Abstract:
Techniques for high throughput parallel separation, filtration and plate-to-plate transfer are described. Cells, proteins, chemical compounds and the like are being developed in multi-well, small volume well plates, such as 1536-well plates having wells of a volume on the order of 1 μl. A mechanism for clamping such plates together so that the wells will be aligned allows rapid separation or transfer by simply centrifuging the assembly. A membrane may be clamped between the plates. Alternatively, a membrane may replace one of the plates. Centrifuge dependent and independent designs are described.

Description:
This application is a division of allowed U.S. application Ser. No. 09/055,855 filed Apr. 6,1998 now U.S. Pat. No. 6,274,008. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to improvements in high throughput assays of chemical compounds, cells, proteins and the like. More particularly, the present invention relates to advantageous transfer techniques for transferring small volumes of materials from small well plates having a large plurality of wells, such as presently existing 1536-well plates, or performing separations, filtrations or the like in a similar context. 
     BACKGROUND OF THE INVENTION 
     To find lead compounds for drug discovery programs, large numbers of compounds are often screened for their activity as enzyme inhibitors or receptor agonists/antagonists. Large libraries of compounds are needed for such screening. As a result of developments in this field, it is now possible to simultaneously produce combinatorial libraries containing hundreds of thousands of small molecules for screening. With the availability of such libraries, however, has come a need for large scale, rapid screening methods. 
     For example, the libraries may be contained on microbeads, each compound being present in a picomolar amount. Because the amount of compound is very small, it is advantageous to conduct the high throughput screening method in very small volumes, e.g., on the order of 1 μl. Such assays can be performed in the 1536-well plate described in PCT Application Serial No. PCT/US98/00494 entitled Multi-Well Plate, filed Jan. 8, 1998 and incorporated by reference herein. 
     Receptor binding assays used in high throughput screening typically involve three steps. First, a labeled ligand is incubated with a target receptor in the presence of a compound to be tested for inhibition of ligand/receptor binding. Second, the receptor and ligand (and compound) are separated using filtration and/or washing of an immobilized receptor. Finally, the amount of labelled ligand bound to the receptor is quantified. This conventional screening is a ‘separations-mode’ assay, i.e., one in which the bound ligand is physically separated from the free ligand using either a filtration membrane or the selective adhesion of either bound or free component to a surface (e.g., the surface of a microtiter plate). 
     Separation, however, is typically time consuming and therefore slows high throughput screening. If fluid handling steps employed are not sufficiently precise, separation also can create variations in the signal generated in the assay and can disturb equilibrium binding conditions. Furthermore, separation has been difficult to automate and is potentially hazardous when radioactive materials are involved. These problems are particularly acute in assays conducted in microvolumes using small amounts of test compound. 
     Many screening assays require the transfer of cells or proteins onto membranes for evaluation of radiosotope levels, fluorescence or activity. For further details of various aspects of such assays, see U.S. patent application Ser. No. 08/868,280 filed Jun. 3, 1997 and entitled High-Throughput Assay which is incorporated by reference in its entirety herein. The small well size and volume used in the 1536-well plates preclude the use of conventional cell harvesting equipment. 
     For larger wells and lower throughput, Millipore provides a filtration system for use with 96-well format plates designated the MULTISCREEN® ASSAY SYSTEM. In that system, individual membranes are sealed to 96 independent wells for use with a vacuum manifold. The membranes are relatively complex and expensive, and the vacuum manifold is relatively slow. Overall this system is not advantageously scaleable to the small volumes used with high throughput plates such as 1536-well plates. 
     Other conventional plate-to-plate transfer systems are inadequate at the small volumes used in 1536-well plates. Typical systems involve physical removal of the sample into a cannula or syringe, either with concomitant dispensing of a wash stream from a plate washer or in an aspirate/dispense mode. This modality is impractical using 1536-well plates due to the number of parallel operations that are needed to maintain an acceptably high throughput. In order to extend the prior art to 1536-well plates, a technique would need to be developed (at considerable expense) for fabricating parallel aspirate/dispense channels which might have disposable tips or washable cannulae to prevent contamination. 
