WO2014198836A1 - Elisa system and related methods - Google Patents

Abstract

The invention relates to an enzyme-linked immunosorbent assay (ELISA) device and system comprising an upper element to which multiple protruding elements are bound, wherein the surface of said protruding elements is coated with capture affinity reagent. The system comprises a corresponding receptacle, an illuminating detection unit and/or an adaptor unit suitable for use with a mobile image acquisition and computing device. The invention also relates to corresponding methods and kits for carrying out said methods.

Description

ELISA SYSTEM AND RELATED METHODS

DESCRIPTION

The invention relates to an enzyme-linked immunosorbent assay (ELISA) device and system comprising an upper element to which multiple protruding elements are bound, wherein the surface of said protruding elements is coated with capture affinity reagent. The system comprises a corresponding receptacle, an illuminating detection unit and/or an adaptor unit suitable for use with a mobile image acquisition and computing device. The invention also relates to corresponding methods and kits for carrying out said methods. The invention relates to the fields of biotechnology, immunoassays, medical diagnostics, and diagnostic devices and methods based on in vitro immune reactions.

BACKGROUND OF THE INVENTION

The Enzyme-Linked Immunosorbent Assay, or ELISA, is a well accepted and widely utilized method used in both qualitative and quantitative assays for biomolecules. The most commonly and widely used sandwich ELISA is based on the initial detection of analyte by the capture antibodies against that analyte, which is followed by the binding of analyte to the biotinylated detection antibody against that analyte. Thereafter, enzyme-labeled streptavidin binds to the biotinylated detection antibody in sandwich immune complex, which is subsequently provided with the enzyme substrate, thereby leading to the formation of colored product. The formation of colored product can be qualitatively observed or quantitatively measured by absorption spectrophotometry or other means. The rate and extent of color formation is proportional to the concentration of antigen. There are numerous other variations of this assay, using various enzymes and chromogenic substrates. Typically, a microtitre plate is used to immobilize capture affinity reagents, which subsequently bind the analyte (such as an antigen) from a liquid sample. A further affinity reagent coupled to an enzyme is then used to bind the analyte and enzyme activity assayed.

The conventional washing procedure in such a method after each step requires the microtitre plate (MTP) to be inverted, or requires a sophisticated instrument, such as an ELISA washer. The commercially-available sandwich ELISA procedure is a relatively complex and time intensive procedure, as it requires many steps and requires the MTP to be inverted after each step. Therefore, the conventional sandwich ELISA, which is currently used in commercial in vitro diagnostic (IVD) kits, requires a highly skilled analyst. The plate inversion can also lead to significant cross-contamination between wells, if the analyst is less skilled.

Recently, further methods and devices have been developed in order to improve various steps of the procedure. Alternative technical solutions address the decrease in time of an ELISA assay using a microfluidics approach, such as used by Siloam, USA (www.siloamsci.com), by designing a microfluidics-capable MTP. Invitrogen has alternatively developed the wash-free AlphaLISA using donor and acceptor beads that are bound to detection and capture antibodies, respectively. However, these attempts at improving the ELISA procedure have significantly increased the production cost through the microfluidics-based MTPs and the use of beads. Moreover, these approaches still require the sample analytical procedures of dispensing, blocking and washing, which require a high level of competence and experience with regard to the analyst's skills.

Alternatively, the ELISA procedure has been modified by finding improved approaches towards immobilisation of capture affinity agents. Various documents of the prior art describe means for developing multisubstrate-compatible multistep ELISA procedures based on the covalent and leach-proof binding of antibodies for ELISA-based IVD (WO/2010/044083; WO/2009/066275; Nature Protocols 6(4), 439-445, 201 1 ; Analytical Chemistry 82(16), 7049-52, 2010; Diagnostics 2, 23-33, 2012; Procedia Chem. 6, 184-193, 2012; Procedia Chem. 6, 141 -148, 2012).

Attempts have been made in related technical fields to develop alternative systems to conventional ELISA. For example, devices are disclosed in EP 0154687 A2, WO 03/085401 A1 and WO 03/042697 A1 for use in immunoassays that implement attachment of a capture reagent to solid phase protrusions, which are suitable for immersion in a reservoir containing an analyte to be captured.

These known systems are however characterised by a number of disadvantages. Earlier attempts at alternative ELISA systems do not employ means for alignment of the protruding elements with the reservoir in which the element is to be submerged. This lack of structural guidance during use of the device in an ELISA method leads to physical contact between the protruding elements and reservoirs, resulting in scratching, loss of capture reagent and subsequently significant non-uniformity between the multiple reactions.

Especially in the context of clinical applications, where a potentially large number of samples must be quantitatively and reliably assayed, loss of capture reagent on any one of the protruding elements will introduce significant false-negative results in the affected wells.

Furthermore, reliable means for aligning the protruding elements with the sample reservoirs is of particular importance in a mobile analytic device, intended for quick and easy application, potentially by non- or low-skilled practitioners wishing to carry out an immunoassay quickly and effectively.

Further disadvantages of previously published ELISA systems of the prior art relate to the absence of a simple, portable light source for illumination of the samples to be analysed, which is integrated into the device, or could be easily combined with or integrated into the device. The systems of the prior art were designed to be analysed using complex microplate readers, which themselves comprise suitable light sources, but are bulky and non-portable. No suggestion for improved portable, and preferably integrated, light sources are provided for alternative ELISA systems in the prior art.

Portable systems for sample analysis based on light emission have been disclosed in the art for non-ELISA applications. For example WO 01/31341 A1 discloses a portable microplate with a substrate attached thereon comprising a light-emitting compound. According to this disclosure, a sample is provided onto the microplate, wherein the analyte within the sample then directly reacts with the light-emitting compound, thereby enabling light emission, which is used as signal readout. This system is however not suitable for an ELISA reaction and no mention is made of a light source separate from the reaction itself, which may be used to illuminate a sample well for subsequent analysis.

The prior art also fails to disclose or suggest improved means for preferably portable and/or disposable devices and systems for carrying out ELISA procedures without the need for highly- skilled personnel. Some inroads have been made in the art with respect to adapting cameras and smart phone technology with portable analytic approaches. For example Coskun et al (Lab Chip, 2013, 13, 636) disclose the use of a personalised food allergy testing platform utilising a mobile phone camera capable of analysing colourimetric assays in test tubes. The mechanical attachment required for cell phone analysis is however bulky and non-portable, and the system is incapable of analysing multiple samples in parallel.

This prior art has provided improvements with respect to antibody immobilisation procedures over traditional approaches and the provision of ELISA systems comprising protruding elements, to which capture reagents may be attached. However, no significant improvements have been achieved with regard to reducing and simplifying ELISA incubation and washing procedures till date, in order to speed up the method, reduce risk of contamination, simplify the assay procedure, and maintain accuracy and low costs. Furthermore, the systems known in the art are not portable and do not enable efficient and reliable ELISA testing without the need for highly skilled personnel or complex image analysis systems.

SUMMARY OF THE INVENTION

In light of the prior art, the technical problem underlying the invention was the provision of means, devices and/or methods for reducing complexity of and therefore simplifying existing ELISA procedures, reducing washing and/or complexity of washing, enabling a faster ELISA procedure and/or reducing cost of the components required for ELISA procedures.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

Therefore, an object of the invention is to provide an enzyme-linked immunosorbent assay (ELISA) system comprising

- an upper element to which multiple protruding elements are attached, said protruding elements positioned parallel to one another on one side of said upper element, wherein said protruding elements are bound on their surface with capture affinity reagent and said capture affinity reagent is capable of binding an analyte in solution; and

- a receptacle, said receptacle comprising multiple wells suitable for holding a solution, wherein said wells are of dimensions that enable the insertion of said protruding elements of the upper element into said wells without contact between the surfaces of the interior of said wells and said protruding elements, and one protruding element may be inserted per well; wherein

- means for alignment of the protruding elements with the receptacle wells are present on the upper element and/or receptacle, wherein said means for alignment enable insertion of said protruding elements into the wells of said receptacle in a (pre-) determined position.

The system may also be referred to as a device. The device or system of the present invention enables the improved washing procedures, thereby reducing processing time and risk of contamination, whilst increasing efficiency. It was an entirely surprising and beneficial development that the device as described herein would enable reduced washing times and complexity in existing ELISA procedures. Until the present time, no such approach has been suggested in the art, as a skilled person would have assumed that more extensive and time intensive washing and incubation steps are required for ELISA methods.

According to the present invention, the upper element may also be referred to as a lid. The term upper refers to the relative positioning of the protruding elements (which protrude downwards during use), which are of a relatively lower position than the lid. The protruding elements may be referred to as rods or antigen or antibody binding elements or structures.

The "easy ELISA" or "eELISA" format of the present invention is based in a preferred embodiment on the dipping of a lid, containing antibody-bound structures, into MTP wells. The present invention therefore completely obviates the traditional washing procedure as it doesn't require the MTP to be inverted at any stage. The washing is executed by simple dipping of the structures attached to the lid into the washing buffer in MTP wells.

The invention as described herein is highly-simplified, low-cost, removes the need for a skilled analyst, removes the complex washing procedure, shows no cross-contamination and is compatible with conventional ELISA reader detection systems (MTP reader-based). The technology has been demonstrated by way of example herein for the development of rapid C- reactive protein (CRP) sandwich ELISA.

The device and system of the present invention solve the technical problems of reducing the time and complexity required for the washing and/or incubation steps in sandwich ELISA procedures as currently known. It was an unexpected finding that a receptacle designed to correspond to the dimensions of the protruding elements would provide sufficient incubation and binding, and washing, of bound analyte. Typical approaches in the art involve extensive agitation or inversion of bound samples. The use of an additional receptacle suitable for comprising sample and wash solutions has not previously been proposed as a suitable format for an ELISA system.

In one embodiment, the system of the present invention is characterised in that means for alignment of the protruding elements with the receptacle wells are present on the device and/or receptacle, or on a separate device such as receptacle holder, for example support structures attached on the receptacle for positioning the upper element. Alignment means may relate to one or more alignment posts and/or alignment jigs and corresponding apertures or depressions, that are present on the upper element and receptacle, wherein said means for alignment enable insertion of said protruding elements into the wells of said receptacle in a pre-determined position.

