GB2423581A

GB2423581A - Particulate sets carrying a unique identifier for assays

Info

Publication number: GB2423581A
Application number: GB0504100A
Authority: GB
Inventors: Caroline Garey; Peter Swarbrick
Original assignee: SMARTBEAD TECHNOLOGIES Ltd
Current assignee: SMARTBEAD TECHNOLOGIES Ltd
Priority date: 2005-02-28
Filing date: 2005-02-28
Publication date: 2006-08-30
Also published as: GB0504100D0; WO2006090165A1

Abstract

A method is provided for the detection of analytes and makes use of a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets. Each set of supports is partitioned from the other sets of supports. The method comprises the steps of: <SL> <LI>(a) attaching a primary analyte (9) to the supports (1) of all the sets, <LI>(b) introducing respective samples to the sets of supports so that each sample is associated with a corresponding unique identifier, each sample potentially containing a secondary analyte (12) which binds to the primary analyte, <LI>(c) combining the sets of supports to form a support mixture containing supports from all of the sets, <LI>(d) introducing a reporter system to the support mixture, the reporter system (13) being responsive to the presence of secondary analyte bound to the supports via the primary analyte, and <LI>(e) interrogating the support mixture to determine the unique identifiers of supports which have an activated reporter system in order to identify which samples contain the secondary analyte. </SL> The application of the system to isotype antibodies in a sample - the antigen being the primary analyte and anti-isotype antibodies being the secondary analyte is described.

Description

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DETECTION OF ANALYTES IN SAMPLES

Field of the Invention

The present invention relates to a method, kit and apparatus for the detection of analytes in samples.

Background of the Invention

There is considerable interest in techniques and associated systems for determining large numbers of analyte characteristics through serial and parallel testing.

Previously, tests for detecting analyte characteristics were performed manually in a sequential manner in laboratories.

However, more recent technological developments relating to analyte characterisation have evolved towards greater automation with associated higher detection throughput.

In areas such as the development, testing and clinical trials of new vaccines, therapeutics, theranostics and diagnostics, the understanding and analysis of the immune response of patients, animal models or cells is of great importance.

There are several known experimental techniques for determining characteristics of analytes relating to the immune response, immunoassays being the most commonly used approach.

One common way of performing immunoassays is by enzyme linked immunosorbance assays (ELISAs) . ELISAs are used to determine the concentration of an analyte in a liquid sample or its concentration at a particular surface.

In a typical ELISA method to detect the presence, or measure the concentration of an analyte of interest, especially in a liquid sample, which may be a body fluid such as blood, plasma, serum, urine, saliva, sputum, etc., the sample is introduced to a first binding partner, and the binding partner and the analyte are incubated for a sufficient time to allow analyte contained in the sample to bind to the binding partner. An example of such a binding partner is an analyte- specific antibody, e.g. a monoclonal antibody with specificity for the analyte in question. Certain adaptations of the ELISA method immobilise the analyte (i.e. bind it to a solid phase such as a substrate or support) via e.g. a linker substance.

However, a disadvantage of the various ELISA techniques is the difficulty associated with binding different analytes in an appropriate manner to the bottom of the ELISA plate. In certain situations, for example when working with novel therapeutics, it is not until the first binding partner (e.g. a specific type of epitope) binds to the analyte in the ELISA plate well that it can be seen if the binding strategy has been successful. When working with limited amounts of sample and expensive reagents this can result in invalid experiments and can be costly and time consuming.

A disadvantage of known immunoassay techniques is the difficulty of measuring low analyte concentrations, particularly when fluorescent labelled reporters are used.

In US 6723524, an ELISA method for determining an analyte adsorbed at a surface or present in a liquid sample is described. The method comprises binding the analyte to a solid phase, attaching a marker to the analyte, and detecting marker attached to the solid-phase. An enzyme-substrate combination produces a precipitate on the solid phase which carries the marker. Detection of the binding of analyte to the solid phase then involves an in-situ determination of the change in surface mass of the solid phase due to the formation of the precipitate. Ellipsometry is an example of a technique suitable for determining the change of surface mass. The invention is said to shorten the assay time and/or improve the assay sensitivity, and reportedly allows the measurement of extremely low surface concentrations of analytes of interest.

However, disadvantages of the method are the high cost of investigating many samples and the costly instrumentation which is required.