     Thus, it would be highly desirable to have simpler, faster and more economical alternatives for transfers and separations of materials from a multiplicity of vessels, such as the wells of a microtiter plate. Further, to provide a rapid separation of cells or proteins from solutions within low volume high throughput plates, such as 1536-well plates, and to provide a mechanism for rapid plate-to-plate transfer of samples from one plate to another will be recognized as highly advantageous. 
     SUMMARY OF THE INVENTION 
     In one aspect, methods and apparatus in accordance with the present invention advantageously use presently existing disposables to transfer samples in parallel. In other words, a whole plate is transferred at once. No other additional disposables are needed except for an optional membrane which can be selected from a number of conventionally available products. 
     Alternatively, the transfer may be from a plate to a membrane. 
     Centrifugation is employed to achieve such rapid separation either from one plate to another (plate-to-plate transfer) or from a plate onto a transfer membrane (“harvesting”). To achieve this separation, apparatus to secure and align and clamp two plates is provided. Both centrifuge dependent and independent designs are described. The approach of the present invention is also applicable to parallel filtration operations and the like. 
     A more complete understanding of the present invention, as well as further features and advantages of the invention, will be apparent from the following Detailed Description and the accompanying drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates a first embodiment of the present invention for high throughput plate-to-plate or plate to membrane transfer which is centrifuge independent; 
     FIG. 2 illustrates a second embodiment of the present invention for high throughput plate-to-plate or plate to membrane transfer which is centrifuge dependent; 
     FIG. 3 illustrates a third embodiment of the present invention for high throughput plate-to-plate or plate to membrane transfer; 
     FIGS. 4A and 4B show data from an exemplary transfer of labeled cells to a PVDF membrane utilizing 1536-well plates; 
     FIGS. 5A-D illustrates details of compression plates which may be suitably used in conjunction with the embodiments of FIGS. 1-3; 
     FIG. 6 illustrates a centrifuge and transfer assembly which may suitably effect plate-to-plate or plate to membrane transfer, separation, filtration or the like in accordance with the present invention; and 
     FIG. 7 is a flowchart illustrating the process steps of one method of plate-to-plate or plate to membrane transfer or separation in accordance with the present invention. 
    
    
     DETAILED DESCRIPTION 
     The present invention advantageously achieves rapid separations and transfer by centrifugation, either from one plate to another (plate-to-plate transfer) or from a plate onto a transfer membrane (harvesting). To achieve this end, apparatus is provided to adequately secure two aligned 1536-well plates, optionally with a membrane between them, for centrifugal transfer. These 1536-well plates are designed symmetrically so that when one is inverted on top of another, the wells are in alignment. For further details of a presently preferred 1536-well plate see PCT Application Serial No. PCT/US98/00494 filed Jan. 8,1998. While the discussion to follow is in the context of 1536-well plates, it will be recognized that the invention will also be applicable to other high throughput, low volume well plates having a larger number of wells. It will also be applicable to plates having a smaller number of wells, such as 96, so long as those wells are low volume, for example, approximately 1 μl. An important design characteristic is adequate pressure on the plates and membranes to prevent leakage of samples from one well to the next while maintaining the alignment defined by the alignment holes. 
     A first embodiment shown in FIG. 1 is centrifuge independent. As shown in FIG. 1, transfer apparatus  100  consists of two compressing plates  110  and  120  of adequately stiff material. One example of a suitable material is aluminum. It will be recognized that other materials may also be utilized. Optionally, a compression spring (not shown) and plate combination may be employed to apply a uniform normal force between the plates  110  and  120  that are assembled with the membrane  130  and 1536-well plates  140  and  150  to form transfer apparatus  100 . Alignment is maintained by the posts  161  and  162  and pressure on the plates is adjustably provided by appropriate tightening of nuts  163  and  164 . While two posts are shown with tightening nuts, it will be recognized that additional posts or an alternative compression or clamping mechanism, such as an air bladder arrangement to provide clamping force using air pressure or a spring-loaded clamp, may also be utilized. A torque wrench may be used to tighten the nuts  163  and  164  with a torque of 6 inch pounds (in.-lbs.). As addressed further below in conjunction with FIG. 6, the assembly  100  is then mounted in a centrifuge which may suitably be a Forma model 5681 three liter swinging bucket plate centrifuge and rotated at 1500 rpms for one minute to achieve bulk parallel separation. 
