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WO2000075663A1 - Methode et kit de bioanalyse de ligand - Google Patents

Methode et kit de bioanalyse de ligand Download PDF

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Publication number
WO2000075663A1
WO2000075663A1 PCT/EP2000/004381 EP0004381W WO0075663A1 WO 2000075663 A1 WO2000075663 A1 WO 2000075663A1 EP 0004381 W EP0004381 W EP 0004381W WO 0075663 A1 WO0075663 A1 WO 0075663A1
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WO
WIPO (PCT)
Prior art keywords
oligomer
nucleic acid
biotinylated
binding partner
antibody
Prior art date
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PCT/EP2000/004381
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English (en)
Inventor
Denis François HOCHSTRASSER
Jean-Charles Sanchez
Catherine Gabrielle Zimmermann
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Universite De Geneve
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.)
Filing date
Publication date
Application filed by Universite De Geneve filed Critical Universite De Geneve
Priority to AU53942/00A priority Critical patent/AU5394200A/en
Publication of WO2000075663A1 publication Critical patent/WO2000075663A1/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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention relates to a method of assay of a ligand, especially an antigen or hapten present in a low concentration in a body fluid. It also relates to a kit for this purpose. Background of the invention
  • ⁇ LISA enzyme-linked immunosorbent assay
  • Various methods of detection of the enzyme label have been used, of which enhanced chemiluminescent assay is one of the best.
  • ELISA is not sensitive enough to detect very low concentrations .
  • the polymerase chain reaction (PCR) has been coupled to the immunosorbent assay to provide a more sensitive assay. In this "immuno-PCR" , a DNA oligomer is attached to the antibody in place of the enzyme label .
  • the invention provides a method of assay of an analyte ligand, which comprises amplifying a nucleic acid oligomer to which a specific binding partner of the analyte ligand is attached and determining the presence or amount of the amplified nucleic acid by capillary electrophoresis .
  • the invention provides a kit for carrying out a method of assay of the invention, comprising (1) a nucleic acid to which a specific binding partner of a ligand is attached, or the nucleic acid and binding partner components thereof, (2) coated capillaries and (3) a molecular sieve for inserting into the capillaries, preferably replaceably.
  • assay means any qualitative, semi-quantitative or quantitative determination and thus covers both detection and measurement. Brief description of the Figures
  • Figures 1 and 2 are plots of the fluorescent signal generated upon detection of the immuno-PCR products in capillary electrophoresis , by laser-induced fluorescence .
  • Figure 1 relates to an assay of alpha-fetoprotein, described in Example 1.
  • Figure 2 relates to assays of prion protein described in Example 2.
  • the fluorescent signal on the y-axis is plotted against time on the x- axis .
  • the assay of the invention is applicable to any ligand and a binding partner specific to that ligand, i.e. to any analyte to which ELISA can be applied. It is principally applied to antigens and haptens .
  • a hapten is a small molecule against which an antibody can be raised. It can also be applied to antibodies . Most preferably it is of interest for detecting antigens and antibodies present in body fluids, such as blood, urine, saliva, tears, nasal excretions and cerebrospinal fluid. Since this assay is more time-consuming than ELISA, it is preferably applied to analytes that are diagnostic for rare diseases , such as prion proteins associated with transmissible spongiform encephalopathies in humans or animals. For this purpose, antibodies specific to the abnormal prion protein (PrP sc ) should be used. An example is 15B3 , the preparation of which is described by C. Korth et al , Nature 390, 74-77 (1997) and commercially available from Prionics AG, Zurich.
  • Two particularly preferred formats are (a) those wherein the analyte ligand is an antigen or hapten and the assay comprises forming a sandwich between an immobilised first antibody to the antigen and a second antibody to the antigen, attaching the second antibody to a nucleic acid oligomer, amplifying the oligomer and determining the presence or amount of the amplified oligomer by capillary electrophoresis ; and (b) those wherein the analyte ligand is an antigen or hapten and the assay comprises immobilising the antigen, binding it to an antibody thereto, attaching this antibody to a nucleic acid oligomer, amplifying the oligomer and determining the presence or -amount of the amplified oligomer by capillary electrophoresis .
  • the assay will usually be carried out in the normal manner of an ELISA, a "universal" second antibody being used where appropriate.
