WO2022219312A2

WO2022219312A2 - Sieve plate and apparatus for performing an automated elispot process

Info

Publication number: WO2022219312A2
Application number: PCT/GB2022/050903
Authority: WO
Inventors: Rene ODA; Matt Quinn; Shadi Barakat
Original assignee: Oxford Immunotec Limited
Priority date: 2021-04-16
Filing date: 2022-04-12
Publication date: 2022-10-20
Also published as: WO2022219312A3

Abstract

A sieve plate is provided for use in an automated ELISPOT assay process, the ELISPOT assay process comprising collecting cells on surfaces of magnetic beads to isolate the cells from a sample liquid. The sieve plate comprises a frame and a membrane supported by the frame, the membrane comprising a plurality of pores. The size of the pores is such that the cells and magnetic beads are able to pass through the membrane, but cellular clumps cannot pass through the membrane. An apparatus for performing an automated ELISPOT assay process comprising a sieve plate is also provided.

Description

SIEVE PLATE AND APPARATUS FOR PERFORMING AN AUTOMATED ELISPOT

PROCESS

TECHNICAL FIELD

The present disclosure relates to a sieve plate for use in an automated ELISPOT process, and to methods and apparatus for performing an automated ELISPOT process.

BACKGROUND

Assays such as ELISPOT processes are commonly used to investigate properties of a sample, for example for disease diagnosis. Typically, an assay process may include a number of aspects, including sample preparation, target entity isolation, and target entity measurement.

Each of these aspects can be time consuming and laborious. It is known to automate certain aspects of the procedure, for example using a liquid handling robot to automate movement of liquid. However, many aspects are still performed by a human operator, slowing the assay process and limiting throughput.

An example of a diagnostic assay process is the T-SPOT^®test for identifying infection with tuberculosis (TB) designed by Oxford Immunotec. In this process, peripheral blood mononuclear cells (PBMC) are isolated from a blood sample using magnetic (paramagnetic) beads. PBMCs are collected on the surfaces of the magnetic beads by binding cell specific biotinylated antibodies which in turn bind to the streptavidin coating on the magnetic beads. Alternatively, cells can be bound directly to antibody-coated magnetic beads. A magnetic field is applied to gather the magnetic beads and the cells attached to them, whilst removing the residual sample liquid. A buffer liquid is then used to re-suspend the magnetic beads and cells, enabling the isolated cells to be moved to a downstream process for counting, plating, testing and hence detection of TB infection. In total, this process takes two days to perform, and conventionally requires manual intervention at a number of aspects, limiting the throughput of the process.

It is therefore desirable to find ways of increasing the automation and throughput of assay processes.

As used herein, a tube may be a well or microwell of an assay sample plate. The term assay process may include preparatory aspects preceding the investigative process.

SUMMARY

According to a first aspect, provided herein is a method for isolating target entities from a sample liquid in an assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube with magnetic beads, such that the target entities collect on the surfaces of the magnetic beads; applying a magnet field for a first period to a lower section of the tube to collect the magnetic beads within the lower section of the tube; extracting a first portion of supernatant from the lower section of the tube, the supernatant comprising the sample liquid less the target entities collected on the surfaces of the magnetic beads; subsequent to extracting the first portion of supernatant, applying the magnetic field for a second period to collect further magnetic beads; and extracting a second portion of supernatant from the lower section of the tube.

The assay process may be a diagnostic assay process, such as an ELISPOT process, and in particular an ELISPOT process for identifying TB infection. The assay process may be an automated assay process. The target entities may comprise cells and/or cellular components.

This multi-stage approach to supernatant extraction means that the portion of the supernatant closest to the magnet is extracted first. Due to the proximity of the magnetic field, more magnetic beads will have been collected from that lower portion of supernatant than the supernatant sitting higher in the tube, further away from the magnet field. After the lower portion of liquid is removed, the upper portion(s) of liquid fall into the lower part of the tube. This brings more magnetic beads into proximity of the magnetic field. The method provides time for those beads to be collected before the now-lower supernatant is extracted. In this way, liquid of higher magnetic bead density is cycled into proximity of the magnetic field for efficient collection. This process can be repeated simultaneously and automatically for many different tubes, each containing a different sample, allowing high throughput of samples.

Moreover, this method allows the magnetic field to be applied to only a portion of the length of the tube, without reducing the number of beads, and hence target entities, collected by the field. Such a magnetic field can be applied by a small-sized magnet co-located with the sample preparation stage of the assay apparatus. Thus unlike in conventional systems, the sample does not have to be moved to a separate magnetic plate. This again allows for a higher throughput of samples, as no time is wasted moving the samples. In particular embodiments, applying the magnetic field may comprise raising the magnet from a position where all of the magnet is below the tube into the position around the tube, for example using a lift. In such embodiments the magnetic field can be applied only when required, without having to move the tubes.

In some embodiments, the magnetic field may be applied by a magnet positioned around the tube such that the lower section of the tube extends below an upper surface of the magnet. The aspects of extracting supernatant may comprise extracting supernatant from the tube below the level of the upper surface of the magnet.

Such embodiments collect the magnetic beads at the side of the tube, rather than at the bottom of the tube as in some conventional systems which place the tube on a flat magnetic plate. This means the target entities on the magnetic beads are collected on the walls, away from the centre of the tube where supernatant will be extracted. This can minimise damage, activation, or loss of the target entities caused by the supernatant extraction, for example due to disruption caused by a pipette tip.

In some embodiments, the magnet may comprise an annulus of magnetic material extending around a portion of the lower section of the tube. The lower section of the tube may extend below the annulus of magnetic material, and supernatant may be extracted from below the annulus of magnetic material. This further removes the target entities from the site of supernatant extraction, and so further protects the collected target entities from damage or loss caused by the supernatant extraction process.

The method may further comprise any embodiment of one or more of the fourth aspect and/or fifth aspect and/or ninth aspect as the same are described herein.

According to a second aspect, there is provided a liquid handling robot comprising one or more pipettes for injecting or extracting liquid from tubes, wherein the liquid handling robot is configured to perform the method of any embodiment of the first aspect. The liquid handing robot may be part of an apparatus for performing an automated assay process. The apparatus may further comprise the features of any embodiment of one or more of the third aspect and/or sixth aspect and/or seventh aspect and/or eighth aspect and/or tenth aspect and/or eleventh aspect.

According to a third aspect there is provided an apparatus for performing an assay process, the apparatus comprising: a sample plate holder for receiving an assay sample plate, the sample plate comprising a plurality of tubes; a magnet plate comprising a respective magnet for each tube of the sample plate, each magnet being a ring magnet comprising an annulus of magnetic material defining a central cavity; a lift configured to raise or lower the magnetic plate between an upper position and a lower position, wherein: in the upper position, the magnetic plate is raised such that, when a sample plate is received within the sample plate holder, each tube extends into the cavity of its respective magnet; and in the lower position, the magnetic plate is lowered such that the tubes do not extend into their respective magnets.

This apparatus allows the tubes to remain in one place throughout multiple stages of the assay process, in particular throughout target entity isolation aspects. A magnetic field can be applied to or removed from the tubes quickly and automatically. In contrast, conventional systems require the tubes (or their contents) to be moved to a separately located magnet, lengthening the assay process and so limiting throughput. The lift allows magnets to be applied to many tubes simultaneously, providing high throughput. Due to their ring shape, each magnet is able to make close contact with its respective tube, providing efficient collection of magnetic beads and attached target entities, whilst allowing placement by one-dimensional motion, simplifying the lifting mechanism.

In some embodiments, the apparatus may further comprise a liquid handling robot comprising one or more pipettes, wherein the liquid handing robot is configured to inject or extract liquid from tubes of a sample plate when the sample plate is in position in the sample plate holder or one of the sample plate holders. Thus the apparatus may be able to automatically perform multiple aspects of an assay process without moving the sample plate, increasing throughput.

The apparatus may further comprise the features of any embodiment of one or more of the second aspect and/or sixth aspect and/or seventh aspect and/or eighth aspect and/or tenth aspect and/or eleventh aspect, as further provided herein.

According to a fourth aspect, there is provided a method for isolating target entities from a sample liquid in an assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube or a plurality of tubes of a sample plate with magnetic beads such that the target entities collect on the surfaces of the magnetic beads; and collecting the magnetic beads in the tube by raising a magnet plate from a lower position to an upper position using a lift, the magnet plate comprising a respective magnet for each tube of the sample plate, each magnet being a ring magnet comprising an annulus of magnetic material defining a central cavity, wherein: in the upper position, the magnet plate is raised such that each tube of the sample plate extends into the cavity of its respective magnet; and in the lower position, the magnetic plate is lowered such that the tubes do not extend into their respective magnets.

The method may further comprise any embodiment of one or more of the first aspect and/or fifth aspect and/or ninth aspect.

According to a fifth aspect, there is provided a method for isolating target entities from a sample liquid in an automated assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube with magnetic beads, such that the target entities collect on the surfaces of the magnetic beads; applying a magnet field to collect the magnetic beads within the tube, the magnetic field applied by a magnet comprising an annulus of magnetic material defining a central cavity, wherein a portion of the tube is positioned within the cavity; extracting supernatant from the tube, the supernatant comprising the sample liquid less the target entities collected on the surfaces of the magnetic beads; and dispensing a buffer liquid into the tube, the buffer liquid for suspending the magnetic beads and/or target entities, wherein dispensing the buffer liquid into the tube comprises: determining a plurality of dispensing positions, each dispensing position located, when projected onto a plane orthogonal to a longitudinal axis of the tube, at a respective point on a predetermined closed loop; dispensing the buffer liquid into the tube at a first dispensing position of the plurality of dispensing positions; and for each subsequent dispensing position of the plurality of dispensing positions: aspirating a portion of the buffer liquid from the tube; dispensing the portion of the buffer liquid into the tube at the respective dispensing position.