An approach for performing simultaneous testing, so called multiplexing, uses several coded arrays in one vessel.

Multiplexing can include the use of programmable matrices with memories as described in WO 96/36436. A recording device records which molecules and biological materials are linked to the matrix material of each programmable matrix. Several matrices can also be arranged in a microarray. Other particle microarrays are described in WO 00/63419 and WO 00/01475.

Other conventional methods for investigating analytes are chromatography, electrochemiluminescence radio immunoprecipitation and western blots. All these methods have limitations when it comes to performing multiparameter analysis, especially when the number of samples increases, or a large variety of different types of analytes are required to be investigated, or the sample volume is limited.

However, there remains a need for improved high throughput analyte testing during research and development of new chemical entities (NCEs) in the biotechnology and pharmaceutical industry. Also, in the diagnostics industry there is a need for improved methods of testing multiple parameters in a cost efficient and rapid way.

Summary of the Invention

The present invention is at least partly based on the realisation that the capability of high throughput analyte testing can be significantly extended when test sample identity is tracked using the unique identifiers of multiplexed particulate supports.

Thus a first aspect of the invention provides a method for the detection of analytes in samples which makes use of a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and each set of supports being partitioned from the other sets of supports, the method comprising the steps of: (a) attaching a primary analyte to the supports of all the sets, (b) introducing respective samples to the sets of supports so that each sample is associated with a corresponding unique identifier, each sample potentially containing a secondary analyte which binds to the primary analyte, (c) combining the sets of supports to form a support mixture containing supports from all of the sets, (d) introducing a reporter system to the support mixture, the reporter system being responsive to the presence of secondary analyte bound to the supports via the primary analyte, and (e) interrogating the support mixture to determine the unique identifiers of supports which have an activated reporter system in order to identify which samples contain the secondary analyte.

In effect there is a double interrogation involving the unique identifier and the reporter system. The pooling of the sets of supports to form the support mixture provides benefits associated with multiplexing, such as parallel processing.

However, the method is also compatible with multi-well plate procedures, allowing the use of conventional and widely- available multi-well testing equipment. Thus, preferably each set of supports is contained in a respective well of a multi- well plate to partition it from the other sets of supports.

By a "reporter system which is responsive to the presence of secondary analyte bound to the supports via the primary } 5 analyte", we mean any reporter system which allows the user to discriminate between supports to which secondary analyte is bound and supports to which secondary analyte is not bound.

Usually, such systems will be activated by secondary analyte bound to the supports. However, there may be systems which are introduced to the support mixture in an activated state and are deactivated by secondary analyte bound to the supports. Such systems nonetheless allow discrimination and are suitable for performing the method.

Preferably, in step (e), the reporter system allows not only the identity of the samples which contain the secondary analyte to be ascertained, but also allows quantification of the amount of secondary analyte contained in the respective test sample (e.g. via quantification of the amount of secondary analyte bound to the supports) Preferably, the primary analyte is a biologically active agent.

Preferably, the secondary analyte is a biologically active agent which binds to the primary analyte, and more preferably is an antibody which binds to the primary analyte.

Typically, the method further comprises the step between steps (a) and (b) of removing excess primary analyte from each set of supports. This avoids the problem that such excess primary analyte could prevent secondary analyte from binding to the supports.

The method may further comprise the step between steps (c) and (d) of: partitioning the support mixture into a plurality of groups, each group containing supports representative of the support mixture, and wherein steps (d) and (e) are performed on the support mixture of each group, a respective reporter system being 1) 6 introduced to each group, with each reporter system being responsive to the presence of a different secondary analyte, whereby the groups relate to respective and different secondary analytes. Preferably, each group of supports is placed in a respective well of a multi-well plate to partition it from the other groups of supports.

A given test sample may contain a plurality of secondary analytes. It is possible to introduce a plurality of respective reporter systems into the unpartitioned support mixture, and interrogate the mixture simultaneously or sequentially for signals from for each of the systems.

However, in such a scenario, the signals from different reporter systems can mask or interfere with each other.

Therefore, it may be desirable to use a range of reporter systems that vary by signal type, such as wavelength, so that different secondary analytes can be distinguished within the same partitioned group. Alternatively, by partitioning the support mixture and performing steps (d) to (e) with a different reporter system for each partitioned group, this potential difficulty can be overcome.

