EP1877238A1 - Novel supports, in particular for immunodetection of molecules of interest - Google Patents
Novel supports, in particular for immunodetection of molecules of interestInfo
- Publication number
- EP1877238A1 EP1877238A1 EP06721551A EP06721551A EP1877238A1 EP 1877238 A1 EP1877238 A1 EP 1877238A1 EP 06721551 A EP06721551 A EP 06721551A EP 06721551 A EP06721551 A EP 06721551A EP 1877238 A1 EP1877238 A1 EP 1877238A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- container
- detection
- polymer
- plasma
- pcr
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C59/00—Surface shaping of articles, e.g. embossing; Apparatus therefor
- B29C59/14—Surface shaping of articles, e.g. embossing; Apparatus therefor by plasma treatment
- B29C59/142—Surface shaping of articles, e.g. embossing; Apparatus therefor by plasma treatment of profiled articles, e.g. hollow or tubular articles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
Definitions
- Novel carriers particularly for the immunodetection of molecules of interest
- the present invention relates to novel types of carriers designed primarily for the detection and assay of molecules of interest, particularly those present at very low concentrations. These supports can also allow the transfer, storage and manipulation of molecules of interest.
- the target areas of application are numerous.
- the present invention relates more particularly to immuno-detection, selective capture, manipulation as well as storage of molecules of interest. These supports can therefore be used in the industrial sectors of
- the preparation of the samples and more particularly the protocol for extracting the protein from the matrix; the supports (tubes, pipettes, tips, etc.), which must be treated in such a way as to improve the fixation of the molecule of interest (antibodies or antigen, etc.);
- the detection method which must be ultrasensitive (eg quantitative immuno-PCR).
- the aim of the invention is to improve the preparation of the sample as a function of the type of antigen that it is desired to detect, more particularly as regards the support.
- Prion diseases or transmissible spongiform encephalopathy are fatal neurodegenerative diseases that affect both humans and animals (Bovine and Feline Spongiform Encephalopathy, Scrapie of Sheep and Goats for Animals, Creutzfeldt-Jakob Disease, Gerstmann Disease). St Hurssler-Scheinker, fatal familial insomnia or Kuru for humans). All prion diseases share the same molecular mechanism that involves the conversion of a normal cellular prion protein (PrPc) into an infectious form, insoluble in nonionic detergents and partially resistant to proteases, generally called PrPsc.
- PrPc normal cellular prion protein
- This infectious protein has a secondary structure particularly rich in b sheets, which gives it a very hydrophobic character; it therefore tends to aggregate to form insoluble fibrils in water.
- PrPrec normal prion protein
- vCJD Creutzfeldt-Jakob
- kits for detecting an infectious protein usually begins with a development of the technology on the recombinant form of this protein, non-infectious and therefore easier to handle.
- the present invention provides a detection kit using ELISA and iPCRq technologies on the recombinant prion protein, PrPrec.
- Immunodetection sandwich In this case, it is necessary to coater a capture antibody on the surface of the support for carrying out the diagnostic test. Depending on the type of antibody used, the nature of the support should be optimized; an antibody being a soluble molecule in an aqueous medium (serum), it will therefore prefer to attach to a support having a hydrophilic nature.
- the prion protein being highly hydrophobic in the infectious form, it is therefore preferable to store it in containers whose wall has a hydrophilic character.
- the prion protein will be more likely to be pushed away from the walls and will not tend to adhere to it. There will be less loss of protein of interest.
- the invention therefore proposes new types of supports for detecting and dosing molecules of interest. These supports can also be used for the storage of (complex or simplified) samples as well as for the manipulation of the molecules of interest (sampling systems reducing the loss of samples on the walls). These supports will be different in that the wall has undergone an electromagnetic plasma treatment and a polymer deposit that modifies the nature of its surface, depending on the application that one wishes to make.
- i-PCR L 1 immuno-Polymerase Chain Reaction
- i-PCRq quantitative i-PCR
- micro- (and nano-) miniaturization of technologies becomes possible and allows microdosing of prion proteins, and more generally of proteins.
- the miniaturization of the detection and / or assay tests by the use of samples of volumes and concentrations always lower and lower, causes an exacerbation of the importance of the surface of the support. This is also true in the field of sample handling, as well as that of storage, in which it is preferable that the support attracts antigen or regrowth, depending on the intended applications.
- the invention therefore aims at controlling the functionality, the uniformity as well as the reproducibility of the support surfaces, in particular for microdosing and for the storage of certain molecules.
- “Plasma treatment of polymeric materials to enhance immobilization of analytes” (Patent No. WO9534814).
- This invention provides a method of surface treatment of a polymeric material with a gas plasma (ionized oxygen). This treatment makes it possible to increase the immobilization of the antigen used to carry out the diagnosis.
