WO2010044083A2

WO2010044083A2 - A multi-well plate for biological assays

Info

Publication number: WO2010044083A2
Application number: PCT/IE2009/000072
Authority: WO
Inventors: Sandeep Kumar Vashist; Stephen A. O'sullivan; Feidhlim T. O'neill; Harry Holthofer; Brian O'reilly; Chandra Kumar Dixit
Original assignee: Dublin City University
Priority date: 2008-10-14
Filing date: 2009-10-14
Publication date: 2010-04-22
Also published as: WO2010044083A3; IE20090797A1

Abstract

A multi-well plate (1) for a biological assay comprising a plate frame (4) attached to a support substrate (7) through a gasket (5) wherein the plate frame (4) defines side walls for a plurality of wells (2) and the gasket (5) comprises a plurality of holes (6), whereby the holes (6) of the gasket (5) are substantially aligned with the wells (2) defined by the plate frame (4) such that the support substrate (7) forms a base for the wells (2) and wherein the side walls of the wells (2) comprise an absorbed layer of blocking agent. The substrate (7) may comprise functional surface amine groups for binding to a protein.

Description

"A multi-well plate for biological assays" Introduction

This invention relates to a multi-well plate for biological assays. In particular, the invention relates to a multi-well plate for enzyme linked immunosorbent assays and a method for preparing such plates.

Conventional multi-well plates are known in the art and include plates with 6-, 24-, 96- 384- well and the like formats. Multi-well plates are used in a number of biological assay formats as they are particularly well suited for screening a number of samples at the same time. With the development of automated laboratory equipment that have been specifically designed for the multi-well plate format for example plate readers, high throughput screening apparatus, plate shakers and the like, the multi-well plate has become a standard format for biological assays.

There are numerous types of multi-well plate on the market which are suited to different applications. Conventional multi-well plates are generally formed from a plastics material and have either a plastic or glass bottom. It is known that for some assays, the biological sample does not absorb or adhere to the base of multi-well plates very effectively and for this reason modified multi-well plates have been developed having for example a base with a charged surface or a base that has been pre-coated with a protein such as poly-lysine to improve the absorption or adherence properties of the plate. Such modified multi-well plates are not suitable for all biological assays and in some cases may result in false positives being detected for control wells. There is therefore a need for an improved modified multi-well plate.

Statements of Invention The invention provides a multi-well plate for a biological assay comprising a plate frame attached to a support substrate through a gasket wherein the plate frame defines side walls for a plurality of wells and the gasket comprises a plurality of holes, whereby the holes of the gasket are substantially aligned with the wells defined by the plate frame such that the support substrate forms a base for the wells and wherein the side walls of the wells comprise an absorbed layer of blocking agent.

The support substrate may comprise two or more different substrate materials. The different substrate materials may be arranged side by side to provide a substantially flat support surface. The substrate may be selected from the group comprising: Zeonex™, Zeonor™, polystyrene, polycarbonate, PMMA, cellulose acetate and glass.

The substrate may comprise functional surface amine groups for binding to a protein. The protein may be an affinity protein such as an Fc binding protein and/or an antibody.

The substrate may comprise an immobilised Fc binding protein. The Fc binding protein may be selected from: Protein A, Protein G and Protein A/G. A capture antibody may be linked to the immobilised Fc binding protein.

The invention also provides a method for preparing a multi-well plate for a biological assay comprising the steps of:

- providing a multi-well plate frame defining side walls of a plurality of bottomless wells; - blocking the side walls of the wells with a blocking agent; providing a gasket comprising a plurality of holes corresponding in size and number to the wells of the multi-well plate frame; aligning the holes of the gasket with the wells of the multi-well plate frame;

- attaching the gasket to the base of the multi-well plate frame; and - attaching a support substrate to the gasket such that the gasket is sandwiched between the multi-well plate frame and support substrate thereby providing a water tight seal.

In one instance the steps are carried out sequentially. Alternatively, the steps may be carried out non-sequentially, for example the gasket may be attached to the substrate material prior to attaching to the multi-well plate frame.

The substrate may comprise function surface amine groups for binding to a protein. The protein may be an affinity protein such as an Fc binding protein and/or an antibody. The substrate may comprise an immobilised Fc binding protein. The Fc binding protein may be selected from: Protein A, Protein G and Protein AJG. A capture antibody may be linked to the immobilised Fc binding protein.

The invention further provides a method for preparing a multi-well plate for a biological assay comprising the steps of:

- providing a multi-well plate comprising a plurality of wells, each well having a base; blocking the wells with a blocking agent;

- removing the base of the wells; - providing a gasket comprising a plurality of holes corresponding in size and number to the wells of the multi-well plate;

- aligning the holes of the gasket with the wells of the multi-well plate;

- attaching the gasket to the base of the multi-well plate; and

- attaching a support substrate to the gasket such that the gasket is sandwiched between the multi-well plate and support substrate thereby providing a water tight seal.

The invention further provides a support substrate for a biological assay comprising functional surface amine groups for binding to a protein. The protein may be an affinity protein such as an - A -

Fc binding protein and/or an antibody. The Fc binding protein may be selected from protein A, protein G and protein A/G. The substrate may comprise a capture antibody linked to the Fc binding protein.

The invention also provides a support substrate for a biological assay comprising an immobilised Fc binding protein. The Fc binding protein may be selected from: Protein A, Protein G and Protein A/G. The substrate may comprise a capture antibody linked to the immobilised Fc binding protein.

The invention further provides a method for immobilising a protein on a multi-well plate for a biological assay comprising the steps of: a) providing a multi-well plate as described herein; b) cleaning the surface of support substrate; c) generating hydroxyl groups on the surface of the support substrate; d) inducing functional amine groups on the surface of the support substrate; and e) cross linking a protein to the thus formed functional amine groups.

Step (b) may comprise treating the support substrate with absolute ethanol at a temperature of about 37⁰C for about 5 minutes.

Step (c) may comprise treating the support substrate with about 1% (w/v) potassium hydroxide. The substrate may be treated at a temperature of about 37⁰C. The substrate may be treated for about 10 minutes.

Step (a) may comprise treating the support substrate with about 2% (w/v) 3-APTES. The substrate may be treated at about room temperature. The substrate may be treated for about 1 hour.

Step (e) may comprise the steps of: i) forming a cross-linking solution; ii) mixing the protein with the cross-linking solution; and iii) incubating support substrate with the protein-cross-linking solution.

The cross-linking solution may comprise 1 -ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The cross-linking solution may comprise sulfo-N Hydroxysuccinmide. The protein and cross-linking solution may be mixed for about 15 minutes prior to step (iii).

Step (iii) may comprise incubating the support substrate with the protein cross-linking solution at a temperature of about 37°C. The support substrate may be incubated with the protein-cross- linking solution for about 1 hour.

The invention also provides a method for performing a sandwich ELISA comprising the steps of: a) providing a multi-well ELISA plate; b) cleaning the surface of the support substrate of the multi-well plate; c) generating hydroxyl groups on the surface of the support substrate; d) inducing functional amine groups on the surface of the support substrate; e) cross-linking a primary antibody to the thus formed functional amine group; f) blocking non-specific antibody binding sites; g) incubating a sample containing a protein specific for the primary antibody; h) adding a detection antibody; and i) detecting the detection antibody.

Steps (a) to (i) may be performed as described herein. The multi-well ELISA plate may be a commercially available ELISA plate or a modified ELISA plate as described herein.

The invention further provides a method of immobilising a protein to a support substrate comprising the steps of: providing a support substrate; - cleaning the surface of support substrate;

- generating hydroxyl groups on the surface of the support substrate;

- inducing functional amine groups on the surface of the support substrate; and

- cross linking a protein to the thus formed functional amine groups. The step of cleaning the surface of the support substrate may comprise treating the support substrate with absolute ethanol of about 37⁰C for about 5 minutes.

The step of generating hydroxyl groups on the surface of the support substrate may comprise treating the support substrate with about 1% (w/v) potassium hydroxide. The substrate maybe treated at a temperature of about 37°C. The substrate may be treated for about 10 minutes.

The step of inducing functional amine groups on the surface of the support substrate may comprise treating the support substrate with about 2% (w/v) 3-APTES. The substrate may be treated at about room temperature. The substrate may be treated for about 1 hour.

The step of cross-linking a protein to the thus formed functional amine groups may comprise the steps of: i) forming a cross-linking solution ii) mixing the protein with the cross-linking solution; and iii) incubating the support substrate with the protein-cross-linking solution.

The cross-linking solution may comprise 1 -ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The cross-linking solution may comprise sulfo-N-Hydroxysuccinmide. The protein and cross-linking solution may be mixed for about 15 minutes prior to step (iii).

Step (iii) may comprise incubating the support substrate with the protein cross-linking solution at a temperature of about 37⁰C. The support substrate may be incubated with the protein-cross- linking solution for about 1 hour.