Within each set of particulate supports, the supports may form a plurality of subsets, the supports of each subset carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, and the surface chemistry of the supports of each subset being different from the surface chemistries of the supports of the other subsets so that, in step (a), the primary analyte may preferentially attach to the supports of one or more of the subsets. The interrogation of step (e) then also determines the further unique identifiers of supports which have an activated reporter system in order to identify which surface chemistries cause preferential attachment of the primary analyte. Thus, a plurality of surface chemistries can be tested at one time, which is Particularly advantageous if an optimum or effective surface chemistry for primary analyte attachment is not known in advance.

Extending this concept further, subsets of supports, each s carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, can also be used to study different primary analytes in parallel. In this case, the subsets are initially held apart and step (a) involves attaching a respective primary analyte to the supports of each subset. The subsets are then combined, and steps (b) to (e) are performed as above. However, interrogation step (e) also involves determining the further unique identifiers of supports which have an activated reporter system in order to identify the primary analytes which cause secondary analyte binding.

A further aspect of the invention provides a kit for the detection of analytes in samples, the kit comprising a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and the supports of each set being partitioned from the supports of the other sets; the kit further comprising instructions for the performance of a method for the detection of analytes in samples according to the first aspect of the invention.

The kit may comprise a multi-well plate with each set of supports held in a respective well. The instructions would typically be written instructions.

A further aspect of the invention provides a kit for the detection of analytes in samples, the kit comprising a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and the supports of each set being partitioned from the supports of the other sets, wherein, within each set, the supports form a plurality of support subsets, the supports of each subset carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, and the surface chemistry of the supports of each subset being different from the surface chemistries of the supports of the other subsets so that an analyte may preferentially attach to the supports of one or more of the subsets.

The kit of this aspect of the invention may comprise a multi- well plate with each set of supports held in a respective well. The kit is particularly suitable for performing embodiments of the method of the first aspect of the invention in which the interrogation of step (e) determines the further unique identifiers of supports which have an activated reporter system in order to identify which surface chemistries cause primary analyte preferential attachment. The kit may further comprise instructions for the performance of a method for the detection of analytes in samples according to the first aspect of the invention.

A further aspect of the invention provides an apparatus for the detection of analytes in samples, the apparatus comprising: a robotics system for the automated manipulation and interrogation of the contents of the wells of multi-well plates, and a computer for controlling the actions of the robotics system, the computer being programmed to perform a method for the detection of analytes in samples according to the first aspect of the invention.

Further aspects of the invention provide: a computer for controlling the actions of a robotics system for the automated manipulation and interrogation of the contents of the wells of multi-well plates, the computer being programmed to perform a method for the detection of analytes in samples according to the first aspect of the invention; a computer program product carrying code for the performance of a method for the detection of analytes in samples according to the first aspect of the invention; and computer code for the performance of a method for the detection of analytes in samples according to the first aspect of the invention.

Brief Description of the Drawings

Embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which: Figures la and b respectively show schematic side and edge views of a microparticle support, Figure 2 shows schematically a primary analyte attached to a section of the support of Figure 1, Figure 3 shows a flow diagram for a method according to an embodiment of the invention, Figures 4a to f show schematically different analyte/reporter attachment techniques, and Figures 5a to d depict schematically stages in an experiment which takes place in a 96 well plate and uses eight sets supports to generate immunogenicity results for 96 test samples against eight different reporter systems.

Detailed Description

A. Particulate Supports Figures la and b respectively show schematic side and edge views of a single microparticle support 1 for use in a method according to the invention. The support 1 can be fabricated from virtually any insoluble or solid material, for example one or more of polymers, silicates, glasses, fibres, metals or metal alloys. In preferred embodiments of the invention, the support 1 is fabricated from a metal, such as gold, silver, copper, nickel, zinc or most preferably aluminium. it is also possible for the support 1 to be partially or totally coated in either of the above-mentioned materials.