After collecting target entities and magnetic beads on the inner surface of a tube as part of the isolation aspects of an assay process, it can be difficult to then remove the beads and target entities from the tube for downstream processing. In conventional methods, a buffer liquid is added and the tube is moved to an orbital shaker to mix the buffer liquid with the beads/target entities. However, this shaking can overstimulate the target entity, for example activating cells or otherwise damaging the target entity. In contrast, the method of the fifth aspect uses the buffer dispensing and aspiration process itself to generate the mixing, potentially avoiding the need for a shaker and so limiting damage to the target entity. The method dispenses and then aspirates buffer liquid at a sequence of points around a closed loop. It has been found that this allows the bead bound cells collected at the sides of the tube to be gently resuspended as buffer liquid is dispensed, with the process of aspiration providing mixing of the beads/target entities and buffer liquid. Not only does this method cause less damage to the target entity than conventional processes, making the ultimate investigative aspect of the assay process more efficient, it allows the bead re-suspension to occur in-situ. The tube does not have to be moved to a separate shaker, saving time and so increasing throughput. The method can be performed simultaneously for many tubes by a pipette head of a liquid handling robot, further increasing throughput. Throughput can be particularly enhanced when combined with the in-situ magnetic plate discussed in the third and fourth aspects, allowing all the target entity isolation aspects to be performed automatically without moving the sample.

In some embodiments buffer liquid may be dispensed into the tube at a dispensing rate, and buffer liquid may be aspirated from the tube at an aspiration rate, wherein the aspiration rate is less than the dispensing rate. It has been realised that turbulent aspiration causes more shear stress forces than predominantly laminar dispensing. A slower aspiration rate is therefore used to reduce damage to the target entity during mixing, whereas a faster dispensing rate can be used to avoid unnecessary delay to the process.

In some embodiments the tube may comprise a conical shaped bottom. This method may be particularly effective for conical shaped tubes, where removal of the beads from the smooth tube wall can be particularly difficult. Conventional methods avoid this by using pyramidal bottomed tubes. The present method allows conical shaped tubes to be used, which enable the tubes to sit in wells formed by annular magnets, such as those used in the third and fourth aspect.

The method may further comprise any embodiment of one or more of the first aspect and/or fourth aspect and/or ninth aspect.

According to a sixth , there is provided an apparatus for performing an automated assay process, the apparatus comprising a liquid handling robot comprising one or more pipettes for dispensing or aspirating liquid from tubes, wherein the apparatus is configured to perform the method of any embodiment of the fifth aspect.

The apparatus may further comprise the features of any embodiment of the second aspect and/or third aspect and/or seventh aspect and/or eighth aspect and/or tenth aspect and/or eleventh aspect.

According to a seventh aspect, there is provided a sieve plate for use in an automated ELISPOT assay process, the ELISPOT assay process comprising collecting cells on surfaces of magnetic beads to isolate the cells from a sample liquid, the sieve plate comprising: a frame; and a membrane supported by the frame, the membrane comprising a plurality of pores; wherein the size of the pores is such that the cells and magnetic beads are able to pass through the membrane, but cellular clumps cannot pass through the membrane.

In particular, able to pass may mean that about 90% or more, or preferably about 95% or more, or more preferably about 99% or more of (non-clumped) cells and magnetic beads pass through the membrane. Not able to pass may mean that about 90% or more, or preferably about 95% or more, or more preferably about 99% or more of cellular clumps are prevented from passing through the membrane. A cellular clump may be a clump of about 20 or more cells, or about 30 or more cells; or a combination of cells and/or cellular material forming a clump with a size equivalent to about 20 or more cells or about 30 or more cells.

It has been realised that cell clumping causes particular problems in ELISPOT processes, such as ELISPOT processes for identifying TB, such as the T-SPOT.ZB test. Such processes require cells to be counted. Cell counters often contain small capillaries, which cellular clumps can block, hindering the counting process. It would be desirable to prevent cell clumping, but this may not be possible in an automated system and/or may slow the assay process, reducing throughput. However, the inventors have found that the cellular clumps can be filtered prior to the cell counting stages, whilst still providing sufficient cells for counting in the counting aspects of the ELISPOT process.

The sieve plate of the present disclosure provides a simple filter than can be inserted into an automated ELISPOT apparatus. It is pressure-fed, so there is no need for additional pumping systems - a liquid handling robot can simply pipette liquid through the filter.

The present inventors have found that the size of the pores in the filter is of crucial importance to both its ability to reduce downstream blockages, and to maintain high throughput of the system. The inventors have found that pores having a diameter selected from a range with an upper limit of about 300 pm or preferably about 250 pm, or more preferably about 215 pm are particularly advantageous. This upper limit on the size of pores has been found to be sufficient to block the most problematic clumps, avoiding downstream blockages.

Additionally, the inventors have found that the lower limit of the range from which the pore size may be selected should preferably be higher than about 100 pm , or more preferably about 150 pm or more preferably still about 180 pm. Such lower limits may be considered high compared to the size of a cell, and so it may be assumed that even smaller sizes would be better, to ensure that only individual cells (and beads) can pass through the filter. However, the present inventors have realised that smaller sizes reduce the flow rate through the filter, and so reduce the overall throughput of the assay process or require active methods of causing the fluid to move through the sieve, such as vacuum or positive pressure manifold. The inventors found that a minimum pore size of about 100, about 150, or about 200 pm allows for a good balance between throughput and clump-filtering.

The pores all have the same or approximately the same size, or may be of different sizes, each spore being within the range of sizes with the upper and lower limits discussed herein.

In some embodiments, the sieve plate may comprise a plurality of membranes, each membrane supported by the frame, the membranes being positioned within the frame at positions which correspond to positions of pipettes in a pipette head of a liquid handling robot, the liquid handing robot for performing the automated ELISPOT assay process.

According to an eighth aspect, there is provided an apparatus for performing an automated ELISPOT assay process, the apparatus comprising: a cell isolation system for isolating cells from a sample liquid, the cell isolation system comprising: a sample plate holder for receiving a sample plate, the sample plate comprising a plurality of tubes, each tube for receiving magnetic beads and a sample liquid for testing as part of the ELISPOT assay process, the magnetic beads having surfaces configured to collect the cells from the sample liquid; a magnet for collecting the magnetic beads in each tube; a liquid handling robot comprising at least one pipette, the liquid handling robot configured to: inject a buffer liquid into each tube of the sample plate when in position in the sample plate holder, to mix the buffer liquid with the magnetic beads and cells in that tube; and extract the mixture of buffer liquid and magnetic beads from each tube; a downstream assay system for performing an ELISPOT on the isolated cells; and a sieve plate according to any of embodiment of the eighth aspect positioned between the cell isolation system and the downstream assay system; wherein the liquid handling robot is configured to inject the extracted mixture of buffer liquid and magnetic beads for each tube onto the sieve plate for filtering prior to processing of the cells by the downstream assay system.

The apparatus may further comprise the features of any embodiment of one or more of the second aspect and/or third aspect and/or sixth aspect and/or tenth aspect and/or eleventh aspect.

According to a ninth aspect, there is provided a method for isolating cells from a sample liquid in an automated ELISPOT assay process, the method comprising: mixing the sample liquid comprising the cells in a tube with magnetic beads, such that the cells collect on the surfaces of the magnetic beads; applying a magnet field to collect the magnetic beads within the tube; extracting supernatant from the tube, the supernatant comprising the sample liquid less the cells collected on the surfaces of the magnetic beads; adding a buffer liquid to the tube, the buffer liquid for suspending the magnetic beads; injecting the mixture of buffer liquid and magnetic beads through a sieve plate of any of claims 1 to 10 to filter the mixture; and inputting the filtered mixture of buffer liquid and magnetic beads into a downstream assay system to test the cells collected on the surfaces of the magnetic beads.

The method may further comprise any embodiment of one or more of the first aspect and/or fourth aspect and/or fifth aspect.

According to a tenth aspect, there is provided a liquid waste collector for use in an automated apparatus for performing an assay process, the apparatus comprising a liquid handling robot comprising a pipette head, the pipette head comprising a plurality of pipettes extending from the pipette head, the liquid waste collector comprising: a funnel section comprising a funnel wall defining a first opening, a second opening, and a passage between the first opening and the second opening; a receiving section comprising a shroud wall, the shroud wall extending from the funnel wall at the first opening away from the funnel section, the shroud wall surrounding the first opening; wherein the receiving section is shaped to receive the pipette head of the liquid handling robot such that, when received in the receiving section, at least a lower portion of the plurality of pipettes of the pipette head is surrounded by the shroud wall. At various stages in an assay process such as an ELISPOT process there is a need to dispose of waste liquid, which may include contaminated liquid such as blood. As such, care must be taken when disposing of liquid, to avoid splashing or aerosol generation. Conventional automated assay systems use complex liquid waste collectors, in which pumps are used to force waste liquid under pressure into a waste receptacle.

However, such systems add complexity to the automated system, and increase the time taken to dispose of liquid waste, reducing the throughput of the assay process. Conventional active waste disposal systems are susceptible to clogging of waste lines that can lead to system blockage and contamination of work surfaces. The use of peristaltic pumps in such systems to provide vacuum and pressure on input and output lines also can cause premature failure of flexible waste line tubing due to deformation of the tube wall and subsequent reduction in tube inner diameter. The complex systems of pumps also have large physical footprints, taking up space that could otherwise be used to test more samples, further reducing potential throughput.

In a large testing lab containing many automated systems, each performing an assay process, this can amount to a significant amount of physical space taken up by liquid waste removal, reducing the number of systems the lab is able to fit in, and hence the total rate of testing the lab can provide.

In contrast, the present disclosure provides a simple, compact liquid waste collector. Liquid waste is simply pipetted into the collector, where it flows under gravity through the collector and out to the second opening, which can be connected to a waste bin. The pipettes are received within the receiving section, so that the point at which liquid is injected into the collector is lower than the top of the shroud wall. This substantially prevents liquid splashing back out of the collector, for example after impact with wall of the passage between the first and second openings. The shroud wall also minimises aerosol escape, which is generally trapped by the shroud wall and pipette head of the liquid handling robot. This gravity-fed, compact waste collector allows waste liquid to be quickly removed by a liquid handling robot, reducing the time and space required for handling liquid waste compared to conventional systems.

According to an eleventh aspect, there is provided an apparatus for performing an assay process, the apparatus comprising: a liquid handling robot comprising a pipette head, the pipette head comprising a plurality of pipettes extending from the pipette head; and a liquid waste collector according to any embodiment of the tenth aspect; wherein the liquid handing robot is configured to: collect waste liquid from the assay process in the plurality of pipettes; position the plurality of pipettes within the receiving section of the liquid waste collector such that at least a lower portion of the plurality of pipettes is surrounded by the shroud wall of the liquid waste collector; and inject the waste liquid from the pipettes into liquid waste collector.

