EP2140266A1 - Quantification de molécules d'analyte à l'aide de multiples molécules de référence et fonctions de corrélation - Google Patents

Quantification de molécules d'analyte à l'aide de multiples molécules de référence et fonctions de corrélation

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Publication number
EP2140266A1
EP2140266A1 EP08715628A EP08715628A EP2140266A1 EP 2140266 A1 EP2140266 A1 EP 2140266A1 EP 08715628 A EP08715628 A EP 08715628A EP 08715628 A EP08715628 A EP 08715628A EP 2140266 A1 EP2140266 A1 EP 2140266A1
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EP
European Patent Office
Prior art keywords
binding
analyte
binding partners
partners
sample
Prior art date
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Application number
EP08715628A
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German (de)
English (en)
Inventor
Anpan Han
Cornelia Steinhauer
Hanne Kaas
Kristine Garde
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Toxispot AS
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Toxispot AS
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Publication date
Application filed by Toxispot AS filed Critical Toxispot AS
Publication of EP2140266A1 publication Critical patent/EP2140266A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to an assay method for quantifying analyte molecules in a sample.
  • the present invention also relates to the use of two or more reference binding partners in an assay for quantifying analyte molecules in order to minimise the risk of false positive results.
  • the method is useful for quantifying microcystin LR.
  • Biochemical assays are methods to analyse chemical substances through the application of biological molecules, e.g. proteins or DNA.
  • An immunoassay is a biochemical assay involving antibodies which are proteins with the property of being able to bind a substance, an antigen, with great specificity.
  • the antigen may be a protein, or a peptide, a carbohydrate, a small organic molecule etc. This property is utilised in an immunoassay, where antibodies are used to bind corresponding antigens that are present in a sample.
  • immunoassays are used to quantify analytes such as biomarkers, toxins, drugs, pesticides, viruses etc.
  • heterogeneous assays which are also referred to as solid-phase assays.
  • heterogeneous immunoas says the antibody or antigen is immobilised onto a surface.
  • sandwich assays There are two general kinds of heterogeneous immunoassays; competitive and non-competitive, the latter of which includes direct and sandwich assays.
  • a typical, traditional immunoassay requires at least an antibody-antigen pair and commonly also a secondary antibody capable of binding either of the binding partners of the antibody-antigen pair and which secondary antibody is equipped with a tag allowing direct or an indirect detection.
  • the antigen is normally the analyte of interest.
  • the binding partners of the antibody-antigen pair interact this interaction can be detected by letting the secondary antibody bind to its corresponding binding partner and detecting this binding via the tag.
  • the antigen e.g. competitive assay
  • the antibody e.g. sandwich or direct assay
  • the detection system may consist of an enzyme linked to the secondary antibody (an indirect detection) or a label allowing direct observation, such as radioactive elements or fluo- rescent probes.
  • Typical enzymes used as labels are horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • binding of the secondary antibody to its corresponding antigen may be detected by adding a chromogenic substrate of the enzyme and measuring the extent of the formation of the coloured product of the enzymatic reaction.
  • analyte i.e. a quantitative measurement of the antigen concentration in the sample. This is also true in environmental monitoring and many other domains of immunoassay applications.
  • To determine the absolute concentration it is necessary to gen- erate a standard curve or a dose response curve, which is a function that relates the signal produced by the label to the absolute concentration of the antigen.
  • standards of the antigen with well-defined concentrations are typically assayed in parallel with the antigen in the sample. The results of the parallel assays with standards will enable the user to determine a function that relates the assay signal to the absolute concentration of the antigen. Using this function, it is then possible to relate the signal generated by the sample with an unknown amount of antigen.
  • a standard curve is required. In the microarray format, this is usually carried out by incubating several arrays with known concentrations of analyte target molecules. Examples of competitive immunoassays in microarray formats are the works of Han et al. (2003) and Belleville et al. (2004) who determined the absolute concen- trations of pesticides. To measure one sample up to 8 arrays were needed for the standard curve and control experiments. For assays using labelled primary antibodies, 8 different solutions with spiked antigen were prepared and the mixture was pipetted onto the arrays. Du et al. (2005) reported competitive immunoassays that detected drugs from se- rum samples. Also here a number of arrays were used to generate the standard curves.