  • a first antibody specific to it especially a mouse monoclonal antibody
  • a further incubation is carried out with an anti-mouse "universal" second antibody.
  • this second antibody is labelled with an enzyme .
  • immuno-PCR it is labelled with an oligonucleotide ("oligo") .
  • a high affinity couple especially biotin with either avidin or streptavidin, of which streptavidin is preferred because it is unglycosylated, minimising the possibility of side-reactions.
  • the oligo is biotinylated, i.e. biotin-labelled. It may contain uracil bases introduced deliberately to facilitate biotin-labelling. In principle, it can be of RNA or DNA. It is preferably double-stranded, so that it can be primed simultaneously with forward and reverse primers in the PCR.
  • the specific binding partner is biotinylated and the avidin or streptavidin group is attached bivalently to the biotinylated specific binding partner and the biotinylated oligo.
  • the oligo used for the immuno-PCR can be any arbitrary nucleic acid, so long as it does not self- anneal , or contain sequences which bind to proteins . It is preferably of DNA, most suitably non-human DNA, and preferably double-stranded. It may be of any appropriate length, but is preferably from 150 to 500bp, more preferably 180 to 350bp, most preferably from 200 to 300bp.
  • the product is preferably purified to eliminate low molecular weight products such as primers and primer-product dimers .
  • PCR ligase chain reaction
  • rolling circle amplification is described in PCT Publication WO 97/19193.
  • the purified product is then subjected to an electrophoretic method which includes capillary flow of the buffer as well as molecular sieving.
  • the molecular sieve matrix which is inserted into the capillary is preferably a hydrophilic polymer, especially a hydrophilic cellulosic polymer and most especially hydroxypropylmethylcellulose, which has been found to give better results in terms of signal to noise than cross-linked polyacrylamide .
  • Other preferred hydrophilic polymers are hydroxyethylcellulose , methylcellulose, liquid agaraose (agar) or uncrosslinked polyacrylamide.
  • polyvinyl alcohol polyethylene oxide, polyethylene glycol, poly [ (N- acryloylamino) ethoxyethanol] , P°l ⁇ (N,N- dimethylacrylamide) , poly [ (N-acryloylaminoethoxy) ethyl- beta-glucopyranoside] , poly (N-acryloylaminopropanol) and hydroxypropylcellulose .
  • Application of a time- temperature gradient to the capillary electrophoresis e.g. as disclosed in PCT Publication WO 96/08715, is not required and is therefore not preferred in this invention because it introduces unnecessary complexity.
  • the electrophoresis may be conducted in any vessel which provides for a thin layer of the molecular matrix, so as to permit capillary action without electroosmotic flow (EOF) under an applied voltage.
  • EEF electroosmotic flow
  • it is carried out in a capillary provided with a window to enable the laser light to be shone through it for the preferred laser-induced fluorescent (LIF) detection.
  • LIF laser-induced fluorescent
  • the internal surface of the capillary is coated or treated to eliminate EOF.
  • the dye is preferably one which detects the oligo by intercalation between the strands .
  • suitable such dyes are those listed by Molecular Probes Inc .
  • the laser is tuned to the wavelength of absorption of the dye, e.g. about 488 nm.
  • the dye is pre-mixed with the sieving matrix and the resulting mixture inserted into the capillary as a plug. Additional dye can be added to the -immuno-PCR product after purification , before electrophoresis .
  • the capillary electrophoresis is run with an internal standard of any arbitrary oligo which has a molecular weight well separated from that of the immuno-PCR product.
  • kits of the invention at least components (1) and (2) are provided in separate containers.
  • the molecular sieve (3) is also provided separately from the capillaries (2) , rather than the capillaries being pre-filled.
  • the kit can include any of the other components preferred or illustrated in the Examples for the assay, as described herein.
  • a 282bp length oligonucleotide was generated from a pBluescript II phagemid SK (+) Sac to Kpn (Stratagene, CA, USA), pBSII, using primers containing 20nt of a phagemid-specific sequence, and 20nt of a synthetic sequence ligated to it (underlined) .
  • the primers were pBSIIlL (5'-biotin ATCGTTACGGCTATCCTTAGATTCAGGCTGCGCAACTGTT-3' , SEQ ID NO:l, containing nucleotides 469-488 of the phagemid and pBSIIlR (5' -CCTAGGGTTACTAATCGTACGCAGGAATTCGATATCAAGC-3 ' SEQ ID NO: 2, containing nucleotides 710-691 of the phagemid.
  • the synthetic sequences are substantially arbitrary
  • PCR amplification was carried out in a thermocycler (Perkin Elmer Gene Amp PCR System TC 9600) .