The alignment means enable reduction of misplacement of the upper element into the wrong wells and may provide a fixed depth of insertion and/or lateral displacement, thereby reducing the risk of unwanted contact between the receptacle wells and the protruding elements. In particular, the combination of upper element, receptacle, hoisting element and means for alignment, especially in the context of manual handling, provides a synergistic effect by enabling a relatively unskilled practitioner to carry out the ELISA method at high throughput (multi-well assays) with high efficiency and reduced risk of sample contamination. Clinical settings or robotic or complicated automated systems can additionally be avoided.

The system is therefore preferably characterised in that the means for alignment are one or more alignment posts (alignment jigs) and corresponding apertures (or depressions), wherein said posts and apertures (or depressions) are present on the receptacle and upper element, respectively, or vice versa, and enable insertion of said posts into said apertures (or

depressions). The posts may therefore be present on either the upper element or receptacle, and vice versa.

In one embodiment, the system of the present invention is characterised in that the alignment posts (jigs) are of a length greater than the length of the protruding element, in order to position the upper element, prior to potential contact with the wells, in a (pre-) determined position for immersion.

In one embodiment, the system of the present invention is characterised in that (preferably multiple) different shapes of the alignment posts (alignment jigs) and corresponding apertures (or depressions) are employed at different locations on the upper element and receptacle. Such embodiments eliminate confusion regarding orientation of the upper element with respect to the receptacle.

In a preferred embodiment of the invention, the system is characterised in that the upper element is of a planar structure. Said protruding elements are preferably positioned in multiple rows, wherein each row comprises multiple protruding elements, for example positioned according to the nodes of a grid. The protruding elements must not necessarily be positioned in straight lines or in a grid shape, but also potentially in any given positions distributed in two dimensions upon a surface of the planar structure of said upper element. Similar systems of the prior art are limited to single rows of samples, such as combs, thereby significantly limiting throughput and reproducibility of an assay. The planar upper element enables larger numbers of samples to be analysed and reliable and uniform positioning, thereby increasing efficiency and reproducibility of the analysis of large numbers of samples. In one embodiment, the system of the present invention is characterised in that the protruding elements comprise of material, to which the capture affinity reagent is immobilized, wherein said material is preferably selected from polystyrene (PS), polypropylene (PP), polyethylene PE), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), water- resistant photopolymer, such as Watershed® XC 1 1 122, cyclic olefin copolymer such as

TOPAS®, cyclo-olefin polymer such as Zeonor® and Zeonex®, cellulose and its derivatives, silicon and its derivatives, glass, quartz, or metals.

In a preferred embodiment, the system of the present invention is characterised in that the capture affinity reagent is a protein, such as an antibody, antibody fragment, a recombinant protein, Fc-binding protein (protein A, protein G, protein A/G), streptavidin, or recombinant proteins or fragments thereof, nucleic acid, polysaccharide, glycoprotein, peptidoglycans, molecular imprinted polymer or aptamer molecule. Various biological affinity agents may be applied in the invention. An antibody is the preferred affinity agent, as is commonly used in ELISA systems.

In one embodiment, the system of the present invention is characterised in that the upper element comprises means for securing and/or hoisting said upper element, thereby enabling vertical displacement of the upper element from the receptacle, wherein said means for hoisting are preferably positioned on the opposite side of said upper element to the protruding elements. The means for securing and/or hoisting may refer to a handle, latch, hook and/or protruding segment of the upper element, which the user may manually hold or integrate into an automated or robotic microtitre plate system. The combination of upper element, receptacle and hoisting element leads to a synergistic effect, providing vastly reduced processing times with regard to binding and washing multiple samples in an ELISA format.

In one embodiment, the system of the present invention is characterised in that the protruding elements are arranged to fit in a receptacle that is a microtitre plate, preferably a microtitre plate comprising 24, 48 or 96 microtitre plate wells, a microtitre disc, comprising preferably 72 microtitre wells, a microtitre platform suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 36 microtitre wells, or a microtitre cartridge suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 24 microtitre wells. The upper element may therefore comprise of preferably 1 , 6, 9, 18, 24, 48 or 96 protruding elements, depending on the scale or number of samples to be processed.

The upper element may comprise a number of protruding elements different from the number of wells comprised in the receptacle. For example, the upper element may comprise fewer protruding elements than wells. This embodiment is particularly relevant in situations where a single 96 well receptacle (microtiter plate) is used for analysis of 24 samples. In such an embodiment, the upper element may comprise 24 protruding elements, wherein the 24 protruding elements are incubated in sample, washed twice in two separate sets of 24 wells, and then immersed in detection substrate before analysis, enabling analysis of 24 samples in a fast and efficient manner using a single 96 well microtiter plate. In one embodiment, the system of the invention is characterised in that the receptacle is a microtiter plate in the shape of a disc, preferably a round or oval disc, comprising preferably 72 microtiter wells. In this embodiment, the means for alignment preferably relate to an

approximately centrally located post, to which the upper element is attached and about which the upper element may rotate, wherein said post is aligned with the wells of the receptacle, preferably in a fixed position. The post may be additionally characterised by notches or other structural features that determine alignment of the protruding element with the receptacle wells. Examples of such positioning mechanisms, based on particular angles of rotation, and the corresponding wells, can be observed in the figures.

In one embodiment, the system of the invention is characterised in that the microtiter plate is suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 36 microtiter wells, or a microtiter cartridge suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 24 microtiter wells.

In further embodiments, the system of the present invention is characterised in that the protruding elements are cylindrical or of rectangular shape and are solid or hollow. The rods of the device may have the lesser, same or greater functional surface area as being used in a standard 96-well microtitre plate. Based on the different formats provided, for example a microtitre plate, microtitre disk, microtitre smartphone platform or microtitre smartphone cartridge, the size of the rods can be changed or customized to match the desired assay sensitivity.

The invention also relates in further embodiments to a system, wherein the capture affinity reagent is bound to the protruding elements by:

covalent attachment of said affinity reagent with said protruding element, preferably via an amide bond, or

by magnetic force, wherein said protruding elements are magnetic and bind magnetic beads that are attached to the capture affinity reagent.

In one embodiment, the system of the invention is characterised in that said system comprises an illuminating detection unit, wherein said unit comprises an artificial light source and a platform for positioning the receptacle (detection plate).

In one embodiment, the system of the invention is characterised in that the illuminating detection unit is positioned below the receptacle and is of dimensions and illumination strength suitable for providing essentially uniform illumination to the wells of the receptacle to be analysed. Examples are provided below. For example, 24 LEDs may be arranged with even spacing, preferably on a printed circuit board, and whereby essentially each LED provides illumination to 4 wells of a 96 well plate. Preferably, a diffusion element is present between receptacle and light source, thereby providing even lighting to all wells. In a further embodiment 8 LEDs may be employed to illuminate 24 wells. In one embodiment, the system of the invention is characterised in that said system comprises a perforated covering element positioned between the light source and the receptacle, wherein the perforations correspond to the size and/or shape of the wells of the receptacle to be analysed. The perforated covering element provides surprisingly good definition of signal boundaries for subsequent analysis, especially when analysis is conducted using a mobile image acquisition and computing device (smart phone).

The perforated covering element may also be one aspect of the receptacle, if so required. The receptacle may comprise of two parts, relating to a dish compromising the wells of the receptacle (such as a MTP) and additionally a dish holder comprising the means for alignment (for example refer to figure 1 ). The dish holder may in one embodiment represent the perforated covering element positioned between the light source and platform for positioning the receptacle, wherein the perforations correspond to the size and/or shape of the wells of the receptacle to be analysed.

The invention also relates to an illuminating detection unit, as such or in combination with the system described herein, suitable for an ELISA assay.

In one embodiment, the system of the invention, or illumination unit itself, is characterised in that the illuminating detection unit comprises LEDs as an artificial light source, preferably white LEDs.

In one embodiment, the system of the invention, or illumination unit itself, is characterised in that the illuminating detection unit comprises an array of uniformly arranged or spaced LEDs below the wells of the receptacle to be analysed.

In one embodiment, the system of the invention, or illumination unit itself, is characterised in that the illuminating detection unit comprises an LED light source, wherein the light from said LED is directed onto a reflective surface (mirror), wherein said reflective surface directs the light from the LED to the wells of the receptacle to be analysed.

In one embodiment, the system of the invention, or illumination unit itself, is characterised in that the illuminating detection unit comprises one or more light diffusing elements, in order to provide essentially uniform illumination to the wells of the receptacle to be analysed.

The illuminating device may also be described as a backlit illuminating device for providing sufficient light for analysis and evaluation of a receptacle after an ELISA reaction. In a preferred embodiment, 24 LEDs are placed beneath a 96-well MTP plate in an array of 6 x 4 matrix. It is preferred that each LED is placed in the middle between four MTP wells for even illumination. The LEDs are preferably connected in parallel to the voltage source, preferably through a variable resistor. Examples of circuits for driving a RGB LED with a pulse-width modulation (PWM) controller are provided below.

In a preferred embodiment, the system of the present invention comprises a mobile image acquisition and computing device with a camera (smart phone), the use of such a device. In a preferred embodiment, the system of the present invention or the illuminating detection unit comprises an adaptor unit for use of a mobile image acquisition and computing device with a camera (smart phone), wherein the adaptor comprises a hollow sealed enclosure that is suitable for preventing interference from ambient light, such as a light impermeable box, preferably to be positioned between the light source and said image acquisition device, preferably between the receptacle comprising the wells to be analysed and said image acquisition device.

In a preferred embodiment, the system of the present invention or the illuminating detection unit is characterised in that the adaptor unit comprises means for alignment of the mobile image acquisition and computing device with the adaptor, such as alignment posts and/or alignment jigs, wherein the camera of the mobile image acquisition and computing device can be positioned at a fixed distance and position from the receptacle to allow acquisition of an image of one or more, preferably all, of the wells of the receptacle to be analysed.

The invention therefore relates to an adaptor unit, as described herein, for the application of a mobile image acquisition and computing device with a camera (smart phone) in the analysis or evaluation of ELISA results. A mobile image acquisition and computing device with a camera is to be understood as any device capable of image acquisition, such as a camera, mobile phone, tablet, portable entertainment system, which also contains sufficient computing hardware to process the acquired images and enable measurement and/or quantification of the reaction products of an ELISA test. A smartphone is a preferred example of a mobile image acquisition and computing device. The term smartphone is known in the art and commonly considered to encompass devices with or without telephoning capabilities, defined generally but not exclusively by mobile, preferably handheld, devices comprising computer processors capable of executing software and in some embodiments capabilities for wireless internet connection.