  • WO2005/090546 describes an- tibody microarrays for identifying, quantifying and qualifying analyte molecules with a special focus on expression analysis.
  • the principles of WO2005/090546 are based on traditional immunoassays. In order to improve normalisation of data the assays are further supplied with spots containing internal control molecules corresponding to molecules nor- mally unexpressed in the target samples.
  • WO2006/020126 also relates to microarrays for the determination of protein concentrations.
  • a target protein of unknown concentration
  • control proteins of known concentration
  • the tag may then be quantified by binding a binding pair member of the tag to the tag and then quantifying these interactions.
  • the quantification of the target protein is only indirect as it is the tag which is quantified and this quantification is then used for calculating the concentration of the target protein.
  • a preferred tag in WO2006/020126 is the protein glutathione-S-transferase.
  • US-application US2005/0250109 relates to methods for the detection, identification and quantification of multiple target molecules using microarray technology.
  • the methods of US2005/0250109 employ immobilised sets of capture molecules capable of binding two different sets of target molecules, so that the binding of a set of target molecules may be quantified relative to the binding of a second set of molecules with the aid of a "correction factor".
  • This correction factor is "calculated through the use of at least one additional identical target molecule (able to bind first set of capture molecules and second set through an adaptor molecule) simultaneously quantified on both capture molecules".
  • the methods are mainly intended for use with nucleotides, though despite mention of how the method may be employed also with proteins and peptides, the methods appear ill-suited for this purpose.
  • This estimate of internal variation may then be employed to improve the reading of the signal from the analyte molecule with respect to variation.
  • the positioning of the reference spots may function as a map of the array for automated reading of the array.
  • Microarrays for the detection and quantification of antibodies are described in US-application US2006/0154299.
  • the arrays contain antigens for the analyte antibodies of interest and a series of deposited antibodies from the same species as the analyte anti- bodies.
  • the deposited antibodies are used for constructing standard curves for the interaction between the immobilised antibodies and a fluo- rescently labelled, secondary antibody from another species (specific for antibodies from the target organism).
  • the present invention relates to an assay method for quantifying analyte molecules in a sample comprising a) providing a substrate containing two or more reference regions containing different immobilised reference first binding partners and one or more analyte regions containing immobilised analyte first binding partner(s), where the reference first binding partners and the analyte first binding partner(s) have affinities for binding different second binding partners; b) applying a mixture containing reference second binding partners and a sample suspected of containing one or more analyte first or second binding partner(s), in which mixture the concentrations of the reference second binding partners are known; c) allowing the mixture to interact with the substrate; d) measuring the binding of the analyte binding partner(s) in the mixture to the corresponding immobilised analyte binding part- ner(s) and the binding of the reference second binding partners to the immobilised reference first binding partners; and e) quantifying the concentration of analyte binding partner(s) in the sample, where
  • This assay method is particularly suited for use in an immunoassay format where the binding partners comprise antibodies and their corresponding antigens.
  • the analyte molecule may be either the antigen or the antibody part of the ana- lyte binding partner pair.
  • the assay method is not limited to antigen-antibody binding partners but may well also include binding partners comprising other pairs of proteins capable of interacting specifically, proteins interacting specifically with non-proteinaceous molecules; or pairs of non-proteinaceous molecules.
  • the binding partners com- prise microcystin LR (MC-LR), human chorionic gonadotropin (hCG), insulin and (Tyr[SO 3 H] 27 ) cholecystokinin fragment 26-33 (CCK) and antibodies raised against MC, hCG, insulin and CCK.
  • a competi- tive assay format in which the mixture containing the reference second binding partners and the sample comprises analyte first and second binding partners.
  • the mixture containing the sample contains both the binding partner of the immobilised binding partner as well as a solubilised form of the binding partner immobilised on the substrate.
  • the method of the present invention may advantageously be employed in a microarray format, although other formats, such as mi- crotiter plates of e.g. 96, 384 or other numbers of wells are also suitable.