  • a sample was subjected to a PCR program starting with 10 PCR cycles, each with the following temperature profile: 30 seconds at 94°C, 5 seconds at 60°C with a decrease of l°C/cycle until 50°C and 20 seconds at 72°C. Then the sample was subjected to 20 cycles, each with the following temperature profile: 30 seconds at 94°C, 5 seconds at 50°C and 20 seconds at 72°C followed by a final 10 min extension at 72°C.
  • biotin-282 50 ⁇ l of the amplified PCR product (“biotin-282") were diluted in 450 ⁇ l of TBE buffer consisting of 89mM Tris-89mM borate- 2mM EDTA pH 8.3 and puri ied three times by centrifugation through a Millipore "Microcon” 50 microconcentrator with a cut off at 50,000g. The filtrate was reconstituted in 50 ⁇ l of TBE. The biotin- 282 was then blotted as follows. lO ⁇ l of the thus purified sample were subjected to agarose gel electrophoresis , stained with ethidium bromide , photographed, and transferred onto a nylon membrane.
  • a chemiluminescence blotting kit for peroxidase determination (Boehringer Mannheim) was used.
  • the membrane was blocked with 10% blocking reagent (BM kit) in 0.1M maleic acid - 0.15M NaCl pH 7.5 (maleic buffer) .
  • a solution of 1/3000 peroxidase-labelled streptavidin (Dako) , 0.33ppm in 5% blocking reagent in maleic buffer was added.
  • a single band corresponding to the biotin- labelled oligo was visualized following exposure of the membrane to X-OMAT S film (Kodak) and development.
  • the purified samples were pooled and quantitated by measuring UV absorption at 260 nm, giving an optical density reading corresponding to 50 ⁇ g/ml ds-DNA.
  • a 96-well polycarbonate microplate was coated overnight at 4°C with lOO ⁇ l of 1/74 polyclonal rabbit anti-human AFP (Dako) , 15ppm in 0.1M sodium carbonate buffer pH 9.6.
  • the plate was automatically washed 5 times with PBS buffer (15mM Na 2 P0 4 - 120mM NaCl-2.7mM KCl, pH 7.4, Sigma) . All subsequent washings were with fresh PBS buffer unless otherwise stated.
  • Non-specific protein binding sites were blocked with 200 ⁇ l carbonate buffer-4% BSA for 2h at 37°C.
  • lOO ⁇ l of diluted AFP sample (analyte) were added and incubated for 2h at 37°C.
  • lOO ⁇ l of 1/6120 mouse anti-human AFP monoclonal antibody (Sigma), 0.15ppm in PBS-1% BSA were incubated for lh at room temperature
  • herring-sperm DNA (Boehringer Mannheim) in 1ml PBS was sonicated for 5 minutes, denatured for 10 minutes at 95°C and chilled quickly at 4°C. Then this herring-sperm DNA was added to 9ml of PBS containing 0.2gBSA, to prepare the blocking buffer II . After again washing the wells , lOO ⁇ l of Biotin-282 (50zmol/ ⁇ l in H 2 0) were incubated for 30 min at R.T. without shaking. The microplate was automatically washed 10 times with distilled water. Then the wells were aspirated to dryness under vacuum. 2 ⁇ l of water were added to each well thus prepared and to another well serving as negative PCR control .
  • a positive PCR control well was filled with 2 ⁇ l of Biotin-282 and another negative PCR mixture control well was left empty. Then 18 ⁇ l of PCR containing 1.5mM MgCl 2 , 0.5 ⁇ m of each dNTP, 2 ⁇ m pBSII5L and 2 ⁇ m pBSII5R, 0.01M Tris, pH 8.3- 55mM KCl and 0.5U rTaq polymerase was added into each well.
  • the primers were pBSII5L (5' -ATCGTTACGGCTATCCTTAG- 3', SEQ ID NO: 3) and pBSII5R (5' -CCTAGGGTTACTAATCGTAC-3' , SEQ ID NO: 4), which amplified the Biotin-282 sequence, the primers being of the respective synthetic sequences conatined in SEQ ID NO : 1 and 2 above .
  • the microplate was sealed with a "Microseal" A system cap (MJ Research) , a thermosensitive cap which needs a high temperature to seal the cap correctly on to the plate, and the plate was positioned onto the thermocycler with a spacer block
  • PCR amplification was carried out with the program described above. lO ⁇ l of the amplified PCR product with 0.1% Bromophenol blue were electrophoresed in the same way as for the biotin-282, to confirm the successful amplifications . Purification of amplified product The remaining immuno-PCR product was purified to eliminate residual primers and primer-dimers . Using a Boehringer Mannheim kit, an 8 ⁇ l sample of the immuno-PCR product, with 2 ⁇ l of a 425bp oligo as internal standard, were mixed with 90 ⁇ l TBE buffer to have a lOO ⁇ l final volume .
  • nucleic acids of length at least lOObp were eluted from the glass fibres in a buffer of lOmM Tris-lmM EDTA, pH 8.5, with spinning for 30 seconds at 13000g.
  • lOO ⁇ l samples of the eluate were concentrated by centri ugation through a "Microcon" 10 microconcentrator with a cut off at 50,000g (Millipore, MA, USA) and lO ⁇ l of samples were ready to be analyzed by capillary electrophoresis .
  • Capillary electrophoresis was performed in a Beckman P/ACE System 5500 (Beckman, Fullerton, CA, USA) instrument equipped with an "eCap DNA" 47cm x lOO ⁇ m i.d. coated fused silica capillary (Beckman) .
  • the capillary was made with a fused silica surface and an external polyimide coating, 365 ⁇ m total diameter, to enhance its resistance.
  • the interior surface of the capillary was presumably coated with a polyacrylamide network .