The apparatus may further comprise the features of any embodiment of one or more of the second aspect and/or third aspect and/or sixth aspect and/or seventh aspect and/or eighth aspect.

BRIEF DESCRIPTION OF THE DRAWINGS Certain embodiments will now be described, by way of example only, with reference to the accompanying drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Fig. 1 illustrates a method of extracting supernatant in an assay process;

Fig. 2 schematically illustrates an example of the method of Fig. 1 in use;

Fig. 3 schematically represents an apparatus comprising a lift for raising a magnet as part of an assay process;

Fig. 4 shows a top-down view of an example magnet plate;

Fig. 5 illustrates a method of collecting magnetic beads and target entities;

Fig. 6 illustrates a method of dispensing buffer liquid as part of an assay process;

Fig. 7 shows an example pattern of dispensing positions;

Fig. 8 illustrates the dispensing positions within tubes of a sample plate;

Fig. 9 schematically represents a sieve plate for use in an assay process;

Fig. 10 schematically represents an automated assay apparatus;

Fig. 11 illustrates a method of performing an assay process using the sieve plate of Fig. 9; Fig. 12 schematically represents an example of a liquid waste collector, in side view (Fig. 12(a)) and isometric view (Fig. 12(b)); and

Fig. 13 schematically represents pipettes received within the liquid waste collector of Fig.

12

DETAILED DESCRIPTION

The embodiments described below illustrate examples of methods and apparatus components that may be used in an automated assay process, such as an ELISPOT process. Although described separately, it will be appreciated that each of the methods and components described below can be combined with any number of the other methods/components. Individually, each of the methods and components described below helps increase the throughput at a particular stage of the automated assay process. When used in combination, the methods and components provide a streamlined, efficient process that allows many samples to be concurrently and automatically processed, greatly increasing the throughput of the overall assay process. This means, for example, more diagnostic tests can be performed in a given period, which could be of great importance to the health outcomes of the patients being tested.

The term “about” as used herein in reference to a number is used herein to include numbers which are greater, or less than, a stated or implied value by 1%, 5%, 10%, or 20%.

Multi-part isolation of magnetic beads and target entities

Fig. 1 shows an example method 100 for isolating target entities from a sample liquid in an assay process, such as an ELISPOT process. The target entities may be cells and/or cellular components, or any other macromolecule that is to be tested as part of the assay process. The method may be performed by a liquid handling robot, i.e., a robot configured to move and control a pipette head from which one or more pipettes extend, such as pipette 203 illustrated in Fig. 2. The liquid handling robot may be part of an automated assay process. The method may be performed simultaneously for a plurality of different sample liquids (e.g., different blood samples), each sample liquid in a respective tube. For example, the method may be performed simultaneously for each well in an assay sample plate, e.g., for 20 or more wells. In a standard sample plate, the number of wells may be 24. The pipette head of the liquid handling robot may comprise a corresponding number of pipettes to perform the method for each well simultaneously. The liquid handling robot, or an apparatus of which it is a part, may comprise a controller configured to control the liquid handling robot to perform the method 100.

The sample liquid is the sample containing the target entities of interest, such as a blood sample or any other biological sample suitable for the specific assay process. The sample liquid may have been processed prior to performing method 100.

Method 100 starts at 101, at which the sample liquid (containing the target entities) is mixed in a tube or well with magnetic beads. The magnetic beads are designed to collect target entities on the bead surfaces. For example, the magnetic beads may be functionalised to collect the target entities, as is known in the art. In some embodiments, a bridging substance is used to bind the target-entities to the (optionally functionalised) magnetic beads. For example, the target- entities may bind to target-entity specific biotinylated antibodies, which in turn bind to a coating on the magnetic beads. The bridging substance may be mixed into the sample liquid. Alternatively, cells can be bound directly to antibody-coated magnetic beads.

The tube/well may generally be an elongated container closed at one end. The term lower section is used herein to refer generally to a portion of the tube closest to the closed end (and in use, closest to the magnet). Method 100 then proceeds to 102, at which a magnetic field is applied for a first period to a lower section of the tube to collect the magnetic beads within the lower section of the tube.

An example of this process is shown in Fig. 2(a). Fig 2(a) shows a cross-section of a tube 201 with a magnet 202 positioned around the tube 201 such that a lower section 201b of the tube 201 extends below an upper surface of the magnet. In this example, the magnet 202 comprises an annulus 202a of magnetic material extending around the lower section 201b. The base 202b of the magnet 202, from which the annulus 202a extends, may or may not be formed of magnetic material. The magnet 202 may extend along the tube (i.e., in a direction parallel to a longitudinal axis of the tube) over a distance less than 25% or less than 10% of a total length of the tube.

Other arrangements of magnet may be used, for example a magnet comprising only a magnetic base positioned under the tube 201. The magnet 202 may be an electromagnet, and applying the magnetic field may comprise activating the electromagnet. Alternatively, the magnet may be a permanent magnet.

Applying the magnetic field may comprise raising the magnet 202 on a lift 400, as discussed herein in relation to Figs. 3-5.

The tube 201 comprises a liquid which is a mixture of magnetic beads 205 (including target entities captured on the magnetic beads 205) and a supernatant 204. The supernatant 204 is the original sample liquid, less the target entities that are now captured on magnetic beads 205. For clarity, only some of the magnetic beads 205 are labelled in the figure. Similarly, although only a small number of magnetic beads are illustrated, it will be appreciated that in practice many more beads will be used.

With the magnetic field applied to the lower section 201b of the tube 201, the magnetic beads 205 (and attached target entities) are attracted to the inner surface of the tube 201, in accordance with the pattern of the magnetic field applied. This process is represented in Fig. 2(a) by the magnetic beads 205 captured at the tube wall in the lower section 201b. This substantially depletes the liquid in the lower section 201b of magnetic beads, leaving only supernatant 204.

The magnetic field is applied to the tube 201 for a first period of time, to allow time for the magnetic beads 205 in the lower section 201b to be collected at the sides of the tube 201. The first period may for example be between 5 minutes and 20 minutes. As will be appreciated, the length of the first period may be varied depending on the specific assay process in question, the number of target entities that need collecting, the size of the magnetic beads 205, the strength of the magnetic field, etc.

After waiting the first time period, the method 100 proceeds to 103, at which a first portion of supernatant 204 is extracted from the lower section 201b of the tube 201 (and is not replaced). In the example illustrated in Fig. 2(a), the supernatant 204 is extracted using a pipette

203 of a liquid handing robot.

As discussed herein, the supernatant 204 in the lower section 201b (i.e., the section to which a magnetic field has been applied) is significantly depleted of magnetic beads 205. In contrast, magnetic beads 205 are still suspended in supernatant 204 in an upper section 201a of the tube 201. Extracting supernatant from the lower section 201b therefore removes depleted supernatant 204 that is now superfluous to the assay process. Importantly, only a first portion of the supernatant is removed 204 - there is no continuous removal of all the supernatant 204. This allows non-depleted supernatant 204 in the upper portion 201a of the tube 201 to fall down the tube 201 into the region of magnetic field, and be held in the magnetic field long enough to collect magnetic beads.

The first portion of supernatant that is removed may be half of the total supernatant 204, or a quarter of the total, or less. The volume of the first portion of the supernatant 204 removed may match the volume of the lower section 201b of the tube 201 to which the magnetic field is applied, or it may be more or less than this volume.

In the illustrated example, the point of extraction (i.e., the location of the tip of pipette 203) is below the level of the upper surface of the magnet 202, but within the region of the annulus 202a of the magnet 202. In other embodiments, the magnet 202 may be shaped such that the tube 201 extends below the magnet 202 (e.g., where the magnet 202 is just a ring around the tube 201). In such embodiments, the point of extraction may be below the magnet 202. This may minimise any impact of supernatant 204 extraction on the magnetic beads 205 collected at the tube wall.

After extracting the first portion, the method 100 proceeds to 104, at which the magnetic field is applied to the lower portion 201b of the tube for a second period to collect further magnetic beads. This process is illustrated in Fig. 2(b). Fig 2(b) shows a portion of supernatant

204 which was previously in the upper section 201a of the tube 201, but which fell into the lower section 201b when the first portion of supernatant 204 was extracted. Upon application of the magnetic field, further magnetic beads 205 are collected at the tube wall from this portion of supernatant 204.

The duration of the second period may be the same as the duration of the first period, e.g., between 5 and 15 minutes. Alternatively, a different duration may be used. Where the magnetic field is applied by a permanent magnet, 104 may comprise waiting for the second period with the magnet 202 in position around the tube. After the second period has ended, the method proceeds to 105. At 105, a second portion of the supernatant 104 is removed from the lower section 201b of the tube. The process of extraction may be the same as for 103.

In some embodiments, the method 100 may be a two-part process for extracting supernatant 204. In such cases, the second portion of supernatant 204 extracted in 105 may comprise all of the supernatant 204 remaining after extraction of the first portion.

Alternatively, more than two supernatant extractions may be used. In such cases, the second portion of supernatant will be less than the total supernatant remaining after extraction of the first portion. The method then comprises additional applications of the magnetic field for a period of time, and subsequently extracting a respective portion of supernatant 204, until all the supernatant has been removed. In general, the method may comprise n extractions, each with a preceding applying of the magnetic field for a period of time to collect magnetic beads 205. The volume of supernatant 204 removed in each extraction may be approximately 1 In of the initial volume of supernatant 204. However, embodiments with only two extractions may provide an optimal balance between magnetic bead collection effectiveness and time taken to perform the method 100.

The method 100 thus provides a simple and effective automated process for isolating and collecting target entities on magnetic beads. Importantly, the method 100 can be performed without moving the tube 201, eliminating the time this wastes, and so increasing the throughput of the assay process.

Lift for magnet plate

In Fig. 2(a) and (b) the magnet 202 was shown as being already in position around the tube 201. However, there are some sample preparation or other assay aspects where it is not desirable to have a magnetic field applied. For example, when initially mixing the magnetic beads 205 with the sample liquid, an applied magnetic field may attract the beads 205 before target entities have time to collect on the bead surfaces. Conventionally, therefore, it is necessary to perform certain aspects of the assay before (or after) moving the tube 201 to a dedicated magnet plate. This wastes time in moving the tube 201, limiting throughput.

