Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2000-07-24
2002-10-22
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C422S050000, C422S098000, C422S063000, C422S064000, C422S091000, C422S105000, C422S105000, C422S105000, C210S695000, C210S088000, C210S416100, C210S224000, C210S232000, C435S287100, C435S288600, C435S288400
Reexamination Certificate
active
06468810
ABSTRACT:
The invention relates to a method for transfer of a substance immobilised to magnetic or magnetisable material with the aid of a magnet. The invention is characterised in that the magnet used for transfer is separated from the material to be transferred by an extendable membrane, shapable membrane or by coating of the magnet.
TECHNICAL BACKGROUND
In the traditional method for separating magnetisable material a magnet which is outside the vessel is used. The magnetisable material is left in the vessel and surrounding solution is removed from the vessel.
By introducing the magnet into the solution great advantages are obtained when collecting magnetisable material compared to the traditional methods. It is especially remarkable that in this case the magnetisable material can simply and efficiently be removed from the vessel. When introducing the magnet into the solution the distance of the magnet from the magnetisable material is shorter than when using an outside magnet. Also, due to the fluid's surface tension collecting magnetisable material left in the solution interface is more efficiently accomplished when the magnet is introduced into the solution.
Patent literature presents numerous devices for separating magnetisable material. The international patent publication WO 87/05536 discloses a separation device within which a permanent magnet moving inside a plastic sleeve can be used to transfer magnetisable material from one vessel to another. The Finnish patent publications (FI 86 05002, FI 95 03669, FI 97 01665, FI 97 01666, FI 97 01667 and FI 97 01668) likewise disclose various methods based on the use of a permanent magnet for transfer of magnetisable material from one vessel to another. The patent publications U.S. Pat. Nos. 4,272,510, 4,649,116 and 4,751,053 disclose magnetic material transfers based on the use of an electromagnet mainly in RIA and EIA assays. The patent publication U.S. Pat. No. 5,567,326 discloses equipment for separation of magnetic particles from the non-magnetic reaction solution with the aid of a steel pin magnetisable with a permanent magnet. Typically, the equipment would include a multiwell reaction plate where magnetic particles can be separated concomitantly in many neighbouring reaction wells using a transfer device with many magnetisable pins. The method described in the patent publication U.S. Pat. No. 5,567,326 is very tedious to use. The unprotected steel pins need to be washed or sterilised in between each time of use. There is a serious risk of contamination in the aforementioned method should the washing not be sufficient.
Magnetic particles have been described in numerous patent publications, for example U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; 4,659,678; 4,978,610; 5,200,084; and 5,705,628. Particle using technology became very popular in for instance immunoassays. Using magnetisable particles for separation of bound antigen-antibody complex from the unbound fraction in immunoassays offered major advantages both in reaction speed and practicality of separation.
Magnetic particles in reaction solution with bound biological material, e.g. cells or an antibody, have been, after the reaction has taken place, secured with the aid of the magnet outside the vessel into a certain location whereupon the solution can be removed without magnetic particles leaving the vessel.
Use of magnetic particles is beneficial because when handling samples, no expensive or space consuming instruments are needed, such as centrifuges, vacuum pumps or chromatographic columns. Magnetic particle applications are simple to perform and volumes used thereupon can vary according to use from small to large.
For the present, magnetic particles are used amongst others in immunoassays, separating cells and bacteria, isolating nucleic acids as well as purification of proteins.
In molecular biology many operations such as isolating and/or transferring nucleic acids as well as using restriction or nucleic acid modifying enzymes pose problems. Among those encountered are inactivation of enzymes, extraction with solvents and star-activity.
Traditionally nucleic acids are isolated and transferred by means of various precipitations and solvent extraction. Some compensatory methods have been presented as aid in nucleic acid management However, these methods are in general expensive and require centrifugation steps. In addition, in some of these methods recovering the nucleic acid in a sufficiently small volume after the operation is difficult.
In methods of molecular biology, where DNA or RNA is manipulated, use is made of restriction enzymes as well as of DNA and/or RNA modifying enzymes. The use of these enzymes is of essential importance in almost all work in the field of molecular biology. The most pre-eminent enzymes in molecular biology labs are the restriction enzymes. These enzymes have made possible major developments in the field. Using restriction enzymes or nucleic acid modifying enzymes in molecular biology applications is mainly routine work which in many cases involves tedious intermittent stages. A good example is provided by the operations needed to eliminate restriction enzyme activity after their use. Many restriction enzymes require phenol extraction in order to inactivate them after use. Phenol extractions are very tedious and from the point of view of the user unpleasant processes. Furthermore, a lot of hazardous waste is generated in these extractions. Commercial manufacturers suggest for many restriction enzymes inactivation by heat treatment whereas in practice users often perform a phenol extraction to insure inactivation of the enzyme. After heat treatment a large percentage of enzyme activity may still remain. Because one has not been able to remove restriction enzymes with currently known techniques the problem has been solved by inactivating enzymes, e.g. by heat or phenol extraction. Another disadvantage is that the used, expensive enzyme can not be reused. Less time consuming but otherwise problematic are various spin columns for purifying DNA from reaction solution. The use of these columns is very expensive, and they are not applicable for removal of many enzymes from DNA solution. Even in this case the retracted enzyme can not be reused.
Phenol extraction is required for inactivation of even many other enzymes commonly used in the field of molecular biology. As examples can be mentioned CIP (Calf Intestinal Phosphatase) and Proteinase K.
No unproblematic means have been presented for transferring and washing restriction enzymes or nucleic acid modifying enzymes. As an example of a problem might be mentioned the star-activity caused by the glycerol used in restriction enzyme storage solution. By this is meant the capacity of restriction enzymes to cut DNA unspecifically, i.e. in places where cutting is not wanted. Commercial restriction enzymes are generally provided as 50% glycerol containing solution. In normal use, a very small amount of restriction enzyme is added to the reaction, even less than 1 &mgr;l. If the glycerol content in reaction mixture is too high, it poses, in many cases, a big problem mainly because of the occurrence of star-activity. This sets limits for many molecular biology applications in regard of restriction enzyme use. Another important fact is that it is recommendable to maintain the total volume of the reaction mixture as low as possible in order to have a sufficiently fast enzyme reaction. Commercial restriction enzymes are generally available in one, or at the most two standard concentrations (U/ml). If a great amount of restriction enzyme is wanted in the reaction the glycerol content in reaction solution reaches too high a level. As a result there is star-activity and reaction kinetics are markedly slowed down.
Patent literature suggests preparations in which restriction, or other in molecular biology commonly used, enzymes have been immobilised on a solid support. International patent publication WO 92 15674 suggests immobilising restriction enzymes as well as nuc
Bio-Nobile Oy
Do Pensee
Le Long V.
Lydon James C.
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