Patent Application
20110256025
The use of gel electrophoresis is currently the ubiquitous technique for the separation of biological materials. Nonbiological materials can also be separated using gels or other chromatographic supports as well, but the scope of effort with regard to biologicals is greater. Typical applications include separation of nucleic acid fragments of various sizes either in the context of sequence determination; in the detection of polymorphisms; or verification of sizes in other contexts. Also frequently conducted are separations of proteins, glycoproteins, protein fragments and application of gel separations as verification of homogeneity or purity, identification of post translational modifications and confirmation of molecular weight.
In all of these procedures, mixed samples of biological entities are applied to electrophoretic gels and the components are separated by application of an electric field across the gel. Regardless of the manner in which the gel is developed, the resulting pattern of migration of the substances contained in the sample must be detected in some manner.
To conduct this detection, typically the gel support is contacted with a blotting membrane to which the substances are transferred in the same pattern in which they appeared on the gel. The “spots” are then detected, at a minimum, by blocking the membrane with a protein or detergent solution to reduce non-specific binding (which otherwise leads to a high level of noise and low level of detection). Typical blocking agents include casein, bovine serum albumin (BSA), non-fat dry milk (generally about 1-5%) in a Tris buffer saline solution with TWEEN® surfactant (TBS-T solution) or phosphate buffer saline solution with TWEEN® surfactant (PBS-T solution). The biological entity is then incubated with an antibody specific for the antigen on the membrane. The membrane is then extensively washed to remove any contaminants, unbound blocking proteins or antibodies and the like. The membrane is then treated and incubated with a secondary enzyme-, radioisotope-, fluorfluor-, or biotin-conjugated antibody specific for the primary antibody. The membrane is then extensively washed again to remove any unbound secondary antibody. Then a detection reagent, generally a chromogenic, chemiluminescent, fluorescent, radiological, or streptavidin-labeled material, is applied which either binds to, or is a substrate of the enzyme-conjugate. Lastly, the appropriate detection device is used to determine the presence, absence, position, quantity, etc. of the biological entity. The last six steps generally take from 3-6 hours to overnight depending on the speed of the reaction between the selected reagents, the membrane and the biological entity. The process requires multiple incubation periods of the membrane on a rocking or other suitable mixing platform. It is a lengthy process that most researchers dislike and which consumes (wastes) a large volume of reagents.
Some researchers have suggested the use of the capillary action of an absorbent material such as filter paper placed below the membrane to draw the remaining fluids through the membrane and improve the speed of the process especially the washing steps.
U.S. Pat. No. 5,155,049 mentions a system called the Hybrid-Ease™ hybridization chamber marketed by Hoefer Scientific Instruments. This chamber is comprised two grids between which the membrane is sandwiched. The grid plates are snapped into position surrounding the membrane, and syringes fitted into the open space created by the grids. One syringe is used to apply reagents and wash, and the other to withdraw excess. The system requires large volumes of liquid in order to operate, is cumbersome to employ and is still quite time consuming. It also mentions that in some particular assays, such as ELISA assays, in small volume wells (such as 96 well microtiter plate), others have used vacuum to draw liquids through a membrane in a washing step. However, they discount this effort as it is only available in small volume applications and still is uncontrollable. They suggest instead that the better method is to use a manual press having the membrane on top of a filter paper and cover layer and then pressing the membrane sandwich between two plates to squeeze the liquid through the membrane and into the paper.
In U.S. Ser. No. 60/732,994, filed Nov. 3, 2005 it is suggested that one use a device formed of several layers including a porous support layer below the one or more layers of blotting membrane, a flow distributor above the blotting membrane(s) and a well on the flow distributor to contain the liquid to the desired area and to allow for lower starting volumes of such liquid. Preferably, the flow distributor is a non-binding or low binding porous membrane such as a 0.22 micron membrane. The device layers are assembled in order and then subjected to vacuum or pressure filtration to wash and detect the biological entities on the membrane.
It is clear that a more efficient method for detection of the biological materials or entities on blotting membranes is required. The present invention permits a more effective and efficient detection of biological entities in a blotting membrane.