The support 1 incorporates a unique identifier 2. Examples of identifiers 2 may be sequential identification codes, varied shape and size of the supports, transponders (for example Radio Frequency identification Chips, RFIDs) attached to the support 1, or different colours of the supports. Preferably, the identifier 2 takes the form of a sequence of grooves, notches, depressions, protrusions, projections, and most preferably holes. Such an identifier 2, being an integral part of the support 1, is advantageous in that there is no need to label each support 1 postmanufacture. The sequential identifier 2 shown in Figure la is a transmission optical barcode, and this particularly preferred. A sequential identification code can thereby be recorded in the support 1 as a series of holes using coding schemes similar to those found on conventional bar code systems, for example as employed for labelling merchandise in commercial retailing outlets. Such a code allows the use of existing reader technology for the determination of the unique identity of each support 1.

The support 1 can be of many different types of shape, but preferably has a substantially planar form with two main attachment surfaces 6 as illustrated in Figures la and b, which allows the supports i to settle flat with one main surface on the bottom of a well of multi-well plate (which is preferably a microtjtre plate of SBS standard having 24, 48, 96, 384 or 1536 wells) . Each support i of a preferred type has a largest dimension 3. The largest dimension 3 may be less than about 300 pm, preferably less than about 200 pm, more preferably less than about 100 pm, and most preferably less than about 50 pm. Clearly, the largest dimension of the support should allow it to settle at the bottom of a well of the multi-well plate. Furthermore, the number, shape and size of the supports 1 are preferably such that multiplexing can take place in a well without too much overlapping of the supports i. The characteristics of the liquid handling unit (e.g. syringe capacity, bore size etc.) used to manipulate the supports may also impact on support shape and size.

The miniaturjsation of the supports i reduces the need for large amounts of reagents, and primary and secondary analytes in experiments. The support 1 suitably has a width 4 to length 3 ratio in a range of about 1:2 to about 1:20, although a ratio in the range of about 1:5 to about 1:15 is preferred.

This promotes the flow of the supports 1 during liquid handling and dispensing. it also allows efficient utilisation of the Support's surface area by the identification means 2.

Moreover, the support 1 has a thickness 5 which is preferably less than about 3 pm, and more preferably less than about 1 pm. Thicknesses of 1 pm and slightly under have been shown to provide sufficient mechanical robustness, rendering the support 1 useable in harsh experimental conditions. Preferred embodiments of the invention employ supports 1 having a length 3 of about 100 pm, a width 4 of about 10 pm and a thickness 5 of about 1 pm; such supports are capable of storing more than 100,000 different identification sequence barcodes. The support preferably includes error checking and directional orientation features to prevent incorrect identification of the support i. Also end markers 8 may be used to indicate the intactness or reading direction of the support 1.

Around ten million such supports i can be fabricated on a single 6-inch diameter semiconductortype wafer, for example a silicon wafer, using contemporary established manufacturing techniques such as photolithography and dry etching processes.

Advantageously the shape of the support 1 optimises the number of supports i manufactured per wafer and also substantially optimises the number of identification codes possible on the supports i.

An example fabrication process for manufacturing a plurality of supports similar to the support 1 may involve the following steps: deposit a soluble release layer onto a planar wafer; deposit a layer of support material onto the release layer remote from the wafer; define support features, including the identifier 2, in the support material layer by way of photolithographic processes and etching processes; remove the release layer using an appropriate solvent to yield the supports released from the planar wafer; and optionally treat the support material to improve its attachment properties.

Fabrication and example uses of such supports is further described in patent applications wo 00/16893, WO 02/165123, WO 02/064829, WO 03/091731, WO 04/015418 and WO 04/011940 which are hereby incorporated by reference.

Many methods of chemically treating or physically altering the support material may be used for the optional treatment step to facilitate the attachment of an analyte to the support 1.

The treatment of the supports 1 can be performed after the release from the wafer as described above or alternatively prior to the release from the wafers or earlier in the manufacturing process steps.

Figure 2 shows schematically a primary analyte 9 attached to a section 7 of the support 1. In the following examples the primary analyte 9 is also referred to as a query molecule or compound. By modifying the attachment surface 6 of the Supports i, the attachment between the primary analyte 9 and Supports i can be improved. Anodising the attachment surface 6 of the supports i is one way of providing such improved attachment enhancement. Aluminium is a preferred material for the supports 1, and there are known methods of growing porous surfaces through aluminium anodisatjon to improve the attachment properties thereof. Likewise, processes for sealing such porous surfaces are also known.