Brief Description of the Drawings

The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which: -

Fig. 1 A to G are isometric views showing the steps for manufacturing a modified multi- well plate in accordance with an embodiment of the invention; Fig. 2 is an isometric view of a hand punch tool in accordance with an embodiment of the invention;

Fig. 3 is an isometric view of a mould tool in accordance with an embodiment of the invention;

Fig. 4 is an isometric view of a vice tool in accordance with an embodiment of the invention;

Fig. 5 is an isometric view of a polymer substrate section and a pressure sensitive adhesive layer in accordance with an embodiment of the invention;

Fig. 6 is an isometric view of a punching tool in accordance with an embodiment of the invention;

Fig. 7 is an isometric view of an upper punching tool in accordance with an embodiment of the invention;

Fig. 8 is an isometric view of a cutting and punching tool in accordance with an embodiment of the invention;

Fig. 9 is an isometric view of a lower assembly tool in accordance with an embodiment of the invention;

Fig. 10 is an isometric view of an upper assembly tool in accordance with an embodiment of the invention;

Fig. 11 is an isometric view of an alternative arrangement of the lower assembly tool of Fig. 9;

Fig. 12 is an isometric view of an alternative arrangement of the upper and lower assembly tools of Fig. 9 and 10; Fig. 13 is a side view of an assembly device in accordance with an embodiment of the invention.

Fig. 14 is an isometric view of an upper surface of an ELISA plate assembly component;

Fig. 15 is an isometric view of a lower surface of an ELISA plate assembly component;

Fig. 16 is an isometric view of a pressure sensitive adhesive gasket for use with a 96 well modified ELISA plate;

Fig. 17 is an isometric view of an upper surface of an ELISA plate assembly component in use;

Fig. 18 is an isometric view of a lower surface of an ELISA plate assembly component in use;

Fig. 19 is an isometric view of an upper surface of an ELISA plate assembly component in use in accordance with an embodiment of the invention;

Fig. 20 is an isometric view of a lower surface of an ELISA plate assembly component in use in accordance with an embodiment of the invention;

Fig. 21 is a graph of an ELISA performed using a commercially available multi-well plate (comparative example);

Fig. 22 is a graph of an ELISA performed using a modified multi-well plate in accordance with an embodiment of the invention;

Fig. 23 is a schematic of an antibody immobilization procedure in accordance with an embodiment of the invention;

Fig. 24 is a graph showing the results of a developed human fetuin A sandwich ELISA performed on a modified ELISA plate in accordance with an embodiment of the invention (n=3); Fig. 25 is a graph showing the results of a conventional human fetuin A sandwich ELISA (comparative example) performed on a commercially available microtitre plate (n=3);

Fig. 26 is a graph showing the results of a developed human fetuin A sandwich ELISA performed on a commercially available ELISA plate (n=3); and

Fig. 27 is a bar chart showing the results of an ELISA performed on a modified multi- well plate having different base substrates (1. Zeonex, 2. Zeonor, 3. PMMA, 4. Polycarbonate, 5. Cellulose acetate, 6. Polystyrene) in accordance with an embodiment of the invention.

Detailed Description We describe a modified multi-well plate format which comprises a pre-blocked multi-well plate frame defining a plurality of sample wells; and a base portion for attaching to the multi-well plate frame. The base portion may be formed from one or more substrate materials including Zeonex™, Zeonor™, polystyrene, polycarbonate, polymethylmethacrylate (PMMA), cellulose acetate, and glass. The base portion may be formed from a combination of two or more different substrate materials.

The modified multi-well plates described herein may be used for a number of different biological assays. In particular, the modified multi-well plates are particularly well suited for use in enzyme linked immunosorbant assays (ELISAs).

Conventional ELISA plate formats are based on multiple well formats such as 96-well or 384- well formats. The modified ELISA plate described herein may be based on a conventional ELISA plate well format.

Advantageously, the substrate material forming the base portion of the modified ELISA plate may be selected by the end user depending on the experimental parameters required. The choice of substrate material may vary depending on the immobilization strategy used for a particular ELISA. The immobilization strategy used will differ depending on the specific antibody to be used and the experimental parameters required. For example, if the antibody is to be immobilised in an orientated site-directed manner, a covalent binding group, such as an Fc binding protein, may be used.

Referring to Fig. 1 A to G, an overview of a method for preparing a modified ELISA plate is shown. Briefly, a modified ELISA plate may be prepared using the following steps in which the letters A to G refer to the Fig. 1 :

A. Providing an ELISA plate 1 comprising a plurality of sample wells 2;

B. Blocking the sample wells 2 of the ELISA plate 1 to prevent non-specific binding of a sample;

C. Removing the base of the pre-blocked sample wells 2 with a punch tool 3 to form:

D. An ELISA plate frame 4 defining a plurality of bottomless sample wells 2;

E. Providing a gasket 5 comprising a plurality of holes 6;

F. Providing a base substrate 7 comprising one or more substrate materials; and G. Assembling the modified ELISA plate by attaching the base substrate 7 to the ELISA plate frame 4 through a gasket 5 such that the holes 6 of the gasket 5 are aligned with the wells 2 of the ELISA plate frame 4 and the base substrate 7 forms a base for the bottomless wells 2.

In an alternative embodiment, a commercially available bottomless multi-well plate, for example those available from Greiner, may be used. In this case, the step of removing the base of the pre- blocked sample wells may be omitted.

The invention will now be described in more detail and will be more clearly understood from the following examples thereof.

Examples

Materials including the chemicals used with their concentrations

Example 1 — Manufacture of a modified ELISA plate

The plates used in this Example were black Greiner multi-well plates, however it will be appreciated that any suitable conventional ELISA plate or multi-well microtitre plate can be used with the method described herein.

Pre-blocking wells

The wells of the plate were pre-blocked by treating with blocking buffer to block the base and side-walls of the wells to reduce the likelihood of non-specific binding of reagents and/or samples. To pre-block the well, each well of the plate was incubated with 300μl of 1% (w/v) BSA in PBS, pH 7.4 at room temperature for about 1 hour and 30 mins. Following pre-blocking, wells were washed five times with 300μl of PBS.

Preparing plate frame Following pre-blocking, the base of each well was removed. In this Example, the base of each well was removed by manually punching through the base material. Referring to Fig. 2, a suitable tool 3 for removing the base is illustrated. The tool 3 has a handle portion 8 and a base punching portion 9. The base punching portion 9 comprises a substantially tubular structure. The base removing portion 9 is configured so that the circumference of portion 9 is complimentary with the inner dimensions of a well. For example, a tool 3 designed for removing the base of wells of a standard 96-well plate will have a base removing portion 9 with a diameter of 6.4 mm which corresponds to the standard circumference of a well of a 96-well plate. It will be appreciated that the circumference of the base punching portion 9 of a tool designed to remove the base of a well of a 384-well plate will have a smaller diameter than a tool 3 designed to be used with a 96-well plate. In the embodiment of Fig. 2, the base punching portion 9 has an open end 10, the edges 11 defining the open end 10 may be sharpened to enable the base of a well to be cleanly punched from the plate when manual pressure is applied to the tool. By cleanly punched we mean that the base is removed from the well without leaving any sharp or jagged edges. In an alternative embodiment, the open end 10 of the tool 3 may comprise a substantially concave roof. The length of the base punching portion 9 is sufficient so that wider handle portion 8 of the tool 3 does not interfere with the removal of a base from a well.

Alternatively, the base can be removed from a well using the flat end of a standard 5mm spatula by engaging the flat end of the spatula with the base of the well and applying pressure. However, the spatula method of removal does not give as clean a punch as the tool described above as with the spatula method some sections of the polymer base can remain attached to the underside of the plate. Therefore, an additional step is required to remove the remaining base portions for example by manually cutting the remaining base portions using a scalpel with No. 11 blade.

In an alternative embodiment, a variety of bottomless multi-well plates can be purchased off the shelf for example from Greiner Labortechnik of Frickenhausen, Germany. When using bottomless multi-well plates the step of removing the base of the wells is omitted and the side walls of the wells can be pre-blocked by immersing the entire plate in a bath of blocking solution or else by covering one of the open ends of the wells with a disposable, removable seal.

Preparing base substrate

In this Example, the following substrate materials were used: Zeonex™, Zeonor™, Polystyrene, Polycarbonate, PMMA, and Cellulose Acetate.

40 mm by 10 mm sections of each substrate were fabricated. The fabrication methods were differed for the different substrate materials. Zeonex™ and Zeonor™ are cyclic polyolefin copolymers and as such cannot be cut or diced using a CO₂ laser. Therefore, to fabricate the substrates, larger templates were injection moulded and then cut to size using a 3D milling center. Initially the templates were fabricated in-house. A single cavity mould 12, compatible with the Babyplast 6/10 injection moulder was prepared from brass (Fig 3). The mould 12 produces a flat substrate with dimensions of about 40 mm by about 40 mm by about 1.5 mm. To achieve this, a nozzle temperature of about 215° C to about 235° C was required, with barrel and heating chamber temperatures of about 5° to about 10° C or higher. An injection volume of about 20.5 mm³ was required when using an injection pressure of about 1300 to about 2000 bar.

