WO2007098755A1 - Procédé d'obtention d'un microréseau et analyse - Google Patents

Procédé d'obtention d'un microréseau et analyse Download PDF

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Publication number
WO2007098755A1
WO2007098755A1 PCT/DK2006/000125 DK2006000125W WO2007098755A1 WO 2007098755 A1 WO2007098755 A1 WO 2007098755A1 DK 2006000125 W DK2006000125 W DK 2006000125W WO 2007098755 A1 WO2007098755 A1 WO 2007098755A1
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WO
WIPO (PCT)
Prior art keywords
binding partner
linking moiety
toxins
detection area
substrate
Prior art date
Application number
PCT/DK2006/000125
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English (en)
Inventor
Hanne Kaas
Kristine Garde
Lars Christian Von Gersdorff
Original Assignee
Toxispot A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Toxispot A/S filed Critical Toxispot A/S
Priority to PCT/DK2006/000125 priority Critical patent/WO2007098755A1/fr
Publication of WO2007098755A1 publication Critical patent/WO2007098755A1/fr

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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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

  • the present invention relates to a method for obtaining a sub- strate having a first binding partner immobilized in a region of a detection area of the substrate surface and an assay for detecting a binding partner in a fluid using the substrate.
  • an attachment technology applicable for a non-nucleic acid microarray is developed.
  • a microarray generally requires a plurality of different probes to be attached in spatially separated regions on a substrate surface.
  • Mi- croarrays of DNA probes were introduced commercially in the last dec- ade of the past century. The development has progressed rapidly since towards a miniaturisation of the spots to obtain a higher density.
  • the rigid solid support may be glass, silicon, oxidised silicon, fused silica, poly(methyl-meta acrylate) (PMMA), poly(dimethylsiloxan)(PDMS), glas, etc.
  • contacting, non-contacting, and in situ synthesis of microarrays There are three fundamental ways to fabricate a DNA micro array: contacting, non-contacting, and in situ synthesis of microarrays. Only the contacting and non-contacting technology is of interest as background of the present invention. According to the contacting and non-contacting technology pre-synthesised DNA is spotted onto a platform, usually a microscope slide. Microarray fabrication using contact printing is based on high definition pins that, upon contact with the microarray substrate deposit a small amount of probe solution. The pins are attached to a robotic arm that moves the pins between the different probe solutions, the glass slides where the microarray is created 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, bubblejet or piezo actuation technology and can usually dispense in the range of 100 pL to 2 mL.
  • Contact printing generally results in spot densities of 2000-4000 spots/cm 2 while non-contact printing can have slightly higher spot density.
  • the solid support is modified for attach- ing the DNA strands.
  • Unmodified DNA has been immobilised on solid supports treated with e.g. polylysine, amine, epoxy, diazonium ion, SU- 8, or agarose.
  • DNA modified with amine groups has been immobilised on solid supports treated with aldehydes, epoxy, or isothiocyanate.
  • DNA modified with thiol groups may be immobilised on solid supports modi- fied with gold, mercaptosilanes, maleimide, or iodoacetyl.
  • DNA strands modified with a silane group may be immobilied on unmodified glass support.
  • the protein to be immobilised may be expressed together with a tag or subsequently provided with a tag which may be recognised by an immobilised capturing entity.
  • the tag may be a hexa histidine-tag which binds to solid support coated with NTA-agarose or the tag may be biotin which binds to a solid support coated with avidine or streptavidine.
  • An immune array has been pre- parred in which biotinylated capture antibodies were immobilised onto an avidin-coated surface of a solid support (Rowe CA, Scruggs SB, FeId- stein MJ, Golden JP, Ligler FS: An array immunosensor for simultaneous detection of clinical analytes, Anal. Chem.
  • the present invention aims at providing a method applicable for immobilisation of a binding partner to a substrate without the need for prior preceding chemical modification.
  • the binding partner to be immobilised can be of a versatile chemical nature, including nucleic acid, protein, and small molecules.
  • the present invention relates to a obtaining a substrate having a first binding partner immobilized in a region of a detection area of the substrate surface, comprising the steps of:
  • step (a) of the method a detection area of the substrate surface is covered with a linking moiety.
  • the linking moiety is evenly distributed on the surface.
  • the surface may or may not be modified for enhancing the compatibility to the linking moiety.