In a preferred embodiment, the invention is characterised in that the system is of a length and breadth essentially that of a 96 well microtiter plate. For example, the system may be exhibit a length or breadth of approximately 100 mm greater than the length or breadth of a microtiter plate, preferably 80 mm, or more preferably 50 mm or 20 mm greater than the length or breadth of a microtiter plate. 96 well microtiter plates are known to a skilled person and are

approximately 128 mm x 86 mm in length and breadth. A microtiter plate may also be referred to as a microplate. The microplates used herein may relate to any given size or design. Preferably standard microplates are used according to ANSI/SLAS 1-2004 (formerly recognized as ANSI/SBS 1-2004). The depth of the plate (and wells of the receptacle) and the system itself may be adjusted according to sample size, and size of the protruding element.

A further aspect of the invention relates to a system as described herein characterised by software designed for the detection of immune complex formation in an ELISA assay. The invention therefore also relates to a software as such as described herein.

In one embodiment, the system of the invention, or software itself, is characterised in that the software detects colour (for example, colour intensity and/or colour formation) in an acquired image. In one embodiment, the system of the invention, or software itself, is characterised in that the detection of colour encompasses processing of the acquired image by an algorithm in which the colour image is split into red, green and blue channels, wherein said image processing procedure comprises (automatic) determination of the centre of each receptacle well, extraction of mean pixel value of its neighbouring pixels and providing the composite pixel value (for example 1 - 256) by assessing the contributions from the individual red, green and blue channels, wherein the processing of the acquired image enables prediction of optical density from the corresponding composite pixel value.

A further aspect of the invention relates to an enzyme-linked immunosorbent assay (ELISA) method comprising

provision of an upper element (lid) to which multiple protruding elements (rods) are attached, said protruding elements positioned parallel to one another on one side of said upper element, wherein said protruding elements are bound on their surface with capture affinity reagent, said capture affinity reagent being capable of binding an analyte in a liquid sample, - incubation via submersion of said protruding elements in one or more solutions comprising the analyte to be detected, preferably for 5 to 30 minutes, more preferably 10 to 20 minutes, thereby binding said analyte to said protruding elements via the capture affinity reagent, wherein

^■ said incubation is carried out in a receptacle, said receptacle comprising multiple wells holding analyte solution, wherein said wells are of dimensions that enable the insertion of said protruding elements into said wells without contact between the surface of the interior of said wells and said protruding elements, and one protruding element may be inserted per well, wherein means for alignment of the protruding elements with the receptacle wells are present on the upper element and/or receptacle, wherein said means for alignment enable insertion of said protruding elements into the wells of said receptacle in a (pre-) determined position, and wherein

^■ said analyte-comprising solution comprises preferably additional detection

reagents, such as biotinylated anti-analyte-affinity reagent (detection antibody) pre-conjugated to horse radish peroxidase (HRP) labeled-streptavidin, vertical displacement of the upper element from the receptacle to remove the protruding elements from wells containing analyte solution,

washing of the protruding element upon which analyte is bound to said immobilized capture affinity reagent, and

detection of immune complex formation between said analyte, said immobilized capture affinity reagent, and said detection reagent either by microplate reader or by a mobile image acquisition and computing device with a camera (smart phone). The eELISA sandwich immunoassay procedure, as described herein, has fewer process steps compared to traditional ELISA, as the immune complex is formed preferably in a single step, whereby the analyte sample is provided to the MTP wells containing biotinylated detection antibody preconjugated to HRP-labeled streptavidin. This is followed by immersing the lid containing capture antibody-bound structures into the MTP. Subsequently, the procedure employs 2 wash steps followed by dipping in enzymatic substrate for colorimetric reaction. The eELISA of the present invention obviates the cross-contamination issues because the liquid cannot flow from one to another well as the MTP is not inverted at any stage.

The absence of inverting the MTP after all process steps is, in comparison to conventional sandwich ELISA, a novel development of the prior art associated with unexpected advantages. The method as described herein, enabled by the device as described herein, requires less time, is less likely to show contamination or failure and is therefore more efficient. The costs for implementing the device and methods of the present invention are significantly reduced compared to other approaches to solve these technical problems.

The invention also relates to a method in which the protruding elements are of magnetic nature, capable of attracting magnetic particles through magnetic force. A skilled person is familiar with magnetic beads, to which affinity reagents may be attached. The use of magnetic protruding elements, preferably covered with disposable polymer/composite/material, allows binding of beads that are bound to the analyte and/or ELISA sandwich complex, thereby providing means for binding capture affinity reagent to the protruding elements of the device of the present invention. According to the method described herein, the binding of the capture affinity reagent to the protruding element can be induced after incubation of the capture affinity reagent with analyte, when for example said affinity reagent is coupled or bound to magnetic beads. The analyte can then be bound by the bead-affinity reagent in solution and the protruding elements subsequently inserted into the incubation wells, the magnetic force induced and then the protruding element withdrawn and washed. This method can also be carried out with affinity reagent bound detection enzyme (HRP) in order to form the ELISA sandwich complexes in solution between bead-affinity reagent, analyte and affinity reagent-detection enzyme, followed by penetration of the protruding element into the reaction well, induction of magnetic force, removal of protruding element and subsequent washing.

In a preferred embodiment of the method, the washing of the protruding element is carried out by submersion of protruding elements in wells of the receptacle containing wash buffer, so that the affinity agent of the protruding agent is immersed in analyte containing solution, followed by vertical displacement of the upper element from the receptacle to remove the protruding elements from wells containing wash buffer. The non-specifically bound reagents and molecules are thereby washed off from the protruding elements.

In one embodiment of the method, 1 to 3 washes (wash steps), preferably 2 washes, of the protruding elements are carried out, wherein each submersion of the protruding elements in wash buffer is carried out for 1 second to 5 minutes, preferably 2 seconds to 1 minute, more preferably 5 seconds to 30 seconds. In a preferred embodiment, no inversion of the receptacle is required for washing. Agitation of the receptacle is unnecessary but may be conducted in some embodiments of the invention.

In one embodiment of the method, detection of immune complex formation between said analyte, said immobilized capture affinity reagent, and said detection reagent is carried out by - submersion of protruding elements in wells of the receptacle comprising detection substrate, such as HRP substrate, preferably 3,3',5,5'-Tetramethylbenzidine (TMB),

incubation of said protruding elements in wells comprising detection substrate, preferably for 1 to 10 minutes, more preferably 2 to 5 minutes, followed by

vertical displacement of the upper element from the receptacle to remove the protruding elements from wells containing detection substrate, and

subsequent detection of signal in the well comprising detection substrate, preferably via TMB substrate assay and measurement of optical density and/or colour.

The enzymatic reaction is ended by removal of the protruding element from the detection solution in the receptacle.

The sequential displacement of protruding elements from one to another set of wells in the receptacles, meant for binding, washing and detection steps, can be automated, preferably using microcontroller-controlled stepper motors for movement along horizontal and vertical axis.

The detection of immune complex formation can in one embodiment of the method be carried out by detecting color formation after an enzyme-substrate reaction using the system according to any one of the preceding claims and a mobile image acquisition and computing device with a camera (smart phone), comprising

positioning the receptacle on an illuminating detection unit of claim 8,

image acquisition of the wells of the receptacle to be analysed using an adaptor unit of claim 9 with the camera of the mobile image acquisition and computing device,

- processing of the acquired image by an algorithm, in which the color image is split into red, green and blue channels, the image processing procedure comprising automatic

determination of the centre of each receptacle (reaction) well, extraction of mean pixel value of its neighbouring pixels and providing the composite pixel value (for example 1 - 256) by assessing the contributions from the individual red, green and blue channels, wherein the processing of the acquired image enables prediction of optical density from the

corresponding composite pixel value after normalization.

Processing of the acquired images could also be carried out by the following steps: image capture, split color channels (RGB), identify the centre of the well, find the mean of the defined neighboring pixels, compute composite pixel value (10% red, 20% green, 70% blue), plot the pixel value against its corresponding standard analyte concentration, and quantify the analyte concentration, for example from a clinical sample, using a calibration curve.

An alternative image processing algorithm could be applied either separately from or together with the image processing methods described herein, for example, comprising weight grey scale conversion, defining pixel scan area, averaging the pixel area, plot the pixel value of calibration points with sigmoid function, calculate the analyte concentration based on a calibration plot and display the analyte concentration in SI units i.e. g/L along with the corresponding absorbance units (optical density).

The present invention therefore relates to software or a computer programme for execution on a computing device, which carries out the image analysis methods as described herein.

The invention also relates to a kit for carrying out the method as described herein, comprising the enzyme-linked immunosorbent assay (ELISA) device or system of the present invention, and preferably additional components for carrying out the method as described, such as

one or more suitable receptacles, preferably in a microtitre format, such as one or more microtitre plates, disks, or platforms or cartridges, preferably suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone),

immunoassay components, whereby the immunoassay components are preferably pre- stored in said receptacles, disks, platforms and/or cartridges, which are preferably covered with a sealing film and/or foil suitable for sterile packaging,

illuminating detection unit according to claim 8 suitable for the aforementioned microtitre formats,

software for a mobile image acquisition and computing device with a camera (smart phone) for executing the image processing algorithm of claim 14, and/or

wash buffers and/or reagents for detection of the analyte-affinity reagent interaction.

The easy ELISA (eELISA) technology as described herein represents a key technology for the development of in vitro diagnostic (IVD) kits for healthcare, industrial, food safety, environmental monitoring and many other bioanalytical applications. The easy and highly simplified assay technology and formats will enable a layman with basic skills to perform the sandwich assays i.e. ELISA, chemiluminescent immunoassay (CIA), fluorescent immunoassay (FIA) and nanoparticle/beads-based naked eye immunoassay at the point-of-care, point-of-need, bioanalytical, home or remote settings. Depending on the analyte to be measured, the device and method of the present invention could be used in the medical and diagnostic industry.

DETAILED DESCRIPTION OF THE INVENTION

The technology of the present invention can be carried out in various eELISA technology formats. The first format employs the covalent and leach-proof immobilization of capture antibodies on the structures designed in the preferably disposable lid structure. The second format employs the use of magnets in the structures designed in the preferably disposable lid structure. The magnets will bind the capture antibody-bound magnetic beads/particles to the structure.

The immobilisation of the affinity reagent to the protruding element (or binding structure) can be carried out in a preferred embodiment by a method for covalent immobilisation comprising contacting the protruding element with an oxidising agent, thereby producing an activated surface comprising carbonyl and/or hydroxyl groups, and contacting the activated surface with a binding solution, comprising a mixture of the affinity reagent to be immobilised and one or more silane components, optionally together with one or more cross-linking agents, thereby producing a covalent attachment of said affinity reagent with said protruding element via an amide bond.

