to occur. The purpose hereof is to charge the nucleic acid with the relevant ions. In the illustrated instance of page 520 replacement of monovalent Na
+
by divalent Ca
2+
occurs. In addition the polystyrene has to be dialysed against the relevant salt containing liquid (page 520) and thus needs pretreatment prior to linkage to the macromolecule being possible. The highest concentration of salt solution applied by Yamaoka is 0.01M and this is a concentration at which the amount of DNA more or less reaches a maximum. On page 523 it is further reported the Na-salt of DNA can hardly be linked. Yamaoka et al believe that binding of divalent anions that bind on one hand to negative groups on DNA and on the other hand to negative groups in the polystyrene is responsible for the linkage of DNA to the matrix. The divalent cation is to be regarded as a spacer molecule (page 519). The removal of the divalent cation for example with EDTA results in desorption of DNA from the matrix. The linked DNA produced by Yamaoka et al can easily be removed from the latex (p522). Obviously the DNA binding achieved with the method described by Yamaoka et al results in such weak linkage of the DNA to polystyrene that no applications with extreme circumstances such as used in hybridisation assays are possible.
In European patent Application 0.389.063 filed Sep. 26 1990 the isolation of nucleic acid via binding thereof to a solid phase in the presence of a chaotropic substance is described. KI, NaI, guadinium(iso)thio cyanate, guanidinehydrochloride and ureum are given as examples of chaotropic substances and latex particles such as polystyrene and glycylmethacrylate particles are provided as examples of solid phase to be used. Page 4 lines 10-13 of the application illustrates that the nucleic acid is easily removed from the carrier for example by treatment with TE buffer or aqua bidest. The process described by Akzo cannot provide nucleic acid sufficiently strongly bound to solid phase to remain bound under conditions generally applicable for nucleic acid assays.
BRIEF DESCRIPTION OF THE INVENTION
The subject invention is capable of providing such linkage. The subject invention comprises a cheap, simple, efficient and reliable method to directly bind strongly negatively charged nucleic acid or glycosaminoglycan macrobiomolecules to plastic carriers such as polystyrene, polyethylene, latex, polyvinylidene difluoride or polycarbonate. These plastics need not be subjected to chemical activation or modification and can thus be applied as such which offers an appreciable advantage over the existing methods. The subject method for preparing the macrobiomolecules of the invention with strong negative charge linked to plastic is characterized in that the macrobiomolecules and plastic are contacted with each other in the presence of a non chaotropic solution capable of removing the water coat of the molecule and/or shielding the negatively charged groups of the macrobiomolecule such that direct linkage occurs between molecules and plastic i.e. without a spacer molecule and without requiring an activation step of the plastic. The linkage step is followed by removal of the solution. The conditions of the solution used depend on the applications. The method can be carried out by applying a solution with 20-100% salt saturation which provides direct linkage between molecules and plastic. Preferably a solution with a degree of saturation of more than 50% is applied, with more preference for a degree of saturation of more than 60%. The optimal results are obtained with a solution with a degree of saturation between 70-100%. The suspected mechanism of operation is as follows. The salt removes the water coat around the negatively charged molecules and shields the negative charges such that the interaction with plastic, for example polystyrene is strongly improved.
Preferably a non chaotropic salt belonging to the Hofmeister series of salts is used for the solution according to the subject invention. A non chaotropic salt of a metal from group I or II of the Periodic Table of Elements or a NH+
4
salt are examples of effective salts. A
Van de Lest Christiaan Hendrikus Adriaan
Van Kuppevelt Antonius Henricus Minardus Severus Marie
Veerkamp Jacobus Henricus
Angiomed GmbH & Co. Medizentechnik KG
Darby & Darby
Houtteman Scott W.
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