Following injection moulding of the substrate material, the material was clamped in a vice 13 (Fig. 4) and mounted onto a Datron 3D milling centre. The substrate material was cut to a specific size, such as sections of about 40 mm x about 10 mm, using the Excalibur CAD /CAM package. The CAD design was appended with cutting information, which also includes the tools to be used and the feed rates and revolutions per minute (rpm) of the tools. The output from Excalibur was a macro file of code which the Datron understands to produce the design part. Carbide cutting tools (either 2mm or 3mm diameter from Jabro (Catalogue #'s 905002-MEGA-T and 905003-MEGA-T)) were used to scribe out the design from the template. Low feed-rates (ca. 5 mm/min) and rpm (ca. 25000) were used to ensure optimum cut quality.

Polystyrene sections, with a thickness of about 1.5mm have also been fabricated using an identical technique with base material from Nova Chemicals.

Zeonex™ and Zeonor™ have also been purchased in 1.5 mm slide format from Microfluidic Chip-shop (Catalogue #'s 10-0667-0000-04 and 10-0667-0000-05 respectively). A separate slide holder or vice to fit in the 3D milling Centre was required but once fabricated, 40 mm by 10 mm by 1.5 mm sections of Zeonex™ and Zeonor™ were made in the same way as described above.

Sections of PMMA and Lexan Polycarbonate can be laser cut using a CO₂ laser. However, if the sheets are sufficiently thin (thickness less than about 300 μm) it is more efficient to simply cut the sections out of the sheet material using a paper guillotine with graduations to control size. Alternatively, the sections can be cut with a sharp pair of scissors or shears. These methods are also used for cutting the polystyrene and cellulose acetate sections with thickness less than about 300 μm.

If the base is to be formed from the same substrate material, the substrate material can be formed as described above in a suitable size for example about 108 mm by about 80 mm by about 1 mm. Attaching base substrate to plate frame

The substrate sections are attached to the underside of the pre-blocked bottomless multi-well plate frame to form a new base for the wells using a pressure sensitive adhesive (PSA) from Adhesives Research (Catalogue No. AR Care 8890). Sections of PSA were fabricated to have a perimeter corresponding with the size of the sections of base substrate materials for example PSA with a perimeter of about 40 mm by 10 mm were used with 40 mm by 10 mm substrate material sections.

Holes corresponding in number, size and arrangement to the wells of the bottomless multi-well plate were formed in the PSA material. For example, for 96-well plates, the holes had a diameter of about 6 mm and were evenly spaced at a pitch of about 9 mm to match the format of the wells of a 96 well-plate.

In this Example, the PSA sections were fabricated by scribing from a sheet of PSA using a CO₂ laser. The laser used was Micro Master CO₂ Laser from OptecSA. The design of the desired PSA section was generated using the AutoCAD design package. The output from AutoCad was a drawing exchange format (dxf) in Release 14 version. The file format can then be uploaded using the Laser station Micromaster software. A sheet of PSA of a size not greater than about 120 mm by about 100 mm was adhered to the bed laser and the Z-focus of the laser was adjusted so that the head was approx 1 mm above the sheet. The laser was 0.245 mJ - 0.400 mJ in power. The power was adjusted using a manual dial to the indication for PSA (approx 5% of max power) prior to cutting to prevent burning of the PSA material. To further aid cut quality, the release valve for compressed air supply at 5 bar was opened as the air assisted cutting and removed smoke and debris during the cutting process.

Referring to Fig. 5, cut PSA sections 5 were adhered to the base polymer sections 7 by first removing the liner on one side of the PSA 5 and placing the active PSA side on the substrate 7. Exact alignment of the edges of the base polymer section 7 and the PSA section 5 is not critical, however care should be taken to ensue that there is a margin of base polymer 7 surrounding the holes 6 in the PSA section 5. Manual pressure was applied to the base polymer section 7 and PSA section 5 to ensure adequate bonding.

To assemble the modified ELISA plate, the pre-blocked bottomless ELISA plate frame was inverted, the liner paper on the top surface (surface opposite the surface attached to the base substrate material) of the PSA-substrate assembly was removed and the PSA-substrate assembly was positioned on the bottomless ELISA plate frame. Manual pressure was applied to ensure sufficient adhesion to give a water tight seal between the bottomless plate frame and the base support material. Care must be taken during the assembly step to ensure that the holes 6 of the gasket are substantially aligned with the wells of the ELISA plate frame.

It will be appreciated that the modified ELISA plate may comprise a base made from a single substrate material or a combination of two or more support materials selected from the group: Zeonex™, Zeonor™, polystyrene, PMMA, polycarbonate, cellulose acetate, glass and the like.

Example 2 - Manufacture of a modified ELISA plate

The plates used in this Example were black Greiner well plates, however it will be appreciated that any suitable conventional ELISA plate or multi-well microtitre plate can be used with the method described herein.

Pre-blockinR wells

Preparing plate frame

A tool was designed to punch through the bases of multiple wells of a plate at the same time to enable more efficient production than the method of Example 1.

Referring to Fig. 6, the tool 14 comprises a recessed channel 15 to accommodate a perimeter 18 of a multi-well plate 1. In a preferred embodiment, in use the perimeter 18 of the multi-well plate 1 forms a snug fit with the recessed channel 15 to substantially prevent movement of the plate 1 during the punching process. The tool 14 also comprises a portion 16 for receiving the base of the multi-well plate 1. Portion 16 may comprise alignment means 17 to assist with the correct positioning of the multi-well plate 1. Portion 16 may also comprise holes 24 to accommodate punching elements 20 of an upper tool 19 in use. Referring to Fig. 7, the upper punching tool 19 comprises a number of upstanding projections 20. The upstanding projections 20 can be considered as punching elements which in use are placed into the wells of a multi-well plate to punch out the bottom of the wells. The punching elements 20 have a substantially tubular structure. The punching elements 20 are configured so that the circumference of each punching element 20 is complimentary with the inner dimensions of a well. For example, a punching tool 19 designed for removing the base of wells of a standard 96- well plate will have punching elements 20 with a circumference of about 6.4 mm to correspond to the standard circumference of a well of a 96-well plate. It will be appreciated that the circumference of the punching elements 20 designed to remove the base of a well of a 384-well plate will have a smaller diameter to correspond with the diameter of the wells. In the embodiment of Fig. 7, the 7 punching elements 20 have an open end 21, the edges 22 defining the open end 21 may be sharpened to enable the base of a well to be cleanly punched from the plate when manual pressure is applied to the tool. By cleanly punched we mean that the base is removed from the well without leaving any sharp or jagged edges. In an alternative embodiment, the open end 21 of the punching elements may comprise a substantially concave roof. The length of the punching elements 20 is sufficient so that when the tool 19 is in position, the punching elements can pass through the wells (including the base) without the support element 23 contacting the upper surface of the multi-well plate.

Both tool 14 and upper punching tool 19 are configured so that they are compatible with a standard punching press. In use, a multi-well plate 1 is positioned on tool 14 so that the perimeter 18 of the plate fits snugly into the recessed channel 14 provided in the tool 14. The plate 1 can be manually pushed down onto tool 14 until it stops, in this position the bases of the wells rest against the recessed pocket 16 and are aligned just above the holes 24 provided to take the punching elements 20 of the upper tool 19. The upper punching tool 19 is inverted such that the punching elements 20 are pointing in a substantially downward direction ready for engagement within the wells of a multi-well plate 1. The upper tool 19 is then lowered towards the plate 1 until the punching elements 20 enter the wells and contact the base of the wells. When pressure is applied for example by actuating a punching press, the punching elements 20 will be forced to pass through the base of the multi-well plate 1 and into holes 24 thereby punching the base out of the wells. Tool 14 may comprise holes 25 to receive pins 26 that project from the support element 23 of the upper punching tool 19. Engagement of pins 26 and holes 25 assists in the correct alignment of the upper punching tool 19 with tool 14. In an alternative embodiment, bottomless multi-well plates can be purchased off the shelf. When using bottomless multi-well plates, the step of removing the base of the wells is omitted and the side walls of the wells can be pre-blocked by immersing the entire plate in a bath of blocking solution or else by covering one of the open ends of the wells with a disposable, removable seal.

Preparing base substrate

In this Example, the following substrate materials were used: Zeonex™, Zeonor™, Polystyrene,

Polycarbonate, PMMA, and Cellulose Acetate.