  • the capillary was connected to the cathodic reservoir with the anodic reservoir connected to earth (electrically grounded) .
  • Each electrophoretic reservoir was filled with sieving buffer, described below.
  • the electrophoresis was driven by an applied voltage through the capillary toward the anodic reservoir .
  • On-column detection was by argon laser at 488nm excitation wavelenghts and 520nm emission wavelength through a 2mm glass window in the capillary, the window being without polyimide coating and present at 7cm from the anodic reservoir.
  • Data were collected using an integrator, by the GOLD system purchased from Beckman. All buffer solutions were degassed immediately before use.
  • the fluorescent dye absorbs light at around 491nm and emits at about 509nm. Light is only absorbed when the dye is intercalated between the strands of ds DNA.
  • mice recombinant prion protein The mouse recombinant prion protein (rPrP) and polyclonal rabbit anti-mouse rPrP (R340) were kindly supplied by workers at the University of Zurich.
  • Mouse recombinant prion protein can be prepared as described by S. Hornemann et al . FEBS Letters 413, 277-281 (1997), Nature 390, 74-77 (1997) at page 77 and is commercially available from Prionics AG, Zurich. Polyclonal antibodies thereto were raised in rabbits by a conventional technique.
  • a "biotin-282" ds DNA oligomer was prepared as in Example 1. Spiked CSF sample
  • the CSF samples were "spiked", as follows. rPrP at different concentrations from 10 -11 M to 10 ⁇ 19 M were added to cerebrospinal fluid from healthy volunteers. Then, each sample was diluted 10 times in 3M guanidine thiocyanate (GuaSCN) in PBS (15mM Na 2 P0 4 120mM NaCl-2.7mM KCl pH 7.4, Sigma). The final range of concentrations was 10 "12 M to 10 "20 M of PrP in CSF.
  • This "spiked CSF sample” was prepared so as to provide accurate concentrations of PrP, along with the "dirty background" of a sample of CSF, so that the assay conditions are more representative of those encountered in clinical samples .
  • a centrifugal filter device "Ultrafree-DA" (Millipore) was used to extract 100-10, OOObp PCR product. 5 ⁇ l of internal standard 425bp (home made) added to lO ⁇ l of each immuno-PCR sample were spun at 5,000g for 10 min. The filtrate was collected and was ready to be analyzed by CE-LIF. Capillary electrophoresis
  • ELISA and Western blotting were applied to rPrP detection .
  • a series of samples of diluted rPrP in aqueous solution with a range of 10 ⁇ 7 M to 10 ⁇ 15 M with a step of one logarithmic unit between each were analyzed.
  • the limit of rPrP detection was 0.9 X 10 _9 M by ELISA.
  • a guanidine thiocyanate buffer was used to dilute the rPrP in order to enhance protein interaction between the polystyrene microplate and the prion protein.
  • a high improvement of signal to noise ratio was observed compared with using the conventional coating buffer, 0.1M carbonate buffer pH 9.6.
  • a background was observed due essentially to interactions between the biotin-labelled reagents, the polymeric surface of the plate and the antibodies . (Indeed, a higher background was observed with polypropylene microplate as than a polycarbonate one . )
  • a biotin-282 concentration of 100 zmol/ ⁇ l involved no background with the agarose visualization of immuno-PCR products .
  • a sensitivity limit of PrP in aqueous solution by such immuno-PCR was clearly 10 ⁇ 14 M and very slightly 10 "16 M.
  • Immuno-PCR products were analyzed by CE-LIF. Each immuno-PCR sample was purified before CE analysis to eliminate excess of primers using the most efficient and the most reproducible method. "Ultrafree-DA" from Millipore used in this example gave a higher reproducibility, with a sufficiently efficient purification, than the Boehringer Mannheim product used in Example 1. The latter gave better purification, due to a complete elimination of primer peaks : however, the reproducibility of this purification was lower and the procedure was more time-consuming. After purification, -immuno-PCR products were analyzed by CE-LIF. A sieving buffer containing a low concentration of hydroxypropylmethycellulose (HPMC) was successfully filled into the capillary to separate ds-DNA by CE-LIF.
  • HPMC hydroxypropylmethycellulose
  • HPMC gave better LIF detection than the short chain non-cross linked polyacrylamide usually used for ds-DNA separation between 20 and lOOObp. It is believed that the high mobility of fragments in HPMC leads to rapid separations without loss of resolution compared with polyacrylamide.
  • the sieving buffer contained the YOYO-1 iodide (see Example 1) .
  • a molar ratio of 5:1 DNA bp: dye was applied to optimise the fluorescent intensity of the DNA.
  • a small plug of TBE containing a relatively high concentration of YOYO-1 iodide (250nM) was filled into the column just before loading of sample. It was found best to mix the HPMC with the YOYO-1 iodide and then fill the capillary with the mixture .

