Abstract:
The invention describes accurate and flexible methods and kits for conducting multi-analyte microarrays through the use of Tag-specific antibodies and analyte-specific Tag-anchored antibodies.

Description:
BACKGROUND 
       [0001]    The multi-analyte microarray (MAMA) is an effective tool for the simultaneous detection and quantification of multiple molecules in various sample matrices. The advantages of the biochip compared to traditional single analyte analysis include reduced sample volume and high-throughput capability. It has important uses in drug development, environmental testing, residue monitoring, veterinarian practice, and especially, clinical chemistry and basic biological research. The first successfully automated commercialisation of the MAMA concept was Randox&#39;s biochip technology (for the purposes of the invention the words biochip and microarray are synonymous and are used interchangeably) incorporated on the Evidence™ and Evidence Investigator™ platforms (EP0874242; EP0902394; EP0988893; EP0994355; EP1227311; EP1273349 ; Clinical Chemistry  2005, 51(7): 11665-1176). The biochip is a ceramic-coated substrate to which a number of specific binding partners are either covalently attached or passively adsorbed at specific locations. The specific binding partners are molecules capable of binding to target analytes in a sample, often antibodies or fragments thereof. The use of various labeling techniques enables the visualization of the interaction of a specific binding partner and an analyte and calibrators enable analyte quantification. Generally, MAMAs are commercialized as a manufacturer-defined end product i.e. a set of analyte-specific antibodies chosen by the company commercializing developing, manufacturing and selling the product. Currently, the combination of analytes chosen for a MAMA is scientifically coherent, being grouped, for example, by physiological function or association with a specific disease state e.g. Randox Cytokine I supports antibodies to IL-1α, IL-1β, IL-2, Il-4, IL-6, IL-8, IL-10, IFN-γ, TNF-α, MCP-1, EGF and VEGF, proteins recognized by the scientific community as important in inter-cellular signaling during an immune response. However, research scientists by definition are constantly testing new ideas and theories and may require biochips that recognize analyte combinations that are scientifically speculative. Although MAMAs can be developed to order, such development is time-consuming and costly. In the context of the current invention, reference will be made to static and flexible MAMAs. Static implies a format with a manufacturer-defined analyte specificity while flexible implies that the end-user can both define the analyte specificity and acquire it off-the-shelf. The static and flexible protein MAMA concepts were proposed by Ekins (1990; EP0749581). The EP0749581 flexible MAMA is underpinned by the DNA hybridization reaction; various capture agents in the form of short-chain oligonucleotides are tethered to the solid support and bind to their complementary sequenced oligonucleotides which are attached (as ‘tails’) to specific analyte-binding agents such as antibodies. The flexible MAMA format based on oligonucleotides attached to a solid support as capture agents has disadvantages compared to the format described by the current invention which uses Tag-specific antibodies attached to the solid support as capture agents. Firstly, the temperatures required to gain oligonucleotide specificity in a multi-analyte array (approximately 40-60° C. based on oligonucleotides of 18-20 base pairs) would likely have a sample denaturing effect and/or affect the conjugation of antibodies to the oligonucleotides (Breslauer et al. 1986; Rychlik et al. 1990). Secondly, due to cross-hybridisation the oligonucleotide format is less accurate. For example, in EP20040759957, a cross-hybridisation level of no greater than 0.1% is offered as an assay attribute, yet in the current invention, the cross-reactivity values of the various monoclonal antibodies of the invention have cross-reactivities for non-target tag molecules of less than 0.00001% (the limit of measurement of the cross-reactivity assay). Thus the MAMA format described by the current invention has no reagent problems related to increased temperatures, reduces the risk of cross-reactivity, and therefore enables a more accurate assessment of the analyte content of a patient sample. There is no mention in EP0749581 of using a solid support supporting Tag-specific antibodies as the capture agent. Although the oligonucleotide tail MAMA was proposed in 1990 the Inventors are not aware of any commercialization of the method. What is required, and which is solved by the current invention, is the provision of off-the-shelf, customisable reagents for flexible MAMA development that retains or betters the performance characteristics of the traditional static MAMA. The invention is underpinned by Tag-specific antibodies attached to a solid support that bind analyte-specific Tag-anchored antibodies. The invention is applicable to traditional competitive and sandwich assay formats, as well as a mixture of the two formats. 
       REFERENCES 
       [0000]    
       