  • step (b) a solution of a first binding partner is spotted onto a region of the detection area provided with the linking moiety.
  • the spot does usually not interfere with other spots, which may appear in the detection area and the spot is generally surrounded by the layer of linking moieties distancing the spot from other optionally appearing spots.
  • step (c) the de- tecting area harbouring the spot is treated with an agent forming a coupling between the linking moiety and the first binding partner.
  • the coupling is a covalent linkage for an attachment reliable under various conditions to be obtained.
  • first binding partner refers to a molecule being immobilised.
  • the first binding partner may also be referred to herein as probe.
  • second binding partner refers to the molecule being captured by the first binding partner.
  • the second binding partner may also be referred to herein as target.
  • substrate denotes the solid support onto which the binding partner is immobilised.
  • platform is usually used herein to denote the assembly of substrate and immobilised binding partner.
  • the substrate may be any conventionally material and may have any convenient design. To be able to fit into a standard scanning apparatus the dimension of the substrate usually resembles that of a microscope slide (25mm x 76mm). Suitable materials for the substrate include glass, poly(methyl-meta acrylate) (PMMA), and poly(dimethylsiloxan)(PDMS). However, any rigid, impervious transparent or non-transparent material may be used.
  • the detection area is pre-treated with an agent providing a chemical group capable of reacting with a chemical group of the linking moiety.
  • a chemical group or compound selected from polylys ⁇ ne, amine, epoxy, diazonium ion, SU-8, agarose, gold, mercap- tosilanes, maleimide, iodoacetyl, aldehyde, isothiocyanate, thiol, silane, aldehyde, keton or NO x .
  • the chemical group provided on the substrate is selected from the group consisting of aldehyde, isothiocyanate, and epoxy.
  • the chemical group attached to the substrate by pre-treatment is an aldehyde group.
  • the aldehyde groups may be provided by a number of chemical reactions.
  • the agent providing the chemical group on the substrate is a polysaccharide reacted with 1O 4 " , thereby forming aldehyde groups.
  • An example of a method of providing aldehyde groups involve heat treatment of an aqueous agarose solution with NaIO 4 , and subsequent spreading on the detection area of a substrate. Specific embodiments are disclosed in Dufva M, et al.
  • the linking moiety preferably comprises a chemical group capable of reacting with the chemical group of the substrate, thereby forming a linkage between the substrate surface and the linking moiety.
  • the linkage is preferably a covalent bond, but may be established due to hydrophobic/hydrophilic interactions or hydrogen-bonds.
  • the chemical group of the linking moiety is preferable selected so as to provide for a chemical reaction to take place. Examples of chemical groups of the linking moiety include: amine, epoxy, diazonium ion, mercaptosilane, maleimide, iodoacetyl, aldehyde, isothiocyanate, thiol, silane, aldehyde, keton and NO x .
  • the chemical group of the linking moiety is preferably a nucleophilic group.
  • the linking moiety comprises an amine group.
  • the linking moiety may be selected from a wide group of compounds. However, an important feature of the linking moiety is the ability, apart from binding to the substrate, also to be able to bind to a first part of a binding partner. Therefore, preferred linking moieties are bi- functional molecules having the ability to react at one part of the mole- cule with a chemical group of the detection area to provide for attachment of the linking moiety to the substrate and to react at another part of the molecule with the first part of a binding partner.
  • the linking moiety is peptide or amino acid.
  • the linking moiety is selected as an amino acid
  • said amino acid suitably has a side chain comprising an amine group.
  • the amino acid is arginine.
  • a peptide is generally preferred as the linking moiety because a variety of functional groups are available for reaction with the first binding partner.
  • the functional groups include hydroxyl groups from tyro- sine, serine, and threonine; thiol groups from cysteine; amine groups from lysine, arginine, asparagines, glutamine, and histidine; and car- boxylic acids from aspartate and glutamate.
  • the functional groups may be treated with a suitable reagent to provide for a coupling to be formed to the first binding partner.
  • Suitable linking moieties include bovine se- rum albumin (BSA) or ovalbumin (OA).
  • the linking moiety may be provided on the detection area in a zone beneath the spot or a group of spots. However, the full benefit of the invention is obtained when the linking moiety is attached to the entire surface of the detection area.
  • the entire surface of the substrate can be covered with a layer of the linking moiety.