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Abstract

L'invention concerne une bioanalyse haute sensibilité de substances présentes en de faibles concentrations dans les liquides organiques, obtenue lorsque le produit d'immuno-PCR est soumis à une électrophorèse capillaire et, de préférence, détecté par fluorescence induite par laser.
PCT/EP2000/004381 1999-06-02 2000-05-10 Methode et kit de bioanalyse de ligand WO2000075663A1 (fr)

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AU53942/00A AU5394200A (en) 1999-06-02 2000-05-10 Method and kit for ligand assay

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GBGB9912743.3A GB9912743D0 (en) 1999-06-02 1999-06-02 Method and kit for ligand assay
GB9912743.3 1999-06-02

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WO2000075663A1 true WO2000075663A1 (fr) 2000-12-14

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006086799A2 (fr) 2005-02-11 2006-08-17 Novartis Vaccines And Diagnostics Inc. Reactifs de peptide specifiques au prion
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
WO2011057029A1 (fr) 2009-11-04 2011-05-12 Novartis Ag Espèces chargées positivement utilisées en tant que réactifs de liaison dans la séparation d'aggrégats protéiques à partir de monomères
CN104777211A (zh) * 2015-03-26 2015-07-15 浙江医学高等专科学校 一种检测α-眼镜蛇神经毒素的方法
CN113125708A (zh) * 2019-12-31 2021-07-16 暨南大学 一种基于核孔膜的微孔板及其制备方法与应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7932060B2 (en) 2003-04-18 2011-04-26 Becton, Dickinson And Company Immuno-amplification
US8372605B2 (en) 2003-04-18 2013-02-12 Becton, Dickinson And Company Immuno-amplification
US9499858B2 (en) 2003-04-18 2016-11-22 Becton, Dickinson And Company Immuno-amplification
WO2006086799A2 (fr) 2005-02-11 2006-08-17 Novartis Vaccines And Diagnostics Inc. Reactifs de peptide specifiques au prion
WO2011057029A1 (fr) 2009-11-04 2011-05-12 Novartis Ag Espèces chargées positivement utilisées en tant que réactifs de liaison dans la séparation d'aggrégats protéiques à partir de monomères
CN104777211A (zh) * 2015-03-26 2015-07-15 浙江医学高等专科学校 一种检测α-眼镜蛇神经毒素的方法
CN113125708A (zh) * 2019-12-31 2021-07-16 暨南大学 一种基于核孔膜的微孔板及其制备方法与应用

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