Chemistry: molecular biology and microbiology – Apparatus – Including condition or time responsive control means
Reexamination Certificate
1999-07-16
2002-05-28
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including condition or time responsive control means
C435S287200, C435S288300, C435S040500, C435S091200
Reexamination Certificate
active
06395536
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a support cell for processing samples, particularly biological samples, supported on solid supports. In addition the invention relates to automated apparatus for processing samples supported on solid supports, which automated apparatus may be used with the support cell of the invention. The invention also relates to a method of processing a sample supported on a solid support.
BACKGROUND OF THE INVENTION
It is common practice, in certain technical fields, for samples of interest to be subjected to a particular process to be supported on solid supports. For example, protein or nucleic acid samples may be supported on filters or membranes: typically such filters or membranes are made of nylon or nitrocellulose. Samples, especially biological samples (e.g. cell suspensions or tissue sections) may also be supported on slides. Typically the slides are of transparent glass.
Samples supported on slides may be processed by a number of various techniques known to those skilled in the art, which include: standard staining techniques (e.g. Gram staining of micro-organisms, haemotoxylin/eosin staining of tissue sections); immuno- and enzyme cytochemistry (e.g. peroxidase/anti-peroxidase staining); ligand binding studies; in situ hybridisation of labelled DNA or RNA probes: and in situ amplification of nucleic acids present in a sample by means of polymerase chain reaction (PCR).
Typically, such processing is at present performed manually by laboratory personnel. This renders the processing slow and subject to individual variation (from one sample to the next, and/or from one day to the next). and is tedious for the personnel concerned. Some attempts have been made to provide apparatus to facilitate the immuno-cytochemistry process. Thus, for example, Shandon Scientific Limited manufacture the “Sequenza”™ and “Cadenza”® immuno-staining apparatus. Use of the “Sequenza”™ apparatus involves attaching a coverplate to the slide to be processed, thereby forming a narrow channel between the slide and coverplate. The coverplate comprises a funnel-like open-ended portion which, when attached to a slide, forms an open-ended chamber, into which staining reagents are manually introduced. The reagent then flows, under the influence of gravity, into the channel between the slide and the attached coverplate. When a new reagent is introduced into the well it displaces the previous reagent into a waste collection tray at the bottom of the apparatus.
The “Cadenza”® apparatus utilises the same coverplate system but additionally provides programmable automated control means for applying reagents to the open-ended well of the assembled slide/coverplate.
In relation to processing of samples supported on membranes, such samples are commonly preparations of proteins (termed Western blots) or DNA (Southern blots) or RNA (Northern blots). The molecular species of interest is visualised on the supporting membrane using specific antibodies and an appropriate detection systems (in the case of Western blots) or by hybridisation with a labelled probe (in the case of Southern and Northern blots). These processing techniques currently require considerable manual processing of the membranes.
The prior art apparatus for processing samples on solid supports is therefore very simple and limited in the functions which it can perform. The present invention aims to provide apparatus capable of more general application.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a support retaining member for use in processing a sample supported on a support, the member being such that when assembled together with a support it forms a support cell, the support cell comprising a substantially sealed chamber, the chamber being provided with a fluid inlet and a fluid outlet for the introduction and removal respectively of fluids used in processing the sample.
In a particular embodiment, part of the internal surface of the substantially sealed chamber is defined by the sample bearing surface of the support, which arrangement has the advantage of minimising the number of components involved.
Conveniently the support is a slide, or a membrane of the type known to those skilled in the art.
Where the support is a slide, the support retaining member will typically be of such dimensions that it may be used with slides of conventional size, (i.e. slides which typically are about 25.4 mm by 76.2 mm). Where the support is a membrane, the dimensions of the retaining member will similarly be of such dimensions that it may be used with membranes of conventional size (typically 80 mm by 120 mm), although membranes are rather more variable in size than slides.
In some embodiments the support retaining member comprises two opposed portions, the support being positioned between the opposed portions of the support retaining member when the support cell is assembled. Preferably at least that portion of the support retaining member which comes into contact with the fluids used to process the sample on the support is made of an inert material, such as a synthetic moulded plastics material. Perspex is found particularly suitable. The remainder of the support retaining member may generally be of the same or other material. e.g. glass, perspex or metal (such as duraluminium).
The components of the support cell are typically held together by clamping means. In certain embodiments the clamping means is capable of clamping together the portions of a plurality of support cells. Typically, from one to around twelve or sixteen support cells may be clamped simultaneously by a single clamping means. The support cells may preferably be arranged in the clamping means in a substantially horizontal or substantially vertical manner, although any position intermediate between these two positions may be possible. The support cells may, for example, be arranged side-by-side, or one in front of the other.
As an alternative, or in addition, to clamping means the support cell may be provided with biasing means, which biasing means tends to urge together the components of the support cell. Conveniently the biasing means will comprise one or more sprung biasing members. In a particular embodiment, the support retaining member is attached to clamping means by spring-loaded mounting pins, such that formation of the support cell places the springs of the spring-loaded mounting pins under compression, which springs therefore urge together the components of the support cell.
In preferred embodiments, the force applied to the support cell by the clamping means and/or the biasing means helps to ensure a fluid-tight seal between the support and the support retaining member.
It is generally preferred that the support cell additionally comprises sealing means to assist in the formation of the substantially sealed chamber. Conveniently the sealing means forms a fluid-tight seal, in the first instance with the sample bearing surface of the support and, in the second instance, with the support retaining member. Alternatively, the sealing means may effect a seal directly between opposed portions of the support retaining member, where the support retaining member is of two-part construction. The sealing means may be an integral part of the support retaining member, or may be provided as a separate component of the support cell. The sealing means typically comprises a gasket, which may be made of silicon rubber or other suitable material. In one embodiment the sealing means comprises an O-ring gasket, the shape of which is generally that of a frame-like surround seated in a groove in one portion of the support retaining member. In an alternative embodiment the sealing means comprises a flattened frame-like surround gasket (about 100 to 150 &mgr;m thick). Either type of gasket may be discarded after a single use (if, for example, contaminated with a radioactive probe) or may be re-used if desired. The flattened gasket embodiment is particularly suitable as a disposable gasket, to be discarded after a single use. It will be apparent that t
Beisner William H.
Medical Research Council
Pillsbury & Winthrop LLP
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