         Ekins R. P. et al. (1990). The development of microspot, multi-analyte radiometric immunoassay using dual fluorescent-labeled antibodies.  Anal Chim Acta,  227:73-96 
         Fitzgerald S. P. et al. (2005).  Clinical Chemistry  51: 11665-1176 
         Breslauer K. J. et al. (1986). Predicting DNA duplex stability from the base sequence.  PNAS,  83: 3746-3750. 
         Rychlik W. et al. (1990). Optimization of the annealing temperature for DNA amplification in vitro.  Nuc. Ac. Res.,  18: 6409-6412. 
       
     
       SUMMARY OF THE INVENTION 
       [0006]    The invention described herein is a method, underpinned by antibody capture agents attached to a solid support and analyte-specific Tag-anchored antibodies that is sensitive, accurate and overcomes the inflexible, expensive nature of current static biochips enabling the end-user to choose and develop personalised MAMAs. Besides empowering the end-user, the system reduces the costs of manufacture leading to a more affordable end-product and is more accurate than the oligonucleotide-based flexible MAMA. This is achieved through the enhancement of the traditional biochip system by using Tag-specific antibodies attached to a solid support and analyte-specific Tag-anchored antibodies ( FIG. 3 ). The invention also provides a kit based on the Tag-specific antibodies and analyte-specific Tag-anchored antibodies for detecting and/or determining two or more analytes in a sample. 
     
    
     
       DRAWINGS 
         [0007]      FIG. 1  Synthesis of Tag 9 
           [0008]      FIG. 2  Synthesis of Tag 11 
           [0009]      FIG. 3  Sandwich immunoassay format for traditional biochip and Tag biochip 
           [0010]      FIG. 4  Examples of Tag molecules microarrays. Empty shapes are analytes to be detected; empty shapes with flower motif are labelled analytes for detection; filled shapes are Tags 
       
    
    