Figures 3(a) and 3(b) illustrate an apparatus in which magnets 202 are positioned onto respective tubes 201 during the assay process, rather than moving the tubes 201 themselves. This enables many aspects of the assay process, and in particular all target entity isolation aspects, to be performed without moving the tubes 201 containing samples.

The apparatus is for performing an assay process, and in particular a process in which target entity is collected on (functionalised) magnetic beads. For example, the apparatus may be for performing an ELISPOT process. The apparatus may comprise a controller configured to control components of the apparatus to perform the assay process, and in particular to perform method 500 discussed herein.

The apparatus comprises a sample plate holder for receiving an assay sample plate, the sample plate comprising a plurality of tubes. In the example illustrated in Fig. 3(a) and (b), two tubes 201-1, 201-2 are shown. It is to be appreciated that the sample plate may comprise any number of tubes/wells, for example 10 or more or 20 or more. A standard sample plate may comprise 24 tubes. It is noted that for clarity, the sample plate holder and elements of the sample plate are not shown in Fig. 3(a) and (b). However, an example sample plate 800 is shown in figure 8, discussed in more detail below. The sample plate holder may take any form suitable for receiving a sample plate, and suspending the sample plate above the magnets 202.

Although not shown, the apparatus may also comprise a robotic arm configured to position a sample plate on the sample plate holder, and/or a liquid handling robot. In particular, the liquid handling robot may be configured to perform method 100, or method 600 discussed herein, whilst the sample plate is in position on the sample plate holder.

The apparatus further comprises a magnet plate 300. The magnet plate 300 comprises a base 301 holding respective magnets 202-1, 202-2 for each tube 201-1, 202-1 of the sample plate. Each magnet is an example of magnet 201 in Fig. 2, and all features of that magnet may also apply to the magnets 202-1, 202-2. In particular, each magnet 202-1, 202-2 comprises a ring magnet comprising an annulus 202a of magnetic material defining a central cavity 202c. The annulus may have discontinuities - i.e., it may not extend fully around the circumference of the tube 201. The cavity 202c may be a through -hole or, as in the illustrated example, closed cavity. The magnets 202 of the magnet plate may for example be any of the magnets described in W02020/041339 Al, W02020/041345 Al, and/or WO2016/061285 Al, which are incorporated herein by reference.

A top-down view of an example magnet plate 300, in this case comprising 24 magnets 202, is shown in Fig. 4. For clarity only some of the magnets 201 are labelled.

The magnet plate is placed on (or otherwise held by) a lift 400, such as a pneumatic lift. The lift 400 is configured to raise or lower the magnet plate between a lower position and an upper position. Fig. 3(a) shows the magnet plate 300 in the lower position, where the magnet plate 300 is lowered such that the tubes 201-1, 201-2 do not extend into their respective magnets 202-1, 202-2. In particular, in the lower position, the magnet plate 400 is distanced from the sample plate holder such that the strength of the magnetic field at the sample plate holder due to the magnet plate 400 is negligible - i.e.. insufficient to hold magnetic beads 205 at an inner wall of their tube 201. In the lower position, the vertical distance between the magnet plate 300 may be at least 2 cm or at least 5cm or at least 10 cm below the sample plate holder or lower end of the tubes/wells 201 of the sample plate.

Fig. 3(b) shows the lift 400 and magnet plate 300 in the upper position. In the upper position, the magnetic plate is raised such that, when a sample plate is received within the sample plate holder, each tube extends into the cavity of its respective magnet. Thus, as shown in the figure, a lower portion of each tube 201-1, 202-1 is (at least partially) surrounded by magnetic material of its respective magnet 202-1, 202-2. In this position, magnetic beads 205, and the target entities caught on them, are attracted to the sides of the tube 201-1, 202-2 in accordance with the magnetic field applied by the magnets 202-1, 202-2. In this way, magnetic beads 205 and target entities can be isolated from the supernatant 204. The supernatant 204 may be extracted using method 100.

The apparatus thus allows a magnetic field to be applied to the tubes 201-1, 201-2 only when required, without having to move the tubes 201-1, 201-2 across an assay apparatus to a fixed magnet plate. Multiple aspects of the assay process, and in particular aspects of the cell isolation stage, can be carried out with the tubes 201-1, 201-2 in situ. These aspects can be performed concurrently and automatically for a large number of tubes 201, without any risk of collisions that can occur when tubes 201 have to be moved. Thus, a large number of samples can be processed within minimal downtime, increasing the throughput of the apparatus.

In some embodiments, the apparatus may comprise a plurality of sample plate holders, each for receiving a respective sample plate, and respective magnet plate 300 for each sample plate holder. Thus, even where a standard sample plate comprises a limited number of tubes 201, such as 24, the apparatus can still process larger numbers of samples concurrently by using multiple sample plates. In such embodiments, the lift 400 may be configured to raise or lower each magnet plate 300 between the upper and lower position. Alternatively, the apparatus may comprise a plurality of lifts 400, each lift 400 configured to raise or lower a respective one of the magnet plates 300 between a respective upper position and a lower position (which may or may not be the same for each magnet plate 300).

Fig. 5 illustrates a method 500 that may be performed by the apparatus of Fig. 3. Method 500 is a method for isolating target entities (e.g., cells and/or cellular material) from a sample liquid in an assay process (e.g., an ELISPOT process).

Method 500 begins at 501, at which the sample liquid(s) comprising the target entities is mixed in a tube or a plurality of tubes of a sample plate with magnetic beads 205 such that the target entities collect on the surfaces of the magnetic beads 205. This aspect may be the same as 101 discussed herein.

The method then proceeds to 502, at which the magnetic plate 300 is raised from a lower position to an upper position using lift 400.

The method then proceeds to 503, at which magnetic beads 205 are collected in the tube. For example, this aspect may comprise waiting a fixed period, with the magnet plate 300 in the upper position, to give the magnetic beads 205 time to collect in the tube. This aspect may correspond to aspectl02 of method 100. After collecting the beads 205, the now depleted supernatant 204 may be extracted from the tube/s 201. This extraction may use the process of method 100 - i.e., method 500 may further comprise 103-105 of method 100. Once the supernatant 204 has been extracted, the magnet plate may be lowered to the lower position, so that the magnetic field does not interfere with subsequent aspects of the assay process.

After extracting the supernatant (using method 100 or otherwise), the magnetic beads 205 and target entities attached to them may be removed from the tube(s) 201, and passed to a downstream assay process (e.g., a counting process). Removing the magnetic beads 205 may comprise adding a buffer liquid to suspend the magnetic beads 205, for example using the method 600 discussed herein - i.e., the method 500 may further comprise 604-608 of example method 600.

The apparatus may comprise a controller configured to control the apparatus to perform the method 500. In particular, the lift 400 may comprise control circuitry to raise or lower the lift in accordance with the method 500.

Resuspension of isolated magnetic beads and target entities

In a typical assay process in which target entities are isolated using magnetic beads 205, it is necessary to remove the magnetic beads 205 and target entities from the tube 201 used for isolation, to process the target entity in the diagnostic stage of the assay process. This may involve adding a buffer liquid to a tube 201 in which magnetic beads 205 are captured at a tube wall, to re-suspend the magnetic beads 205 and attached target entities. However, it can be difficult to displace the magnetic beads 205 from the wall of the tube 201. The smoother the wall of the tube 201, the more difficult it is to displace the magnetic beads 205. For this reason, conventional techniques may use tubes 201 with a pyramid-shaped lower end, to provide a rougher surface from which magnetic beads 205 may be more easily displaced. However, in the present disclosure conical bottomed tubes 201 are preferredto make better contact with the ring magnets 202 discussed herein. Further, in conventional techniques the buffer-filled tube 201 is generally moved to an orbital shaker to shake the magnetic beads 205 off the walls of the tube 201. This limits the throughput of the assay process, as tubes 201 must be moved, and generally only a limited number of tubes can be processed simultaneously in the orbital shaker. Furthermore, forceful shaking risks damaging or activating the target entities attached to the magnetic beads 205, which can limit the effectiveness of the overall assay process.

Fig. 6 illustrates a method 600 that may be used to displace the magnetic beads 205 without applying a damaging amount of force to the captured target entities. Method 600 is a method for isolating target entities from a sample liquid in an automated assay process. The target entities may comprise cells and/or cellular components, and the assay process may be an ELISPOT process, such as an ELISPOT process for detecting TB infection.

The method 600 may be performed by an apparatus for performing an automated assay process. The apparatus comprises a liquid handling robot comprising one or more pipettes 203 for dispensing or aspirating liquid from tubes, wherein the apparatus is configured to perform method 600. The apparatus may be apparatus 1000, or cell isolation system 1001 of an assay process, as discussed herein.

Method 600 starts at 601, at which the sample liquid comprising the target entities is mixed in a tube 201 with magnetic beads 205, such that the target entities collect on the surfaces of the magnetic beads 205. This aspect may be the same as aspects 101 or 501 discussed herein. Method 600 may be performed simultaneously for a plurality of different sample liquids, each sample liquid in a respective tube 201. For example, the tubes 201 may be tubes/wells 201-1, 202-2 of a sample plate.

The method 600 then proceeds to 602, at which a magnet field is applied to collect the magnetic beads 205 within the tube 201. The magnetic field is applied by a magnet 202 comprising an annulus 202a of magnetic material defining a central cavity 202c, wherein a portion of the tube 201 is positioned within the cavity 205. In embodiments, the magnet 202 comprises a base portion 202b from which the annulus 202a of magnetic material extends. The base portion comprises a recess shaped to receive a conical shaped bottom of the tube 201, where conical -bottomed tubes 201 are used. Where the method 600 is performed for multiple tubes

201-1, 201-2, each tube 201-1, 201-2 is received in the cavity 202c of a respective magnet 202-1,

202-2. 602 of method 600 may comprise performing aspects 502 and 503 of method 500, using a lift 400 to raise the magnet 202.

The method 600 then proceeds to 603, in which supernatant 204 is extracted from the tube 201. This aspect may use the multi-part extraction process of method 100. After extracting all the supernatant 204, the magnetic field may be removed from the or each tube 201 prior proceeding to 604 of method 600. Removing the magnetic field may comprise lowering the or each magnet away from the or each tube, e.g., with lift 400.