Following injection moulding of the substrate material, the material was clamped in a vice 13 (Fig. 4) and mounted onto a Datron 3D milling centre. The substrate material was cut to a specific size, such as sections of about 40 mm x about 10 mm, using the Excalibur CAD /CAM package. The CAD design was appended with cutting information, which also includes the tools to be used and the feed rates and revolutions per minute (rpm) of the tools. The output from Excalibur was a macro file of code which the Datron understands to produce the design part. Carbide cutting tools (either 2mm or 3mm diameter from Jabro (Catalogue #'s 905002-MEGA-T and 905003-MEGA-T)) were used to scribe out the design from the template. Low feed-rates (ca. 5 rnm/min) and rpm (ca. 25000) were used to ensure optimum cut quality.

Polystyrene sections, with a thickness of about 1.5mm have also been fabricated using an identical technique with base material from Nova Chemicals. Zeonex™ and Zeonor™ have also been purchased in 1.5 mm slide format from Microfluidic Chip-shop (Catalogue #'s 10-0667-0000-04 and 10-0667-0000-05 respectively). A separate slide holder or vice to fit in the 3D milling Centre was required but once fabricated, 40 mm by 10 mm by 1.5 mm sections of Zeonex and Zeonor were made in the same way as described above.

Sections of PMMA and Lexan Polycarbonate can be laser cut using a CO₂ laser. However, if the sheets are sufficiently thin (thickness less than about 300 μm), it is more efficient to simply cut the sections out of the sheet material using a paper guillotine with graduations to control size. Alternatively, the sections can be cut with a sharp pair of scissors or shears. These methods are also used for cutting the polystyrene and cellulose acetate sections with thickness less than about 300 μm.

If the base is to be formed from the same substrate material, the substrate material can be formed as described above in a suitable size for example about 108 mm by about 80 mm by about 1mm.

Attaching base substrate to plate frame

Holes corresponding in number, size and arrangement to the wells of the bottomless multi-well plate were formed in the PSA material. For example, for 96-well plates, the holes had a diameter of 6.4 mm and were evenly spaced at a pitch of about 9 mm to match the format of the wells of a 96 well-plate.

Holes in the PSA layer can be formed by punching with a tool such that multiple holes are created simultaneously along with the sheet perimeter being cut to size. Referring to Fig. 8, a suitable cutting and punching tool 27 is illustrated. Tool 27 comprises an inlet 28 for PSA film so that PSA film can be fed directly from a source external to tool 27 to the cutting and punching area 29. The cutting and punching area 29 comprises a portion 30 defining the dimensions of the perimeter of a multi-well plate and holes 31 arranged in the same layout of the wells of a multi- well plate. In use, a layer of PSA is fed from the inlet 28 to cover the punching area 30. The upper portion 32 is inverted and arranged on top of tool 27 so that the punching elements 33 project substantially downwardly for engagement into holes 31. As described above in relation to upper tool 19, the punching elements 33 have a substantially tubular structure. The punching elements 33 are configured so that the circumference of each punching element 33 is complimentary with the inner dimensions of a well. The punching elements 33 have an open end 34, the edges 35 defining the open end 34 may be sharpened to enable holes to be cleanly punched in the PSA layer when pressure is applied to the tool. In an alternative embodiment, the open end 34 of the punching elements may comprise a substantially concave roof. The length of the punching elements 33 is sufficient so that when the tool is in position, the punching elements 33 can pass through the layer of PSA and into holes 31 when pressure is applied for example by actuating a punching press. At the same time as punching holes through the PSA layer, the PSA layer is simultaneously cut to the correct size for example about 72 mm by about 109 mm corresponding to the size of the 96- well ELISA plate. In addition to the 96 tubular punching elements which are configured to punch the 96 holes in the PSA, the punching tool may also comprise a rectangular punching element or knife surrounding the tubular punching elements. In use, the rectangular punching element or knife will impinge on the PSA sheet at the same time as the 96 tubular punching elements impinge on the PSA sheet with the result that the perimeter of the PSA sheet can be cut to size as the holes are being punched. Tool 27 may comprise holes 36 to receive pins 37 that project from the upper portion 32. Engagement of pins 37 and holes 36 assists in the correct alignment of the upper portion 32 with tool 27.

When a layer of PSA has been cut to size and punched, tool 27 and upper portion 32 can be disassembled and the shaped and punched PSA layer 38 can be removed from tool 27

In the embodiments of Figs 6 to 12, tools for a 96-well plate are illustrated. However, it would be apparent to a person skilled in the art that these tools could be adapted for multi-well plates containing a different number of wells by adjusting the number and arrangement of punching elements 20 and 33.

Assembling modified ELISA plate

The modified ELISA plate may be assembled with the aid of an assembly tool such as the one illustrated in Figs 9 to 12 using the following steps: " " I / f h t~WW v» ^• \J XT \J \f f Im

- 21 -

Step 1:

Remove the liner from one face of the PSA film 38 and locate the PSA film 38 in the lower assembly tool 39 so that the PSA film 38 sits within the area 40 between the alignment features 41 with the exposed adhesive facing upward (Fig. 9).

Step 2:

Mount the pre-blocked bottomless ELISA plate frame 4 in the upper assembly tool 42 so that the elastomeric member 43 grips the upper side faces 44 of the ELISA plate frame 4 (Fig. 10).

Step 3:

Align the upper 42 and lower 39 assembly tools so that the alignment pins 45 engage to alignment holes 46. Apply pressure to compress the upper 42 and lower 39 tools together so that the PSA layer 38 adheres to the underside of the ELISA plate frame 4.

Step 4:

Release the pressure and separate upper 42 and lower 39 tools. Remove the ELISA plate frame 4 from the upper tool 42. Remove the remaining liner layer from the PSA layer 38 to expose an adhesive surface.

Step 5:

Place the base substrate layer 7 in the lower assembly tool 39 so that it sits in the area 40 between the alignment features 41 (as with Step 1 — Fig. 11).

Step 6:

Re-mount the ELISA plate frame 4 in the upper assembly tool 42 so that the elastomeric member 43 grips the upper side faces 44 of the ELISA plate frame 4 (as with Step 2).

Step 7: Align the upper 42 and lower 39 assembly tools so that the alignment pins 45 engage with alignment holes 46. Apply pressure to compress the upper 42 and lower 39 tools together so that the PSA layer 38 adheres to the substrate base 7 to form a water tight seal.

Step 8: Remove pressure and separate upper 42 and lower 39 tools. Remove the fully assembled modified ELISA plate.

Referring to Fig. 13, it can be seen that the upper 42 and lower 39 assembly tools are configured to mate with the ELISA plate frame 4. The elastomeric member 43 of the upper assembly tool 42 is profiled to compliment the profile of the upper side faces 44 of the ELISA plate frame to ensure that the ELISA plate frame 4 is firmly gripped during assembly. The lower assembly tool 39 comprises a stationary portion 47 and a moveable portion 48. The moveable portion 48 is configured to engage with the ELISA plate frame 4 during assembly of the modified ELISA plate. The alignment features 41 can be considered to have a dual functionality as not only do they define the area into which the PSA layer 38 or substrate base 7 is placed during assembly of the modified ELISA plate, the alignment features 41 are designed to engage with the underside of the plate frame 4 during the assembly process. When the upper 42 and lower 39 portions are brought together and pressure is applied, the alignment features 41 will engage with portion 52 of the underside of the ELISA plate frame 4. Engagement of portions 52 and alignment features 41 will assist in maintaining the plate frame 4 in the correct position during assembly of the modified ELISA plate.

The alignment feature 41 comprises an extending portion 50 to engage with the stationary portion 47. A resilient means 51, for example a spring in the embodiment of Fig. 13, is located between the moveable portion 48 and the stationary portion 47 so that in use, when pressure is applied, the moveable portion 48 and the stationary portion 47 of the lower assembly tool 39 are brought into closer proximity which results in the substantially flat support 49 located between the alignment features 41 of the moveable portion 48 projecting above the level of the alignment features 41 thereby exerting a pressure on the underneath of the ELISA plate frame 4 through the PSA layer 38 or substrate base 7. The substantially flat support 49 exerts an even pressure on the underneath of the ELISA plate frame 4 which ensures good contact between the underneath of the ELISA plate frame 4 and the PSA layer or the substrate base 7 and the PSA layer.

It will be appreciated that the modified ELISA plate may comprise a base made from a single substrate material or a combination of two or more support materials selected from the group: Zeonex™, Zeonor™, polystyrene, PMMA, polycarbonate, cellulose acetate, glass and the like. The methods described herein use planar substrates. However, almost any type of substrate could be used with these methods. Possible substrates include, but are not limited to, micro-structured surfaces, v-shaped and round bottomed surfaces. Many different fabrication techniques could be utilised to produce these substrate including mechanical milling, photo-lithography and hot- embossing techniques.

Example 3 - manufacture of a modified ELISA plate

The plates used in this example were bottomless 96-well ELISA plates from Greiner. However, any suitable conventional bottomless multi-well ELISA / microtitre plate can be used with the method described herein.