The mixture of silane component and affinity reagent leads to an effective "one-step" immobilisation of the affinity reagent without the need for EDC or sulfo-NHS (SNHS) -based chemistry. In one embodiment, the method is carried out in the absence of EDC and/or the absence of SNHS based linkage chemistries. In one embodiment, the method of immobilisation is characterised in that the hydroxide-containing solution comprises of sodium hydroxide, potassium hydroxide and/or ammonium hydroxide, preferably potassium hydroxide, whereby said hydroxide is preferably present at a concentration of 0.1 to 10%, preferably 0.5 to 5%, more preferably 1 %. In one embodiment, the immobilisation method is characterised in that said silane component is 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-trimethoxysilane

(APTMS), (3-mercaptopropyl)trimethoxysilane (MPTMS) and/or 3-glycidoxypropyltriethoxysilane (GOPTS), whereby said binding solution comprises preferably of APTES at a concentration of 0.1 to 10%, preferably 0.5 to 5%, more preferably 1 %. The immobilisation method therefore comprises the steps of: generation of hydroxyl groups on substrate by KOH pre-treatment; one- step covalent binding of capture reagent (dispersed in 3-aminopropyltriethoxysilane (APTES)) to the protruding element; preferably followed by blocking the non-specific protein binding sites by bovine serum albumin (BSA).

The protruding elements may also be magnetic, capable of attracting magnetic particles through magnetic forces. A skilled person is familiar with magnetic beads, to which affinity reagents may be attached. The use of magnetic protruding elements, preferably covered with disposable polymer/composite/material, allows binding of beads that are bound to the analyte and/or ELISA sandwich complex, thereby providing means for stopping the reaction if the magnetic force is applied after the reaction of the final method step, or for binding capture affinity reagent to the protruding elements of the device of the present invention.

One major application of the developed eELISA technology is the sandwich ELISA-based IVDs as it accounts for >95% of the market size of immunodiagnostics. A further main application of eELISA is for the smartphone-based point-of-care/ point-of-need diagnostics. The invention provides formats for smartphone-based IVDs in the form of customized well plates having uniform illumination zones for colorimetric readout; detachable round wells for inserting into the smartphone attachment for colorimetric readout; detachable cuvette-shaped rectangular/square wells for absorbance readout; and POC cartridge or tube formats for colorimetric/absorbance readout. Mobile phones have more than 6.5 billion users world-wide. The smartphone-based IVD kits, as enabled by the technology herein, provide an enormous advantage in ease of use and independence from previously essential clinical or diagnostic settings that required complicated hardware and expert skills.

The developed formats provided herein have the advantage of avoiding all the limitations of the current state-of-the-art in microfluidic and centrifugal microfluidic platforms. These limitations include the loss of analyte by non-specific binding to the microfluidic channels, loss due to bead transfer, cross-contamination of liquid chambers, requirement of costly disk readers for rotation and readout, lack of high-throughput for sample analysis, and increased production cost for miniaturized channels and structures.

Microarray-based approaches utilising the invention as disclosed herein, based on a solid substrate that can be attached at the tip of the structures on the disposable lid using double- sided pressure sensitive adhesive, for the multiplex detection of biomarkers/analytes is a further aspect of the invention.

Table 1. Comparison of eELISA format of the present invention with conventional sandwich ELISA format and the most recent AlphaLISA format by InVitrogen. eELISA format AlphaLISA (InVitrogen) Conventional sandwich ELISA

(Commercially-available established technology)

Put pre-capture antibody-bound Add acceptor beads and Add capture antibody and protruding elements in biotinylated anti-analyte incubate (4 h - overnight) proposed lid in receptacle (e.g. antibody

96-well TP) containing all Wash 5-6 times

sandwich ELISA components +

analyte sample

Incubate for 15 min Add analyte and incubate for Add blocking solution and

30-60 min incubate for 1-2 h

Wash 2 times

Wash 5-6 times

Enzyme-substrate reaction - 4 Add donor beads and Add analyte and incubate for 1-2 h min incubate for 30-60 min

Wash 5-6 times

Read Read Add biotinylated detection

antibody and incubate for 1-2 h

Wash 5-6 times

Add streptavidin-HRP and incubate for 20 min - 1 h

Wash 5-6 times Add TMB substrate and incubate for a few min (usually 5-20 min). Stop TMB substrate reaction

Read

Table 2. Comparison of developed eELISA format of the present invention with other previously developed sandwich ELISA formats. eELISA format Covalent sandwich Covalent sandwich Conventional

ELISA** ELISA* (Multistep sandwich ELISA antibody

(One-step antibody immobilization (Commercially- immobilization procedure) available established procedure) technology)

Put pre-capture Covalent capture Covalent capture Add capture antibody antibody-bound antibody immobilization antibody and incubate (4 h - protruding elements in (30-40 min) immobilization (2.5 h) overnight) proposed lid in

receptacle (e.g. 96-well Wash 5-6 times Wash 5-6 times Wash 5-6 times MTP) containing all

sandwich ELISA

components + analyte

sample

Incubate for 15 min Add blocking solution Add blocking solution Add blocking solution and incubate for 30 min and incubate for 30 and incubate for 1-2 h Wash 2 times min

Wash 5-6 times Wash 5-6 times

Wash 5-6 times

Enzyme-substrate Add analyte and Add analyte and Add analyte and reaction - 4 min incubate for 1 h incubate for 1 h incubate for 1-2 h

Wash 5-6 times Wash 5-6 times Wash 5-6 times

Read Add biotinylated Add biotinylated Add biotinylated

detection antibody and detection antibody detection antibody incubate for 1 h and incubate for 1 h and incubate for 1-2 h

Wash 5-6 times Wash 5-6 times Wash 5-6 times

Add streptavidin-HRP Add streptavidin-HRP Add streptavidin-HRP and incubate for 20 min and incubate for 20 and incubate for 20 - 1 h min - 1 h min - 1 h

Wash 5-6 times Wash 5-6 times Wash 5-6 times

Add TMB substrate and Add TMB substrate Add TMB substrate incubate for a few min and incubate for a few and incubate for a few (usually 5-20 min). Stop min (usually 5-20 min (usually 5-20 TMB substrate reaction min). Stop TMB min). Stop TMB

substrate reaction substrate reaction Read Read Read

^*Nature Protocols 6(4), 439-445, 201 1 ; Analytical Chemistry 82(16), 7049-52, 2010; Biosensors and Bioelectronics 40, 397-402, 2013; Diagnostics 2, 23-33, 2012; Procedia Chem. 6, 141-148 2012; Procedia Chem. 6, 184-193, 2012; WO 2010/044083, ^** WO/2014/056896. Advantages and technology features of the developed eELISA format are as follows:

Obviation of complex washing procedure in conventional format: The invented eELISA format is based on the dipping of proposed lid, containing antibody-bound protruding elements, into the receptacle i.e. MTP wells. Therefore, it completely obviates the traditional washing procedure as it doesn't require the MTP to be inverted at any stage. The washing is done by simple dipping of the structures in lid into the washing buffer in MTP wells.

Simplicity: The eELISA is one of the most simplified ELISA format presently known. The eELISA sandwich immunoassay procedure has fewer process steps as the immune complex is formed in one step, when the analyte sample is provided to the MTP wells containing biotinylated detection antibody preconjugated to HRP-labeled streptavidin. This is followed by immersing the proposed lid containing capture antibody-bound structures into the MTP. Subsequently, the procedure employs 2 washes by dipping the structure (lid) into the washing buffer in MTP wells and lastly, dipping the protruding elements of the upper element in enzyme specific substrate for colorimetric reaction.

Obviates cross-contamination: The eELISA obviates cross-contamination issues because the liquid cannot flow from one well to another. The MTP is not inverted at any stage.

Obviates the stopping of enzyme-substrate reaction: The format as described herein avoids the requirement of having to stop the enzyme-substrate reaction. The proposed protruding element, comprising antibody-bound protruding structures, can be removed after the desired enzyme- substrate reaction, which stops the reaction instantly.

Obviates high-end analyst skills required for performing sandwich ELISA: The present invention requires only very basic skills from the end user. There is no requirement for a highly-trained analyst due to the avoidance of the conventional washing procedure, in addition to the use of simplified sandwich assay procedure.

Reduced assay time: The eELISA takes -20 min for an immunoassay.

Reduced cost: The novel formats described herein lead to >50% cost reduction due to the reduced amount of analyte sample required, fewer process and washings steps.

Ideal for existing and remote settings: The eELISA format is ideal for existing bioanalytical labs in healthcare, industrial and other settings. Moreover, the simplicity, rapidness and cost- effectiveness of eELISA format will make this a potential format for remote settings such as for veterinary applications, in developing countries and even for resource-limited small-scale bioanalytical labs. The point-of-care eELISA cartridge format will be highly significant for personalized mobile Healthcare, where the users can do the assay independently at their convenience without any need of going to a clinical setting.

Requires no additional instrument: The existing sandwich ELISA users don't require any other instrument other than the standard microtiter plate reader. Therefore, there is no added instrumentation cost.

Providing next-generation mobile Healthcare (mHealthcare) diagnostics: The smartphone-based eELISA formats will lead to next-generation of mHealthcare diagnostics, which will obviate the requirement of costly microtiter plate reader for sandwich ELISA and will enable the

development of cost-effective smartphone attachments that will incorporate the technologies for colorimetric and absorbance measurements.

Based on the above-mentioned advantages, the eELISA format represents a beneficial format for the development of in vitro diagnostic (IVD) kits. It is fully compatible with conventional (Microtiter plate reader-based), ongoing (detector-based) and next-generation formats

(smartphone/camera/scanner/naked eye-based).

FIGURES

The figures provided herein represent examples of particular embodiments of the invention and are not intended to limit the scope of the invention. The figures are to be considered as providing a further description of possible and potentially preferred embodiments that enhance the technical support of one or more non-limiting embodiments.

Figure 1. eELISA format - 24-well MTP. (A) Alignment jig, (B) Alignment jig containing 96-well MTP, (C) Capture antibody-functionalized protruding elements in the lid and its alignment using alignment jig, and (D) lid with capture antibody-functionalized protruding elements.