The support's 1 surface 6 may be treated with a polymer material such as silane and/or biotin, to further enhance attachment properties. The Supports 1 preferably have a silane layer coated onto their surfaces 6. Alternatively or additionally, the analyte may be modified to promote attachment to the supports 1. For example, an avidin-biotin system can be used to improve chemical binding between the supports 1 and their associated analyte 9. Also, a crosslinker system may be used which interacts with both the silane coated support and the analyte.

The enhanced attachment is preferably achieved through having electrostatic, hydrophobic or more preferably covalent bonds between attachment surface 6 of the support 1 and the primary analyte 9, thereby fixedly attaching the analyte 9 to the supports i. Covalent bonds prevent the analyte 9 from being dislodged from the supports i and causing background noise during analysis.

There is also a potential problem that loose (unbound) analyte 9 can hinder the identification of reactions that have occurred. it is therefore desirable to wash the supports 1 after the supports 1 have had analyte 9 attached thereto, to remove excess analytes 9 that could otherwise increase experimental noise during analysis.

B. Analyte Detection Figure 3 shows a flow diagram for a method according to an embodiment of the invention. The method is suitable for performing immunogenicity experiments to determine the type of antibody response against NCEs. At preparation step 20, a large number of particulate supports are provided. The supports are partitioned into different sets by being kept in respective wells of a 96 well plate. The supports of each set share a unique identification code for that set. The number of individual wells containing uniquely coded supports is equal to the number of test samples to be tested. Thus, in this example, up to 96 samples can be simultaneously analysed.

To attain robust experimental data, each set contains several hundred supports.

Each well also contains appropriate buffers and regents (see e.g. A.R. Mire-Sluis et al. (2004), Recommendations for the design and optimisation of immunoassays used in the detection of host antibodies against biotechnology products. J. Immunol. Methods, 289 (1-2) : 1-16) . The same primary analyte, e.g. a novel NCE in clinical trials or late stage screening, is then added to each of the wells allowing the primary analyte to attach to the supports therein. Following appropriate incubation each well is washed to remove any excess of the primary analyte which is not attached to the supports. This can be achieved by using a filter based bottom multi-well plate. Such a plate has filter apertures which are smaller than the supports, allowing the supports to remain in the well for future re-suspension.

At dispensing step 30, a programmable, computer-controlled, liquid handling system, such as a PerkinElmer Mu1tipROBETM or Qiagen BioRobot3OlJOTM, is then used to dispense a respective test sample into each of the prepared wells. Each test sample potentially contains a secondary analyte, such as an antibody produced in response to the NCE. Following appropriate incubation, the secondary analyte, if present, binds with the primary analyte on the supports in the wells. Blocking buffer is added to each well to prevent additional reactions occurring. Supports from different wells are then mixed together in the same well of an analysis substrate to form a multiplexed support mixture. If there is a large number of test samples, it may be desirable to form more than one such mixture, so that each mixture does not overload the reporter system (see below) . Thus, for example, if 96 test samples are being analysed, twelve mixtures may be formed with the supports of eight samples in each mixture.

Next, analysis step 40 makes use of a reader e.g. as described in WO 02/165123 and WO 02/064829. To detect interaction between the primary and secondary analyte, a reporter system is added. The reporter system is preferably a fluorescent reporter molecule linked to a matching pair analyte which binds to the supports where the primary and secondary analytes have bound to each other. The reader can simultaneously identify the identification code of each support and measure the reporter system's signal from the interaction between the secondary analyte and the primary analyte.

In a second embodiment of the invention, suitable for immunogenicity testing, it is preferred to measure the response in respect of a number of different reporter systems.

This can be achieved by using a specific signal, e.g. a fluorescent or Qdot signal, for each system. This can however be problematic when the number of reporter systems increases above four or five. Therefore, preferably the support mixture is divided into several wells. This allows a reporter system for each immunoglobulin class/isotype (e.g. IgA, IgD, IgE, IgGi, IgG2, IgG3, IgG4 and 1gM) to be measured in a separate well. In each well the reaction conditions can be optimised for the specific reporter system.

A problem associated with looking at a new primary analyte, such as an NCE, is that an optimum or effective method for attaching the analyte to the support and/or detecting the result may not be known. A way of increasing the success rate in respect of such analytes is to use a number of different attachment and/or detection methods with a further unique identifier (e.g. a further identification code) to identify the particular attachment/detection method used for each support. Thus at the outset of the experiment, the supports of each well each belong to one of a plurality of support subsets, each subset relating to a respective attachment or detection method. Different attachment regimes can be obtained by changing the surface chemistry and/or physical structure of the supports.