Pre-blocking wells

The 96-well bottomless ELISA plate was pre-blocked by putting the ELISA plate in a container containing 1% (w/v) BSA in PBS, pH 7.4 at 37°C for 30 min. The pre-blocked ELISA plate was then washed extensively with PBS by immersing in another container containing PBS for 5 min.

Preparing base substrate

In this Example, the following substrate materials were used: Zeonex™, Zeonar™, Polystyrene,

Polycarbonate, PMMA₃ and Cellulose Acetate.

110mm x 75mm pieces of each substrate were fabricated, except in the case of Zeonex™ which was sourced in microscope slide format, from Microfluidic ChipShop (Catalogue #'s 10-0664- 0000-04), and implemented in this format. Each substrate was cut to size from a larger sheet and prepared for assembly, using the Micro Master CO₂ Laser from OptecSA. Care was taken that the edges were de-burred after cutting to avoid leaking wells when the plate is assembled.

Alternatively a section of the polymer, if sourced as thin sheets 0.5mm thick or less, may be cut to size using a paper guillotine with graduations to control size or sections can be cut with a sharp pair of scissors or shears.

Preparation of patterned PSA

Pressure sensitive adhesive (PSA) from Adhesive Research (Catalogue No. AR Care 8890) was loaded on to the bed of the Micro Master Laser system and aligned for ablation using a CO₂ laser. Holes corresponding to the pattern of the wells of the 96-well ELISA plate, having 9mm distance from the centre of one well to the centre of an adjacent well, were then patterned on the PSA.

The PSA sections were fabricated from a sheet of PSA by scribing using a CO₂ laser. The laser used was Micro Master CO₂ Laser from OptecSA. The design of the desired PSA section was generated using the AutoCAD design package. The output from AutoCad was a drawing exchange format (dxf) in Release 14 version. The file format can then be uploaded using the Laser station Micromaster software. A sheet of PSA of a size not greater that about 120 mm by about 100 mm was adhered to the bed laser and the Z-focus of the laser was adjusted so that the head was approx 1 mm above the sheet. The laser was 0.245 mJ — 0.400 mJ in power. The power was adjusted using a manual dial to the indication for PSA (approx 5% of max power) prior to cutting to prevent burning of the PSA material. To further aid cut quality, the release valve for compressed air supply at 5 bar was opened as the air assisted cutting and removed smoke and debris during the cutting process.

Attaching base substrate to plate frame

An assembly component was prepared in-house. The upper surface 60 of the assembly component is shown in Fig. 14. The lower surface 61 of the ELISA plate assembly component is shown in Fig. 15. Briefly, a base substrate sheet of the same dimensions as the 96-well ELISA plate was placed on the lower surface 61 of the assembly component. Thereafter, the 96-well ELISA plate with a PSA gasket attached to the bottom was brought in contact with the base substrate sheet. The assembly components shown in Figs. 14 and 15 may be machined from one block of polymer to further improve the alignment accuracy.

In more detail, substrate sections are attached to the underside of a pre-blocked bottomless 96- well plate frame to form a new base for the wells using patterned PSA. Sections of PSA were fabricated to have a perimeter corresponding with the size of the sections of base substrate materials. Referring to Fig. 16, a patterned PSA sheet 62 corresponding to the base of the bottomless 96-well ELISA plate was used.

Holes corresponding in number, size and arrangement to the wells of the bottomless 96-well plate were formed in the PSA material. For example, for 96-well plates, the holes had a diameter of about 6 mm and were evenly spaced at a pitch of about 9 mm to match the format of the wells of a 96 well-plate. Referring to Fig. 17, cut PSA sections 62 were aligned with the bottomless multi-well plate 63, using the pins 64 on the upper surface of the assembly component by first removing the liner on one side of the PSA 62 (the active side) and placing the inactive PSA side on the alignment jig 60. The bottomless plate 63 was then placed over the alignment pins 64 to contact the active side of the PSA 62 and pressure was applied to ensure adequate bonding of PSA 62 to the bottomless plate 63.

The assembly component 60 was then inverted to remove the bottomless plate - PSA assembly. Referring to Fig. 18, the substrate 66 was aligned on surface 65, ready for the introduction of the bottomless plate with the PSA attached.

To assemble the modified ELISA plate, the protective liner on the top surface (surface opposite to the PSA surface attached to the frame of the 96-well plate, previously the inactive side) of the PSA-bound 96-well ELISA plate assembly was removed and the PSA-bound 96-well ELISA plate assembly is positioned on the substrate 66 with pressure to ensure adequate bonding. Manual pressure was applied to ensure sufficient adhesion to give a water tight seal between the bottomless 96-well ELISA plate frame and the base support material.

An alternative embodiment is shown in Figs. 19 and 20 in which a number of different types of substrate materials are arranged side by side to form the base of wells of an ELISA plate. Referring to Fig. 19, cut PSA sections 162 were aligned on an alignment jig 160 using the pins 164 positioned on the upper surface of the assembly component 160.

The bottomless multi-well plate 163 was then placed over the alignment pins to contact an active side of the PSA 162 and pressure was applied to ensure adequate bonding of PSA 162 to the bottomless plate 163. The assembly component 160 was then inverted to remove the bottomless plate - PSA assembly. Referring to Fig. 20, the substrates 166 were aligned on the surface 165 of the alignment jig 161. In the embodiment shown in Fig. 20, surface 165 of the alignment jig 161 is configured to receive the different substrate materials (four different substrate materials can be accommodated in the embodiment of Fig. 20). To assemble the modified ELISA plate, a protective liner was removed from the top surface of the PSA (surface opposite to the PSA surface attached to the frame of the multi-well plate) attached to the multi-well plate. The PSA - bound multi-well plate assembly was positioned on the substrate 166 and pressure was applied to ensure adequate bonding of the PSA 162 and substrate 166. A water tight seal should be formed between the frame of the multi-well plate and the substrate base support material.

Thus formed modified ELISA plates were stored at 4°C until needed.

Example 4 — preparing substrate for immobilisation of antibodies in a site directed, orientated manner

Using a modified ELISA plate formed as described in any of Examples 1 to 3 above, the wells of the plate were prepared for the immobilization of antibodies using the following methodology:

Surface cleaning

The surfaces of the base substrates were cleaned by treating with absolute ethanol for 10 min and then washing five times with 300 μl of deionized water (DIW).

Generation of hydroxyl groups

The surface of the base substrate is functionalized by treating the cleaned substrate with 100 μl of 1% potassium hydroxide (KOH) for 10 min and then washing five times with 300 μl of DIW. The modified microtiter plate was placed in the Oxygen plasma for 3 min, to generate hydroxyl groups on the surface of the support.

Induction of amine groups by treatment with 3-aminopropyltHethoxysilane (APTES) The hydroxyl group functionalized modified microtiter plate wells were provided with 100 μl of 2% 3 -APTES (in DIW) per well at room temperature inside the fume hood. Thereafter, the modified microtiter plate was placed inside the glass desiccator and a vacuum was created inside the desiccator prior to placing it in an oven at about 80° C for about 6 hours. After 6 hours, the desiccator was removed from the oven and placed at room temperature for 20 min to cool down then washed five times with 300 μl of DIW.

Optionally, crosslinking of induced amine groups on base substrates to the carboxyl group on Fc binding proteins

In an eppendorf tube, 0.4 mg EDC and 1.1 mg Sulfo NHS were added and dissolved in 100 μl of 0.1 M MES, pH 4.7. In a second eppendorf tube, 990 μl of an Fc binding protein such as Protein A, Protein A/G, or Protein G (10 μg/ml in PBS) was prepared and 10 μl of the crosslinking solution from the first eppendorf tube was added to the second eppendorf tube. The mixture was left for 15 min at room temperature. 1.4 μl of 2-mercaptoethanol (20 mM) was then added to quench the EDC. Finally, 100 μl of this crosslinking solution i.e. EDC-sulfo NHS-Fc binding protein was added to each well of the amine functionalized modified microtiter plate wells and incubated at room temperature for 2 hours. Wells were then washed five times with 300 μl of PBS.

Example 5 - Immobilising antibodies to the base support material

Using the plates of any of Examples 1 to 4 antibodies were immobilised onto the base of the wells using the following methodology:

Immobilization of Mouse IgG

Mouse IgG was immobilized on to the base of the wells by adding 100 μl of mouse IgG (12.1 μg/ml in PBS) to each modified microtiter plate well and leaving it overnight at 4° C. Thereafter, the modified microtiter plate wells were washed five times with 300 μl of PBS.

Blocking of the base substrate in modified microtiter plate wells

The modified microtiter plate wells were incubated with 1% BSA (in PBS, pH 7.4) for 1 hour and 30 min at room temperature to block non-specific binding sites on the base substrate and then washed five times with 300 μl of PBS.