Reference characters (Fig. 1 ):

(1 ) upper element (lid)

(2) protruding elements (rods)

(3) receptacle (detection plate)

(4) wells

(5) means for securing and/or hoisting

(6) means for alignment

Figure 2. eELISA format - 48-well MTP. (A) Alignment jig, (B) Alignment jig containing 96-well MTP, (C) Capture antibody-functionalized protruding elements in the lid and its alignment using alignment jig, and (D) lid with capture antibody-functionalized protruding elements.

Figure 3. eELISA format - 96-well MTP. (A) Alignment jig, (B) Alignment jig containing 96-well MTP, (C) capture antibody-functionalized protruding elements in the lid and its alignment using alignment jig, and (D) lid with capture antibody-functionalized elements. Figure 4. eELISA format - Smartphone (/camera-enabled device). The design of the cartridge for the Smartphone (/camera-enabled device) containing 9 (Upper) and 6 (Lower) analyte detection wells. The capture antibody-functionalized protruding elements in the lid will be circular (Upper) or rectangular (Lower).

Figure 5. eELISA format - Smartphone (/camera-enabled device). (A) Smartphone attachment for immunoassay cartridge and optics, (B) Smartphone attachment (Upper view), (C, D) Putting the cartridge in smartphone attachment, (E, F) Optical readout of cartridge, and (G)

immunoassay cartridge.

Figure 6. eELISA format - Disk. (A) Alignment jig, (B) Alignment jig containing Disk, (C) Capture antibody-functionalized protruding elements in the lid and its alignment using alignment jig, and (D) lid with capture antibody-functionalized protruding elements.

Figure 7. eELISA format - Disk. (A) Alignment jig, (B) Alignment jig containing Disk, (C) Capture antibody-functionalized protruding elements in the lid and its alignment using alignment jig, and (D) lid with capture antibody-functionalized protruding elements.

Figure 8. Types of Proof-of-concept experiments performed. (A) Capture anti-CRP antibodies were bound to the outside of the 200 \L Eppendorf pipette tips by a 1 -step antibody

immobilization procedure, (B) Capture anti-CRP antibodies were bound to the inside of the 200\L Eppendorf pipette tips using the same procedure. (C) Capture anti-CRP antibodies- functionalized dynabeads were bound to the outside of the 200 μί Eppendorf pipette tips having the magnets inside.

Figure 9. Proof-of-concept experiment where capture anti-CRP antibodies were bound to the outside of the 200 \L Eppendorf pipette tips by a 1 -step antibody immobilization procedure. The full procedure consists of four steps as mentioned in the schematic in (A). The anti-CRP antibody-bound and BSA blocked pipette tip is put into the solution containing biotinylated anti- CRP detection antibody pre-conjugated to streptavidin-HRP, and analyte sample. The presence of CRP leads to the formation of immune complex after 15 min of incubation. The immune complex-bound tip is then washed by dipping in washing buffer twice. Thereafter, the tip was dipped in TMB for 3 min for enzyme-substrate reaction that leads to the formation of colored product. The absorbance was read after stopping the reaction. (B) The CRP immunoassay curve plotted by SigmaPlot version 1 1 .2 software using four-parameter logistic-based standard curve analysis.

Figure 10. Proof-of-concept experiment where capture anti-CRP antibodies were bound to the inside of the 200 μί Eppendorf pipette tips by a 1 -step antibody immobilization patent

(WO/2014/056896). B) The CRP immunoassay curve plotted by SigmaPlot version 1 1 .2 software using four-parameter logistic-based standard curve analysis.

Figure 11. Proof-of-concept experiment where capture anti-CRP antibodies-functionalized dynabeads were bound to the outside of the 200 μί Eppendorf pipette tip having the magnets inside. The experiment was performed only for one concentration i.e. 27 ng/mL of CRP and the negative control.

Figure 12. Types of antibody-bound structures for the proposed eELISA formats shown in the previous sections. (A) solid cylindrical rod structures, where antibodies are bound to the outside of the solid structures, and (B) hollow cylindrical rods where antibodies are bound to the outside as well as inside the structures.

Figure 13. (A) Schematic of the previously developed 1 -step antibody immobilization procedure for sandwich ELISA for CRP (European Patent Application No. WO/2014/056896 - this scheme is applied to immobilise the affinity capture reagent to the protruding element, not the microtitre plate as shown in the diagram), (B) Detection of CRP in PBS buffer (with 0.1 % BSA) and CRP- spiked dilute human serum using conventional microplate reader-based readout. (C)

Smartphone-based CRP sandwich ELISA based on the imaging of colorimetric sandwich ELISA by iPhone 4 under ambient conditions.

Figure 14. (A) Schematic illustration of the optical setup for the detection of 24 wells (involved in eELISA) in the 96-well MTP, (B) Integral components, (C) MTP platform with alignment jigs, (D) 96-well MTP placed on the optical setup, and (E) Illumination of MTP wells (24 wells format).

Figure 15. Image processing algorithm for smartphone-based calculation of target analyte concentration in clinical sample.

Figure 16. Smartphone-based detection of 1 -step kinetics-based immunoassay for the detection of C-reactive protein (CRP) in diluted whole blood using the image processing algorithm mentioned in Fig. 15. (A) Gray scale image of blue channel, and (B) Immunoassay curve.

Figure 17. Smartphone-based immunoassay for the highly-sensitive point-of-care detection of human C-reactive protein in whole blood and serum. Smartphone-based human C-reactive protein (hCRP) immunoassay in 20 min. (A) Schematic, (B) Smartphone-based detection of CRP in human serum and whole blood (inset shows the results obtained by the microtiter plate reader), (C) Overlay of smartphone-based predictive absorbance readings and the microtiter plate reader-based actual absorbance readings in human whole blood (inset shows the overlay of same curves for serum), (D) Experimental process controls (anti-CRP1 and anti-CRP2 are the capture and detection antibodies, respectively), and (E) Correlation of developed smartphone-based CRP immunoassay in whole blood with the microtiter plate reader-based immunoassay.

Figure 18. Smartphone-based colorimetric readout device involving a low-cost optical attachment and an image processing algorithm for bioanalytical sciences. A developed

Smartphone-based colorimetric readout device is shown. (A) Low-cost optical attachment, (B) Smartphone-based image processing procedure, (C) Image of colorimetric solution, obtained after 1 -step kinetics-based human CRP sandwich immunoassay, captured by the smartphone's camera at a defined height, (D) Smartphone-based human CRP immunoassay in human whole blood, and (E) Smartphone-based prediction of absorbance versus the actual absorbance readings obtained by microtiter plate reader. Figure 19: Schematic illustration of the light source developed for the smartphone-based colorimetric readout device for 96-well microtiter plate. (A) 24 LEDs placed beneath the MTP plate in an array of 6 x 4 matrix, (B) Each LED is placed in the center of 4 MTP wells for even illumination, (C) Circuit diagram of 24 LEDs connected in parallel to the voltage source through a variable resistor, and (D) Circuit for driving a RGB LED with pulse-width modulation (PWM) controller.

EXAMPLES

The examples provided herein represent practical support for particular embodiments of the invention and are not intended to limit the scope of the invention. The examples are to be considered as providing a further description of possible and potentially preferred embodiments that demonstrate the relevant technical working of one or more non-limiting embodiments.

The initial experimental prototypes of the proposed easy ELISA (eELISA) formats are presented below. The initial proof-of-concept experiments were carried out using the 200 [\L Eppendorf pipette tips' exterior and interior, and the commercially-available C-Reactive Protein (CRP) sandwich ELISA kit components from RnD Systems, USA.

The antibody-functionalized protruding elements in the proposed lid for the eELISA formats are shown in the figures in the form of solid cylindrical rods. However, it is also encompassed by the present invention to use hollow cylindrical rods, where antibodies are bound to the exterior as well as interior for increasing the surface area. Apart from these, the magnets can also be used as solid cylindrical rods. But in this case, they are covered with disposable polymer such as polystyrene. The rapid magnetic bead immunoassay using the 1 -step kinetics has been developed by us previously (Anal. Biochem. 456, 32-37, 2014).

The following ELISA devices and systems have been developed as prototypes:

eELISA format - 24-well microtiter plate (MTP) (Fig. 1 ): The device shown therein is intended for Point-of-care/ Point-of-need/ existing bioanalytical settings that require fewer numbers of analyte samples to be analyzed by sandwich immunoassays.

eELISA format - 48-well MTP (Fig. 2): The device shown therein is intended for Point-of-care/ Point-of-need/ existing bioanalytical settings that require medium number of analyte samples to be analyzed by immunoassays. eELISA format - 96-well MTP (Fig. 3): The device shown therein is intended for Point-of-care/ Point-of-need/ existing bioanalytical settings that require large number of analyte samples to be analyzed by immunoassays. eELISA formats - Smartphone (/camera-enabled device) (Fig. 4): The device shown therein is intended for Point-of-care/ Point-of-need/ existing bioanalytical settings. eELISA format - Disk (Fig. 5): The device shown therein is intended for Point-of-care/ Point-of- need settings.

Light Source developed for the smartphone-based colorimetric readout device for 96-well microtiter plate (Fig. 19)

The light source of the optical unit is preferably powered by an array of light emitting diodes (LEDs) (Fig. 19A) that provides an even illumination of 96-well MTP by placing a LED in the middle of every four MTP wells (Fig. 19B). The height between the LEDs and the MTP can be adjusted to cover the extreme ends of 4 wells based on the angle of light emission. The LEDs are connected in parallel to have a constant current flowing across them, thereby leading to an even illumination. The brightness of the illumination is controlled by a potentiometer (/variable resistor) (Fig 19C). In case of driving RGB LEDs, a pulse-width modulation controller is used to obtain the desired color from the light source (Fig. 19D).

Example 1 : Proof of concept experiments

The initial proof-of-concept experiments were carried out using the 200 μί Eppendorf pipette tips' exterior and interior, and the commercially-available C-Reactive Protein (CRP) sandwich ELISA kit components from RnD Systems, USA. The method was carried out as described herein using the device and system of the present invention. The results are shown in Figures 8 to 16.