With the supernatant 204 removed, the next aspects are to dispense buffer liquid into the tube 201 to re-suspend the beads. Rather than simply adding a buffer liquid and relying on an orbital shaker to dislodge magnetic beads 205 from the tube 201 walls, the method uses the actual process of dispensing (and aspirating) the buffer liquid to wash the magnetic beads 205 off the walls, and to mix the beads 205 with the buffer liquid. As a result, much reduced orbital shaking is needed, or the orbital shaker can be eliminated completely.

The dispensing process starts at 604, at which a plurality of dispensing positions are determined at which buffer liquid will be dispensed. Each dispensing position is located, when projected onto a plane orthogonal to a longitudinal axis (i.e., the length) of the tube 201, at a respective point on a predetermined closed loop. In other words, a pattern of dispensing points is determined such that, when looking at the tube 201 from a top-down/horizontal cross-section perspective, the pattern of dispensing points appears as a closed loop shape such as a circle.

Fig. 7 illustrates an example dispensing pattern 700. Dispensing pattern 700 is made up of a plurality of dispensing points 701, 702, 703.. 70n, where n is the number of dispensing points in the pattern 700. n=40 in this case, but any number can be used, in particular, about 5 or more, or about 8 or more, or about 10 or more, or about 20 or more, about 30 or more, or about 40 or more. Optimally, the number of dispensing positions may be in the range about 15-25, providing a balance between time taken and the effectiveness of re-suspension. For clarity, only three dispensing points 701-703 are labelled in the drawing. Fig. 7 shows the locations of the dispensing points 701-70n as projected onto a horizontal plane, in a coordinate space defined by the centre of the tube 201 in a horizontal cross-section. The projections of the dispensing points 701-70n all lie on a closed loop, represented by the solid line in Fig. 7. In this example, the closed loop is a circle. In general, the shape of the closed loop may conform to the shape of the tube 201 in horizontal cross-section.

Fig. 8 illustrates the dispensing pattern 700 within tubes 201. Fig. 8 shows an example sample plate 800 comprising a plurality of tubes (or wells) 201-1, 201-2, 203-3 in a top-down view. For clarity only three tubes 201-1, 201-2, 203-3 are labelled in the drawing. Although sample plate 800 comprises 12 tubes 201-1, 201-2, 203-3, it will be appreciated that alternative sample plates may have any number of tubes 201, for example 20 or more.

A dispensing pattern 700 is shown within each tube 201-1, 201-2, 201-3. The dots of the dispensing pattern represent the horizontal locations of the dispensing points 701, 702,

703.. 70n. The closed loop on which the dispensing points 701-70n lie matches the shape of the tubes 201, but with a smaller diameter. The diameter of the closed loop may be at least about 70%, or at least about 80% of an inner diameter of the respective tube 201-1, 201-2, 201-3 in the region of the tube 201 at which magnetic beads 205 are collected. The distance between a dispensing point 701-70n and the inner wall of the tube 201 may be about 3mm or less, or preferably about 2mm or less. These values mean that there is space between the closed loop and the tube wall to fit in the pipette dispensing the buffer liquid; but the dispensing positions 701- 70n are close enough to the tube wall that some buffer liquid creates turbulence at the periphery of the tube wall near where it is dispensed, washing magnetic beads 205 off that part of the wall. This provides a gentle way of washing magnetic beads 205 off the wall, reducing the risk of the target entity on the beads 205 being damaged.

In the longitudinal plane (i.e., in a vertical cross-section), the dispensing points 701-70n may be at different heights in the tube. Alternatively, the dispensing points 701-70n may lie in the horizontal plane itself, i.e., may all have the same height in the tube 201.

Determining the dispensing pattern 700 at604 of the method 600 may comprise defining the dispensing pattern 700. Alternatively, it may comprise accessing a previously determined dispensing pattern 700, for example from a memory associated with a controller of the apparatus performing the automated assay process.

With the dispensing pattern 700 determined, the method 600 proceeds to 605, at which the buffer liquid is dispensed into the or each tube 701 at a first dispensing position 701 of the plurality of dispensing positions 701-70n. For example, the pipette 203 of a liquid handling robot is moved such that its tip is at the first dispensing position 701, at which buffer liquid is released from the pipette 203. The full volume of buffer liquid in the pipette 203 may be dispensed at the first dispensing position 701, or only a portion of the full volume may be dispensed.

The method then proceeds to 606, at which at least a portion of the buffer liquid is aspirated from the tube(s) 201. The portion may be at least about 40%, or at least about 50%, or at least about 60% of the volume of buffer liquid dispensed in 605. The aspiration may take place at the first dispensing location 701, or alternatively the pipette 203 may first be moved to the second dispensing location 702.

This aspiration performs two purposes. Firstly, it takes in buffer liquid which can be dispensed again at subsequent dispensing positions 702-70n to wash magnetic beads 205 off further sections of the tube wall. Secondly, the process of aspirating introduces a turbulent flow, mixing the buffer liquid and magnetic beads 205, providing a suspension of beads 205 in the buffer liquid. The present inventors have realised that the turbulent flow of the aspiration, whilst useful for mixing, can damage or activate the target entities. Conversely, the smooth laminar flow of the dispensing is unlikely to damage target entities. Therefore, liquid may be aspirated at a lower rate than it is dispensed, to make limit damage due to aspiration, whilst avoiding unnecessary delay in the dispensing.

After aspirating the portion of buffer liquid, the illustrated method 600 proceeds to 607. At 607, buffer liquid is dispensed at the second dispensing position 702. Where aspiration in 606 was performed at the first dispensing position 701, 607 may comprise moving the pipette 203 to the second dispensing position 702 prior to dispensing the buffer liquid.

The volume of buffer liquid dispensed at the second dispensing position 702 may be the full volume of the portion of buffer liquid aspirated at 606. Alternatively, it may be a smaller volume than that portion, or a greater volume (where some buffer liquid was retained in the pipette 203 at 605).

The Fig.7 method 600 then proceeds to 608. At 608, a portion of buffer liquid is aspirated from the tube 201, and the or a portion of buffer liquid is dispensed for each subsequent dispensing position 703-70n. As above, the aspiration may be performed before or after moving the pipette 203 to the next dispensing position 703-70n.

Aspect 608 continues until buffer liquid has been dispensed at each position of the plurality of dispensing positions 701-700n. In this way, the full circumference of the tube wall is washed with dispensed buffer liquid, suspending the magnetic beads 205 and target entities without requiring use of an orbital shaker.

Method 600 may be performed for a plurality of tubes 201 simultaneously, for example for all the tubes 201-1, 201-2, 201-3 of a sample plate 800, or even for multiple sample plates. Method 600 is particularly useful where it is performed by a liquid handling robot comprising a pipette head, from which a plurality of pipettes 203 extend. The pipettes 203 of the pipette head typically form a rectangular pattern, matching the arrangement of tubes 201-1, 201-2, 203-3 in the sample plate 800. Such pipette heads are generally movable in the x, y, or z direction; but generally cannot be (easily) tilted. Method 600 provides a process in which the pipette head need only move in the x and y directions to move through all the dispensing points 701-70n, washing the sides of the tubes 201-1, 201-2, 201-3 without having to tilt the pipette head to direct buffer liquid towards the tube walls. In this way, re-suspension of magnetic beads 205 can be performed concurrently for a large number of samples, increasing the throughput of the assay process.

Sieve plate

After isolating and re-suspending the magnetic beads 205 and captured target entities, the next part in the assay process is typically to provide the target entities to a downstream assay system, such as a system for performing diagnostic tests. For example, in an ELISPOT process, the target entities (typically cells) are passed to a system for counting the target entities. Such systems often involve small capillaries, which can become blocked, limiting the effectiveness and throughput of the assay process.

The present inventors have realised that these blockages occur because of clumps of cells and/or other cellular material. The inventors have further realised that this clumping particularly occurs during the cell isolation stage of the assay process. It would therefore be desirable to minimise clumping, to avoid downstream problems. However, reducing the process of clumping itself can be difficult, and may hinder the progress of the cell isolation aspects. Instead of eliminating the clumps, the inventors have found that it is instead possible to filter out the clumps before introducing the sample to the downstream assay system, whilst still yielding good ELISPOT results.

Fig. 9 illustrates a sieve plate 900 that may be used for this process. Sieve plate 900 is for use in an automated ELISPOT assay process, the ELISPOT assay process comprising collecting cells on surfaces of magnetic beads 205 to isolate the cells from a sample liquid. In particular, the ELISPOT process may involve the example methods 100, 500, 600 discussed herein. The cells may be peripheral blood mononuclear cells. The ELISPOT process may be a process for identifying tuberculosis.

The sieve plate 900 comprises a frame 901 holding a plurality of membranes 902. For clarity, only three membranes 902-1, 902-2, 902-3 are labelled in the drawing. The number and arrangement of membranes 902 may match the number and arrangement of tubes in a sample well. In the case of sieve plate 900, the membranes 902 are arranged to match the tubes 201 of example sample plate 800. It is to be appreciated that in other examples the sieve plate can comprise any number of membranes 902, including one. The membranes 902 may be formed of nylon.

Each membrane 902-1, 902-2, 902-3 comprises a plurality of pores. The pores may be gaps between the thread of the membrane 902-1, 902-2, 902-3. The size of the pores is selected such that the cells and magnetic beads 205 of the assay process are able to pass through the membrane 902-1, 902-2, 902-3, but cellular clumps (optionally of a minimal size) cannot pass through the membrane 902-1, 902-2, 902-3. In particular, the size of the pores may be such that cellular clumps comprising about 20 or more cells, or about 30 or more cells cannot pass through the membrane (or clumps of cells and cellular material with equivalent sizes). In such cases, smaller clumps may still be able to pass through the membrane 902-1, 902-2, 902-3. As used herein, able to pass means substantially able to pass, e.g., > about 90% or > about 95% of cells and magnetic beads 205 are able to pass through the membrane 902-1, 902-2, 902-3. Clumps not being able to pass means clumps (of the predetermined size) are substantially blocked from passing, e.g., > about 90% or > about 95% of clumps are blocked from passing through the membrane 902-1, 902-2, 902-3.