Binding of goat anti-mouse IgG HRP labeled

100 μl of goat anti-mouse IgG (in varying ng/ml concentrations in PBS) was provided to each of the modified microtiter plate wells and thereafter, the modified microtiter plate was left at room temperature for 1 hour. The modified microtiter plate wells were then washed five times with 300 μl of PBS.

Example 6 - TMB substrate assay

TMB substrate solution was made by mixing equal amounts of TMB solution (0.4 g/L) and Peroxide solution (containing 0.02 % hydrogen peroxide in citric acid buffer) as per the instructions of the TMB substrate kit from Pierce. 100 μl of this TMB substrate solution was added to all modified microtiter plate wells of plate prepared using the methodology of Example 5 above. The peroxidase enzyme, in the presence of H₂O₂, catalyses the oxidation of colorless TMB substrate to a blue colored product. After a fixed reaction time (30 min), the reaction was stopped with 100 μl of IN H₂SO₄ and the absorbance of the solution was measured at 450 nm with reference at 650 nm.

Example 7 - ELISA using a standard ELISA plate (comparative example) A conventional 96-well microtiter plate was used. Mouse IgG was immobilized directly onto the base of the wells using the method of Example 5 above. Following antibody immobilization, the microtiter plate was treated with blocking buffer (1% BSA in PBS, pH 7.4) for 1 hour and 30 min at room temperature and wells were then washed five times with 300 μl of PBS. Goat anti- mouse IgG was added to the wells using the method of Example 5 above.

The detection of goat anti-mouse IgG HRP labeled by TMB substrate assay was performed using the method of Example 6 above.

The ELISA performed was sensitive for goat anti-mouse IgG HRP from 44.81 to 1210 ng/ml (as shown in Fig. 21) with coefficient of variance (% CV) in the range of 1.99 - 16.98 for various cone, of goat anti-mouse IgG HRP.

Example 8 - ELISA on modified microtiter plate

An ELISA was performed using a modified multi-well plate to demonstrate the advantages of the modified microtiter plate technology and approach. A modified multi-well plate was prepared according to the method of Example 1 above using three different base support materials: Zeonex™ , Zeonor™ and polystyrene. The substrates were prepared according to

Example 4 above prior to the immobilization of antibodies according to Example 5 above and detection of bound goat-anti -mouse IgG was performed using the TMB substrate assay described in Example 6 above.

It was found that the detection range of mouse IgG immobilized on Zeonex™, Zeonor™ and polystyrene for goat anti-mouse IgG HRP labeled were 1.66 - 1210 ng/ml, 1.66 - 1210 ng/ml and 14.94 - 1210 ng/ml respectively, as shown in Fig. 22 and Table 1 below. The coefficient of variance (% CV) on Zeonex™, Zeonor™ and polystyrene were in the range of 0.2 - 4.39, 0.15 — 4.92 and 0.31 - 5.13 respectively for various concentrations of goat anti-mouse IgG HRP.

Table 1 - Sensitivity and variability of modified ELISA plate.

S. No. Base substrate for modified Detection Range Variability (% CV)

The normal ELISA procedure on polystyrene microtiter plates has a % CV (variability) in the range of 1.99 - 16.98 which is much higher than the %CV of 0.31 - 5.13 obtained on polystyrene wells of the modified microtiter plate. Also, the ELISA in polystyrene wells of the modified microtiter plate has a higher detection range of 14.94 - 1210 ng/ml for goat anti-mouse

IgG HRP compared to the detection range of ELISA in the normal polystyrene ELISA plate i.e.

44.81 — 1210 ng/ml. Thus, the ELISA procedure adopted on the modified microtiter plate with our particular antibody immobilization strategy has much less variability and more detection range than the normal polystyrene based ELISA plate.

Additionally, the ELISA procedure on the modified microtiter plate allowed for antibodies to be immobilized on a range of commercially relevant base substrates such as Zeonex™ and Zeonor within the same assay format. Thus, the modified ELISA plate technology allows for various base substrates to be screened in one assay which is useful for determining the optimum support material to be used for a particular biosensor, diagnostic or other application.

Example 9 - ELISA using a modified microtitre plate

Materials

In this example, we performed a human fetuin A sandwich ELISA on a modified microtiter plate prepared in accordance with Example 3 above.

Preparation of modified ELISA plate

In this example, a modified ELISA plate was prepared in accordance with Example 3 above. Briefly:

Preblocking of 96-well microtiter plates A Greiner 96-well microtiter plate was incubated with 300 μl of 1% Blocker BSA (in PBS, pH 7.4) for 30 min at 37° C and then washed five times with 300 μl of PBS.

Fabrication of modified 96-well microtiter plates with different substrates A modified microtiter plate, having different base substrates (zeonex, polystyrene, and PMMA) were fabricated by the procedure described in Example 3, briefly:

96-well bottomless microtiter plates from Greiner were attached to different base substrates (zeonex, polystyrene and PMMA) through a double sided pressure sensitive adhesive (PSA). A Laser with power 0.245 mJ - 0.400 mJ was used to generate the hole pattern on PSA corresponding to the pattern of wells on the bottomless 96-well ELISA plate. One side of PSA was then applied to the bottom of the plate using the developed assembly component while the other side was attached to the substrates.

Sandwich ELISA

Surface cleaning and silanisation

The surfaces of the base substrates employed in the modified microtiter plate wells were cleaned by treatment with 100 μL of absolute ethanol for 5 min at 37⁰C and then washed five times with 300 μL of DIW. The cleaned surface was treated with 100 μL of 1.0% (w/v) KOH at 37⁰C for 10 min and then washed five times with 300 μL of DIW. The surface-treated microtiter plate wells were then functionalised with amino groups by incubating with 100 μL of 2% (w/v) 3- APTES per well for 1 hr at room temperature. The amine-functionalised microtiter plate wells were subsequently washed five times with 300 μL of DIW.

Cross-linking of induced amino groups on amine-functionalised microtiter plate wells to the carboxyl groups of anti-human Fetuin A

990 μL of anti-human fetuin A (4 μg/mL) was incubated with 10 μL pre-mixed solution of EDC (4 mg/mL) and sulfo-NHS (11 mg/niL) for 15 min at 37⁰C. 100 μL of the resulting EDC cross- linked anti-human fetuin A solution was then added to each of the functionalised wells and incubated for 1 hr at 37⁰C. The anti-human fetuin A-coated wells were then washed five times with 300 μL of PBS.

Fig. 23 is a schematic of the antibody immobilization procedure on modified microtiter plate. In an alternative embodiment the induced amino groups on the surface of the substrate may be cross linked to the carboxyl groups of an Fc binding protein such as protein A, protein G or protein A/G (as described in Example 4 above). The antibody can then be bound to the Fc binding protein in a site-directed orientated fashion.

As a control, anti-human fetuin A was immobilized by passive absorption as follows. 100 μL of anti-human fetuin A (4 μg/mL) was added to each well to be used and then the plate was incubated overnight at room temperature (RT). The anti-human fetuin A adsorbed ELISA plate was later washed five times with 300 μL of PB S .

Human fetuin A sandwich ELISA procedure on modified microtiter plate coated with anti- human fetuin A by covalent cross-linking The anti-human fetuin A-coated modified microtiter plate was blocked with 1% v/v BSA for 30 min at 37⁰C and subsequently washed five times with 300 μL of PBS. Varying concentrations of human fetuin A (from 4.8 pg/mL to 20 ng/mL) were prepared and 100 μL of each of these concentrations were then incubated onto the antibody-coated plate for 1 hr at 37⁰C and subsequently washed five times with 300 μL of PBS. 100 μL of biotinylated anti-human fetuin A detection antibody (200 ng/mL) was then added and incubated for 1 hr at 37⁰C followed by five washes with 300 μL of PBS. lOOμL of HRP-conjugated streptavidin at a dilution of 1:200 was provided to each of the used microtiter plate wells and then incubated for 20 min at 37⁰C followed by washing five times with 300 μL of PBS. TMB substrate was subsequently added to each of the used microtiter plate wells. The HRP enzyme-TMB substrate reaction was then stopped after 20 min by adding 50 μL of IN H₂SO₄ and ODs were taken according to the manufacturer's guidelines at a wavelength of 450nm with reference wavelength of 540nm. This dual wavelength system is well established and very specific in eliminating optical imperfections at well to well level whereas measurements taken at 450 run alone may result in non-specific absorbance.

