Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-03-26
2001-07-10
Swartz, Rodney P. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S007100, C435S262000, C435S270000, C436S518000, C436S528000, C436S807000, C530S412000, C530S413000, C530S415000
Reexamination Certificate
active
06258531
ABSTRACT:
Subject matter of the invention is a method of isolating a biological material by binding said biological material to a solid phase, and releasing said material in a special procedure and a system suitable for isolating said biological material.
Recently, biological materials have gained increasing interest in many fields. This is facilitated by the fact that it has become possible over the last few decades to separate biological materials from other materials. Biological materials are generally available in a complex mixture together with other materials. Moreover, most biological materials are also present in very minute amounts compared to other components of a biological individual. Changes of biological materials with respect to its normal condition serve to diagnose the condition of the biological individual. Methods of analyzing biological materials are, hence, of particular interest in the field of molecular biology and health care. Depending on the required analysis, a more or less specific method of isolation is selected. There exists a multitude of isolation methods for biological materials which depend on the type of biological material to be isolated and its subsequent use. In the method used in the analysis of antigens and antibodies, the biological material (e.g. an antigen, antibody or nucleic acid) is bound to the non-porous inner wall of a cuvette made of glass or polystyrene. In this case, the binding of the biological material is so specific that the biological material to be detected is immobilized on the surface. This method does not propose to again release the biological material; this would even be adverse to the subsequent quantitative determination.
A second type of method uses swellable, porous materials to separate biological materials, e.g. according to their molecular weight. In this method, there is no binding between a biological material and a solid phase. The separation is essentially accomplished by using the different penetration properties of biological materials based on their different sizes.
A third kind of isolation method is based on the varying degree of binding of different biological materials to porous materials which exhibit a particular affinity to the biological material when specificity-enhancing groups are attached. This method makes use of column-like accumulations of particle-type affinity materials. A liquid which contains the biological material is made to pass through this accumulation of material. The biological material to be isolated is then bound to the surface of the porous particles on the groups with the affinity for the biological material; all other components emerge from the column together with the remaining liquid. In a subsequent step, the biological material is released from the porous material by allowing an elution to flow through the column in exactly the same direction which then releases the binding to the porous material. The biological material is then contained in the elution liquid.
In presently known methods, the elution liquid was passed through the porous matrix either by applying pressure in flow direction of the liquid, or even by centrifuging the column to spin the elution liquid out of the porous matrix. This method, however, requires the use of vacuum pumps or centrifuges, hence, instruments that are often used in medical routine diagnostics for this particular purpose. Moreover, the use of these instruments, especially centrifuges, is time-intensive and does not allow the continuous processing of samples.
It is an object of the present invention to provide a new method of isolation for biological material which brings about improvements with respect to those known in prior art.
Subject matter of the invention is a method of isolating a biological material by providing a biological material bound to a compressible porous matrix, and compressing the matrix under conditions where the biological material is released from the surface of the matrix into an elution liquid. Another subject matter of the invention is a system for isolating the biological material.
An isolation method as understood in the invention is a method where one or several components of the mixture are separated from the remaining components of this mixture. This covers in particular those methods where the one or several components to be separated are bound to a solid phase, where the remaining liquid is removed and the one or several bound components are subsequently released into another liquid.
Biological material is understood to be organic compounds that are in a relation to beings such as animals, humans, viruses, bacteria, or plants. They include, for example, the components found in such beings. Particularly preferred components are those that are present either in a dissolved form or can be dissolved in liquid, but can also be bound to a solid matrix. They include low-molecular substances, on the one hand, (with a molecular weight of less than 2000 D), such as vitamins, but also therapeutically active substances and hormones; further, they also include high-molecular substances (with a molecular weight of more than 2,000 D), such as biopolymers that consist of monomer units. They include, for example, proteins and nucleic acids. The method in accordance with the invention is particularly suitable for isolating nucleic acids. When dealing with proteins, the immunologically active substances, such as antigens and antibodies are particularly preferred.
Biological materials which can be isolated in a preferred manner are recovered from a liquid of the being in question and bound to a matrix. Depending on the part of the being from which the biological material is to be isolated, the being may be subject to a pretreatment. Such a pretreatment serves to release the biological material from the being. Such a pretreatment is necessary, for example, when the being is a bacterium or a group of bacteria. In this case, it is preferred to destroy the cells to allow the components of the being to be released into the liquid. Subsequently, the solid substances that may have formed can be separated. If the biological material is present in a liquid in an already accessible form, e.g. in a body fluid, cells or other substances that may interfere with the isolation of the biological material are often separated. This can be accomplished by means of filtration or by using affinity materials. In any case, the result of a pretreatment is always a sample liquid which contains the biological material in a form where it can be bound to a matrix.
In one step of the method of the invention, the biological material is provided in a form where it is bound to a compressible porous matrix. The matrix as understood in the invention is a non-soluble material present in the liquid containing the biological material. The chemical composition of the matrix is determined by the fact that the matrix must be compressible and porous, for which reason organic or inorganic polymers are used. Organic polymers include, for example, plastics such as polystyrene, but also cellulose, e.g. paper. Inorganic polymers are in particular substances which contain a certain percentage of glass. The matrix, can, however, also consist of a metal.
The compressible porous matrix in accordance with the invention is a spatially extending structure having a non-soluble part made of the above-mentioned material and a part which can be filled with liquid. This fillable part is in the following referred to as the inner volume. The inner volume extends between the non-soluble part thus forming a system of coherent porous and/or empty spaces. This system could preferably also be referred to as an open pore system.
The non-soluble part in the system also forms a spatially extending structure, which, in a preferred manner, has a coherent structure. It is possible, but not preferred, that the matrix be a multi-component system, e.g. a system where the particle-type component is sealed between two particle-type components in direction toward the interior (C14) and in direction of a
Bienhaus Gerhard
Fritz Michael
Geisler Edda
Harttig Herbert
Macho Heinz
Arent Fox Kintner Plotkin & Kahn
Roche Diagnostics GmbH
Swartz Rodney P.
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