Other examples of different attachment and detection approaches which may be used are outlined in Figures 4a to f.

Each approach enables the determination of the amount of the secondary analyte.

Figure 4a shows schematically an analyte/reporter attachment technique.The primary analyte 9 (antigen, such as an NCE) of interest is bound to the coded support 1, which is exposed to a test sample which may contain the secondary analyte 12 (immunoglobulin isotypes) of interest. The reporter system 13, e.g. a dye labelled anti-isotype antibody, gives the identity and quantity of the bound isotype, i.e. the specific antibody isotype present in the test sample.

Figure 4b shows schematically an enhanced analyte/reporter attachment technique. Linker systems such as primary analyte antibodies 9a are first attached to the support 1, followed by attachment of the primary analyte 9, such as an NCE, to the linker antibody 9a. The linker antibody 9a improves the attachment of the primary analyte 9 thereto. The support 1 with its bound linker antibody 9a/analyte 9 complex is exposed to the test sample, and secondary analyte 12 (immunoglobulin isotypes) if present bind to the primary analyte 9. The reporter system 13 (dye labelled anti-isotype antibody) is then added, and binds to the linker antibody 9a/analyte 9/analyte 12 complex. However, because in this case the primary analyte 9 is bound to the support 1 via the linker system 9a, which is for example a polyclonal or monoclonal antibody, the concealment of primary analyte 9 antigen epitopes from the test sample immunoglobulin isotypes can be prevented. Depending on the nature of the primary analyte, physically locating the primary analyte 9 away from the surface of the supports 1 should also reduce the likelihood of primary analyte 9 denaturation, and improve its stability and hence its likelihood of binding to the secondary analyte 12 in the test sample. This is particularly the case when the primary analyte 9 is small or would experience steric hindrances at the surface of support 1.

Figure 4c shows schematically a third approach for attachment of analytes, which can be used for the detection of the total amount of specific antibody isotype rather than identifying specific anti-NCE (antigen) isotypes as per the approaches of Figures 4a and b. The primary analyte 9 (isotype specific antibodies rather than NCE antigens as in Figures 4a and b) is bound to the support 1. The secondary analyte 12 (specific immunoglobulin isotypes potentially present in the test samples) binds if present, its presence is then detected with a dye labelled anti isotype antibody reporter system 13. By setting up such a method for each class of immunoglobulin isotypes, this approach allows the total amount of each immunoglobulin isotype to be measured and not just the anti- NCE isotype specific to the NCE (antigen) of interest. This approach does not utilise the NCE (antigens) as part of the attachment or detection system as in the approaches of Figures 4a, b, d, and e.

Figure 4d shows schematically an attachment technique using specific isotype determination via labelled antigen, whereby the primary analyte 9 (isotype specific antibodies as in Figure 4c) is bound to the support 1. The secondary analyte 12 (isotype specific sample antibodies) binds to the primary analyte 9 if present in the test sample. The presence of such a reaction is detected by binding a dye labelled antigen as reporter system 14, which differs from the reporter system used in the approaches of Figures 4a, b, c, and e. The reporter system consists of a dye labelled antigen (NCE) which will bind to the primary analyte 9/secondary analyte 12 complex only if that complex contains specificity for the particular antigen/NcE. This approach enables subtyping of the immunoglobulin isotype specific to the antigen/NcE rather than a general detection of all isotypes as per the approach of Figure 4c.

Figure 4e shows schematically an attachment and detection technique using antibody linked to antigen for specific isotype measurement. The primary analyte 9 (antigen) is linked to the supports 1, as per Figure 4a. In addition, antigen specific antibodies 9a (preferably polyclonal) are bound to the primary analyte 9 (antigen) and additional primary analyte 9 (antigen) is added to occupy a second binding site of the antibody 9a. This approach effectively exposes different binding sites on the primary analyte 9 so that secondary analyte may be detected that could have been overlooked by the approaches of Figures 4a and b, or that would have produced a false positive result by the approach of Figure 4a due to nonspecific binding. Incubation with patient serum binds secondary analyte 12 (antibodies) , and the isotype specific reporter system uses labelled antibodies 13 to detect the occurred reaction. Depending on individual sample immune responses, there may be a range of antibody subtypes interacting with the antigen, which would be known by the person skilled in the art but are not discussed further here.