  • each well of the microtiter plate may comprise one region, or each well may comprise a microarray of regions.
  • the regions for immobilising analyte or reference binding partners are preferably discrete, although by careful choice of detection methods several regions may well be superimposed without jeopardising the measurement of bound binding partners. For example, by employing a combination of principally different detection methods, e.g. fluorescent and radioactive labelling simultaneous reading of superimposed signals may be possible.
  • the regions are discrete in the substrate it may be advantageous to position a plurality of identical regions in close vicinity so that the identical regions can be said to be arranged in sets.
  • a preferred method of detecting interactions between respective binding partners involves using one or more additional binding partners which additional binding partners are capable of binding to the analyte second binding partners and/or to the reference second binding partners, and which additional binding partners are not capable of binding to the immobilised first binding partners, and where the additional binding partners facilitate measurement.
  • these additional binding partners comprise secondary antibodies labelled with a tag allowing detection.
  • This tag may be an enzyme capable of converting a colourless substrate molecule into a coloured or fluorescent form for indirect detection, examples of such enzymes being horseradish peroxidase or alkaline phosphatase, or the tag may be a fluorescent moiety, such as green fluorescent protein, Cy3 or Cy5, or a radioactive atom, such as 125 I, allowing direct detection.
  • Other tags facilitating detection will be well known to those skilled in the art.
  • the present invention also relates to the use of two or more first reference binding partners in an assay for quantifying analyte molecules in a sample in order to minimise the risk of false positive results
  • an assay comprises a) measuring the binding between analyte first and second binding partners and the binding between the two or more reference first binding partners and corresponding reference second binding partners; and b) quantifying the concentration of analyte binding partner(s) in a sample, where the quantification is performed by correlating the measurement of the binding of the analyte partners to the measurement of the binding of the reference binding partners.
  • this assay is a competitive assay.
  • Fig. 1 shows an exemplary schematic illustration of a substrate with mi- croarrays.
  • Fig. 2 depicts the steps in an example of a competitive ELISA assay ac- cording to the method of the present invention.
  • Fig. 3 shows images of microarrays after binding and detection of first and secondary binding partners
  • Fig. 4 shows correlation curves for MC
  • Fig. 5 shows a standard curve for MC Detailed description of the invention
  • the present invention is related to the field of quantification of biochemical analyte molecules. In a preferred embodiment it is related to the measurement of microcystin LR in a competitive immunoassay in a mi- croarray format.
  • binding partner refers to either of two parts of a pair of molecules capable of binding specifically to each other, and it may therefore refer to either the antigen or the antibody of an antigen- antibody pair, to either the carbohydrate or the lectin of a carbohydrate- lectin pair, to either strand of DNA of a double stranded molecule of complementary sequence etc.
  • pairs of binding partners include those formed by antigen-antibody interactions, compound-aptamer interactions, antibody-antibody interactions, protein- small molecule interactions, enzyme-substrate interactions, enzyme- substrate analogue interactions, lectin-carbohydrate interactions, drug- receptor interactions, streptavidin-biotin interactions, nucleic acid- nucleic acid interactions, nucleic acid-protein interactions etc.
  • antibody refers to antibodies of any suitable isotype as well as antibody fragments containing the antigen binding specificity of a complete antibody. Antibodies may be of monoclonal or polyclonal origin, or they may be produced recombinantly.
  • first binding partner refers to the part of a pair of binding partners immobilised on a substrate, whereas the “second binding partner” is not immobilised; the fact that “first binding partner” refers to the immobilised form does not exclude that the same type of molecule is used in a non-immobilised form.
  • analyte binding partner is either part of a pair of binding partners comprising an analyte molecule, whereas the “reference binding partner” refers to either part of a pair of binding partners employed to construct a correlation curve between the binding of analyte binding partners and reference binding partners.
  • the sample to be analysed by the present assay method can be of any origin.