     DETAILED DESCRIPTION OF INVENTION 
       [0011]    In a first aspect, the invention is a method which enables the flexible MAMA for detecting or determining two or more analytes in a sample, the method comprising adding the sample, two or more analyte-specific Tag-anchored antibodies and two or more labeled conjugates to a solid support comprising two or more Tag-specific antibodies at spatially-defined locations on the solid support, each Tag-specific antibody recognizing a different Tag; and detecting or determining the amount of two or more analytes in the sample by detecting and measuring the labeled conjugates and comparing the measurement values with the measurement values from calibrators 
         [0012]    The Tag is a molecule which is, to the best of the Inventors&#39; knowledge, not a biochemical and is structurally distinct from commonly targeted analytes. The Tag can be for example a small molecule, polymer, peptide, polypeptide, oligonucleotide or protein. Preferably the Tag is a small molecule of molecular weight less than approximately 5000 Daltons, preferably less than approximately 1000 Daltons. Exemplary Tag molecules are shown in  FIG. 1 . The Tag-specific antibody is a highly specific polyclonal antibody, a monoclonal antibody, a highly specific antibody fragment such as a scFV, a highly specific antibody hybrid such as a humanized antibody, or any other antibody-based agent. Preferably, the Tag-specific antibody is a monoclonal antibody. For the current invention, the term ‘analyte-specific Tag-anchored antibody’ can mean that the Tag-anchored antibody either recognizes a single analyte or a well-defined group of analytes possessing a common epitope that is recognised by the Tag-anchored antibody. Detecting means qualitatively analyzing for the presence or absence of an analyte or analytes. This may require, for example, exceeding the well known analytical concept the limit of detection, or alternatively, a predefined cut-off concentration(s) set for the analyte(s); the latter can be referred to as a semi-quantitative method. Determining means quantitatively analyzing the amount(s) of analyte(s). Whether detecting or determining analyte(s), as is standard in the art, controls and/or calibrators are required. The calibrators may be previously measured values or may be values derived concurrently with the analytes to be measured. The previously measured calibrator values may be stored in a database. The sample can be any biological fluid but is preferably, oral fluid, urine, serum, plasma or blood. Environmental and food samples, after the appropriate pre-treatment steps if required, can also be tested using the invention. Environmental samples to be tested could be, for example, water and soil samples for the detection and/or quantification of contaminants. The solid support is a material capable of supporting an antibody either through a chemical reaction between the antibody and the molecules making up the solid support surface or a derivatised antibody in which the chemical reaction is between the group constituting the derivative of the derivatised antibody and the molecules making up the solid support surface. The solid support is preferably a biochip but can be various substrates such as, for example, inorganic metal oxides, that includes but is not limited to glass, ceramic and silicon. Additionally, the solid support can be a polymer such as (but not limited to) polypropylene, polyvinylene difluoride, and polytetrafluoroethylene. Furthermore, the inorganic solid support can be activated by the process of silanation to introduce chemical functionality, such as epoxy, amine, amide, chloride, isocynate, etc. The preferred solid support is a ceramic biochip. The solid support can also be beads such as magnetic and polystyrene beads and nanoparticles. The antibody or antibody derivative may also be supported by the solid support surface through non-bonding means using molecular cohesive forces such as van der waal&#39;s forces. The label of the labeled conjugate is a detectable label such as an enzyme, a luminescent substance, a radioactive substance or a mixture thereof. Preferably the label is a peroxidase, most preferably horseradish peroxidase; the conjugate, for a competitive assay, is a labeled small molecule (or ‘hapten’) which can compete with the analyte to be detected/measured in the patient sample for the Tag-anchored antibody while the conjugate for a sandwich assay is a labeled antibody which binds to an epitope of the target macromolecule e.g. peptide or protein. 
         [0013]    The flexible MAMA format, while enabling traditional competitive and sandwich immunoassay formats, also enables a mixture of the two formats. This allows co-analysis of small molecules (usually analysed using a competitive format) and macromolecules such as peptides or proteins (usually analysed using a sandwich format). The invention also includes a method in which the flexible and static MAMA formats are combined; this involves detecting or determining two or more analytes in a sample comprising adding the sample, one or more analyte-specific Tag-anchored antibodies and two or more labeled conjugates to one or more analyte-specific antibodies (static MAMA) and one or more Tag-specific antibodies (flexible MAMA) occupying spatially-defined locations on a solid substrate. 
         [0014]    The invention further provides a method which enables the flexible MAMA for determining two or more analytes in a sample, in which the labeled conjugates are labeled antibodies for sandwich assays and/or labeled haptens for competitive assays. A further aspect of the invention is Tag-specific antibodies and Tag-conjugated analyte-specific antibodies for use in flexible MAMAs, in which both sets of antibodies are monoclonal antibodies. 