Figure 4f shows schematically an analyte and multiple reporter attachment technique. The primary analyte 9 (antigen, such as an NCE) of interest is bound to the coded support 1, which is exposed to a test sample which may contain the secondary analyte 12 (immunoglobulin isotypes) of interest. Multiple reporter systems can be used in this instance to identify a range of different isotypes. The reporter systems 13 and 14, e.g. each a different dye labelled anti-isotype antibody, gives the identity and quantity of the bound isotypes, i.e. the specific antibody isotype present in the test sample. In the schematic of Figure 4f, reporter system 13 is activated, while reporter system 14, which does not attached to secondary analyte 12, is not.

The approach of using coded supports for multiplexed analysis of test samples against NCEs has considerable advantages in that enhanced throughput and good sensitivity can be achieved while making use of established reader/detection technology.

Many different types of analytes may be used. For life science industries the primary and secondary analytes may be e.g. antibodies, antigens, proteins, enzyme substrates, carbohydrates, peptides, nucleic acids, peptide nucleic acids, cell lines, chemical components, oligonucleotides, serum components, drugs or any derivatives or fragments thereof.

For other industries, the analytes can be, e.g. dyes, preservatives, labelling chemicals (for example for tracking the movement of counterfeit products), radioactive labelling chemicals, and food.

Figures 5a to d depict schematically stages in an experiment 0 which takes place in a 96 well plate and uses eight sets of supports to generate immunogenicity results against eight different reporter systems for 96 test samples. Each set of supports bears one of unique codes 1 to 8.

In Figure 5a, the sets of uniquely coded supports are placed in respective wells. Each column of wells 1 to 12 has codes 1 to 8 in the wells of rows A to H respectively. Primary analyte is introduced into each well and attaches to the supports, excess primary analyte being removed as discussed above. A different test sample is then placed in each well, and a reaction takes place when primary analyte attached to a support reacts with secondary analyte in a test sample.

Following incubation and washing, the reacted supports of each column are pooled together into one well, as seen in Figure 5b, to form twelve support mixtures. Each mixture is then divided into eight groups by dispensing it into the eight wells of a respective column of a fresh 96 well plate, as shown in 5c. By ensuring that there is a sufficient number of each coded support in each well at the outset, it is possible to divide the support mixtures in this way and still obtain accurate results for every test sample. Typically, at the outset in each well there are several hundreds of coded supports, and we have shown that after dividing the support mixture into groups, robust results can be obtained with as few as 10 supports per code per well.

Finally, as shown in Figure 5d, a different reporter system is added to each of the rows of the 96 well plate. This avoids interference that could occur if two or more different reporter systems were added to each support mixture. When a reporter system is activated, the identity of the test sample is determined by reference to the unique code of the support to which the activated reporter system is attached and the column number of 96 well plate. An optional approach, possible for reporter systems that vary sufficiently by signal type such that interference between reporter systems can be avoided, is to add a plurality of reporter systems labelled with a range of different reporters in the same well. Due to the risk of interference, this approach may be limited to small numbers of reporter systems in each well, such as less than five.

The adaptability and robustness of the coded supports allows the experimental conditions, such as reaction conditions (chemistry, buffers, ph, etc.), incubation times, number of supports, and shape and size of supports to be freely adapted.

If the sensitivity of the secondary analyte tested for in a particular well is much lower than in other wells, the number of supports placed in that well can be modified. Additional supports having filtering analytes attached thereto instead of primary analytes can be added to the coded supports to filter away unwanted analytes (e.g. proteins, antibodies, antigens) present in the sample (see W004/0l54l8).

A reader system adapted for reading the codes and reporter system signals of supports suspended in liquid solutions in wells is described in WO 02/165123 and WO 02/064829.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

Claims

CLA IMS

1. A method for the detection of analytes in samples which makes use of a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and each set of supports being partitioned from the other sets of supports, the method comprising the steps of: (a) attaching a primary analyte to the supports of all the sets, (b) introducing respective samples to the sets of supports so that each sample is associated with a corresponding unique identifier, each sample potentially containing a secondary analyte which binds to the primary analyte, (c) combining the sets of supports to form a support mixture containing supports from all of the sets, (d) introducing a reporter system to the support mixture, the reporter system being responsive to the presence of secondary analyte bound to the supports via the primary analyte, and (e) interrogating the support mixture to determine the unique identifiers of supports which have an activated reporter system in order to identify which samples contain the secondary analyte.