  • the sample is a water sample and the analyte binding partner it is suspected of containing is selected from the group consisting of bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, enzymes occur- ring in the synthesis pathway of toxins, markers for the occurrence of pathogenic microorganisms, geosmins, plant protection products and pharmaceutical products, and degradation products of the above mentioned group of compounds.
  • a certain group of binding partners of interest in the present invention is algal toxins, such as cyanotoxins, especially hepatoxins and neurotoxins.
  • hepatoxins include microcystin (MC) like microcystin-LR (MC-LR) and microcystin-RR (MC-RR), nodularin, and cylindrospermopsin.
  • MC-LR microcystin-LR
  • MC-RR microcystin-RR
  • nodularin adularin
  • neurotoxins include ana- toxins and saxitoxins.
  • algal toxins of interest include paralytic shellfish toxins/poisoning (PST, PSP, saxitoxins and gonyautoxins), diar- rheic shellfish toxins/poisoning (DST, DSP, ocadeic acid, yessotoxin, etc.), amnesic shellfish toxins/poisoning (AST, ASP, domoic acid), ciguatera fish toxins/poisoning (CFT, CFP, ciguatera etc.), neurotoxins shell- fish toxins/poisoning (NST, NSP, brevetoxin, etc.), and endotoxins (lipopolysaccharides, LPS).
  • PST paralytic shellfish toxins/poisoning
  • DST diar- rheic shellfish toxins/poisoning
  • AST amnesic shellfish toxins/poisoning
  • CFT ciguatera fish toxins/poisoning
  • the reference binding partners are selected from molecules not expected to be present in a sample to be analysed.
  • the reference binding partners may be of the same chemical nature as that of the analyte binding partners, e.g. proteins when proteins are to be analysed or nucleic acids when nucleic acids are to be analysed, or they may be of a different chemical nature.
  • the chemical nature of the reference binding partners is the same as that of the analyte binding partners.
  • Suitable pairs of reference binding partners include proteins known to give an immune response upon immunisation of animals, such as mice, rabbits or goats, and the corresponding antibodies raised against the proteins upon the immunisation.
  • the use of dose response curves and control experiments for measuring and quantifying and analyte molecule is replaced by another set of functions that relate a measured signal to the concentration of an analyte molecule.
  • this set of functions is stored in e.g. a computer, three arrays at the most, preferably a single array is needed to determine the absolute concentration of multiple analytes when analysing a sample.
  • each of the regions are formed in separate wells of a microtiter plate in place of the microarray format the term "microtiter plate” will generally substitute the term "microarray" in the discussions below.
  • the method of the present invention involves the generation of a set of correlation functions correlating the signals obtained from the binding of analyte binding partners to that of the signals of the binding between the two or more respective reference binding partners.
  • the microarray is spotted with (please see also Fig. 1): - two or more (sets of) regions of reference first binding partners; the regions are denoted r mir where the subscripted / is the index denoting the region, r denotes a reference binding partner, and the subscripted m is and index identifying the reference binding partner.
  • control regions may contain only the buffers otherwise used for immobilising the first binding partners, molecules, such as proteins, not expected to bind any of the binding partners or they may contain a substance facilitating detection. Thus, these may serve as negative controls and may further act as landmarks in the microarray landscape for the software employed for analysis or for normalisation pur- poses.
  • Fig. 1 schematically illustrates a substrate with two microarrays (numbered 1 and 2) according to the present invention. Also shown is an enlarged section with a microarray depicting the positions of the analyte and reference binding partners (denoted a and r, respectively as discussed above) as well as of potential control regions (denoted c).
  • the concenctrations of the second binding partners are indicated as [ ⁇ r m ], and [ ⁇ a ⁇ ]/, respectively, where / denotes the index of different concentrations.
  • Preferably 10 such standards are used.
  • the microarray signals are measured: the average signal intensities of the regions caused by the complexes formed between the reference binding partners are denoted Sr m .
  • the average signal intensities of the regions caused by the complexes formed between the analyte binding partners are denoted Sa n .
  • the optional signal intensities from the optional control regions are denoted Sc k .
  • R(Sa n , Sr m ) ⁇ are calculated; in case control regions are used, R(Sa n , Sc k ) ⁇ is also calculated.