All these experiments were performed in triplicate. The control for the experiment was zero ng/mL concentration of human fetuin A. The OD of the control was subtracted from the ODs of all assay points. As a control, a conventional human fetuin A sandwich ELISA procedure on normal microtiter plate coated with anti-human fetuin A by passive adsorption was also performed as follows: the anti-human fetuin A-adsorbed normal microtiter plates was blocked with 1% v/v BSA for 2 hrs at RT and subsequently washed five times with 300 μL of PBS. Varying concentrations of human fetuin A (from 4.8 pg/mL to 20 ng/mL) were prepared and 100 μL of each of these concentrations were then incubated onto the antibody-coated plates for 2 hrs at RT and subsequently washed five times with 300 μL of PBS. 100 μL of biotinylated anti-human fetuin A detection antibody (200 ng/mL) was then added and incubated for 2 hrs at RT followed by five washes with 300 μL of PBS. lOOμL of HRP-conjugated streptavidin at a dilution of 1:200 was provided to each of the used ELISA plate wells and then incubated for 20 min at RT followed by washing five times with 300 μL of PBS. TMB substrate was subsequently added to each of the used ELISA plate wells. The HRP enzyme-TMB substrate reaction was then stopped after 20 min by adding 50 μL of IN H₂SO₄ and ODs were taken according to the manufacturer's guidelines at a wavelength of 450nm with reference wavelength of 540nm. This dual wavelength system is well established and very specific in eliminating optical imperfections at well to well level whereas measurements taken at 450 nm alone may result in non-specific absorbance.

All these experiments were performed in triplicate. The control for these experiments was zero ng/mL concentration of human fetuin A. The OD of the control was subtracted from the ODs of all assay points.

Results

Human fetuin A sandwich ELISA was performed to demonstrate the advantages of the modified microtiter plate technology and antibody immobilisation method described herein. Greiner 96- well microtiter plates were preblocked with 1% (v/v) BSA and three base substrates (Zeonex™, PMMA, and polystyrene) were attached to form the base of wells. Thereafter, the modified ELISA procedure as stated above was employed. Fig. 24 shows the sandwich ELISAs for the different substrates based modified microtiter plates whereas Table 2 shows the various analytical parameters.

The advantages of the modified ELISA procedure using the modified microtiter plate were demonstrated by performing a normal ELISA based on the passive adsorption of mouse anti- human fetuin A and then, comparing the results of the two procedures. Table 2 — detection range and percentage variability for different substrates

The control conventional human fetuin A sandwich ELISA was performed on a normal Nunc 96- well flat bottom plate formed from polystyrene by the procedure described above where anti- human fetuin A were bound passively by adsorption.

Fig. 25 shows the conventional human fetuin A sandwich ELISA which had a detection range of 156.16 - 20,000 pg/mL and %CV of 4.72 - 17.38.

Comparison of the human fetuin A sandwich ELISA in accordance with an embodiment of the invention and the conventional ELISA format

The conventional sandwich ELISA procedure on normal 96-well polystyrene microtiter plates had % CV in the range of 4.72 - 17.38 which is much higher than the % CV of 0.84 - 7.2 obtained with modified microtiter plate having polystyrene at the bottom. Also, the modified microtiter plate based sandwich ELISA had higher detection range for human fetuin A i.e. 19.52

- 20,000 pg/mL. Whereas the detection range of conventional sandwich ELISA was only 156.16

- 20,000 pg/mL. Thus, the modified microtiter plate based sandwich ELISA in accordance with the invention had less variability and a higher detection range and thus higher sensitivity than the conventional sandwich ELISA format.

Additionally, the modified ELISA plate described herein allows ELISAs to be performed on most of the commercially relevant base substrates apart from polystyrene, which are currently being employed in various immunobiosensor applications. The modified ELISA plate technology can be used to screen various potential base substrates for a particular immunobiosensor application and for rapid preconfirmation studies on a particular substrate.

The human fetuin A sandwich ELISA described herein is a rapid assay compared to conventional ELISA. The ELISA procedure described herein takes only about 6 hrs from start to finish whereas the conventional ELISA procedure takes about 24 hrs. Comparison of the developed human fetuin A sandwich ELISA on normal microliter plate with that done on modified microtiter plate

The developed human fetuin A sandwich ELISA procedure on normal 96-well polystyrene microtiter plates had % CV in the range of 1.23 - 9.76 which is higher than the % CV of 0.84 - 7.2 obtained with modified microtiter plate having polystyrene at the bottom. Therefore, the modified microtiter plates had less variability.

The detection range of the developed ELISA on modified microtiter plate was 19.52 - 20,000 pg/mL. Whereas the detection range of the developed ELISA on normal ELISA plate was 9.76 - 20,000 pg/mL. The slightly lower sensitivity of the developed ELISA on modified microtiter plate might be due to the nature of the polystyrene substrate employed as the base. The polystyrene slides used as substrate were meant for microfluidic applications and might not be so good for performing ELISA in comparison to the high-quality polystyrene used in normal microtiter plate.

But the developed ELISA on modified microtiter plate has a very specific advantage based on which it will have tremendous applications in bioanalytical sciences i.e. it is capable of performing ELISA on most of the commercially relevant base substrates apart from polystyrene, which are currently being employed in various immunobiosensor applications. The developed technology can be used to screen various potential base substrates for a particular immunobiosensor application and for rapid preconfirmation studies on a particular substrate. Therefore, the developed ELISA procedure on modified microtiter plate has several applications in different disciplines of bioanalytical sciences, which will significantly increase the number of ELISA users in different disciplines.

Example 10 — ELISA using a conventional niicrotitre plate

Materials

In this example, we performed a human fetuin A sandwich ELISA on a conventional 96-well polystyrene microtiter plate as follows:

Surface cleaning and silanisation

The microtiter plate wells were cleaned by treatment with 100 μL of absolute ethanol for 5 min at 37⁰C and then washed five times with 300 μL of DIW. The cleaned surface was treated with 100 μL of 1.0% (w/v) KOH at 37⁰C for 10 min and then washed five times with 300 μL of DIW. The surface-treated microtiter plate wells were then functionalised with amino groups by incubating with 100 μL of 2% (w/v) 3 -APTES per well for 1 hr at room temperature. The amine- functionalised microtiter plate wells were subsequently washed five times with 300 μL of DIW.

Cross-linking of induced amino groups on amine-functionalised microtiter plate wells to the carboxyl groups of anti-human fetuin A 990 μL of anti-human fetuin A (4 μg/mL) was incubated with 10 μL pre-mixed solution of EDC (4 mg/mL) and sulfo-NHS (11 mg/mL) for 15 min at 37⁰C. 100 μL of the resulting EDC cross- linked anti-human fetuin A solution was then added to each of the functionalised wells and incubated for 1 hr at 37⁰C. The anti-human fetuin A-coated wells were then washed five times with 300 μL of PBS. As a control, anti-human fetuin A was immobilized by passive absorption as follows:

100 μL of anti-human fetuin A (4 μg/mL) was added to each well to be used and then the plate was incubated overnight at room temperature (RT). The anti-human fetuin A adsorbed ELISA plate was later washed five times with 300 μL of PBS.

Human fetuin A sandwich ELISA procedure on normal microtiter plate coated with anti-human fetuin A by covalent cross-linking The anti-human fetuin A-coated normal microtiter plate was blocked with 1% v/v BSA for 30 min at 37⁰C and subsequently washed five times with 300 μL of PBS. Varying concentrations of human fetuin A (from 4.8 pg/mL to 20 ng/mL) were prepared and 100 μL of each of these concentrations were then incubated onto the antibody-coated plate for 1 hr at 37⁰C and subsequently washed five times with 300 μL of PBS. 100 μL of biotinylated anti-human fetuin A detection antibody (200 ng/mL) was then added and incubated for 1 hr at 37⁰C followed by five washes with 300 μL of PBS. lOOμL of HRP-conjugated streptavidin at a dilution of 1 :200 was provided to each of the used microtiter plate wells and then incubated for 20 min at 37⁰C followed by washing five times with 300 μL of PBS. TMB substrate was subsequently added to each of the used microtiter plate wells. The HRP enzyme-TMB substrate reaction was then stopped after 20 min by adding 50 μL of IN H₂SO₄ and ODs were taken according to the manufacturer's guidelines at a wavelength of 450nm with reference wavelength of 540nm. This dual wavelength system is well established and very specific in eliminating optical imperfections at well to well level whereas measurements taken at 450 nm alone may result in non-specific absorbance.

All these experiments were performed in triplicate. The control for the experiment was zero ng/mL concentration of human fetuin A. The OD of the control was subtracted from the ODs of all assay points.

As a control, a conventional human fetuin A sandwich ELISA procedure on normal microtiter plate coated with anti-human fetuin A by passive adsorption was also performed. The anti- human fetuin A-adsorbed normal microtiter plate was blocked with 1% v/v BSA for 2 hrs at RT and subsequently washed five times with 300 μL of PBS. Varying concentrations of human fetuin A (from 4.8 pg/mL to 20 ng/mL) were prepared and 100 μL of each of these concentrations were then incubated onto the antibody-coated plates for 2 hrs at RT and subsequently washed five times with 300 μL of PBS. 100 μL of biotinylated anti-human fetuin A detection antibody (200 ng/mL) was then added and incubated for 2 hrs at RT followed by five washes with 300 μL of PBS. lOOμL of HRP-conjugated streptavidin at a dilution of 1:200 was provided to each of the used ELISA plate wells and then incubated for 20 min at RT followed by washing five times with 300 μL of PBS. TMB substrate was subsequently added to each of the used ELISA plate wells. The HRP enzyme-TMB substrate reaction was then stopped after 20 min by adding 50 μL of IN H₂SO₄ and ODs were taken according to the manufacturer's guidelines at a wavelength of 450nm with reference wavelength of 540nm. This dual wavelength system is well established and very specific in eliminating optical imperfections at well to well level whereas measurements taken at 450 nm alone may result in non-specific absorbance.