In one embodiment, the invention is directed to an apparatus useful in conducting the method of the invention. The device is comprised of a blotting membrane holder formed of a lower porous support layer and an upper flow distributor. The two are held together by a method such as by a hinge, clips, elastic bands, adhesives, ball and socket, pins and recesses, or cooperatively engaging fasteners or other such means. The holder is opened and one or more blotting membranes are placed between the lower and upper layers. The holder is then sealed and placed either onto a manifold or into a special apparatus (described below) to process the samples on the blotting membrane. In one embodiment, the flow distributor has an outer perimeter wall extending upwardly from the flow distributor to form a well to hold reagents and washing fluids.
In another embodiment the well and flow distributor are subdivided into two or more subwells to run parallel blotting membranes or subparts of one blotting membrane, each membrane is typically processed with at least one different reagent.
In another embodiment, the flow distributor is a non-binding or low binding porous membrane such as a 0.22 micron membrane.
In another embodiment, a porous pliable layer, such as a filter paper or glass paper, is placed below the blotting membrane and above the porous support so that when the flow distributor membrane is secured against the blotting membrane the pliable layer deforms to insure a complete mating and uniform flow between the blotting membrane and the flow distributor.
In a further embodiment the holder has an integral well formed above the flow distributor to hold reagents and/or wash fluids during the processing of them.
Additional embodiments include a pressure or vacuum manifold designed to retain the holder and conduct the filtration steps. In one embodiment, a separate well device is placed adjacent to the top of the flow distributor, either directly or through contact when the manifold lid is closed. In another, the well is integrally formed on top of the flow distributor
In another embodiment a rapid, efficient and convenient method to detect one or more biological entities on a blotting membrane is provided. The detection can relate to the position, nature or amount of the biological substance on a membrane. The invention method involves a pressure assisted regiment, selected from positive pressure or a vacuum for the supply and removal of reagents to and from the blotting membrane and permits washing of the contaminants from substances embedded in the membrane that are to be detected using very low volumes of liquid and reagents. This method enables completion of the blocking, washing and antibody binding steps in about 30-45 minutes without compromising blot quality. One simply takes a holder, opens it and places the blotting membrane(s) on one of the surfaces such that the lower surface of the blotting membrane is adjacent the porous support and the upper surface of the blotting membrane is adjacent the flow distributor when the device is closed around the membrane(s). The device is placed on or in a manifold having a pressure or vacuum supply and the process is commenced.
It is an object of the present invention to provide a device for conducting pressure or vacuum assisted immunoassays comprising a holder for the one or more blotting membranes formed of a porous support and a flow distributor which are held together.
It is another object of the present invention to provide an apparatus for conducting pressure or vacuum assisted immunoassays comprising a holder for the one or more blotting membranes formed of a porous support and a flow distributor which are removably held together and the flow distributor having an upwardly extending wall from its upper surface which forms one or more reagent wells on top of the flow distributor.
It is another object of the present invention to provide a device for conducting vacuum assisted immunoassays of one or more blotting membranes comprising a vacuum manifold and a holder for the one or more blotting membranes formed of a porous support and a flow distributor which are held together.
It is another object of the present invention to provide an apparatus for conducting pressure or vacuum assisted immunoassays of one or more blots comprising a vacuum manifold and a holder for processing the blots and a mean of collecting one or more of the antibodies.
It is a further object of the present invention to provide a device for conducting positive pressure assisted immunoassays comprising a manifold, a holder for one or more blotting membranes, the holder being formed of a porous support and a flow distributor which are held together and a positive pressure device removably mounted on top of the flow distributor.