  • this value could be the ratio between Sa n and Sr m , i.e. Sa n /Sr m .
  • R(Sa n , Sr m ) ⁇ is now plotted as a function of [ ⁇ a ⁇ ]/, and functions will be generated by fitting data.
  • F nm the analyte concentrations with the signals from the reference and the analyte regions.
  • the number of functions is equal to the number of reference binding partners. For example if there are 3 analyte binding partner and 3 reference binding partner, there will be 9 functions in total.
  • analyte and reference first binding partners could be capture antibodies, and the corresponding second binding partners would then be antigens with multiple epitopes, so that the analyte molecule(s) would in this case be antigens. Additional bind- ing partners facilitating detection would then be necessary to measure the binding. These additional binding partners could be different antibodies each capable of binding to a different epitope on the antigens (i.e. the second binding partners).
  • each of these additional binding partners would be coupled to e.g. HRP, AP, fluorescent dyes or radioactive labels allowing detection of the interactions between the first and second binding partners via the corresponding additional binding partners.
  • HRP e.g. HRP
  • AP e.g. HRP
  • fluorescent dyes or radioactive labels e.g. fluorescent dyes or radioactive labels
  • the additional binding partners would be antibodies from the same species (if necessary also of the same isotype) raised against the second binding partners. This would allow all additional binding partners to be detected using only a single appropriately labelled antibody raised against antibodies of the origin (and possibly isotype) of the additional binding partners.
  • Fig. 2 illustrates a competitive ELISA according to a preferred embodiment of the present invention.
  • the substrate is func- tionalised with an analyte first binding partner (a ⁇ ), three different reference first binding partners (r lr r 2r r 3 ) and a control region (c) not contan- ing any immobilised binding partners.
  • analyte first and second binding partners a ⁇ and ⁇ a 1 , respectively
  • reference second binding partners ⁇ r 2-5
  • additional binding partners are added to facilitate the measurement of the binding between respective binding partners.
  • reference binding partners When two, or more preferably three or more, reference binding partners are employed to generate the correlation functions further advantages are provided. These reference partners will be selected from molecules not expected to bind to molecules present in a sample to be analysed. However, there is always a risk that cross-binding may occur, but by using two, three or more reference binding partners, the risk of incorrect results caused by cross-binding can be nearly eliminated. This is particularly relevant in the case of competitive assays, where any un- wanted cross-binding may results in false positive measurements which are highly undesirable.
  • Empirical correlation functions in the method of the present invention may be empirical, though as will be appreciated by those of skill in the art, more complex correlations based on theoretical consideration may also be used.
  • An example of such an empirical function is the four parameter logistic function presented below.
  • A is the maximal signal (upper asymptote)
  • E is the minimal signal (lower asymptote)
  • C or IC 5 O is the analyte concentration at the midpoint between A and E ((A + E)II) and B(A - f)/4 is the slope of the four-parameter function at IC 5 O-
  • the substrate material used in the method of the present invention may be any conventional material and may have any convenient design. To be able to fit into a standard scanning apparatus the dimension of the substrate, when a microarray format is employed, usually resembles that of a microscope slide (e.g. 25 mm x 76 mm).
  • the wells of a micro- titer plate may also serve as a substrate for preparation of microarrays and furthermore, the microarrays to be used in the method of the present invention may also be comprised in such devices commonly known as microfluidic devices.
  • the surface of the material may be hydrophobic, hydrophilic, thiophilic or it may contain a contain mixture of areas with these characteristics.
  • Suitable materials for the substrate include any rigid transparent or non-transparent material, however materials, such as glass, silicon, gold or polymers, such as polystyrene, polymethylmethacrylate), polypropylene, cyclic olefin copolymers (COC), polyethylene terephthalate, polycarbonate etc. are preferred.
  • rigid materials soft materials, such as polydimethylsiloxane, agarose or poly- acrylamide gels may also be used.
  • An example of a suitable material is MaxiSorpTM produced by Nunc A/S (Denmark).