The inventors have realised that there is a balance to be struck between blocking cellular clumps, and hence avoiding downstream problems; and the speed at which cells pass through the sieve plate 900. Smaller pores may block more clumps, but they also slow down the rate at which the desired cells pass through the membrane 902-1, 902-2, 902-3, limiting the throughput of the assay process. It has been found that pores sized to block cellular clumps of the sizes disclosed herein, eliminate the clumps that provide most of the downstream problems, whilst still allowing for a high throughput.

In embodiments, pores may have a diameter (or generally maximal size) selected from a range with an upper limit of about 300 pm or preferably about 250 pm, or more preferably about 215 pm. Such values have been found to block the problematic clumps. The pores may have a diameter (or generally maximal size) selected from a range with a lower limit of about 100 pm or preferably about 150 pm or more preferably about 200 pm. Thus, for example, sizes of pores may be in the range about 100 pm to about 300 pm, or about 200 pm to about 215 pm. This lower limit on the size ensures that the desired cells can still pass through the sieve plate at a good rate. The pore sizes referred to here may be mean pore sizes. The pore sizes may be determined by introducing particles of different known sizes (e.g., measured by microscopy) to the membrane, and determining the smallest size of particle for which at least about 70% of particles do not pass through the membrane, as is known in the art.

The sieve plate 900 provides a simple sieve plate in which cells and magnetic beads 205 pass through the membrane due to gravitational force. No external pressure is used to force the cells through the membrane 902-1, 902-2, 902-3. This means there is no need for external pumps, eliminating the complexity of filtering systems used in conventional automated ELISPOT processes.

Fig. 10 schematically illustrates an apparatus 1000 in which a sieve plate 900 is be used. Apparatus 1000 is an apparatus for performing an automated ELISPOT assay process, for example a process involving one or more of methods 100, 500, 600.

Apparatus 1000 comprises a cell isolation system 1001 for isolating cells from a sample liquid. The cell isolation system comprises a sample plate holder for receiving a sample plate 800, the sample plate comprising a plurality of tubes e.g., 201-1, 202-2, each tube for receiving magnetic beads 205 and a sample liquid for testing as part of the ELISPOT assay process. Cell isolation system also comprises a magnet for collecting the magnetic beads 205 in each tube 201- 1, 201-2. The magnet may be any magnet 202 discussed herein, such as the magnets 202-1, 202- 2 of magnet plate 300. The cell isolation system may comprise a lift 400 for raising the magnet plate 300 into position around the tubes 201-1, 201-2.

The cell isolation system 1001 further comprises a liquid handling robot comprising at least one pipette 203, the liquid handling robot configured to: extract supernatant 204 (e.g. using method 100); inject a buffer liquid into each tube 201-1, 201-2 of the sample plate 800 when in position in the sample plate holder, to mix the buffer liquid with the magnetic beads and cells in that tube; and extract the mixture of buffer liquid and magnetic beads 205 from each tube 201-1, 201-2 (e.g. using method 600).

Apparatus 1000 further comprises a downstream assay system 1002. Downstream assay system is for performing an ELISPOT on the isolated cells, for example to count cells or a product of the cells (e.g., cykotine). The downstream assay system 1002 may by any system suitable for the assay being performed, as would be appreciated by the person skilled in the art.

A sieve plate 900 is positioned between the cell isolation system and the downstream assay system. The output of the cell isolation system 1001, namely isolated cells and magnetic beads 205, is input into the downstream assay system 1002 via the sieve plate 900. In particular, the liquid handling robot is configured to inject the extracted mixture of buffer liquid and magnetic beads 205 for each tube 201-1, 201-2 onto the sieve plate 900 for filtering prior to processing of the cells by the downstream assay system 1002.

The liquid handling robot may particularly comprise a pipette head, the pipette head comprising a plurality of pipettes 203, and may be configured to simultaneously inject the extracted mixture of buffer liquid for each tube 201-1, 201-2 onto a respective membrane 902-1, 902-2 of the sieve plate 900 using a respective pipette 203 of the plurality of pipettes. In this way, many sample streams can be filtered concurrently, providing a high throughput, automated assay system.

Fig. 11 illustrates a method 1100 for isolating cells from a sample liquid in an automated ELISPOT assay process, such as an ELISPOT process discussed herein. The method 1100 may be automatically performed by an apparatus such as apparatus 1000.

The Fig. 11 method 1100 begins at 1101, at which the sample liquid comprising the cells is mixed in a tube 201 with magnetic beads 205, such that the cells collect on the surfaces of the magnetic beads 205. Aspect 1101 may the same as 101, 501, or 601 discussed herein. At 1102, a magnetic field is applied to collect the magnetic beads 205 within the tube 201. The magnetic field may be applied by any of the magnets 202 discussed herein. 1102 may comprise raising a magnet plate 300 into position around the tube 201, in accordance with 502 and 503 of illustrated method 500.

At 1103, supernatant 204 is extracted from the tube 201. This may comprise performing the method 100 - i.e., 1103 may comprise 102-105 of illustrated method 100.

At 1105, a buffer liquid is added to the tube, the buffer liquid for suspending the magnetic beads 205. Buffer liquid may be added using the 604-608 of illustrated method 600.

At 1105, the mixture of buffer liquid and magnetic beads 205 is injected onto a sieve plate 900 to filter the mixture.

At 1106, the filtered mixture of buffer liquid and magnetic beads 205 (and cells on the magnetic beads 205) is injected into a downstream assay system 1002 to test the cells collected on the surfaces of the magnetic beads 205.

Liquid waste collector

The supernatant 204 removed using the processes discussed herein must be disposed. It likely contains biological liquids such as blood, and so must be disposed carefully as contaminated waste. Various other aspects of assay processes such as those discussed herein may produce other liquid wastes which must be disposed.

Fig. 12 shows an example of a liquid waste collector 1200, for use in an automated apparatus for performing an assay process (e.g., ELISPOT process), the apparatus comprising a liquid handling robot comprising a pipette head, the pipette head comprising a plurality of pipettes 203 extending from the pipette head. Fig. 12 (a) shows a side-view of the liquid waste collector 1200, whereas Fig. 12(b) shows an isometric view of the liquid waste collector 1200.

Liquid waste collector 1200 provides a simple, small form-factor device for gathering liquid waste simultaneously from multiple pipettes 203, for transporting to a liquid waste storage. Liquid waste collector 1200 avoids the complex pumping systems used in conventional devices, whilst still efficiently transporting liquid waste, minimising any impact on the throughput of the assay process.

Liquid waste collector 1200 may be used as part of an automated assay apparatus, such as those discussed herein. In particular, the apparatus may comprise cell isolation system 1001 and downstream assay system 1002 (with or without sieve plate 900). The apparatus may be configured to perform illustrated methods 100, 500, 600, 1100 discussed herein. Liquid waste collector 1200 comprises a funnel section 1201 comprising a funnel wall 1202 defining a first opening 1203, a second opening 1204, and a passage between the first opening 1203 and the second opening 1204.

In the illustrated embodiment, the liquid waste collector 1200 further comprises an attachment section 1207 extending below the second opening 1204. The attachment section 1207 is configured to attach to a waste container, for example it may comprise a screw thread for attaching to a tube extending from a waste container. The second opening 1204 and attachment section 1207 may generally be shaped to match a connection to the waste container, and in particular may have a substantially circular cross-section.

In contrast, the first opening 1203 is shaped to match the pipette head of the liquid handling robot, so that waste liquid from all the pipettes 203 of the pipette head may be received simultaneously. First opening 1203 may be substantially or approximately rectangular in shape. First opening 1203 may be larger than second opening 1204. Funnel wall 1202 provides a passage with a continuous or constant gradient between the first opening 1203 and second opening 1204. In this way, there is no area within the passage where liquid may pool, in contrast to the generally flat-bottomed collectors used in some conventional systems. Avoiding pooling is particularly important where contaminated waste is being disposed.

Liquid waste collector 1200 further comprises a receiving section 1205 comprising a shroud wall 1206. The shroud wall 1206 extends from the funnel wall 1202 at the first opening 1203, and away from the funnel section 1201. The shroud wall 1206 extends away from the first opening 1203 substantially in the same direction as the longitudinal axis of pipettes 203 when they are dispensing into the liquid waste collector 1200. This may also be described as substantially perpendicular to the plane in which the first opening 1203 lies.

The shroud wall 1206 surrounds the first opening 1203. The receiving section 1205 is shaped to receive the pipette head of the liquid handling robot such that, when received in the receiving section 1205, at least a lower portion of the plurality of pipettes of the pipette head is surrounded by the shroud wall. The lower portion is the portion of a pipette 201 from which liquid is dispensed - i.e., the opposite end to the pipette head. Fig. 13 shows an example of the lower sections of a plurality of pipettes 201a-f received within the receiving section 1205 of a liquid waste collector 1200. Although not shown in the figures, the top end of each pipette 201a- f is held by a pipette head of a liquid handling robot.

The shroud wall 1206 provides a shield around the pipettes 203 of the pipette head as they dispense liquid waste towards the first opening 1203. The shroud wall 1206 ensures all the waste liquid is trapped within the liquid waste collector 1200, and substantially prevents any liquid splashing out of the collector after impact within the funnel wall 1202. Waste collector 1200 therefore provides a simple but secure method of collecting liquid waste. Due to the shape of the liquid waste collector 1200, liquid can be collected safely and efficiently under gravity, without requiring any external and complicated pumping systems.

When using the liquid waste collector 1200, the liquid handing robot is configured to collect waste liquid from the assay process in the plurality of pipettes 203; and to position the plurality of pipettes 203 within the receiving section 1205 of the liquid waste collector 1200 such that at least a lower portion of the plurality of pipettes 203 is surrounded by the shroud wall 1206 of the liquid waste collector 1200. When in position, the liquid handling robot is configured to inject the waste liquid from the pipettes into liquid waste collector. The liquid handling robot may also be configured to similarly inject a cleaning fluid, such as bleach, into the liquid waste collector 1200 for cleaning.