     FIG. 2 illustrates a second embodiment of the present invention which is centrifuge specific. FIG. 2 illustrates a transfer assembly  200  including a rotor bucket  270  designed to fit a particular centrifuge. In the assembly  200 , a base  220  of rotor bucket  270  replaces the bottom compression plate  120  of assembly  100 . Threaded posts  261  and  262  extend from the base  220 . Membrane  230  and 1536-well plates  240  and  250  are sandwiched between a compression plate  210  and the base  220 . Nuts (not shown) may again be tightened on threaded ends of posts  261  and  262  using a torque wrench and applying a torque of 6 inch pounds. The plates  240  and  250  are then preferably rotated at 1500 rpms for one minute to affect the desired separation. 
     In a third embodiment shown in FIG. 3, an assembly  300  includes an enclosing box  370 . Box  370  is a tight-fitting box, which is open at one end. As two 1536-well plates  340  and  350  separated by an optional membrane  330  are inserted into the open end of box  370 , they are forced together by the tight fit (like a deck of cards being slid into its box). Alignment posts  361  and  362  are inserted through holes  371  and  372  in the box  370 , as well as through matching holes in the membrane  330  and the plates  340  and  350  to insure proper alignment throughout centrifugation. Threaded ends of posts  361  and  362  may be utilized to attach assembly  300  to an appropriate centrifuge. Centrifugal force drives liquid from the wells in plates  350 , which may be termed a source plate, and into plate  340 , which may be termed a target plate. The optional membrane  330  may be used to perform a filtration or an adhesion step. 
     FIGS. 4A and 4B show data from a transfer of labeled cells from a 1536-well plate to a polyvinylidine difluoride (PVDF) membrane. In this experiment, a column of empty wells was employed between each column of filled wells to make it easier to see if cross-contamination had occurred. Chinese hamster ovary (CHO) cells containing the interleukin-8 receptor, subtype B (IL8B receptor) were grown to a confluency in a standard T-75 tissue culture flask. The cells were then removed from the flask (trypsin, 5 mm), and washed twice with buffer (BSS/BSA). CHO/IL8B cells were then counted and diluted to 2,000 or 1,000 cells per μL using the same buffer. These suspended cells were incubated with various concentrations of [ 215 I]IL8 (as indicated in FIG. 4A, 1, 0.5 and 0.25 nM) with or without cold competitor (unlabelled IL8, 1 μM) for 1 hour. The total [ 125 ]IL8 bound (the total is seen above the letter T in figs. 4A and 4B) was quantitated without cold competitor, while non-specifically bound 125 I (the non-specifically bound is seen above the letter N in FIGS. 4A and 4B) was measured with competitor. Aliquots (1 μL) of each sample were pipetted in quadruplicate into a 1536-well plate, which was then inverted onto a PVDF membrane and clamped into the centrifugation assembly of the present invention. The apparatus was then centrifuged (1500 RPM, 1 mm) to transfer cells to the membrane, inverted and re-centrifuged to remove the unbound supernatant. The membrane was then removed and sealed in plastic wrap and placed face down onto a phosphor screen for  1  hour. The screen was imaged for 7 minutes on a STORM phosphorimager manufactured by Molecular Devices and quantitated using software adapted to quantify objects by processing captured images of the objects, with the specific software employed in the present exemplary case being the IMAGEQUANT software provided by the manufacturer. The distinct spots seen in FIG. 4A that cross-contamination has not occurred while the desired spatial separation of samples has been achieved. 
     FIGS. 5A-5D show a presently preferred embodiment of compression plates  510  and  520  for use in conjunction with the present invention. FIGS. 5A and 5B show top down views and 
     FIG. 4B shows a graph of the data generated in which error bars represent ±σ. 