  • the surface of the substrate material may be employed in an unmodified or modified form, or it may be activated to carry groups such as amines, thiols, aldehydes, epoxies, amino- or other silanes, polylysine etc. to allow the immobilised first binding partners to couple covalently to the surface.
  • the first binding partners are immobilised on the surface of the substrate material by physical adsorption, though in certain cases it may be necessary to induce covalent links be- tween the first binding partners and appropriate chemical moieties on the surface. Methods and conditions for immobilisation via chemical coupling of binding partners to surfaces are well-known to those skilled in the art.
  • Immobilisation of the first binding partners to the surface of the substrate material is generally performed by applying a liquid comprising the binding partner to the surface and allowing the binding partner to interact with the surface; the liquid may be in the form of a solution or suspension.
  • the liquid of the first binding partners to be used for immobilisation is preferably an aqueous solution, and it may contain salts, polymers, and/or further additives.
  • An organic solvent may be selected in special circumstances, e.g. in the event the binding partner to be immobilised is not sufficiently soluble in water.
  • the content of the organic solvent(s) may be from 0-100%.
  • the immobilisation solution may contain a certain amount of formaldehyde, betaine, or DMSO for reducing the evaporating time or to avoid or inhibit degradation of the first binding partner.
  • microarrays For the preparation of microarrays it is generally desired to add to the immobilisation solution such additives which will provide for a sufficiently high viscosity that reduces mass transport by capillary actions and provide for sufficiently low evaporation for allowing the binding partner to interact with the surface.
  • An adjusted evaporation rate is also important for spot homogeneity and morphology.
  • Various commercial spotting buffers are suitable. An example of such buffers is Genetix' amine spotting buffer.
  • the immobilisation liquid is generally applied in a quantity in the sub- ⁇ l range.
  • the solution of the first binding partner is spotted in an amount of 10 nl or less.
  • each spot is applied in an amount of about 1 nl or less.
  • a deposited amount of solution of 1 nl corresponds to a spot diameter of around 100 ⁇ m.
  • Microarray fabrication using contact printing is based on high definition pins that, upon contact with the microarray substrate deposit a small amount of a probe solution.
  • the pins are attached to a robotic arm that moves the pins between the different probe solutions, the microarray substrate and a washing station.
  • Non-contact printing is similar in terms of robotics but instead of pins, small dispensing systems are mounted on the robotic arm.
  • the dispensing system can be based on inkjet, bubble-jet or piezo actuation technology and can usually dispense in the range of 100 pL to 2 ml_.
  • An example of a suitable apparatus for creating microarrays using contact printing is the QArray2 (Genetix Ltd., New Milton, L)K) which may be operated with a suitable printhead, such as the microarray pins of an SMP3 printhead (Arrayit, USA).
  • a suitable printhead such as the microarray pins of an SMP3 printhead (Arrayit, USA).
  • Other types of contact printing equipment may be equally suited for creating microarrays for use in the methods of the present invention.
  • the substrate may further be inactivated by immobilisation of chemical entities considered inert to the assay concerned.
  • the substrate is blocked with bovine serum albumin (BSA) or ovalbumin (OA) after immobilisation of analyte and reference first binding partners.
  • BSA bovine serum albumin
  • OA ovalbumin
  • the substrate should be handled in a suitable fashion to avoid degeneration or degradation.
  • the substrate may be used immedi- ately upon manufacture or it may be stored under dry or moist conditions at ambient or cold temperatures.
  • the interactions between binding partners may be detected and meas- ured using any appropriate method.
  • the assay method of the present invention involves the use of binding partners linked with enzymes, such as HRP or AP.
  • enzymes such as HRP or AP.
  • reaction products formed in the chromogenic, enzymatic reaction may be measured with an image scanner of sufficient resolution.
  • a flat-bed scanner such as the Cano- Scan 8400F (Canon) with a resolution of 1200 dpi (corresponding to a pixel size of around 20 ⁇ m)
  • Relative quantification of the signals may then be performed using appropriate software, such as ScanAlyze2 (www.rana.lbl.gov/EisenSoftware.htm)
  • Microarray fabrication A microarray was created for use in an assay method according to the present invention for the quantification of the algal hepatotoxin micro- cystin LR (MC).