In some embodiments, the receiving section 1205 may be shaped such that, when the pipette head is received within the receiving section at least a quarter, or preferably at least a third of the length of the plurality of pipettes 1203 extending from the pipette head is surrounded by the shroud wall 1203. For example, the height of the shroud wall 1206 may be at least about 20 mm or preferably at least about 30 mm or more preferably at least about 40 mm. The height of the funnel section 1201 may be between about 30mm and about 60mm. The shroud wall 1203 may preferably extend sufficiently far upwards to contact the pipette head itself, completely enclosing the pipettes 203. Such embodiments provide complete or near complete containment of the liquid waste during dispensing.

It will be appreciated that any of the methods discussed herein, and in particular methods 100, 500, 600 and/or 1100, may be implemented as computer readable instructions which, when executed by a processor of an automated assay apparatus/liquid handling robot, cause the apparatus/robot to perform the associated method. The computer readable instructions may be stored in in a transitory or non-transitory computer readable medium, such as a memory associated with the apparatus/robot. In particular, the apparatus and/or robot may comprise a controller, the controller comprising the processor and memory. The apparatus/robot may be any of the apparatuses or liquid handling robots discussed herein.

The following clauses define further statements of embodiments disclosed herein:

1. An apparatus for performing an assay process, the apparatus comprising: a sample plate holder for receiving an assay sample plate, the sample plate comprising a plurality of tubes; a magnet plate comprising a respective magnet for each tube of the sample plate, each magnet being a ring magnet comprising an annulus of magnetic material defining a central cavity; a lift configured to raise or lower the magnetic plate between an upper position and a lower position, wherein: in the upper position, the magnetic plate is raised such that, when a sample plate is received within the sample plate holder, each tube extends into the cavity of its respective magnet; and in the lower position, the magnetic plate is lowered such that the tubes do not extend into their respective magnets.

2. The apparatus of clause 1, wherein in the lower position, the magnet plate is distanced from the sample plate holder such that the strength of the magnetic field at the sample plate holder due to the magnet plate is negligible.

3. The apparatus of clause 1 or clause 2, wherein in the lower position the vertical distance between the magnet plate is at least 2cm or at least 3 cm or at least 5cm or at least 10 cm below the sample plate holder.

4. The apparatus of clause 1, further comprising a robotic arm configured to position a sample plate on the sample plate holder.

5. The apparatus of any preceding clause, wherein the sample plate comprises at least 20 tubes.

6. The apparatus of any preceding clause, wherein the apparatus comprises a plurality of sample plate holders, each for receiving a respective sample plate, and respective magnet plate for each sample plate holder.

7. The apparatus of clause 6, wherein the lift is configured to raise or lower each magnet plate between the upper and lower position.

8. The apparatus of clause 6, wherein the apparatus comprises a plurality of lifts, each lift configured to raise or lower a respective one of the magnet plates between an upper position and a lower position.

9. The apparatus of any preceding clause, further comprising a liquid handling robot comprising one or more pipettes, wherein the liquid handing robot is configured to inject or extract liquid from tubes of a sample plate when the sample plate is in position in the sample plate holder or one of the sample plate holders.

10. The apparatus of any preceding clause, wherein the apparatus is for performing an ELISPOT process. 11. The apparatus of any preceding clause, wherein the lift is a pneumatic lift.

12. A method for isolating target entities from a sample liquid in an assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube or a plurality of tubes of a sample plate with magnetic beads such that the target entities collect on the surfaces of the magnetic beads; and collecting the magnetic beads in the tube by raising a magnet plate from a lower position to an upper position using a lift, the magnet plate comprising a respective magnet for each tube of the sample plate, each magnet being a ring magnet comprising an annulus of magnetic material defining a central cavity, wherein: in the upper position, the magnet plate is raised such that each tube of the sample plate extends into the cavity of its respective magnet; and in the lower position, the magnetic plate is lowered such that the tubes do not extend into their respective magnets.

13. The method of clause 12, further comprising extracting supernatant from the tube, the supernatant comprising the sample liquid less the target entities collected on the surfaces of the magnetic beads.

14. The method of clause 13, wherein extracting the supernatant comprises, with the magnet plate in the upper position: extracting a first portion of the supernatant from the tube; stopping extraction of the supernatant after the first portion has been extracted; and after a predetermined time period, extracting a second portion of the supernatant from the tube.

15. The method of any of clauses 12 to 14, wherein the sample plate is positioned on a sample plate holder during collection of the magnetic beads, and wherein the method comprises performing additional aspects of the assay process whilst the sample plate is positioned on the sample plate holder.

16. The method of any of clauses 12 to 15, further comprising, after collecting the magnetic beads in the tube, lowering the magnet plate to the lower position.

17. The method of any of clauses 12 to 16, further comprising, after collecting the magnetic beads in the tube: adding a buffer liquid to the tube, the buffer liquid for suspending the magnetic beads and/or target entities; and dispensing a buffer liquid into the tube, the buffer liquid for suspending the magnetic beads and/or target entities, wherein dispensing the buffer liquid into the tube comprises: defining a plurality of dispensing positions, each dispensing position located, when projected onto a plane orthogonal to a longitudinal axis of the tube, at a respective point on a predetermined closed loop; dispensing the buffer liquid into the tube at a first dispensing position of the plurality of dispensing positions; and for each subsequent dispensing position of the plurality of dispensing positions: aspirating a portion of the buffer liquid from the tube; dispensing the portion of the buffer liquid into the tube at the respective dispensing position.

18. The method of any of clauses 12 to 17, wherein the assay process is an ELISPOT process.

19. The method of any of clauses 12 to 18, wherein the target entities are cells and/or cellular components.

Claims

1. An apparatus for performing an automated ELISPOT assay process, the apparatus comprising: a cell isolation system for isolating cells from a sample liquid, the cell isolation system comprising: a sample plate holder for receiving a sample plate, the sample plate comprising a plurality of tubes, each tube for receiving magnetic beads and a sample liquid for testing as part of the ELISPOT assay process, the magnetic beads having surfaces configured to collect the cells from the sample liquid; a magnet for collecting the magnetic beads in each tube; a liquid handling robot comprising at least one pipette, the liquid handling robot configured to: inject a buffer liquid into each tube of the sample plate when in position in the sample plate holder, to mix the buffer liquid with the magnetic beads and cells in that tube; and extract the mixture of buffer liquid and magnetic beads from each tube; a downstream assay system for performing an ELISPOT on the isolated cells; and a sieve plate positioned between the cell isolation system and the downstream assay system the sieve plate comprising: a frame; and a membrane supported by the frame, the membrane comprising a plurality of pores; wherein the size of the pores is such that the cells and magnetic beads are able to pass through the membrane, but cellular clumps cannot pass through the membrane; wherein the liquid handling robot is configured to inject the extracted mixture of buffer liquid and magnetic beads for each tube onto the sieve plate for filtering prior to processing of the cells by the downstream assay system.

2. The apparatus of claim 1, wherein the size of the pores of the membrane of the sieve plate is such that cellular clumps comprising about 20 or more cells, or about 30 or more cells cannot pass through the membrane.

3. The apparatus of claim 1 or claim 2, wherein the pores of the membrane of the sieve plate have a diameter selected from a range with an upper limit of about 300 pm or preferably about 250 pm, or more preferably about 215 pm.

4. The apparatus of any preceding claim, wherein the pores of the membrane of the sieve plate have a diameter selected from a range with a lower limit of 100 pm or preferably about 150 pm or more preferably about 200 pm.

5. The apparatus of any preceding claim, wherein the sieve plate is configured such that the cells and magnetic beads pass through the membrane due to gravitational force.

6. The apparatus of any preceding claim, wherein the membrane of the sieve plate is formed from nylon.

7. The apparatus of any preceding claim, wherein the downstream assay system comprises a spot counting apparatus for counting the cells or a product of the cells.

8. The apparatus of any preceding claim, wherein: the liquid handling robot comprises a pipette head, the pipette head comprising a plurality of pipettes; and the sieve plate comprises a plurality of membranes, the membranes positioned within the frame at positions corresponding to the position of pipettes in the pipette head; wherein the liquid handling robot is configured to simultaneously inject the extracted mixture of buffer liquid for each tube onto a respective membrane of the sieve plate using a respective pipette of the plurality of pipettes.

9. The apparatus of claim 8, wherein the sieve plate comprises about 20 or more, or about 30 or more, or about 50 or more membranes.

10. The apparatus of any preceding claim, wherein the cells are peripheral blood mononuclear cells.

11. The apparatus of any preceding claim, wherein the ELISPOT process is a process for identifying tuberculosis.

12. A method for isolating cells from a sample liquid in an automated ELISPOT assay process, the method comprising: mixing the sample liquid comprising the cells in a tube with magnetic beads, such that the cells collect on the surfaces of the magnetic beads; applying a magnet field to collect the magnetic beads within the tube; extracting supernatant from the tube, the supernatant comprising the sample liquid less the cells collected on the surfaces of the magnetic beads; adding a buffer liquid to the tube, the buffer liquid for suspending the magnetic beads; injecting the mixture of buffer liquid and magnetic beads through a sieve plate to filter the mixture, the sieve plate comprising: a frame; and a membrane supported by the frame, the membrane comprising a plurality of pores; wherein the size of the pores is such that the cells and magnetic beads are able to pass through the membrane, but cellular clumps cannot pass through the membrane; and inputting the filtered mixture of buffer liquid and magnetic beads into a downstream assay system to test the cells collected on the surfaces of the magnetic beads.

13. The method of claim 12, wherein the method is performed simultaneously for a plurality of sample liquids, each sample liquid in a respective tube.

14. The method of claim 13, wherein the method is performed simultaneously for at least about 20 sample liquids.

15. The method of any of claims 12 to 15, wherein the magnetic field is applied by a magnet comprising an annulus of magnetic material defining a central cavity, wherein a portion of the tube is positioned within the cavity.

16. The method of any of claims 12 to 15, wherein the cells are peripheral blood mononuclear cells.

17. The method of any of claims 12 to 16, wherein the ELISPOT process is a process for identifying tuberculosis infection.

18. A sieve plate for use in an automated ELISPOT assay process, the ELISPOT assay process comprising collecting cells on surfaces of magnetic beads to isolate the cells from a sample liquid, the sieve plate comprising: a frame; and a membrane supported by the frame, the membrane comprising a plurality of pores; wherein the size of the pores is such that the cells and magnetic beads are able to pass through the membrane, but cellular clumps cannot pass through the membrane.