Materials and Methods used in the experimental examples:

1.1. One-step kinetics-based CRP Immunoassay inside 200 μί Eppendorf pipette tip

1.1.1 Materials

Phosphate buffered saline (Cat.# 18912-014; PBS, pH 7.4), Tween 20, Nunc microwell 96 well polystyrene plate (Cat.# 12-565-31 1 ) were purchased from Invitrogen, Carl Roth GmbH and Fisher Scientific, respectively, while the 200 μί pipette tips were procured from Eppendorf, Germany. The human CRP Duoset kit's (DY1707) components, i.e. anti-human CRP capture antibody, recombinant human CRP and biotinylated anti-human CRP detection antibody, were procured from RnD Systems, USA. 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate, stop solution, bovine-serum albumin (BSA), streptavidin-conjugated horseradish peroxidase (SA- HRP), potassium hydroxide (KOH), and 3-APTES were bought from Sigma-Aldrich, Germany. The autoclave was from Systec GmbH, Germany, while the MTP reader was Perkin Elmer

Wallac VICTOR 1420 Multilabel Counter. All buffers and solutions were prepared in autoclaved ultrapure water - DNase and RNase free (Cat. # 10977; Gibco, Germany). The binding and washing buffers employed for the developed CRP immunoassay were PBS with 0.1 % BSA and PBS with 0.05% Tween 20, respectively. The working aliquots of commercially supplied lyophilized human CRP were made in 20 rtiM Tris-HCI, pH 8.0 with 0.1 % BSA, while the CRP concentrations for the immunoassay were made in the binding buffer. The biotinylated anti-CRP detection antibody conjugated to HRP-labeled streptavidin was prepared by adding 2 μί of biotinylated anti-CRP detection antibody (0.5 mg/mL) and 2 μί of SA-HRP to 2996 μί of binding buffer, and incubating it for 20 min at RT. Therefore, the concentration of biotinylated anti-CRP detection antibody used was 0.17 μg/mL, while SA-HRP dilution employed was 1 :3000.

1.1.2 CRP immunoassay procedure

The Eppendorf pipette tips were made to suck 100 μΙ_ of 1 % KOH from the 96-well MTP and incubated for 10 min. The excess solution was dispensed out and the tips were subsequently washed five times with 200 μΙ_ of ultrapure water (by sucking & dispensing using multichannel pipette). Thereafter, 100 μΙ_ of 5 μg/mL of anti-CRP capture antibody prepared in APTES, by mixing equal volumes of 10 μg/mL antibody and 2% APTES, was put in the MTP wells and sucked inside the KOH-treated pipette tips. The anti-CRP antibody was bound inside the tips by incubating at RT for 30 min. The excess solution was dispensed out and the tips were then washed with 200 μΙ_ of washing buffer five times. The anti-CRP antibody-bound Eppendorf pipette tip was blocked with BSA by sucking 200 μΙ_ of 5% BSA and incubating for 30 min at RT. The excess solution was dispensed out and the tips were washed five times with 200 μΙ_ of wash buffer. The BSA blocked anti-CRP antibody-bound Eppendorf pipette tip was then sucked with the mixture of 50 μΙ_ of biotinylated anti-CRP detection antibody pre-conjugated to SA-HRP, and 50 μΙ_ of human CRP (varying concentrations; 0.33-81 ng/mL), and left incubated for 15 min at RT. The excess solution was dispensed out and subsequently the tips were washed twice with 200 μΙ_ of wash buffer. Thereafter, 100 μΙ_ of TMB substrate was sucked into the tips from the MTP wells and the enzyme-substrate reaction was allowed to proceed for 4 min before being stopped by 50 μΙ_ of the stop solution. The colorimetric solution was then dispensed out into the MTP wells and the absorbance of the colorimetric solution was measured by MTP reader at 450 nm. All datasets were subjected to 4-parameter logistic fit-based standard curve analysis using SigmaPlot software, version 1 1.2.

1.2. One-step kinetics-based CRP Immunoassay outside 200 μΙ_ Eppendorf pipette tip

1.2.1 Materials

Phosphate buffered saline (Cat.# 18912-014; PBS, pH 7.4), Tween 20, Nunc microwell 96 well polystyrene plate (Cat.# 12-565-31 1 ) were purchased from Invitrogen, Carl Roth GmbH and Fisher Scientific, respectively, while the 200 μΙ_ pipette tips were from Eppendorf, Germany. The human CRP Duoset kit's (DY1707) components, i.e. anti-human CRP capture antibody, recombinant human CRP and biotinylated anti-human CRP detection antibody, were procured from RnD Systems, USA. 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate, stop solution, bovine- serum albumin (BSA), streptavidin-conjugated horseradish peroxidase (SA-HRP), potassium hydroxide (KOH), and 3-APTES were bought from Sigma-Aldrich, Germany. The autoclave was from Systec GmbH, Germany, while the MTP reader was Perkin Elmer Wallac VICTOR 1420 Multilabel Counter. All buffers and solutions were prepared in autoclaved ultrapure water - DNase and RNase free (Cat. # 10977; Gibco, Germany). The binding and washing buffers employed for the developed CRP immunoassay were PBS with 0.1 % BSA and PBS with 0.05% Tween 20, respectively. The working aliquots of commercially supplied lyophilized human CRP were made in 20 rtiM Tris-HCI, pH 8.0 with 0.1 % BSA, while the CRP concentrations for the immunoassay were made in the binding buffer. The biotinylated anti-CRP detection antibody conjugated to HRP-labeled streptavidin was prepared by adding 2 μΙ_ of biotinylated anti-CRP detection antibody (0.5 mg/mL) and 2 μΙ_ of SA-HRP to 2996 μΙ_ of binding buffer and incubating it for 20 min at RT. Therefore, the concentration of biotinylated anti-CRP detection antibody was 0.17 μg/mL, while SA-HRP dilution employed was 1 :3000.

1.2.2 CRP immunoassay procedure

The Eppendorf pipette tips were dipped in 200 μΙ_ of 1 % KOH inside the 96-well MTP, left incubated for 10 min and subsequently washed with ultrapure water. Thereafter, 200 μΙ_ of 5 μg/mL of anti-CRP capture antibody prepared in APTES, by mixing equal volumes of 10 μg/mL antibody and 2% APTES, was put in the MTP wells followed by the immersion of KOH-treated pipette tips in it. The anti-CRP antibody was bound to pipette tips, when incubated at RT for 30 min and subsequently, washed five times by dipping in 300 μΙ_ of washing buffer in MTP wells. The anti-CRP antibody-bound Eppendorf pipette tips were dipped in 300 μΙ_ of 5% BSA for 30 min at RT and then washed five times by dipping in 300 μΙ_ of washing buffer. The BSA blocked anti-CRP antibody-bound Eppendorf pipette tip was immersed in MTP wells containing 100 μΙ_ of biotinylated anti-CRP detection antibody pre-conjugated to SA-HRP, and 100 μΙ_ of human CRP (varying concentrations; 0.33-81 ng/mL) and left incubated for 15 min at RT. Thereafter, 200 μΙ_ of TMB substrate was provided to each MTP well and the enzyme-substrate reaction was allowed to proceed for 4 min before being stopped by 100 μΙ_ of the stop solution. The absorbance of the colorimetric solution was measured by MTP reader at 450 nm. All datasets were subjected to 4-parameter logistic fit-based standard curve analysis using SigmaPlot software, version 1 1.2.

1.3. One-step kinetics-based CRP immunoassay using paramagnetic beads inside 200 μΙ_ Eppendorf pipette tip

1.3.1 Materials

Dynabeads M-280 Tosylated (Cat.# 142.03; 2.8 μιη diameter; concentration 30 mg/mL) and phosphate buffered saline (Cat.# 18912-014; PBS, pH 7.4) were procured from Invitrogen, while Tween 20 and Nunc microwell 96 well polystyrene plate (Cat.# 12-565-31 1 ) were purchased from Carl Roth GmbH and Fisher Scientific, respectively. The human CRP Duoset kit's

(DY1707) components, i.e. anti-human CRP capture antibody, recombinant human CRP and biotinylated anti-human CRP detection antibody, were procured from RnD Systems, USA.

3,3 ,5,5 -tetramethylbenzidine (TMB) substrate, stop solution, bovine-serum albumin (BSA), streptavidin-conjugated horseradish peroxidase (SA-HRP), potassium hydroxide (KOH), and 3- APTES were bought from Sigma-Aldrich, Germany. The microtiter plate shaker was from VWR International, Germany; autoclave was from Systec GmbH, Germany; and, the MTP reader used was Perkin Elmer Wallac VICTOR 1420 Multilabel Counter. All buffers and solutions were prepared in autoclaved ultrapure water - DNase and RNase free (Cat. # 10977; Gibco, Germany). The binding and washing buffers employed for the developed CRP immunoassay were PBS with 0.1 % BSA and PBS with 0.05% Tween 20, respectively. The working aliquots of commercial lyophilized human CRP were made in 20 mM Tris-HCI, pH 8.0 with 0.1 % BSA, while the CRP concentration for the immunoassay i.e. 27 ng/mL was made in the binding buffer. The anti-CRP capture antibody was bound to the tosylated Dynabeads using the standard immobilization procedure provided by the Dynabeads' manufacturer i.e. Invitrogen. The prepared stock solution of anti-CRP capture antibody-bound Dynabeads was then stored at 4°C. The biotinylated anti-CRP detection antibody conjugated to HRP-labeled streptavidin was prepared by adding 2 μΙ_ of biotinylated anti-CRP detection antibody (0.5 mg/mL) and 2 μΙ_ of SA-HRP to 2996 μΙ_ of binding buffer and incubating it for 20 min at RT. Therefore, the concentration of biotinylated anti-CRP detection antibody was 0.17 μg/mL, while SA-HRP dilution employed was 1 :3000. Three magnets were put inside the 200 μΙ_ Eppendorf pipette tip that was used in this experiment.

1.3.2 CRP immunoassay procedure

The MTP wells were initially pre-blocked with BSA by incubating with 5% BSA for 30 min at room temperature (RT) followed by subsequent washing with 300 μΙ_ of wash buffer five times. The Eppendorf pipette tip with magnets inside was then dipped in the MTP well containing 4 μΙ_ diluted stock solution of anti-CRP capture antibody-bound Dynabeads (diluted 1 :10 in binding buffer) and 76 μΙ_ binding buffer. After 3 min incubation, the anti-CRP capture antibody-bound Dynabeads get immobilized on the tip by magnetic force and subsequently washed by dipping in 300 μΙ_ of washing buffer in MTP well. The antibody-bound Dynabeads were then dipped in MTP well containing 40 μΙ_ biotinylated anti-CRP detection antibody pre-conjugated to SA-HRP and 40 μΙ_ of human CRP (27 ng/mL). After 15 min incubation at RT, the magnets specifically captured the sandwich immune complex-bound Dynabeads. The magnetically-captured sandwich immune complex-bound Dynabeads were then washed by dipping the tip twice in 300 μΙ_ of washing buffer. Thereafter, 200 μΙ_ of TMB substrate was provided to each MTP well and the enzyme-substrate reaction was allowed to proceed for 4 min before being stopped by 100 μΙ_ of the stop solution. The absorbance of the colorimetric solution was measured by MTP reader at 450 nm. All datasets were subjected to 4-parameter logistic fit-based standard curve analysis using SigmaPlot software, version 1 1.2.