  • Transparent MaxiSorpTM polymer microarray slides (of 76 x 25 mm 2 size), obtained from Nunc A/S (Denmark), were used as a substrate to immobilise analyte and reference first binding partners.
  • the analyte first binding partner ⁇ a ⁇ was microcystin LR (MC-LR) from DHI Water & Environment, Denmark.
  • MC-LR microcystin LR
  • hCG man chorionic gonadotropin
  • the first binding partners were each mixed with a spotting reagent (phosphate buffered saline, pH 7.2) to a final concentration of 0.5 g/L and spotted using the microarray pins of an SMP3 printhead (Ar- raylt, USA) operated by a microarray robot (Qarray 2, Genetix, UK) according to the manufacturers' guidelines. Following the spotting the slides were incubated for 1 hour at ambient temperature to allow the molecules to adsorb to the substrate. Each binding partner was spotted in a row of 8 replicates, and a row of spotting reagent was spotted between the rows of MC and hCG spots to function as a negative control. Thus, this resulted in an 8 x 5 array of spots, where the rows were: MC, spotting reagent ⁇ c ⁇ , hCG, insulin and CCK (see Fig. 1).
  • a spotting reagent phosphate buffered saline, pH 7.2
  • the created microarray substrates were used for generating correlation functions for quantifying MC in a competitive ELISA format (Fig. 2).
  • the second binding partners were murine monoclonal antibodies to the respective first binding partners.
  • the second binding partners were anti-MC (Alexis, Switzerland), anti-CCK (Statens Serum Institute, Denmark), anti-hCG (Fitzgerald) and anti-insulin (Sigma- Aldrich).
  • a secondary anti-mouse antibody labeled with horseradish peroxidase (anti-mouse-HRP, Sigma-Aldrich) was employed with a peroxidase specific substrate (Sigma-Aldrich). This secondary antibody was capable of binding specifically to the second binding partners but could not bind to the first binding partners.
  • the eight arrays on a slide were each incubated for 1 hour at ambient temperature with the murine antibodies (second binding partners) in a buffer spiked with MC (analyte first binding partner) in different concentrations.
  • the concentrations of MC were 0 ⁇ g/L, 0.1 ⁇ g/L, 0.3 ⁇ g/L, 0.6 ⁇ g/L, 1 ⁇ g/L, 2 ⁇ g/L, 3 ⁇ g/L and 10 ⁇ g/L, respectively.
  • the concentrations of the four murine antibodies (second binding partners) in the mixture were 0.2 ⁇ g/mL for anti-MC and anti-CCK, 0.1 ⁇ g/mL for anti-insulin and anti-hCG.
  • the buffer used was PBS with 0.05% BSA and 0.05% Tween.
  • the volume applied to each array was 50 ⁇ L.
  • spotting reagent itself did not give any signal which indicates that neither of the second binding partners were adsorped to the microarray substrate material after blocking this with BSA.
  • the signal for CCK was very weak.
  • the analyte binding partners in solution did not seem to interact with the immobilised first reference binding partners.
  • the assay signal for the analyte binding partners decreased with an increasing concentration of the analyte first binding partner in the sample solution. This is typical for competitive assays where the assay signal decreases with increasing amount of antigen due to the competition of binding between the second analyte binding partner in solution and the immobilised and dissolved first analyte binding partners. Hence, we show that we have a competitive assay for MC.
  • the mean spot intensities at different MC concentrations are given in Table 1. Using the mean intensities, we calculated the ratios between MC and the reference signals, and plotted the ratios as a function of MC concentration (Fig. 4). A spline interpolation was used in place of curve fitting for data treatment. From the correlation curves we observed that the dynamic range of the assay was between 1 ⁇ g/L to 3 ⁇ g/L.
  • Probing assays The next step is to probe the correlation functions and compare them with the traditional method of conducting immunoassays, i.e. creating a standard curve in parallel with the test of samples.