         [0015]    Also described is a kit for detecting or determining two or more analytes in a sample comprising two or more Tag-specific antibodies at spatially-defined locations on a solid support and two or more Tag-conjugated analyte-specific antibodies. 
         [0016]    Another aspect of the invention is a kit for detecting or determining two or more analytes in a sample comprising one or more Tag-specific antibodies and one or more analyte-specific antibodies occupying spatially-defined locations on a solid support, and one or more analyte-specific Tag-anchored antibodies. The kits of the invention optionally contain one or more labeled conjugates. 
         [0017]    Finally, the invention describes one or more Tag-specific antibodies on a solid support together with one or more analyte-specific Tag-anchored antibodies for use in a MAMA. If there is only one Tag-specific antibody on a solid support and one corresponding analyte-specific Tag-anchored antibody the solid support further supports one or more analyte-specific antibodies, the analyte-specific Tag-anchored antibody and one or more analyte-specific antibodies each recognizing different analytes. The solid support is as previously described, but is preferably a biochip, most preferably a ceramic biochip. 
       General Methods 
     TAG Molecules 
       [0018]    Suitable Tag molecules include low molecular weight organic molecules such as dyes, peptides, and protecting groups used in organic synthesis. 
       Immunogen Preparation by Conjugation of Tag Molecules to Carrier Proteins 
       [0019]    Most antigens are macromolecules that contain distinct antigenic sites or epitopes that are recognized and interact with the various components of the immune system. However, Tag molecules are low molecular weight compounds that can interact with the products of an immune response but cannot induce an immune response on their own. These Tag molecules (Tags can be classed as haptens i.e. pre-immunogenic molecules) can be made immunogenic by coupling them to a suitable carrier protein using conventional methods for conjugation of haptens to proteins. Carriers are, typically, antigens of higher molecular weight that are able to cause an immunological response when administered in vivo. The most commonly used carriers are all highly immunogenic, large molecules that are capable of imparting immunogenicity to covalently coupled haptens (Tags). Some of the more useful carriers are proteins (bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and ovalbumin), but others include liposomes, synthetic or natural polymers and synthetically designed organic molecules. The criteria for a successful carrier molecule are the potential for immunogenicity, the presence of suitable functional groups for conjugation with a hapten (a Tag) and reasonable solubility properties even after derivatisation. The coupling chemistry used to prepare an immunogen from a hapten (Tag) and a carrier protein is an important consideration for the successful production and correct specificity of the resultant antibodies. The choice of cross-linking methodology is governed by the functional groups present on the carrier and the hapten (tag) as well as the orientation of the hapten (Tag) necessary for appropriate presentation to the immune system. An associated concern is the potential for antibody recognition and cross-reactivity toward the cross-linking reagent used to effect the conjugation. The current methods used for conjugation of the hapten (Tag) to carrier molecules include: carbodiimide-mediated hapten-carrier conjugation (EDC Method); NHS ester-mediated hapten-carrier conjugation (NHS Method); maleimide (or haloacetyl)-thiol conjugation; glutaraldehyde-mediated hapten-harrier conjugation (glutaraldehyde method); diazonium conjugation (diazo method); Mannich Condensation (formaldehyde method); homobifunctional cross-linker-mediated hapten-carrier conjugation. 
         [0000]    Tag-Specific mAbs 
         [0020]    The isolation, characterisation and purification of Tag specific monoclonal antibodies is an important component of a MAMA, although highly specific polyclonal antibodies are equally suitable. The monoclonal or polyclonal antibodies may be mammalian derived from, for example, mice or sheep. Other sources of antibodies are insects, plants or antibody library technologies such as phage or yeast display. Alternatively short chain antibody fragments (scFVs) or hybrid antibodies such as humanised antibodies may be used. 
       Coupling Tag to Test-Specific Antibody 
       [0021]    Conjugation of Tag molecules to the test-specific antibody is achieved using a range of coupling methods, some of which are described above (see Immunogen preparation by conjugation of Tag molecules to carrier proteins). The Tag molecules are coupled via the Fc portion of the test antibody using, for example, the periodate method. 
         [0022]    The invention will now be described by way of examples. 
       Examples 
     1. Tag Synthesis 
     Preparation of 3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric acid (Tag 9) 
       [0023]    3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid (1.02 g, 3.38 mmol), methyl 4-aminobutyrate hydrochloride (779 mg, 1.5 eq) and EDC hydrochloride (972 mg. 1.5 eq) in pyridine (20 ml) were stirred at RT O/N. The solvents were removed in vacuo and to the resulting orange residue was added CH 2 Cl 2  (50 ml) and water (20 ml). The organic portion was separated, dried over sodium sulphate, filtered and evaporated to dryness. The crude residue was purified by column chromatography (silica gel: 50% ethyl acetate in hexane) to give the title compound (1.27 g, 90%) as a yellow solid. 