It is a further object of the present invention to provide a process for conducting vacuum assisted immunoassays on one or more membranes comprising the steps of:
It is an object of the present invention to provide a process of passing a wash or reagent-containing liquid through one or more blotting membranes containing one or more biological entities, at least one of which is to be detected wherein the process comprises:
It is an object of the present invention to provide a process of passing a wash or reagent-containing liquid through one or more blotting membranes containing one or more biological entities, at least one of which is to be detected wherein the process comprises:
As shown in
The top 10 and the bottom 4 pieces are preferably attached to each other at least during use to hold the one or more membranes securely in place. As shown in
Optionally and preferably the flow distributor 10 may either have one or more wells 12 for holding washing fluids and reagents during use. In
In
As shown in
Alternatively, the waste collection device 22 can be a waste drain or other similar device as is known to one of ordinary skill in the art. In this instance the holder 2 is formed of a porous support structure 32, such as a plastic or metal grid or a porous sintered sheet of plastic or metal or other similar devices as are well known in the art. The one or more blotting membranes 34 is again placed on top of the support 32, over which is the flow distributor 36 and a well structure 38 (if desired) as described above in relation to the embodiments of
In an additional embodiment as shown in
The flow distributor 10 is a porous structure. The flow distributor not only provides even liquid distribution but it also acts as a flow regulator. It provides for complete and uniform distribution of the liquids as well as allowing sufficient time residence in the membrane for proper interaction between the molecules of the liquid and the specimen. In one embodiment, the entire structure is porous. In another embodiment, such as may be used in conjuncture with the embodiment of
The flow distributor 10 may be any porous structure that provides for even distribution of the liquid across its face and which is sufficiently porous to allow for easy movement under the influence of a vacuum or pressure and which is also capable of filtering out agglomerates, particles and other debris from the liquid.
The flow distributor may be of any desired size. Gels come in a variety of “standard” sizes from about 7 cm by 8 cm to a 20 cm by 20 cm area. The flow distributor should preferably cover the entire blotting membrane to insure complete flow of reagents through all of the blotting membrane.
Such materials include but are not limited to woven, non-woven and fibrous porous filters such as TYVEK® or TYPAR® paper, cellulosic materials such as MF filters available from Millipore Corporation of Billerica, Mass., membranes such as DURAPORE® and MILLIPORE EXPRESS® microporous membranes available from Millipore Corporation of Billerica, Mass., sintered membranes such as POREX® filters and the like. Preferred are membranes, especially plastic microporous membranes.
A preferred pore size of such membranes is between about 0.1 and about 0.65 micrometer, preferably between 0.2 and about 0.45 micrometer and more preferably about 0.22 micrometer.
Additionally, the preferred porous structure has low binding characteristics for the reagents used in order to minimize the amount used. More preferably, as it is generally used with biological materials it is hydrophilic and has low protein binding characteristics. One such distributor is a hydrophilic DURAPORE® membrane formed of PVDF available from Millipore Corporation of Billerica, Mass. Another is a MILLIPORE EXPRESS® hydrophilic PES membrane available from Millipore Corporation of Billerica, Mass.
The porous support 4 may be a simple screen, a grid (as shown in
The outer edges of the support 4 and the flow distributor 10 may be made of the same materials as the support 4. When an integral hinge is used, it must be made of a flexible material such as polyethylene, polypropylene, an elastomer or one of the impact modified materials such as ABS, K-resin and the like. When a separate hinge, clips, elastic bands, adhesive film or other securing means are used they may be made of metal, plastic or elastomers as desired.
Various methods may be used in the present invention. The key factor being that they all rely on a vacuum or positive pressure driven filtration of the liquids to access the large inner surface area of the membrane allowing 3-D interaction of all the molecules throughout the depth rather than only 2-D interaction at the surface as has occurred in the past.
The simplest method is to use the present invention to conduct one or more of the washing cycles. Typically each washing cycle is comprised of one or more washing steps. Generally, 2-5 steps are used per cycle.
Another method is to use the present invention in each step in which liquid needs to be moved through the blotting membrane such as after incubation of the antibodies or in the washing steps.
In all of these processes, any pressure suitable to move the liquid(s) through the device and into the manifold can be used. This can vary depending upon the membranes selected for blotting and the flow distributor, the manifold used, the desired speed of the filtration and the supply of vacuum or positive pressure available to the researcher.