  • the MC concentrations used to generate the standard curve were 0 ⁇ g/L (blank), 0.3 ⁇ g/L, 1 ⁇ g/L, and 3 ⁇ g/L to cover the dynamic range observed above.
  • the other 4 arrays on the same slide were used to test and probe the correlation functions and the standard curve.
  • the probe MC concentrations were 0 ⁇ g/L (blank), 0.6 ⁇ g/L, 2 ⁇ g/L, and 10 ⁇ g/L.
  • the assay conditions and time were the same as for the assays used for generation of the correlation curve.
  • the standard curve (a linear fit) is shown in Fig. 5. From the standard curve it seems that there was a difference between 0 and 1 ⁇ g/L, however, since the errors in our assay was about 15%, the dynamic range for the standard curve was practically also between 1 and 3 ⁇ g/L.

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Abstract

L'invention concerne un procédé pour quantifier des molécules d'analyte dans un échantillon, le procédé comprenant les étapes consistant à se procurer un substrat contenant deux ou plusieurs régions de référence contenant différents premiers partenaires de liaison de référence immobilisés, et une ou plusieurs régions d'analyte contenant un ou des premiers partenaires de liaison d'analyte immobilisés ; appliquer un mélange contenant de seconds partenaires de liaison de référence et un échantillon supposé contenir un ou des premiers ou seconds partenaires de liaison d'analyte, dans lequel mélange les concentrations en seconds partenaires de liaison de référence sont connues ; mesurer la liaison du ou des partenaires de liaison dans le mélange au ou aux partenaires d'analyte immobilisés ; et quantifier la concentration du ou des partenaires de liaison d'analyte dans l'échantillon, la quantification étant effectuée par corrélation de la mesure de la liaison des partenaires d'analyte à la mesure de la liaison des partenaires de liaison de référence. Le procédé est utile pour quantifier la microcystine LR.
EP08715628A 2007-03-26 2008-03-26 Quantification de molécules d'analyte à l'aide de multiples molécules de référence et fonctions de corrélation Withdrawn EP2140266A1 (fr)

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CN102409080A (zh) * 2010-09-20 2012-04-11 中国农业科学院饲料研究所 一种用于转基因大豆检测的两基因标准质粒分子及其构建
CN102409082B (zh) * 2010-09-20 2015-05-20 中国农业科学院饲料研究所 一种用于转基因大豆检测的五基因标准质粒分子及其构建
CN102409081B (zh) * 2010-09-20 2015-08-26 中国农业科学院饲料研究所 一种用于转基因大豆和转基因棉花检测用标准质粒分子及其构建方法
CN102559854B (zh) * 2010-12-20 2015-05-20 中国农业科学院饲料研究所 一种用于转基因大豆、玉米和棉花检测的标准质粒分子及其构建
CN102495212B (zh) * 2011-12-16 2014-02-26 上海海洋大学 快速检测微囊藻毒素的方法和试纸
CN104407039B (zh) * 2014-11-28 2017-02-22 青岛海佑海洋生物工程有限公司 一种用于检测mc‑lr型微囊藻毒素的生物电极传感器及其制备方法和应用
GB201905181D0 (en) * 2019-04-11 2019-05-29 Arrayjet Ltd Method and apparatus for substrate handling and printing

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US20040229226A1 (en) * 2003-05-16 2004-11-18 Reddy M. Parameswara Reducing microarray variation with internal reference spots

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US20030148339A1 (en) * 2001-05-07 2003-08-07 Hrissi Samartzidou Artificial genes for use as controls in gene expression analysis systems
WO2003025580A1 (fr) * 2001-09-19 2003-03-27 Ingenium Pharmaceuticals Ag Dosages immunologiques quantitatifs miniaturisés parallèles
WO2004064482A2 (fr) * 2003-01-22 2004-08-05 Modular Genetics, Inc. Sequences allogenes
EP1912067A1 (fr) * 2006-10-12 2008-04-16 Eppendorf Array Technologies S.A. Procédé de quantification d'un composé cible issu d'un échantillon biologique à l'aide de puces à microreseau

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