  • the linking moiety is applied the surface as an aqueous solution. The solvent is allowed to evaporate and the dry or moist surface is used for spotting.
  • the first binding partner is a member of a binding pair in addi- tion comprising a second binding partner.
  • the first and the second binding partner have a certain affinity for each other.
  • the affinity may be relatively high, i.e. in or above the micromolar range. In a preferred aspect of the invention the affinity is 10 "4 M or higher.
  • the affinity of the binding partners towards each others is usually specific, i.e. the tendency to cross-bind to other substance is low.
  • binding pairs include antigen-antibody interaction, compound-aptamer interaction, antibody-antibody interactions, protein-small molecule interactions, enzyme-substrate interac- tions, nucleic acid-nucleic acid interactions, nucleic acid-protein interactions etc.
  • a first binding partner is selected as an antibody the assay is generally referred to as an immunoassay or antibody microarray and when the first binding partner is a protein the assay is referred to as a protein assay or protein microarray. If the first binding partner is a nucleic acid the assay may be referred to a DNA microarray.
  • the method provides a platform for performing a competitive assay, in which the first binding partner is immobilised on the substrate.
  • the first binding partner may be selected from a variety of sources including bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, enzymes occurring in the synthesis pathway of toxins, markers for the occurrence of pathogenic micro organisms, geosmins, plant protection products, pharmaceutical products and degradation products of the above mentioned group of compounds.
  • a certain group of binding partners of interest is algal toxins, such as cyanotoxins, especially hepatoxins and neurotoxins.
  • hepatoxins include microcystein (MC) like microcystins-LR (MC- LR) and microcystin-RR (MC-RR), nodularin, and cylindrospermopsin.
  • MC- LR microcystins-LR
  • MC-RR microcystin-RR
  • nodularin adularin
  • neurotoxins include anatoxins and saxitoxins.
  • algal toxins of interest include paralytic shellfish toxins/poisoning (PST, PSP, saxitoxins and gonyautoxins), diarrheic 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 shellfish tox- ins/poisoning (NST, NSP, brevetoxin, etc.), and endotoxins (lipopolysac- charides, LPS).
  • PST paralytic shellfish toxins/poisoning
  • DST diarrheic shellfish toxins/poisoning
  • DST diarrheic shellfish toxins/poisoning
  • AST amnesic shellfish toxins/poisoning
  • the first binding partner is a polypeptide, such as a microcystin, or an alkaloid.
  • the solution of the first binding partner to be used for spotting is preferably an aqueous solution.
  • An organic solvent may be selected in special circumstances, e.g. in the event the binding partner to be immobilised is not sufficient soluble in water.
  • the spotting buffer i.e. the solution in which the binding partner is dissolved or dispersed, may comprise salts, polymers, and further additives.
  • the spotting buffer 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.
  • spotting solution such additives which will provide for a sufficient high viscosity that reduces mass transport by capillary actions and provide for sufficient low evaporation for allowing the binding partner to react with the linking moiety.
  • 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's amine spotting buffer.
  • the spotting solution is generally applied in a small quantity in the sub- ⁇ l range.
  • the solution of the first binding partner is spotted in an amount of lOnl or less.
  • each spot is applied in an amount of about InI or less.
  • a deposited amount of spotting solution of InI corresponds to a spot diameter of around lOO ⁇ m.
  • the substrate usually comprises more than a single spot.
  • a plurality of different first binding partners are spotted in corre- sponding discrete regions of the detection area.
  • Such design is generally referred to as a microarray.
  • Manufacture of a microarray according to the invention may be effected using contact printing or non-contact printing.
  • Contact printing implies that a hollow pin is allowed to soak a minor amount of spotting solution. Subsequently, the spotting solution is deposited by the pen on the detection area.
  • Non-contact spotting makes use a small dispenser instead of the pen.
  • the dispensing system can be based on inkjet, bub- blejet or piezo actuation technology.
  • the binding partners attached to the substrate in discrete regions may be of the same or a related chemical nature. As an example two different proteins are regarded to have a related chemical nature. According to a certain embodiment of the invention, at least two of the plurality of first binding partners are chemically unrelated.
  • An example of chemically unrelated compounds is a protein and a small molecule not exclusively comprising naturally occurring amino acids
  • the first binding partner After the first binding partner has been spotted onto the substrate, it is treated with an agent forming a coupling between the linking moiety and the first binding partner.