     FIGS. 5C and 5D show edge on views. Plates  510  and  520  are each preferably formed from a solid piece of aluminum which is approximately 3 ½″×{fraction (5 3/16)}″×¼″. Two 0.05 inch recesses  511  and  521  are cut around the edges of the top surfaces of the plates  510  and  520 . At the top and bottom, each recess is approximately 0.29″ wide, and on the left and right each recess is approximately 0.23″ wide. The creation of the recesses  511  and  521  results in the plates  510  and  520  having raised central portions  513  and  523  which fit into a matching recess in the base of a standard 1536-well plate. 
     For plate to plate transfer, a first 1536-well plate is seated on compression plate  510  an a second 1536-well plate having material to be transferred is seated on compression plate  520 . A threaded dowel pin (not shown) is then screwed into threaded hole  515  in the plate  510 . The two compression plates  510  and  520  are folded or placed together like a sandwich. Because the wells in the 1536-well plate are so small, liquid does not escape or leave those wells even when the second 1536-well plate is turned on its side or upside down to form the sandwich. The present invention is suited to any wells having this characteristic. The end of the dowel pin sticks up into a matching hole  525  in plate  520  to assist in the alignment process. Then, size number  5  flat head screws are passed through holes  526  and  528  of top plate  520  and screwed into threaded holes  516  and  518  of bottom plate  510 . As previously addressed a torque of  6  inch pounds may suitably be employed. As shown in FIG. 5D, a surface  529  of holes  526  and  528  is countersunk so that the underside of the head of the screws seats snugly in these holes. The dashed lines present in  5 A- 5 D help illustrate the relative alignments of the holes in FIGS. 5A and 5B,  5 A and  5 C and  5 B and  5 D. When sandwiched or assembled, the overall unit is approximately 3 ½″×5 {fraction (3/16)}″×2 ¼″ and may be utilized with a standard centrifuge bucket. For plate to membrane transfer, the plate  520  may be replaced by a similarly dimensioned flat block of aluminum. 
     While two screws are shown to compress the plates  510  and  520 , it will be recognized that other compression mechanisms may also be employed. For example, the center portion  513  might be a separate piece which was spring or otherwise loaded so that more uniform pressure on the 1536-well plates might be employed. 
     FIG. 6 illustrates how a high throughput transfer assembly  600 , which might be any of the assemblies of FIGS. 1,  3  or  5 , maybe suitably inserted into a bucket  610  of a centrifuge  650  for centrifuging. The dashed line present in FIG. 6 illustrates the axis of rotation of the centrifuge  650 . The presently preferred centrifuge is a FORMA Model number 5681 three liter swinging bucket plate centrifuge, although other centrifuges may be employed. 
     FIG. 7 shows a flowchart of a method  700  of plate to plate or plate to membrane high throughput transfer or separation in accordance with the present invention. In step  701 , a multi-well microtiter plate which has a large plurality of low volume wells, such as a 1536-well plate, is aligned with a first compression plate, such as plate  520  of FIG.  5 . This microtiter plate has material in liquid in its wells. In step  702 , a receiving membrane or receiving multi-well plate is aligned with a second compression plate, such as plate  510  of FIG.  5 . As discussed above, a membrane may also be disposed in-between multi-well plates. 
     In step  703 , a multilayer sandwich is formed by putting the two compression plates together. Proper alignment is preferably aided and maintained utilizing an alignment mechanism such as the pin and holes  515  and  525  of FIG.  5 . 
     Next, in step  704 , the multi-layer sandwich is clamped together utilizing a clamping mechanism, such as the screws and threaded holes  516 ,  518 ,  526  and  528  of FIG.  5 . In step  705 , the resulting assembly is loaded into a centrifuge bucket. Finally, in step  706 , the assembly is centrifuged to effect transfer or separation. Subsequently, assays or other analysis may be performed. 
     While the present invention has been disclosed in a presently preferred embodiment, it will be recognized that the present teachings and claims may be embodied in a variety of formats adapted to particular applications as necessary to achieve the desired result of high throughput parallel transfer or separation in conjunction with multi-well plates with low volume wells. By way of example, different torques may be employed as needed to prevent leakage from well to well, different clamping mechanisms may be adapted to suit particular applications and environments, and centrifuge speed may be adapted to the materials and operation to be conducted.