Example 2a: Smartphone-based immunoassay for the highly-sensitive point-of-care detection of human C-reactive protein in whole blood and serum

A highly sensitive and cost-effective smartphone-based immunoassay (IA) was developed for the point-of-care (POC) detection of human C-reactive protein (hCRP) in human whole blood and serum, to be carried out in -20 min. The precise determination of CRP is essential for the diagnosis and management of neonatal sepsis, cardiovascular diseases, infectious and inflammatory conditions, meningitis and diabetes. It obviates the need of expensive instruments and special analytical skills, and employs the one-step kinetics-based sandwich IA procedure (Fig. 1 A) that is superior to the commercial kit as it employs a highly simplified procedure with fewer steps and significantly reduced IA duration.

The procedure involves the dispensing of capture anti-CRP antibody (Ab)-bound dynabeads (2.8 μιη dia) and biotinylated anti-CRP detection Ab prebound to horseradish peroxidase (HRP)- labeled streptavidin (SA) to the bovine serum albumin-preblocked 96-well microtiter plate wells. Subsequently, CRP spiked in 1 :100 diluted human whole blood or serum is provided and incubated for 15 min at room temperature that leads to the formation of immune complex, which is then captured by the magnet and washed twice with washing buffer. The hCRP concentration is determined by smartphone-based image capture of the colorimetric product formed after 3,3',5,5'-tetramethylbenzidine (TMB) substrate reaction, and subsequent image processing that provides nearly precise predictive absorbance readings from the pixel intensity of the captured image . The developed hCRP IA in human whole blood has dynamic range, limit of detection, analytical sensitivity, correlation coefficient (R²), percentage coefficient of variance and half- maximal effective concentration (EC₅₀) of 0.33-81 ng/mL, 0.27 ng/mL, 0.50 ng/mL, 0.999, 0.5- 4.6, and 16.8, respectively (Fig. 17). It has the same precision as CRP ELISA (Pearson correlation coefficient of 1 ). The developed technology has immense potential to develop low- cost POC in vitro diagnostic kits/devices to detect analytes in various bioanalytical settings.

Example 2b: Smartphone-based colorimetric readout device involving an optical attachment and an image processing algorithm for bioanalytical sciences

A smartphone-based colorimetric readout device has been developed and applied in

bioanalytical applications. It comprises of a low-cost optical attachment that employs an array of white light emitting diodes (LED) to provide uniform back-illumination of 96-well microtiter plate (MTP). The smartphone-based human C-reactive protein (CRP) immunoassay based on 1-step kinetics-based sandwich ELISA procedure was performed, where the colorimetric product obtained after enzyme-substrate reaction was imaged inside an optically-opaque hood using the smartphone's camera at a defined height (to prevent the parallelex error). The acquired image was then subjected to an image-processing algorithm that extracts the pixel value and plots them against the corresponding CRP concentration using the four-parameter logistic fit (Fig. 18).

The image processing algorithm, programmed in MATLAB, initially determines the center of each MTP well and subsequently calculates the mean of defined neighbouring pixels. The composite pixel value is then computed from 10%, 20% and 70% mean pixel values from red, green and blue channels, respectively. The experiments demonstrate that the smartphone- based composite pixel values can be used to accurately predict the corresponding absorbance values obtained by the MTP reader. The developed low-cost device is ideal for colorimetric readout-based assays in clinical, industrial, veterinary sciences, environmental, food analysis, security and other bioanalytical settings. It obviates the need of an expensive MTP reader, for colorimetric readout-based immunoassays, protein determination and other biochemical assays as demonstrated by the same or increased sensitivity in our obtained results. Single color or tunable RGB LEDs, smart application (software) with integrated image analysis, advanced calibration algorithms for absorbance prediction, and miniaturizing the thickness of optical attachment have also been applied with promising results.

Materials and Methods used in the experimental examples:

2.1 One-step kinetics-based CRP immunoassay in 96-well microtiter plate wells using smartphone readout

2.1 Materials Dynabeads M-280 Tosylated (Cat.# 142.03; 2.8 μιη diameter; concentration 30 mg/mL) and phosphate buffered saline (Cat.# 18912-014; PBS, pH 7.4) were procured from Invitrogen, while Tween 20 and Nunc microwell 96 well polystyrene plate (Cat.# 12-565-31 1 ) were purchased from Carl Roth GmbH and Fisher Scientific, respectively. The human CRP Duoset kit's

(DY1707) components, i.e. anti-human CRP capture antibody, recombinant human CRP and biotinylated anti-human CRP detection antibody, were procured from RnD Systems, USA.

3,3 ,5,5 -tetramethylbenzidine (TMB) substrate, stop solution, bovine-serum albumin (BSA), streptavidin-conjugated horseradish peroxidase (SA-HRP), potassium hydroxide (KOH), and 3- APTES were bought from Sigma-Aldrich, Germany. The human whole blood (Cat.# 232754, HQ-Chex Level 2) was procured from Streck, USA, while CRP-free human serum (Cat.# 8CFS) was purchased from HyTest Ltd., Finland. The magnetic holder (Cat.# Q-19-13-06-LN;

Quadermagnet) containing 24 magnets, with each magnet spaced in the center of four MTP wells, was from Supermagnete, Germany; microtiter plate shaker was from VWR International, Germany; autoclave was from Systec GmbH, Germany; and, the MTP reader used was Perkin Elmer Wallac VICTOR 1420 Multilabel Counter. All buffers and solutions were prepared in autoclaved ultrapure water - DNase and RNase free (Cat. # 10977; Gibco, Germany). The binding and washing buffers employed for the developed CRP immunoassay were PBS with 0.1 % BSA and PBS with 0.05% Tween 20, respectively. The working aliquots of commercial lyophilized human CRP were made in 20 rtiM Tris-HCI, pH 8.0 with 0.1 % BSA, while the CRP spiking was done in diluted human whole blood and serum (diluted in the binding buffer). The anti-CRP capture antibody was bound to the tosylated Dynabeads using the standard immobilization procedure provided by the Dynabeads' manufacturer i.e. Invitrogen. The prepared stock solution of anti-CRP capture antibody-bound Dynabeads was then stored at 4°C. The biotinylated anti-CRP detection antibody conjugated to HRP-labeled streptavidin was prepared by adding 2 [\L of biotinylated anti-CRP detection antibody (0.5 mg/mL) and 2 [\L of SA-HRP to 2996 [\L of binding buffer and incubating it for 20 min at RT. Therefore, the concentration of biotinylated anti-CRP detection antibody was 0.17 μg/mL, while SA-HRP dilution employed was 1 :3000.

2.2 Developed CRP immunoassay procedure

The MTP wells were initially pre-blocked with BSA by incubating with 5% BSA for 30 min at room temperature (RT) followed by subsequent washing with 300 [\L of wash buffer five times. They were sequentially dispensed with 2 μί diluted stock solution of anti-CRP capture antibody- bound Dynabeads (diluted 1 :10 in binding buffer), 38 [\L binding buffer and 40 [\L biotinylated anti-CRP detection antibody (0.17 μg/mL) pre-conjugated to SA-HRP (diluted 1 :3000).

Thereafter, 40 [\L of human CRP (varying concentrations; 0.33-81 ng/mL) spiked in 1 :100 diluted human whole blood or serum is provided to the respective MTP wells in triplicate and left incubated for 15 min at RT. The MTP was put on the shaker at 250 rpm for 1 min and then it is put on the magnetic holder having 24 magnets with each magnet in the center of four MTP wells for 3 min. The magnets specifically capture the sandwich immune complex-bound Dynabeads. The excess reagents are then taken out by sucking back the solution using the 300 μί pipette tip by a multi-channel pipette. The magnetically-captured sandwich immune complex-bound Dynabeads were then washed twice with 300 μΙ_ of washing buffer using the same washing procedure and subsequently suspended in 50 μΙ_ of binding buffer. Thereafter, 100 μΙ_ of TMB substrate was provided to each MTP well and the enzyme-substrate reaction was allowed to proceed for 4 min before being stopped by 50 μΙ_ of the stop solution. The image of the colorimetric solution was then captured by the smartphone camera and processed using the procedure specified below. All datasets were subjected to 4-parameter logistic fit-based standard curve analysis using SigmaPlot software, version 1 1.2.

2.3 Smartphone-based Image capture and processing

A smartphone-based colorimetric readout device has been developed, which comprises of a low-cost optical attachment that employs an array of white light emitting diodes (LED) to provide uniform back-illumination of 96-well microtiter plate (MTP). The colorimetric product obtained after enzyme-substrate reaction in developed CRP immunoassay was imaged inside an optically-opaque hood using the smartphone's camera at a fixed height in order to prevent the parallelex error. The acquired image was then subjected to an image-processing algorithm that extracts the pixel value and plots them against the corresponding CRP concentration using the four-parameter logistic fit. The image processing algorithm, programmed in MATLAB, initially determines the center of each MTP well and subsequently calculates the mean of defined neighboring pixels. The composite pixel value is then computed from 10%, 20% and 70% mean pixel values from red, green and blue channels, respectively.

The composite pixel values were initially normalized and converted to normalized composite pixel values, which were then employed to predict the corresponding absorbance values obtained by the MTP reader. The calibration curve using the actual absorbance values obtained by the MTP reader will be stored inside the smartphone as the absorbance reference curve. The absorbance prediction is carried out by three-point calibration using the relation between normalized composite pixel value and actual absorbance in the absorbance reference curve at the lowest, highest and near half-effective maximal concentration i.e. EC₅o of developed CRP immunoassay.

Claims

1. Enzyme-linked immunosorbent assay (ELISA) system comprising

- an upper element to which multiple protruding elements are attached, said protruding elements positioned parallel to one another on one side of said upper element, wherein said protruding elements are bound on their surface with capture affinity reagent and said capture affinity reagent is capable of binding an analyte in solution; and

- a receptacle, said receptacle comprising multiple wells suitable for holding a solution, wherein said wells are of dimensions that enable the insertion of said protruding elements of the upper element into said wells without contact between the surfaces of the interior of said wells and said protruding elements, and one protruding element may be inserted per well; wherein

- means for alignment of the protruding elements with the receptacle wells are present on the upper element and/or receptacle, wherein said means for alignment enable insertion of said protruding elements into the wells of said receptacle in a (pre-) determined position.