Generally, the vacuum available may vary between 100 and 760 mm Hg (133 millibars and 1013 millibars). The use of valves, pressure restrictors and the like may also be used to keep the vacuum within the allowed ranges for the membranes used. A preferred vacuum manifold of one embodiment of the present invention uses of a vacuum of about 100 mm Hg. Other suitable vacuum manifolds include but are not limited to the MULTISCREEN™ and MULTISCREEN™ HTS vacuum manifolds available from Millipore Corporation of Billerica, Mass.
Generally the positive pressure is supplied by an air line at pressures ranging from about 2 psi to about 15 psi. The use of valves, pressure restrictors and the like may also be used to keep the pressure within the allowed ranges for the membranes used. Such pressure systems include but are not limited to Amicon® stirred cell devices available from Millipore Corporation of Billerica. Mass. and positive pressure filtration units available from Caliper Life Sciences of Hopkinton, Mass.
To use a device according to the invention one simply takes a holder, opens it and places the blotting membrane(s) on one of the surfaces such that the lower surface of the blotting membrane is adjacent the porous support and the upper surface of the blotting membrane is adjacent the flow distributor when the device is closed around the membrane(s) so as to have no air bubbles between the blot and the flow distributor. Bubbles between these two surfaces can cause areas of no flow. The device is placed on or in a manifold having a pressure supply (vacuum or positive pressure). Preferably the blotting membrane(s) has been prewet. The pressure (vacuum or positive pressure) is turned on and a liquid, such as a wash liquid or a reagent, is placed on top of the flow distributor or into the well(s) if used. The pressure continues until the liquid has been moved through the device and membrane(s). Then the pressure is turned off.
When more than one blotting membrane is used, they can be arranged in series on top of each other and sufficient liquid containing the same desired reagents can be easily moved through the multiple layers in one process step. Generally when more than one layer is used it is preferred that one use between 2 and 10 layers, preferably between 2 and 5 layers at a time. Alternatively, one can use a flow distributor having multiple subwells and use more than one blotting membrane in parallel to each other, each with their own well in the flow distributor and each with its own set of reagents as is required for its specific purpose. Also one can mount two or more separate holders each with one or more subwells. One can even use multiple layers in adjacent wells if desired. With two or more separate holders, they may if desired be run independently of each other or together.
The liquid can either be added with the pressure supply being off or the supply being turned on only briefly so as to get the liquid into the membrane(s) and is allowed to incubate (such as may be required with the primary or secondary antibodies). The pressure is then turned on to remove the liquid and/or replace it with another used sequentially. Preferably, during washes, the vacuum is left on and remaining washes are added sequentially.
Optionally, if one wishes, one can place a collection vessel 70 below the device, preferably in the manifold itself or downstream. It can then be used to collect one or more unbound reagents that may be expensive and which can be collected and recycled for use in future assays. The vessel can also be subdivided into multiple chambers that are in alignment and fluid communication with the respective portion of the blotting membrane. One such collection vessel 70 is shown in
Additionally or alternatively, one can place in the downstream flow path below the holder an absorbent matrix that is capable of reversibly binding one or more unbound reagents that are expensive. The matrix is preferably in the form of a monolith, such as a pad, a plug or a paper sheet, that is positioned so that all the liquid passing through the blotting membrane and holder passes through the matrix. It can then either be removed and the reagent eluted or if desired, it can have the bound reagents eluted in situ after completion of the testing of the blotting membrane.
Other processes may also be used with the device of the present invention. The membrane contains, in its interstices, one or more substances to be detected. Generally these substances are present in the interstices either by virtue of having been blotted from a solid support for electrophoresis or chromatography or by direct application, usually to detect the presence, absence, or amount of a particular type of material such as an antibody or specific protein—i.e. a Dot-Blot type assay as described above. The definition of the membrane is not limited, however, to these instances, but applies to any case wherein a membrane contains in its interstices one or more substances to be detected. Included in the types of membranes envisioned for use in the present invention are membranes commonly used to blot electrophoresis gels such as nitrocellulose; nylon; or various other polymeric membranes, such as polyvinylidene fluoride (PVDF), sold as IMMOBILON™ membranes by Millipore Corporation of Billerica, Mass.