19. The sieve plate of claim 18, wherein the size of the pores is such that cellular clumps comprising 20 or more cells, or 30 or more cells cannot pass through the membrane.

20. The sieve plate of claim 18 or claim 19, wherein the pores have a diameter selected from a range with an upper limit of 300 pm or preferably 250 pm, or more preferably 215 pm.

21. The sieve plate of any of claims 18 to 20, wherein the pores have a diameter selected from a range with a lower limit of about 100 pm or preferably about 150 pm or more preferably about 200 pm.

22. The sieve plate of any of claims 18 to 21, wherein the sieve plate is configured such that the cells and magnetic beads pass through the membrane due to gravitational force.

23. The sieve plate of any of claims 18 to 22, wherein the membrane is formed from nylon.

24. The sieve plate of any of claims 18 to 23, wherein the cells are peripheral blood mononuclear cells.

25. The sieve plate of any of claims 18 to 24, wherein the ELISPOT process is a process for identifying tuberculosis.

26. The sieve plate of any of claims 18 to 25, wherein the sieve plate comprises a plurality of membranes, each membrane supported by the frame, the membranes being positioned within the frame at positions which correspond to positions of pipettes in a pipette head of a liquid handling robot, the liquid handing robot for performing aspects of the automated ELISPOT assay process.

27. The sieve plate of claim 26, wherein the sieve plate comprises about 20 or more, or about 30 or more, or about 50 or more membranes.

28. A method for isolating target entities from a sample liquid in an assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube with magnetic beads, such that the target entities collect on the surfaces of the magnetic beads; applying a magnetic field for a first period to a lower section of the tube to collect the magnetic beads within the lower section of the tube; extracting a first portion of supernatant from the lower section of the tube, the supernatant comprising the sample liquid less the target entities collected on the surfaces of the magnetic beads; subsequent to extracting the first portion of supernatant, applying the magnetic field for a second period to collect further magnetic beads; and extracting a second portion of supernatant from the lower section of the tube.

29. The method of claim 28, wherein the target entities are cells and/or cellular components.

30. The method of claim 28 or claim 29, wherein the surfaces of the magnetic beads are functionalised to collect the target entities.

31. The method of any of claims 28 to 30, wherein the magnetic field is applied by a magnet positioned around the tube such that the lower section of the tube extends below an upper surface of the magnet, and wherein the extracting supernatant comprises extracting supernatant from the tube below the level of the upper surface of the magnet.

32. The method of claim 31, wherein the magnet comprises an annulus of magnetic material extending around a portion of the lower section of the tube.

33. The method of claim 32, wherein the lower section of the tube extends below the annulus of magnetic material, and wherein extracting supernatant comprises extracting supernatant from the tube below the annulus of magnetic material.

34. The method of claim 32 or claim 33, wherein the magnet extends in a direction parallel to a longitudinal axis of the tube over a distance less than 10% of a total length of the tube.

35. The method of any of claims 31 to 34, wherein applying the magnetic field comprises raising the magnet from a position where all of the magnet is below the tube into the position around the tube.

36. The method of any of claims 28 to 35, wherein the second portion of supernatant comprises all of the supernatant remaining in the tube after the extraction of the first portion of supernatant.

37. The method of any of claims 28 to 35, further comprising, subsequent to extracting the second portion of supernatant, applying a magnetic field for one or more further periods to collect further magnetic beads and, after each period, extracting a further portion of supernatant from the lower portion of the tube.

38. The method of any of claims 28 to 37, wherein the magnetic field remains applied while extracting supernatant.

39. The method of any of claims 28 to 38, wherein the magnet is an electromagnet, and wherein applying the magnetic field comprises activating the electromagnet.

40. The method of any of claims 28 to 39, wherein the magnet is a permanent magnet.

41. The method of any of claims 28 to 40, wherein the method is performed simultaneously for a plurality of different sample liquids, each sample liquid in a respective tube.

42. The method of claim 41, wherein the method is performed simultaneously for about 20 or more sample liquids.

43. The method of any of claims 28 to 42, wherein the tube is a well of an assay sample plate.

44. The method of any of claims 28 to 43, wherein the assay process is an ELISPOT process.

45. The method of any of claims 28 to 44, wherein the method is performed by a liquid handling robot.

46. A liquid handling robot comprising one or more pipettes for injecting or extracting liquid from tubes, wherein the liquid handing robot is configured to perform the method of any of claims 28 to 45.

47. A method for isolating target entities from a sample liquid in an automated assay process, the method comprising: mixing the sample liquid comprising the target entities in a tube with magnetic beads, such that the target entities collect on the surfaces of the magnetic beads; applying a magnet field to collect the magnetic beads within the tube, the magnetic field applied by a magnet comprising an annulus of magnetic material defining a central cavity, wherein a portion of the tube is positioned within the cavity; extracting supernatant from the tube, the supernatant comprising the sample liquid less the target entities collected on the surfaces of the magnetic beads; and dispensing a buffer liquid into the tube, the buffer liquid for suspending the magnetic beads and/or target entities, wherein dispensing the buffer liquid into the tube comprises: determining a plurality of dispensing positions, each dispensing position located, when projected onto a plane orthogonal to a longitudinal axis of the tube, at a respective point on a predetermined closed loop; dispensing the buffer liquid into the tube at a first dispensing position of the plurality of dispensing positions; and for each subsequent dispensing position of the plurality of dispensing positions: aspirating a portion of the buffer liquid from the tube; dispensing the portion of the buffer liquid into the tube at the respective dispensing position.

48. The method of claim 47, wherein the predetermined closed loop conforms to a shape of a cross-section of the tube.

49. The method of claim 47 or claim 48, wherein the predetermined closed loop is a circle.

50. The method of claim 49, wherein the diameter of the circle is at least 70%, or at least

80% of an inner diameter of the tube.

51. The method of any of claims 47 to 50, wherein buffer liquid is dispensed into the tube at a dispensing rate, and buffer liquid is aspirated from the tube at an aspiration rate, wherein the aspiration rate is less than the dispensing rate.

52. The method of claim 47, wherein the tube comprises a conical shaped bottom.

53. The method of any of claims 47 to 52, wherein the magnet further comprises a base portion from which the annulus of magnetic material extends, wherein the base portion comprises a recess shaped to receive the conical shaped bottom of the tube.

54. The method of any of claims 47 to 53, wherein dispensing and aspirating the buffer liquid are performed by a pipette of a liquid handling robot.

55. The method of any of claims 47 to 54, wherein the method is performed simultaneously for a plurality of different sample liquids, each sample liquid in a respective tube, each tube received within a respective magnet.

56. The method of claim 55 as dependent on or from claim 54, wherein the liquid handling robot comprises a plurality of pipettes, wherein: the dispensing and aspirating buffer liquid are performed for each tube by a respective pipette of the liquid handling robot.

57. The method of claim 55 or claim 56, wherein the method is performed simultaneously for about 20 or more sample liquids.

58. The method of any of claims 47 to 57, wherein the number of dispensing positions in the plurality of dispensing positions is at least about 8, or at least about 10, or preferably at least about 20.

59. The method of any of claims 47 to 58, further comprising removing the magnetic field from the or each tube prior to the adding the buffer liquid.

60. The method of claim 59, wherein removing the magnetic field comprises lowering the or each magnet away from the or each tube.

61. The method of any of claims 47 to 60, wherein extracting the supernatant comprises extracting a first portion of the supernatant and subsequently extracting a second portion of the supernatant from the tube.

62. The method of any of claims 47 to 61, wherein the tube is a well of an assay sample plate.

63. The method of any of claims 47 to 62, wherein the target entities comprise cells and/or cellular components.

64. The method of any of claims 47 to 63, wherein the assay process is an ELISPOT process.

65. An apparatus for performing an automated assay process, the apparatus comprising a liquid handling robot comprising one or more pipettes for dispensing or aspirating liquid from tubes, wherein the apparatus is configured to perform the method of any of claims 47 to 64.

66. A liquid waste collector for use in an automated apparatus for performing an assay process, the apparatus comprising a liquid handling robot comprising a pipette head, the pipette head comprising a plurality of pipettes extending from the pipette head, the liquid waste collector comprising: a funnel section comprising a funnel wall defining a first opening, a second opening, and a passage between the first opening and the second opening; a receiving section comprising a shroud wall, the shroud wall extending from the funnel wall at the first opening away from the funnel section, the shroud wall surrounding the first opening; wherein the receiving section is shaped to receive the pipette head of the liquid handling robot such that, when received in the receiving section, at least a lower portion of the plurality of pipettes of the pipette head is surrounded by the shroud wall.

67. The liquid waste collector of claim 66 wherein the receiving section is shaped such that, when the pipette head is received within the receiving section, at least a quarter or a preferably at least a third of the length of the plurality of pipettes extending from the pipette head is surrounded by the shroud wall.

68. The liquid waste collector of any claim 66 or 67, wherein the receiving section is shaped to receive a pipette head comprising at least about 20 pipettes.

69. The liquid waste collector of any of claims 66 to 68, wherein the height of the funnel wall is at least about 20 mm or preferably at least about 30 mm or more preferably at least about 40 mm

70. The liquid waste collector of any of claims 66 to 69, wherein the funnel section is configured to attach at the second end to a waste container.

71. An apparatus for performing an assay process, the apparatus comprising: a liquid handling robot comprising a pipette head, the pipette head comprising a plurality of pipettes extending from the pipette head; and a liquid waste collector according to any of claims 66 to 70; wherein the liquid handing robot is configured to: collect waste liquid from the assay process in the plurality of pipettes; position the plurality of pipettes within the receiving section of the liquid waste collector such that at least a lower portion of the plurality of pipettes is surrounded by the shroud wall of the liquid waste collector; and inject the waste liquid from the pipettes into liquid waste collector.

72. The apparatus of claim 71, where the apparatus further comprises: a cell isolation system for isolating cells from a sample liquid, the cell isolation system comprising: a sample plate holder for receiving a sample plate, the sample plate comprising a plurality of tubes, each tube for receiving magnetic beads and a sample liquid for testing as part of the assay process, the magnetic beads having surfaces configured to collect the cells from the sample liquid; and a magnet for collecting the magnetic beads in each tube; and a downstream assay system for performing a test on the isolated cells.

73. The apparatus of claim 71 or claim 72, wherein the apparatus is for performing an automated ELISPOT assay process.