Results

The human fetuin A sandwich ELISA procedure was performed on normal Nunc 96-well flat bottom plate made up of polystyrene. Fig. 26 shows the assay curve obtained by the human fetuin A sandwich ELISA. The assay had a detection range of 9.67 - 20,000 pg/ml and a %CV of 1.23 to 9.76.

The advantages of the human fetuin A sandwich ELISA procedure over the conventional ELISA procedure were determined by performing normal ELISA based on the passive adsorption of anti-human fetuin A and comparing the results.

The conventional human fetuin A sandwich ELISA was performed on a normal Nunc 96-well flat bottom plate made up of polystyrene on which the anti-human fetuin A antibody was bound passively by adsorption.

Fig. 25 shows the results of the conventional human fetuin A sandwich ELISA which had a detection range of 156.16 - 20,000 pg/ml and %CV of 4.72 - 17.38. The conventional human fetuin A sandwich ELISA procedure on a normal 96-well polystyrene microtiter plates had % CV in the range of 4.72 - 17.38 which is much higher than the % CV of 1.23 - 9.76 obtained with the modified sandwich ELISA procedure described herein.

The sandwich ELISA method described herein had a higher detection range for human fetuin A i.e. 9.76 - 20,000 pg/mL compared to that of conventional sandwich ELISA which was only 156.16 - 20,000 pg/mL. Thus, the sandwich ELISA described herein had less variability and a higher detection range resulting in a higher sensitivity compared to the conventional sandwich ELISA format.

The human fetuin A sandwich ELISA method described herein is rapid compared to a conventional ELISA. The ELISA procedure described herein takes only about 6 hrs from start to finish whereas the conventional ELISA procedure takes about 24 hrs.

Example 11 - Selection of base substrates for immunoassays

A modified microtiter plate, having various base substrates were made using the method of Example 1 above and used for an ELISA. Mouse IgG was immobilized in an orientated site directed fashion using the method of Examples 4 and 5 above. Thereafter, HRP labeled goat anti-mouse IgG (12 μg/ml) was bound to the mouse IgG using the method of Example 5 above and the amount of bound antibody was detected by taking optical density (O.D.) readings after stopping the TMB substrate reaction (Example 6 above) after 3 min. The average O.D. reading provided an estimate of the suitability of a particular substrate for a specific immunobiosensor application. The results of this experiment are shown in Fig. 27 and Table 3 below.

Table 3 — Results of ELISA on different support materials

The invention is not limited to the embodiment hereinbefore described, with reference to the accompanying drawings, which may be varied in construction and detail.

Claims

1. A multi-well plate for a biological assay comprising a plate frame attached to a support substrate through a gasket wherein the plate frame defines side walls for a plurality of wells and the gasket comprises a plurality of holes, whereby the holes of the gasket are substantially aligned with the wells defined by the plate frame such that the support substrate forms a base for the wells and wherein the side walls of the wells comprise an absorbed layer of blocking agent.

2. A multi-well plate as claimed in claim 1 wherein the support substrate comprises two or more different substrate materials.

3. A multi-well plate as claimed in claim 2 wherein the different substrate materials are arranged side by side to provide a substantially flat support surface.

4. A multi-well plate as claimed in any one of claims 1 to 3 wherein the substrate is selected from the group comprising: Zeonex™, Zeonor™, polystyrene, polycarbonate, PMMA, cellulose acetate and glass.

5. A multi-well plate as claimed in any one of claims 1 to 4 wherein the substrate comprises functional surface amine groups for binding to a protein.

6. A multi-well plate as claimed in claim 5 wherein the protein is an affinity protein.

7. A multi-well plate as claimed in claim 6 wherein the affinity protein is an Fc binding protein and/or an antibody.

8. A method for preparing a multi-well plate for a biological assay comprising the steps of:

- providing a multi-well plate frame defining side walls of a plurality of bottomless wells; - blocking the side walls of the wells with a blocking agent;

- providing a gasket comprising a plurality of holes corresponding in size and number to the wells of the multi-well plate frame; aligning the holes of the gasket with the wells of the multi-well plate frame; attaching the gasket to the base of the multi-well plate frame; and - attaching a support substrate to the gasket such that the gasket is sandwiched between the multi-well plate frame and support substrate thereby providing a water tight seal.

9. A method for preparing a multi-well plate for a biological assay comprising the steps of:

- providing a multi-well plate comprising a plurality of wells, each well having a base; blocking the wells with a blocking agent; removing the base of the wells; - providing a gasket comprising a plurality of holes corresponding in size and number to the wells of the multi-well plate;

- aligning the holes of the gasket with the wells of the multi-well plate;

- attaching the gasket to the base of the multi-well plate; and

10. A method as claimed in claim 8 or 9 wherein the support substrate comprises two or more different substrate materials.

I L A method as claimed in claim 10 wherein the different substrate materials are arranged side by side to provide a substantially flat support surface.

12. A method as claimed in any one of claims 8 to 11 wherein the substrate is selected from the group comprising: Zeonex™, Zeonor™, polystyrene, polycarbonate, PMMA, cellulose acetate and glass.

13. A method as claimed in any one of claims 8 to 12 wherein the substrate comprises functional surface amine groups for binding to a protein.

14. A method as claimed in claim 13 wherein the protein is an affinity protein.

15. A method as claimed in claim 14 wherein the affinity protein is an Fc binding protein and/or an antibody.

16. A support substrate for a biological assay comprising functional surface amine groups for binding to a protein.

17. A support substrate as claimed in claim 16 wherein the protein is an affinity protein.

18. A support substrate as claimed in claim 17 the affinity protein is an Fc binding protein and/or an antibody.

19. A support substrate as claimed in claim 18 wherein the Fc binding protein is selected from: Protein A, Protein G and Protein A/G.

20. A support substrate as claimed in claim 19 wherein the substrate comprises a capture antibody linked to the Fc binding protein.

21. A support substrate as claimed in any one of claims 16 to 20 wherein the support substrate comprises two or more different substrate materials.

22. A support substrate as claimed in claim 21 wherein the different substrate materials are arranged side by side to provide a substantially flat support surface.

23. A support substrate as claimed in any one of claims 16 to 22 wherein the substrate is selected from the group comprising: Zeonex™, Zeonor™, polystyrene, polycarbonate, PMMA, cellulose acetate and glass.

24. A method for immobilising a protein on a multi-well plate for a biological assay comprising the steps of: a) providing a multi-well plate as claimed in any one of claims 1 to 4; b) cleaning the surface of support substrate; c) generating hydroxyl groups on the surface of the support substrate; d) inducing functional amine groups on the surface of the support substrate; and e) cross linking a protein to the thus formed functional amine groups.

25. A method as claimed in claim 24 wherein step (b) comprises treating the support substrate with absolute ethanol at a temperature of about 37°C for about 5 minutes.

26. A method as claimed in claim 24 or 25 wherein step (c) comprises treating the support substrate with about 1% (w/v) potassium hydroxide.

27. A method as claimed in claim 26 wherein the substrate is treated at a temperature of about 37°C.

28. A method as claimed in claim 27 wherein the substrate is treated for about 10 minutes.

29. A method as claimed in any one of claims 24 to 28 wherein step (a) comprises treating the support substrate with about 2% (w/v) 3 -APTES.

30. A method as claimed in claim 29 wherein the substrate is treated at about room temperature.

31. A method as claimed in claim 30 wherein the substrate is treated for about 1 hour.

32. A method as claimed in any one of claims 24 to 31 wherein step (e) comprises the steps of: i) forming a cross-linking solution; ii) mixing the protein with the cross-linking solution; and iii) incubating the support substrate with the protein-cross-linking solution.

33. A method as claimed in claim 32 wherein the cross-linking solution comprises 1 -ethyl 3- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).

34. A method as claimed in claim 32 or 33 wherein the cross-linking solution comprises sulfo-N-Hydroxysuccinmide.

35. A method as claimed in any of claims 32 to 34 wherein the protein and cross-linking solution are mixed for about 15 minutes prior to step (iii).

36. A method as claimed in any one of claims 32 to 35 wherein step (iii) comprises incubating the support substrate with the protein-cross-linking solution at a temperature of about 37°C.

37. A method as claimed in claim 36 wherein the support substrate is incubated with the protein-cross-linking solution for about 1 hour.