         [0024]    3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric acid methyl ester (1.27 g, 3.05 mmol) was dissolved in THF (20 ml). Water was added followed by potassium hydroxide, 85% (623 mg, 3 eq). The mixture was stirred at RT for 4 h. Solvents were removed in vacuo and the remaining aqueous solution was adjusted to pH 3. The mixture was extracted with EtOAc/THF (1:1) (3×100 ml). The organic extracts were combined, dried over sodium sulphate, filtered and evaporated to dryness. The resultant solid was recrystallised from minimum amount of methanol containing ethyl acetate to give 3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric acid methyl ester (840 mg, 69%) as a yellow solid. 
       Preparation of 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (Tag 11) 
       [0025]    4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid (1.02 g, 4.32 mmol), methyl 4-aminobutyrate hydrochloride (995 mg, 1.5 eq) and EDC hydrochloride (1.24 g. 1.5 eq) in pyridine (20 ml) were stirred at RT overnight. The solvents were removed in vacuo and to the resulting orange residue was added CH 2 Cl 2  (50 ml) and water (20 ml). The organic portion was separated, dried over sodium sulphate, filtered and evaporated to dryness. The crude residue was purified by column chromatography (silica gel: 50% ethyl acetate in hexane) to give 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid methyl ester (1.28 g, 88%) as an orange/brown solid. 
         [0026]    4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid methyl ester (1.28 g, 3.79 mmol) was dissolved in THF (20 ml). Water was added followed by, water (20 ml) and potassium hydroxide, 85% (776 mg, 3 eq). The mixture was stirred at RT for 4 h. Solvents were removed in vacuo and the remaining aqueous was adjusted to pH 3. A precipitate resulted which was collected by filtration with water wash. The solid was dried in a dessicator over phosphorous pentoxide giving 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (Tag-9) (1.07 g, 88%) as an off-white solid. 
       2. Tag Immunogen Synthesis 
     Conjugation of TAG 9 to BSA 
       [0027]    To a solution of 3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric acid (14.5 mg, 0.0375 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (8.45 mg, 0.041 mmol) and N-hydroxysuccinimide (4.7 mg, 0.041 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BSA (50 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (5 ml). The mixture was then stirred overnight at 4 C. The solution was then dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give the Immunogen. MALDI results showed 20.2 molecules of TAG 9 had been conjugated to one molecule of BSA. 
       Conjugation of TAG 9 to BTG 
       [0028]    To a solution of 3-[3-nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric acid (52.3 mg, 0.135 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (30.6 mg, 0.1485 mmol) and N-hydroxysuccinimide (17.1 mg, 0.1485 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BTG (150 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (15 ml). The mixture was then stirred overnight at 4 C. The solution was then dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give the immunogen. 
       Conjugation of TAG 11 to BSA 
       [0029]    To a solution of 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (11.88 mg, 0.037 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (8.25 mg, 0.04 mmol) and N-hydroxysuccinimide (4.6 mg, 0.04 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BSA (50 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (5 ml). The mixture was then stirred overnight at 4 C. The solution was then dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give the Immunogen. MALDI results showed 17.6 molecule of TAG-11 had been conjugated to one molecule of BSA. 
       Conjugation of TAG 11 to BTG 
       [0030]    To a solution of 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (43.37 mg, 0.135 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (30.5 mg, 0.1485 mmol) and N-hydroxysuccinimide (17 mg, 0.1485 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BTG (150 mg) in 100 mM sodium bicarbonate solution (pH 8.5) (15 ml). The mixture was then stirred overnight at 4 C. The solution was then dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give the immunogen. 
       3. Antibodies for LSD, Norketamine, NGAL and FABP 
       [0031]    All antibodies were sourced from Randox Laboratories Ltd 
       4. Tag Monoclonal Development 
     Development, Isolation, Characterisation and Purification of Tag Specific Monoclonal Antibodies 
       [0032]    A series of structurally different Tag haptens were developed for sheep immunisation, two examples of which are provided in this application (i.e. Tag 9 and Tag 11). Each hapten was conjugated to BTG as a carrier protein, to increase antigen recognition in the immune system of the sheep. Three sheep were given an initial priming dose of the Tag immunogen followed by a series of monthly maintenance doses. During the maintenance dose immunization period, polyclonal serum was collected monthly and antibody titre assessed. Two animals were then selected for nodal extraction. The two sheep were given two final peri-nodal boosts at monthly intervals and the axillary and prescapular nodes were collected after the final nodal boost. Lymphocytes were extracted, fused with a hetero-myeloma cell line to create hybridoma cells that were then cultured. The supernatant was collected and screened using both specificity and affinity ELISA methods. Specific anti-tag monoclonal antibodies were selected by negatively selecting those clones which had a signal for more than one tag. Positive hybridomas were cloned to stability by means of limit diluting and purified antibody was collected and tested for affinity. The table below shows an example of this affinity data for two Tag 11 clones. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Clone 2 anti-tag 11 was more capable of maintaining &gt;50% 
               