  • the agent may e.g. be of a chemi- cal nature or radiation. Coupling agents of a chemical nature are well- known in the art and include mineral acids, oxidation agents, and reducing agents.
  • the radiation When radiation is used as a coupling agent, the radiation may be ultra violet light, gamma radiation, alpha-radiation, or beta- radiation.
  • the agent used for treating the spotted detection area is ultra violet (UV) light.
  • the formed coupling is a covalent bond.
  • the platform should be handled in a suitable fashion to avoid degeneration.
  • the platform may used immediately upon manufacture or it may be stored under dry or moist conditions at ambient or cold temperatures.
  • the invention also relates an assay for detecting a binding partner in a fluid comprising the steps of contacting the detection area of the substrate with a sample suspected of comprising a free first bind- ing partner and a second binding partner capably of binding to the free and immobilised first binding partner, removing excess non-reacted second binding partner and free first binding partner, and analysing the region comprising the immobilised first binding partner to detect whether the second binding partner has reacted with the immobilised first binding partner.
  • the second binding partner is an antibody or a fragment thereof having retained activity, said antibody or fragment being directed to the first binding partner.
  • This assay design is commonly referred to as competitive immunoassay because the free and the bound binding partner, i.e. antigen, compete for binding with the antibody.
  • the antibody is immobilised and the sample is spiked with a labelled antigen that competes with the unlabelled antigen suspected in being contained in the sample.
  • the second binding partner may also be an aptamer or a protein-displaying phage having affinity for the first binding partner.
  • the second binding partner may be directly labelled.
  • Suitable labels include fluorescence labels like Cy-3 or Cy-5.
  • the secondary antibody comprises a detectable label or an enzyme capable of producing a detectable label.
  • the detectable label of the second label may be a fluorescence label or a radio active label.
  • Preferred fluorescence labels include Cy-3 and Cy-5.
  • reactions between the first and the second binding partner may be measured using scanning electron microscopy or by using colorimetric detection of nanoparticles, such as nanogold particles, similar to the method disclosed in Han A. et al "Detection of analyte binding to microarrays using gold nanoparticle label and a desktop scanner" Lab Chip, 2003, 3, 329-332.
  • a sample comprising a first binding partner may be quantified with respect to the first bind partner.
  • concentration of first binding partner in a sample is calculated by comparing the meas- ured signal of a label in a region comprising an immobilised first binding partner with a standard curve prepared for know concentrations of the first binding partner.
  • the sample to be analysed by the present assay can stem from any source.
  • the sample is a water sample and the first 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 occurring in the synthesis pathway of toxins, markers for the occurrence of patho- genie micro organisms, geosmins, plant protection products and pharmaceutical products, and degradation products of the above mentioned group of compounds.
  • Samples may comprise a variety of compounds to be analysed by the assay of the invention. Therefore a fast parallel method of de- tecting a multitude of compounds is provided.
  • the substrate comprises a plurality of different first binding partners immobilised on the substrate a corresponding number of parallel measurements of these compounds may be conducted.
  • multiplex quantification of a plurality of first binding partners are performed simultaneously.
  • Fig. 1 discloses a schematic representation of the immobilisation method
  • Fig. 2 shows a typical slide and array layout
  • FIG. 3 schematically shows the slide layout used in example 1
  • Fig. 4 shows the principle of the competitive assay
  • Fig. 5 Shows a diagram of the results of example 2.
  • Fig. 6 shows a standard curve obtained in example 2 Examples
  • MIA Bio-Chip MIA Bio-Chip
  • 1% activated agarose solution was prepared by dissolving 1.5 g of UltraPure Electrophoresis grade Agarose (Ivitrogen, UK) and 0.32 g NaIO 4 (Fluka Chemie GmbH, Switzerland) in 150 ml MiIIiQ water (Millipore, Bedford, MA, USA) (sufficient for preparing 100 - 120 slides).
  • the agarose was dissolved in the MiIIiQ water by heating the solution in a microwave owen until the agarose was completely dissolved before the NaIO 4 .
  • 76 x 26 mm unmodified microscope glass slides Superfrost, Menzel-Glaser, Germany
  • microcystin- LR MC-LR
  • negative controls The spots were printed onto the ac- tived slides in a cooled chamber (temperature below 12 0 C).