A variety of materials can be used to replicate the results of electrophoresis gels performed on various samples as is understood in the art. Most commonly, the samples contain biological substances such as individual proteins, antibodies, nucleic acids, oligonucleotides, complex carbohydrates, and the like, but the application of the technique is not limited to these substances. The invention technique is applicable to any membrane containing within it a substance to be detected regardless of the chemical composition of the membrane or of the target substances.
When membranes which represent replicas of electrophoretic results are employed, the transfer of the substances to be detected from the gel to the membrane can be conducted by utilizing membranes containing transfer buffer, by electroelution, or by dry blotting of the gels. Techniques for these transfers are well understood in the art, and do not constitute part of the invention herein.
The liquid to be supplied may contain detecting reagents or may simply be provided as a wash. The nature of the detecting reagent depends, of course, on the substance to be detected. Typically, proteins are detected by immunological reactions between antigen and antibody or immunoreactive portions thereof; typically the presence of nucleic acid fragments is detected by suitable oligonucleotide probes. The detecting substances responsible for the immediate or specific reaction with the substance to be detected may be further supplemented, if needed, with label and a multiplicity of applications of the detecting reagents may be needed—e.g., a protocol may include detection of an antigen by supplying an antibody labeled with an enzyme, e.g., commonly, horseradish peroxidase, and then this binding is detected by means of supplying substrate for this enzyme. In application of reagent, it is possible, though not preferred, to use only a positively pressed donor matrix to expose this component of the membrane for a defined period.
It is most convenient to conduct the method of the invention at room temperature, but elevated and lower temperatures can also be used. This can be effected by heating the device, its surrounding environment (as in a heat box or cooling box) or the liquids used in the system.
Blots can be sequentially analyzed with multiple antibodies or probes in the present device and process by stripping the previously bound antibodies from the blot followed by subsequent incubations with antibodies or other probes specific other target proteins. The stripping process disrupts the antigen-antibody bonds and dissolves the antibodies in the surrounding buffer. This is usually achieved by a combination of detergent and heat or by exposure to either high or low pH. The device, in combination with the flow distributor, enables the stripping of blots using the high or low pH method. The subsequent reprobing of blots either directly (e.g., using the same flow distributor used for striping) or subsequently after storage, would use the same protocol as the initial probing. Suitable kits for strip blotting are available from Chemicon International, Inc under the brand names of ReBlot™ Plus kit (catalogue # 2500), ReBlot Plus-Mild solution (catalogue # 2502) and ReBlot Plus-Strong solution (catalogue #2504).
In standard western blotting, the antigen or target is transferred to a membrane support and probed with a suitable probe such as an antibody, protein (e.g., Protein A) or lectin (proteins or glycoproteins which binding to carbohydrate moieties). In some applications, a reverse format (e.g., reverse array) is used, wherein the antibody or other probes are spotted onto a membrane or other support (typically in an array format) and the antigen or target is presented to the immobilized antibodies on the array. Visualization of a target-probe binding event can be achieved by labeling of the antigens or targets or by using a secondary antibody specific for the target. Reverse arrays often employ mixtures of targets, for example lysates labeled with different fluorescent colors to enable parallel processing. Reverse assays can also be performed with the present invention.
This application is a divisional patent application of U.S. application Ser. No.: 11/582,599, filed on Oct. 18, 2006 and claims the benefit of U.S. Provisional Application No. 60/795,452, filed on Apr. 27, 2006, U.S. Provisional Application No. 60/795,532, filed on Apr. 27, 2006 and U.S. Provisional Application No.: 60/732,994, filed on Nov. 3, 2005. The invention relates to a device and process for the detection and position of substances that are contained in a blotting membrane. More particularly, it concerns a technique for applying reagents and wash solutions to a blotting membrane to accomplish this detection quickly via the use of vacuum or positive pressure.
| Number | Date | Country | |
|---|---|---|---|
| 60795452 | Apr 2006 | US | |
| 60795532 | Apr 2006 | US | |
| 60732994 | Nov 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11582599 | Oct 2006 | US |
| Child | 13078290 | US |