               
                 pH 7 signal across a wider pH range and therefore was selected 
               
               
                 as the clone for further characterization. 
               
             
          
           
               
                   
                 Conditional Formatting = More 
               
               
                   
                 Than 50% from pH 7 
               
               
                   
                 pH 
               
             
          
           
               
                   
                 Target 
                 2.03 
                 3.04 
                 3.95 
                 5.02 
                 6.05 
                 7.2 
                 11.5 
                 12.8 
               
               
                   
                   
               
             
          
           
               
                 Clone 
                 TAG 11 
                 25% 
                 87% 
                 94% 
                 96% 
                 94% 
                 100% 
                 96% 
                 47% 
               
               
                 1 
               
               
                 Clone 
                 TAG 11 
                 27% 
                 92% 
                 98% 
                 104% 
                 99% 
                 100% 
                 99% 
                 83% 
               
               
                 2 
               
               
                   
               
             
          
         
       
     
       5. Tag-Antibody Coupling 
     Coupling of Tag 9 to Norketamine Monoclonal Antibody 
       [0033]    Norketamine monoclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 2 hours at 15-25° C. 3-[3-Nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric Acid (Tag 9) (6.0 mg) was dissolved in N,N-dimethylformamide (0.6 mL). The resulting solution was added to N-hydroxysuccinimide (2.14 mg), and pipette up and down until dissolved (this should take no longer than 30 seconds). The resulting solution was added to N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.56 mg), and the mixture was incubated on the roller at 15-25° C. for 2 hours. N,N-Dimethylformamide (1.4 mL) was added to the resulting solution. Conjugation: 4.1 μL of the pre-activated Tag 9 was added to antibody to give 10:1 molar ratio of Tag 9:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS (pH 7.2). 
       Coupling of Tag 9 to LSD Polyclonal Antibody 
       [0034]    LSD polyclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 2 hours at 15-25° C. 
         [0035]    3-[3-Nitro-4-(2-pyridylthio)phenyl]acrylic acid-aminobutyric Acid (TAG 9) (6.0 mg) was dissolved in N,N-Dimethylformamide (0.6 mL). The resulting solution was added to N-hydroxysuccinimide (2.14 mg), and pipette up and down until dissolved (this should take no longer than 30 seconds). The resulting solution was added to N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (3.56 mg), and the mixture was incubated on the roller at 15-25° C. for 2 hours. N,N-Dimethylformamide (1.4 mL) was added to the resulting solution. 4.1 μL of the pre-activated TAG 9 was added to LSD polyclonal antibody to give 10:1 molar ratio of TAG 9:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS (pH 7.2). 
       Coupling of Tag 11 to LSD Polyclonal Antibody 
       [0036]    LSD polyclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 2 hours at 15-25° C. 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (TAG 11) (6.0 mg) was dissolved in N,N-dimethylformamide (0.6 mL). The resulting solution was added to N-hydroxysuccinimide (2.58 mg), and pipette up and down until dissolved (this should take no longer than 30 seconds). The resulting solution was added to N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4.30 mg), and the mixture was incubated on the roller at 15-25° C. for 2 hours. N,N-Dimethylformamide (1.4 mL) was added to the resulting solution. 3.4 μL of the pre-activated TAG 11 was added to antibody to give 10:1 molar ratio of TAG 11:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS (pH 7.2). 
       Coupling of Tag 11 to Norketamine Monoclonal Antibody 
       [0037]    Norketamine monoclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 2 hours at 15-25° C. 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid (TAG 11) (6.0 mg) was dissolved in N,N-dimethylformamide (0.6 mL). The resulting solution was added to N-hydroxysuccinimide (2.58 mg), and pipetted up and down until dissolved (this should take no longer than 30 seconds). The resulting solution was added to N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4.30 mg), and the mixture was incubated on the roller at 15-25° C. for 2 hours. N,N-Dimethylformamide (1.4 mL) was added to the resulting solution. 3.4 μL of the pre-activated TAG 11 was added to antibody to give 10:1 molar ratio of TAG 11:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS (pH 7.2). 
       Coupling of Tag 11 to NGAL Polyclonal Antibody 
       [0038]    NGAL polyclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 1 hour at 15-25° C. 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid NHS ester (Tag 11) (5 mg) was dissolved in N,N-dimethylformamide (0.5 mL). 3.5 μL of TAG 11 was added to antibody to give 25:1 molar ratio of TAG 11:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS containing 0.1% CAA, 0.01% MIT (pH 7.2). 
       Coupling of Tag 11 to FABP Monoclonal Antibody 
       [0039]    FABP monoclonal antibody (0.5 mg) was dialysed against 50 mM HEPES solution, pH8.0 with stirring for 1 hour at 15-25° C. 4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-4-oxobutanoic acid-aminobutyric acid NHS ester (Tag 11) (5 mg) was dissolved in N,N-dimethylformamide (0.5 mL). 3.5 μL of TAG 11 was added to antibody to give 25:1 molar ratio of TAG 11:antibody. The resulting solution was incubated on the roller at 15-25° C. for 2 hours. Excess hapten was removed with PD-10 column (Pharmacia), pre-equilibrated with PBS containing 0.1% CAA, 0.01% MIT (pH 7.2). 
       6. MAMA Development and Implementation 
       [0040]    Biochip spotting: prior to spotting, biochip surface is silanated (Fitzgerald et al 2005). The Perkin-Elmer BCA spotter was used to spot antibody onto the biochip surface. Monoclonal anti-Tag antibody was spotted (at 10 mL) onto the biochip surface at 1 mg/ml concentration in carbonate buffer (pH—9.6). Spotted biochips were blocked with 2% Casein in carbonate buffer for one hour at room temperature and then washed with water. Biochips were then stabilised using 5% Trehalose in carbonate buffer for 5 minutes at room temperature and finally dried overnight at 37° C. and 25% relative humidity. Biochips were finally chopped and assembled into the carriers and vacuum-sealed for storage at 2-8° C. All immunoassay reagents were sourced from Randox Laboratories Ltd unless otherwise stated. To perform the assay, biochips were loaded with the appropriate volume of assay buffer (100-200 μL), calibrators (25-100 μL) and HRP-labelled conjugate (50-100 μL). Assay buffer was spiked with the appropriate concentration of Tag-conjugated-capture antibody. Biochips were incubated for 30 minutes up to 60 minutes based at 37° C. Finally, biochips were washed using Tris-buffer saline-tween and imaged using 1:1 mixture of luminol/peroxide. For chemi-luminescence imaging luminol/peroxide was mixed 1:1 and 250 μl was added in each well. After two minutes of soak in the dark, biochips were imaged using Randox Evidence Investigator. 
         [0041]    For NGAL, NKet, LSD assay format: assay diluent (150 μl), sample (75 μl) incubated at 33° C. and 370 rpm for 30 minutes, conjugate (75 μl) incubated at 33° C. and 370 rpm for 30 minutes. 
       Results 
       [0042]      
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Competitive Format - Tag 11/LSD + Tag 9/Norketamine 
               