2. A method according to claim 1, wherein, before step (a), each set of supports is contained in a respective well of a multi-well plate to partition it from the other sets of supports.

3. A method according to claim 1 or 2 wherein the primary analyte is a biologically active agent.

4. A method according to any one of the previous claims wherein the secondary analyte is a biologically active agent which binds to the primary analyte.

5. A method according to any one of the previous claims, further comprising the step between steps (a) and (b) of: removing excess primary analyte from each set of supports.

6. A method according to any one of the previous claims, further comprising the step between steps (c) and (d) of: partitioning the support mixture into a plurality of groups, each group containing supports representative of the support mixture, and wherein steps (d) and (e) are performed on the support mixture of each group, a respective reporter system being introduced to each group, with each reporter system being responsive to the presence of a different secondary analyte, whereby the groups relate to respective and different secondary analytes.

7. A method according to claim 6, wherein each group of supports is placed in a respective well of a multi-well plate to partition it from the other groups of supports.

8. A method according to any one of the previous claims, wherein: within each set, the supports form a plurality of support subsets, the supports of each subset carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, and the surface chemistry of the supports of each subset being different from the surface chemistries of the supports of the other subsets so that, in step (a), the primary analyte may preferentially attach to the supports of one or more of the subsets, and the interrogation of step (e) also determines the further unique identifiers of supports which have an activated reporter system in order to identify which surface chemistries cause preferential attachment of the primary analyte.

8. A method according to any one of the previous claims, wherein: within each set, the supports form a plurality of support subsets, the supports of each subset carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, and the surface chemistry of the supports of each subset being different from the surface chemistries of the supports of the other subsets so that, in step (a) , the primary analyte may preferentially attach to the supports of one or more of the subsets, and the interrogation of step (e) also determines the further unique identifiers of supports which have an activated reporter system in order to identify which surface chemistries cause preferential attachment of the primary analyte.

9. A kit for the detection of analytes in samples, the kit comprising a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and the supports of each set being partitioned from the supports of the other sets; the kit further comprising instructions for the performance of a method for the detection of analytes in samples according to any one of claims 1 to 8.

10. A kit for the detection of analytes in samples, the kit comprising a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing them to be distinguished from the supports of the other sets, and the supports of each set being partitioned from the supports of the other sets, wherein, within each set, the supports form a plurality of support subsets, the supports of each subset carrying a further unique identifier allowing them to be distinguished from the supports of the other subsets, and the surface chemistry of the supports of each subset being different from the surface chemistries of the supports of the other subsets so that an analyte may preferentially attach to the supports of one or more of the subsets.

11. The kit according to claim 10, further comprising instructions for the performance of a method for the detection of analytes in samples according to claim 8.

12. An apparatus for the detection of analytes in samples, the apparatus comprising: a robotics system for the automated manipulation and interrogation of the contents of the wells of multi-well plates, and a computer for controlling the actions of the robotics system, the computer being programmed to perform a method for the detection of analytes in samples according to any one of claims 1 to 8.

13. A computer program product carrying code for the performance of a method for the detection of analytes in samples according to any one of claims 1 to 8.

14. Computer code for the performance of a method for the detection of analytes in samples according to any one of claims 1 to 8.

Amendments to the claims have been filed as follows

1. A method for the detection of analytes in samples which makes use of a plurality of sets of particulate supports, the supports of each set carrying a unique identifier allowing s them to be distinguished from the supports of the other sets, and each set of supports being partitioned from the other sets of supports, the method comprising the steps of: (a) attaching a primary analyte to the supports of all the sets, (b) introducing respective samples to the sets of supports so that each sample is associated with a corresponding unique identifier, each sample potentially containing a secondary analyte which binds to the primary analyte, (c) combining the sets of supports to form a support mixture containing supports from all of the sets, (d) introducing a reporter system to the support mixture, the reporter system being responsive to the presence of secondary analyte bound to the supports via the primary analyte, and (e) interrogating the support mixture to determine the unique identifiers of supports which have an activated reporter system in order to identify which samples contain the secondary analyte.