  • EasySpot U-vision Biotech Inc., Taiwan
  • Immobilization of microcystin to the slide surface was ensured by pre-incubating the slide surface in 0.1% BSA for 15 minutes at medium agitation prior to printing, and exposing the printed arrays to UV light after spotting (UV light at a wavelength of 254 nm in a Strata- linker 2400 (Stratagene, USA) for 4 minutes).
  • the final microcystin concentration in the printing solution was 0.5 mg/ml, obtained by diluting a stock solution with Genetix amine spotting buffer (Genetix, UK). Spot volumes were approximately 1 nl/spot delivered by CMP3 Chipmaker spotting pins (TeleChem International, USA). See fig. 2 for array and spotting layout. Two negative controls were spotted: A negative control for the spotting procedure (MiIIiQ water) and a negative control for un- specific binding of the antibodies (Avidin). The spot density was ⁇ 563 spots/cm 2 .
  • the microarray substrate surface not involved in immobilizing the printed spots were inactivated for preventing unspecific background.
  • the microarray substrates were blocked by incubating the slides for 10 minutes in TBST/0.1% BSA, fol- lowed by a 2 minute rinse in MiIIiQ water.
  • the slides were dried by spinning in a Mini Centrifuge (National Labnet co., NJ, USA) for 30 seconds.
  • the 8 individual arrays were separated by applying a Blue Film layer with at a structure cut using a CCVIaser, see Figure 3.
  • the dimensions are (height x width) 80 x 26 mm, designed to match a standard microscope slide with a 5 mm slip at the bottom for swift displacement of the film before scanning. 10 wells are cut in the film, but only well 1 - 6 are used in this experimental setup.
  • the processed microarray substrates were stored in dry conditions at room temperature until use.
  • a water sample was incubated with the primary detection molecule (mAb against MC) on the BioChip.
  • the mAb was the anti-microcystin antibody MC10E7 obtained from Alexis Bio- chemicals.
  • the primary mAb was bound in part to MC in the water sample, and to the MC-LR on the BioChip. After incubation, primary mAb not bound to MC-LR on the BioChip is washed away.
  • the BioChip was incubated with a secondary detection molecule (pAb-Cy3) which binds to the mAb bound on the BioChip. After incubation, secondary pAb-Cy3 not bound on the BioChip was washed away. The BioChip was then subjected to scanning and quantification.
  • pAb-Cy3 secondary detection molecule
  • the standard solutions were mixed with primary antibody in a 1 :1 reaction mix prepared by mixing 10 ⁇ l of standard with 10 ⁇ l of primary monoclonal antibody (mAb-LR) (MC10E7, Alexis Biochemicals), diluted 5,000-fold (200 ng/ml) in TBS incubation buffer, supplemented with concentrations of each 0.05% (w/v %) BSA Fraction V and (v/v %) Tween 20.
  • mAb-LR primary monoclonal antibody
  • M10E7 primary monoclonal antibody
  • BSA Fraction V and (v/v %) Tween 20 were then exposed to an individual array for 60 minutes.
  • the mi- croarrays were incubated uncovered in a humid chamber at room temperature.
  • microarrays were washed for 10 minutes in TBS buffer supplemented with 0.1 % (v/w %) Tween 20, rinsed with MiIIiQ water for 2 minutes and spin-dried.
  • microarrays were then incubated with the secondary anti- body, a Cy3 labelled polyclonal anti-mouse antibody (pAb-Cy3) (Sigma-
  • microarrays were washed for 10 min- utes in TBS buffer supplemented with 0.1 % (v/w %) Tween 20, rinsed with MiIIiQ water for 2 minutes and spin-dried.
  • Fluorescent pAb-Cy3 emissions were acquired with a ScanArray LITE confocal laser scanner (Perkin Elmer, MA, USA) and quantified us- ing the freeware software ScanAlyze 2.50
  • V 2 Fluorescence signal bi: Maximum fluorescence signal (upper asymptote) b 4 : Minimum fluorescence (lower asymptote)
  • V 1 Concentration of analyte
  • b 3 IC50 of signal
  • b 2 Transition between bl and b4. (reference: Rodbard, D. Statistical Quality-Control and Routine
  • the signal intensity from the ES slides were accept- able, however some background fluorescence were noticed.