               
                 flexible MAMA and single analyte comparisons using biochips 
               
             
          
           
               
                   
                 LSD 
                   
                 Norketamine 
               
             
          
           
               
                 Conc n   
                 MAMA 
                 Single analyte 
                 Conc n   
                 MAMA 
                 Single analyte 
               
               
                 pg/ml 
                 RLU 
                 RLU 
                 pg/ml 
                 RLU 
                 RLU 
               
               
                   
               
             
          
           
               
                 0.00 
                 5622 
                 5628 
                 0.00 
                 10292 
                 10033 
               
               
                 30.00 
                 5058 
                 5167 
                 0.98 
                 9690 
                 9819 
               
               
                 50.00 
                 4821 
                 5096 
                 1.95 
                 9357 
                 9144 
               
               
                 100.00 
                 4079 
                 4578 
                 3.91 
                 7859 
                 8200 
               
               
                 156.25 
                 3436 
                 3677 
                 7.81 
                 6052 
                 6327 
               
               
                 312.50 
                 2692 
                 3065 
                 15.63 
                 1753 
                 1780 
               
               
                 625.00 
                 2230 
                 2126 
                 31.25 
                 1238 
                 1362 
               
               
                 1250 
                 1324 
                 1395 
                 62.50 
                 594 
                 790 
               
               
                 2500 
                 1148 
                 1036 
                 125.00 
                 409 
                 398 
               
               
                 IC 50   
                 287.50 
                 396.03 
                   
                 9.39 
                 10.10 
               
               
                 (pg/ml) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Sandwich and Competitive Format - Tag 11/NGAL + 
               