2. System according to the preceding claim, wherein the upper element is of a planar

structure.

3. System according to the preceding claim, wherein said protruding elements are positioned (distributed) in two dimensions upon the planar surface of said upper element.

4. System according to the preceding claim, wherein said protruding elements are positioned in multiple rows, wherein each row comprises multiple protruding elements, for example positioned according to the nodes of a grid.

5. System according to any one of the preceding claims, wherein the means for alignment are one or more alignment posts (alignment jigs) and corresponding apertures (or depressions), wherein said posts and apertures (or depressions) are present on the receptacle and upper element, respectively, or vice versa, and enable insertion of said posts into said apertures (or depressions).

6. System according to any one of the preceding claims, wherein the alignment posts (jigs) are of a length greater than the length of the protruding element, in order to position the upper element, prior to potential contact with the wells, in a (pre-) determined position for immersion.

7. System according to any one of the preceding claims, wherein (multiple) different shapes of the alignment posts (alignment jigs) and corresponding apertures (or depressions) are employed at different locations on the upper element and receptacle.

8. System according to any one of the preceding claims, characterised in that

said protruding elements comprise of material, to which the capture affinity reagent is immobilized, wherein said material is preferably selected from polystyrene (PS), polypropylene (PP), polyethylene (PE), polymethyl methacrylate (PMMA),

polydimethylsiloxane (PDMS), polycarbonate (PC), water-resistant photopolymer, cyclic olefin copolymer, cyclo olefin polymer, cellulose, silicon, glass, quartz, and/or metal.

9. System according to any one of the preceding claims, wherein the capture affinity reagent is a protein, such as an antibody, antibody fragment, a recombinant protein, Fc-binding protein, such as protein A, protein G or protein A/G, streptavidin, or recombinant proteins or fragments thereof, nucleic acid, polysaccharide, glycoprotein, peptidoglycans, molecular imprinted polymer or aptamer molecule.

10. System according to any one of the preceding claims, wherein the upper element

comprises means for securing and/or hoisting said upper element, thereby enabling vertical displacement of the upper element from the receptacle.

1 1. System according to the preceding claim, wherein the means for hoisting are positioned on the opposite side of said upper element to the protruding elements.

12. System according to any one of the preceding claims, wherein the protruding elements are arranged to fit in a receptacle that is a microtiter plate, preferably a microtiter plate comprising 24, 48 or 96 microtiter plate wells,

13. System according to the preceding claim, wherein the receptacle is a microtiter plate in the shape of a disc, preferably a round disc, comprising preferably 72 microtiter wells.

14. System according to the preceding claim, wherein the means for alignment are an

approximately centrally located post, to which the upper element is attached and about which the upper element may rotate, wherein said post is aligned with the wells of the receptacle, preferably in a fixed position.

15. System according to any one of the preceding claims, wherein the microtiter plate is

suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 36 microtiter wells, or a microtiter cartridge suitable for analysis by a mobile image acquisition and computing device with a camera (smart phone), comprising preferably 24 microtiter wells.

16. System according to any one of the preceding claims, characterised in that the capture affinity reagent is bound to the protruding elements by covalent attachment of said affinity reagent with said protruding element, preferably via an amide bond.

17. System according to any one of the preceding claims, characterised in that the capture affinity reagent is bound to the protruding elements by magnetic force, wherein said protruding elements are magnetic and bind magnetic beads that are attached to the capture affinity reagent.

18. System according to any one of the preceding claims, comprising additionally an

illuminating detection unit, wherein said unit comprises an artificial light source and a platform for positioning the receptacle (detection plate).

19. System according to the preceding claim, wherein the illuminating detection unit is

positioned below the receptacle and is of dimensions and illumination strength suitable for providing essentially uniform illumination to the wells of the receptacle to be analysed.

20. System according to any one of the preceding claims, comprising additionally a perforated covering element positioned between the light source and the receptacle, wherein the perforations correspond to the size and/or shape of the wells of the receptacle to be analysed.

21. System according to the preceding claim, wherein the illuminating detection unit

comprises LEDs as an artificial light source, preferably white LEDs.

22. System according to any one of the preceding claims, wherein the illuminating detection unit comprises an array of uniformly spaced LEDs below the wells of the receptacle to be analysed.

23. System according to any one of the preceding claims, wherein the illuminating detection unit comprises an LED light source, wherein the light from said LED is directed onto a reflective surface (mirror), wherein said reflective surface directs the light from the LED to the wells of the receptacle to be analysed.

24. System according to any one of the preceding claims, wherein the illuminating detection unit comprises one or more light diffusing elements, in order to provide essentially uniform illumination to the wells of the receptacle to be analysed.

25. System according to any one of the preceding claims, comprising a mobile image acquisition and computing device with a camera (smart phone).

26. System according to any one of the preceding claims, comprising an adaptor unit for use of a mobile image acquisition and computing device with a camera (smart phone), wherein the adaptor comprises a hollow sealed enclosure that is suitable for preventing interference from ambient light, such as a light impermeable box.

27. System according to the preceding claim, wherein the adaptor unit comprises means for alignment of the mobile image acquisition and computing device with the adaptor, such as alignment posts and/or alignment jigs, wherein the camera of the mobile image acquisition and computing device can be positioned at a fixed distance and position from the receptacle to allow acquisition of an image of one or more, preferably all, of the wells of the receptacle to be analysed.

28. System according to any one of the preceding claims, wherein the system is of a length and breadth essentially that of a 96 well microtiter plate.

29. System according to any one of the preceding claims, comprising software for the

detection of immune complex formation in an ELISA assay.

30. System according to the preceding claim, wherein the software detects colour (for

example colour intensity and/or colour formation) in an acquired image.

31. System according to the preceding claim, wherein the detection of colour encompasses processing of the acquired image by an algorithm in which the colour image is split into red, green and blue channels, wherein said image processing procedure comprises (automatic) determination of the centre of each receptacle well, extraction of mean pixel value of its neighbouring pixels and providing the composite pixel value (for example 1 - 256) by assessing the contributions from the individual red, green and blue channels, wherein the processing of the acquired image enables prediction of optical density from the corresponding composite pixel value.

32. Enzyme-linked immunosorbent assay (ELISA) method comprising

- provision of an upper element to which multiple protruding elements are attached, said protruding elements positioned parallel to one another on one side of said upper element, wherein said protruding elements are bound on their surface with capture affinity reagent, said capture affinity reagent being capable of binding an analyte in a liquid sample, - incubation via submersion of said protruding elements in one or more solutions comprising the analyte to be detected, preferably for 5 to 30 minutes, more preferably 10 to 20 minutes, thereby binding said analyte to said protruding elements via the capture affinity reagent, wherein

^■ said incubation is carried out in a receptacle, said receptacle comprising multiple wells holding analyte solution, wherein said wells are of dimensions that enable the insertion of said protruding elements into said wells without contact between the surface of the interior of said wells and said protruding elements, and one protruding element may be inserted per well, wherein means for alignment of the protruding elements with the receptacle wells are present on the upper element and/or receptacle, wherein said means for alignment enable insertion of said protruding elements into the wells of said receptacle in a (pre-) determined position, and wherein

^■ said analyte-comprising solution comprises preferably additionally detection

reagents, such as biotinylated anti-analyte-affinity reagent pre-conjugated to horse radish peroxidase (HRP) labeled-streptavidin,

- vertical displacement of the upper element from the receptacle to remove the

protruding elements from wells containing analyte solution,

- washing of the protruding element upon which analyte is bound to said immobilized capture affinity reagent, and

- detection of immune complex formation between said analyte, said immobilized capture affinity reagent, and said detection reagent, preferably by a microtiter plate reader or by a mobile image acquisition and computing device with a camera.

33. Method according to the preceding claim, wherein washing of the protruding element is carried out by submersion of protruding elements in wells of the receptacle containing wash buffer, followed by vertical displacement of the upper element from the receptacle to remove the protruding elements from wells containing wash buffer, wherein 1 to 3 wash steps of the protruding elements are carried out, wherein each submersion of the protruding elements in wash buffer is carried out for 1 second to 5 minutes, preferably 2 seconds to 1 minute, more preferably 5 seconds to 30 seconds.

34. Method according to any one of claims 32 or 33, wherein detection of immune complex formation between said analyte, said immobilized capture affinity reagent, and said detection reagent is carried out by submersion of protruding elements in wells of the receptacle comprising detection substrate, such as HRP substrate, preferably 3,3',5,5'-Tetramethylbenzidine (TMB), incubation of said protruding elements in wells comprising detection substrate, preferably for 1 to 10 minutes, more preferably 2 to 5 minutes, followed by vertical displacement of the upper element from the receptacle to remove the protruding elements from wells containing detection substrate, and subsequent detection of signal in the well comprising detection substrate, preferably via TMB substrate assay and measurement of optical density and/or color.

35. Method according to any one of claims 32 to 34, wherein the detection of immune

complex formation is carried out by detecting color formation after an enzyme-substrate reaction using the system according to any one of the preceding claims and a mobile image acquisition and computing device with a camera, comprising positioning the receptacle on an illuminating detection unit of any one of the preceding claims, image acquisition of the wells of the receptacle to be analysed using an adaptor unit of any one of the preceding claims with the camera of the mobile image acquisition and computing device, and processing of the acquired image by an algorithm, in which the color image is split into red, green and blue channels, the image processing procedure comprising automatic determination of the centre of each receptacle well, extraction of mean pixel value of its neighbouring pixels and providing the composite pixel value (for example 1 - 256) by assessing the contributions from the individual red, green and blue channels, wherein the processing of the acquired image enables prediction of optical density from the corresponding composite pixel value.

36. Kit for carrying out the method according to any one of claims 32 to 35, comprising the enzyme-linked immunosorbent assay (ELISA) system according to any one of the preceding claims.

37. Kit according to the preceding claim comprising one or more suitable receptacles in a microtitre format, such as one or more microtitre plates, disks, platforms or cartridges.

38. Kit according to any one of the preceding claims comprising immunoassay components, whereby the immunoassay components are pre-stored in said receptacles, disks, platforms and/or cartridges, which are preferably covered with a sealing film and/or foil suitable for sterile packaging.

39. Kit according to any one of the preceding claims comprising an illuminating detection unit according to any one of the preceding claims.

40. Kit according to any one of the preceding claims comprising software according to any one of the preceding claims for a mobile image acquisition and computing device with a camera for processing an acquired image.