  • the signal intensity from the AG slides was a bit lower than the ES slides, but still acceptable.
  • the background fluorescence from the AG slides was considerably higher than from the ES slides.
  • the higher background level may be explained by the scanning method, as the focus of the laser scanner is very sensitive to the height of the agarose film.
  • a blank unspotted and unprocessed slide was scanned under the same conditions as the spotted processed AG slides. As this gave fluorescence background similar to that observed in the spotted processed AG slides, this seems not to be the case.
  • Other scanners as a CCD-based scanner (e.g. ArrayWoRx) may reduce the background signal since they are less sensitive to the height of the agarose film.
  • Figure 5 shows the results of the microcystin quantification on ES and AG based microarrays, respectively. It is observed that the signal intensity from the ES slides generally is higher than the AG slides. This may be explained by a higher background observed in the AG slides, as the background signal is subtracted from the signal intensity values. The chip-to-chip variation is low for the individual substrates (ES 1, ES 2 and ES 3, respectively AG 4, AG 5 and AG 6). Hence, the method is capable of producing standard curves applicable for quantification of unknown microcystin concentrations.
  • the MIA BioChip is used to quantify the microcystin concentration in an extract made from a natural sample col- lected at a Polish water work.
  • Example 2 The setup of the experiment was identical to Example 1 with the following modifications: The glass carrier was coated with agarose and the arrays were separated by applying hydrophobic borders using a PAP-pen instead of the structure cut in blue film. The sample volume applied in each array was increased to 30 ⁇ l, with the reaction mix ratio still being 1 : 1. Only 5 standard solutions were applied.
  • Array no. AOl - A05 were used for generation of a standard curve, while A06 were used to estimate microcystin concentration in Sl and A07 and A08 were used for a double estimation of S2.
  • the estimated concentrations were compared with correspond- ing results obtained by HPLC analyses of the same sample. This comparison is not straight forward as although primary targeting MC-LR, the applied antibody does also detect other microcystins with similar structure; which other derivates is not obvious. In the comparison the HPLC values is calculated by summing the concentrations of derivates that to our best knowledge can be expected to be included in the immunoassay.
  • Table 3 gives the concentrations estimated using the two methods. The results cooperates the applicability of the described assay (using agarose coated slides and fluorescence detection) with samples ob- tained from natural sources. The results of MIA BioChip were comparable to the HPLC analyses.

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Abstract

L'invention concerne un procédé qui permet d'obtenir un substrat présentant un premier partenaire de liaison immobilisé dans une région d'une zone de détection de la surface du substrat. Le procédé de l'invention comprend les étapes qui consistent : (a) à fixer un fragment de liaison sur une zone de détection de la surface du substrat; (b) à appliquer par points une solution d'un premier partenaire de liaison sur une région de la zone de détection sur laquelle le fragment de liaison est fixé; et (c) à traiter la zone de détection avec un agent formant un couplage entre le fragment de liaison et le premier partenaire de liaison. L'invention concerne également une analyse permettant de détecter un partenaire de liaison dans un fluide.
PCT/DK2006/000125 2006-03-01 2006-03-01 Procédé d'obtention d'un microréseau et analyse WO2007098755A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0088695A2 (fr) * 1982-03-09 1983-09-14 Cytogen Corporation Liaisons d'anticorps
US20040121399A1 (en) * 2002-12-20 2004-06-24 International Business Machines Corporation Substrate bound linker molecules for the construction of biomolecule microarrays
WO2005093419A1 (fr) * 2004-03-26 2005-10-06 Capitalbio Corporation Methodes et puces a adn pour la detection de petits composes moleculaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0088695A2 (fr) * 1982-03-09 1983-09-14 Cytogen Corporation Liaisons d'anticorps
US20040121399A1 (en) * 2002-12-20 2004-06-24 International Business Machines Corporation Substrate bound linker molecules for the construction of biomolecule microarrays
WO2005093419A1 (fr) * 2004-03-26 2005-10-06 Capitalbio Corporation Methodes et puces a adn pour la detection de petits composes moleculaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHOU S-F ET AL: "Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on surface plasmon resonance", BIOSENSORS & BIOELECTRONICS, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 19, no. 9, 15 April 2004 (2004-04-15), pages 999 - 1005, XP002997261, ISSN: 0956-5663 *

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