               
                 Tag 9/Norketamine flexible MAMA and single 
               
               
                 analyte comparisons using biochips 
               
             
          
           
               
                   
                 Norketamine 
               
             
          
           
               
                   
                 NGAL 
                   
                 Single 
               
             
          
           
               
                 Conc n   
                 MAMA RLU 
                 Single analyte 
                 Conc n   
                 MAMA 
                 analyte 
               
               
                 ng/ml 
                 (B/Bmax) 
                 RLU (B/Bmax) 
                 pg/ml 
                 RLU 
                 RLU 
               
               
                   
               
             
          
           
               
                 0.00 
                  58 (1.7) 
                  62 (1.6) 
                 0 
                 4835 
                 5102 
               
               
                 11.40 
                 172 (5.1) 
                 184 (4.8) 
                 0.80 
                 4342 
                 4490 
               
               
                 25.80 
                 231 (6.9) 
                 294 (7.7) 
                 1.60 
                 3976 
                 4318 
               
               
                 53.10 
                  431 (12.8) 
                  483 (12.6) 
                 3.10 
                 3803 
                 4183 
               
               
                 93.30 
                  878 (26.1) 
                  980 (25.6) 
                 6.20 
                 2870 
                 2986 
               
               
                 159.20 
                 1531 (45.5) 
                 1683 (43.9) 
                 12.50 
                 728 
                 743 
               
               
                 395.80 
                 2459 (73.1) 
                 2526 (65.9) 
                 25.00 
                 429 
                 382 
               
               
                 751.40 
                 3278 (97.5) 
                 3282 (85.6) 
                 50.00 
                 229 
                 196 
               
               
                 983.20 
                 3362 
                 3833 
                 100.00 
                 165 
                 131 
               
               
                 IC 50   
                 NA 
                 NA 
                   
                 7.51 
                 7.40 
               
               
                 (pg/ml) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Sandwich and Competitive Format - Tag 11/FABP + Tag 9/Norketamine 
               
               
                 flexible MAMA and single analyte comparisons using biochips 
               
             
          
           
               
                   
                 Norketamine 
               
             
          
           
               
                   
                 FABP 
                   
                 Single 
               
             
          
           
               
                 Conc n   
                 MAMA RLU 
                 Single analyte 
                 Conc n   
                 MAMA 
                 analyte 
               
               
                 ng/ml 
                 (% B/Bmax) 
                 RLU (% B/Bmax) 
                 pg/ml 
                 RLU 
                 RLU 
               
               
                   
               
             
          
           
               
                 0.00 
                  51 (0.2) 
                   0 
                 0 
                 4835 
                 5102 
               
               
                 2.30 
                  363 (1.6) 
                 320 (1.4) 
                 0.80 
                 4342 
                 4490 
               
               
                 4.90 
                 1022 (4.4) 
                 970 (4.1) 
                 1.60 
                 3976 
                 4318 
               
               
                 9.00 
                  2533 (10.8) 
                 2666 (11.3) 
                 3.10 
                 3803 
                 4183 
               
               
                 18.70 
                  6336 (27.1) 
                 6478 (27.5) 
                 6.20 
                 2870 
                 2986 
               
               
                 38.40 
                 13250 (56.7) 
                 13655 (58.0)  
                 12.50 
                 728 
                 743 
               
               
                 58.20 
                 16933 (72.5) 
                 17634 (74.9)  
                 25.00 
                 429 
                 382 
               
               
                 73.70 
                 20742 (88.8) 
                 20765 (88.2)  
                 50.00 
                 229 
                 196 
               
               
                 97.40 
                 23359 
                 23556 
                 100.00 
                 165 
                 131 
               
               
                 IC 50   
                 NA 
                 NA 
                   
                 7.51 
                 7.40 
               
               
                 (pg/ml) 
               
               
                   
               
             
          
         
       
     
         [0043]    The results show that there is no loss in sensitivity in going from the biochip single analyte to the biochip Tag-based flexible MAMA. Furthermore, the sandwich and competitive assay formats can be effectively combined using the Tag concept. The use of Tag-specific antibodies bound to a solid support and analyte-specific Tag-anchored antibodies in effecting flexible MAMAs is thus proven.