- This invention provides a method of modifying a solid surface to improve its biocompatibility. This method uses a biocompatible agent capable of activating covalent bonds with the surface of the solid.
- This invention provides a method of immobilizing biomolecules on a support.
- the biomolecule itself is grafted onto a solid and hydrophilic polymeric support having been previously ionized.
- This technique is applicable to the immobilization of a wide variety of biomolecules such as enzymes, hormones, lectins, drugs, vitamins, antibodies, etc. These products can be used for therapeutic or diagnostic applications.
- the invention therefore proposes to specifically functionalize these supports, using a clean, reproducible and easily industrializable technology known as "electromagnetic plasmas".
- electromagnetic plasmas a plasma gas treatment of varying nature, pressure, power and frequency; followed in some cases and preferably by a physical treatment depositing on the surface of the support of polymers of medical grade or other organic molecules, poly vinylpyrrolidone and celluloses ...
- the present invention therefore proposes, among other things, new types of supports (tubes, tips and multi-well plates) that enable the detection, dosing, manipulation and storage of molecules of interest.
- these supports according to the invention are "activated” by covering their surface with antibodies, antigens, ligands and / or specific receptors fixed by physico-chemical bonds.
- the support is "neutralized” and therefore allows the improvement of the sensitivity of the assay (mainly at low concentrations) but also the improvement of the reproducibility of the assay.
- These supports can in particular be used for the detection and assay of prion proteins, as described in the examples below.
- the invention has been applied more particularly to multi-well supports polypropylene (PP), polystyrene (PS) and polycarbonate (PC), but can be applied to other types of media such as commercial products, consumables laboratory (tips, tubes,.) and medical consumables (catheter,.) and artificial organs.
- PP polypropylene
- PS polystyrene
- PC polycarbonate
- Electromagnetic plasmas These are electrical discharges carried out either at ambient pressure (in this case they are called crown discharges, corona or DBD), or under partial vacuum (approximately 0.1 to 5 torr, ie 1 to 10 mPa). These electric discharges contain excited gas atoms (N2, 02, etc.), but of rather low energy (some eV). These excited species will bombard the surface of the supports and will therefore break some of the chemical bonds constituting this object (-CC-- bonds, -C-0, --CH, etc.), which will give rise to so-called species. "Radicals" (--C 0 ; --0 °, etc.) generally unstable. It has been estimated that the surface density of reactive species is 10 to 100 radicals per square nanometer. As the energy of the bombardment is low, the sites are created in a slice of material (the support) of thickness less than 10 nanometers.
- These groups have a hydrophilic character and gives a possibility of adhesion with something that we will bring: a paint, a glue, bio-compatible polymers. Adhesion will be acquired spontaneously by the establishment of low energy bonds called hydrogen bonds. If they are numerous, the adhesion will be excellent.
- the radicals make it possible to initiate a polymerization, on the condition of bringing into contact with molecules capable of polymerizing, in an oxygen-free medium, and in general by heating for several hours: polymer chains are hooked by strong bonds, covalent type, on the support. This system is over. heavy to achieve but the adhesion is excellent.
- containers or plastic supports can be chosen from the following nonlimiting list:
- PP Polypropylene
- PET polyester terephthalate
- PC Polycarbonate
- PS Polystyrene
- the supports used are commercial supports having the desired dimensional characteristics for the analyzes (boxes, flat supports, multi-well systems, balls, etc.). They are clean and have no pollutants, which can come from manufacturing.
- the plasma treatment containers or plastic materials may be selected 'from the following nonlimiting list: Plasma types: o Carbon Dioxide (CO2); o Carbon tetrafluoride (CF4); o Nitrogen (N2); o Oxygen (02); o Argon (Ar).
- CO2 Carbon Dioxide
- CF4 Carbon tetrafluoride
- N2 Nitrogen
- Oxygen 02
- Argon Ar
- the treatment by polymer deposition on the plastic containers or supports can be effcetué with a solution of non-limiting list of polymer following:
- Polymer Chemical Treatments Polyvinylpyrrolidone (or PVP) K30 and K90; polyvinyl alcohol (or PVA) 88 and 98% in H 2 O; o carboxymethyl cellulose (or CMC); hydroxypropylmethyl cellulose (or HPMC) 0.2%; o Polyethylene glycol (or PEG) E 4000; o Polyhydroxyethyl methacrylate (or PHEMA).
- a 8-well PCR-type polycarbonate strip is used, marketed by the German firm RoboScreen (reference number 0501000102).
- the plasma treatment of the polycarbonate support is carried out in a reactor with a capacity of 20 liters working in radiofrequency.
- the reactor is evacuated to degass the support for 10 minutes, then E2006 / 000030
- the nitrogen (N 2) is introduced into the reactor at a flow rate of 10 cm 3 / min.
- the support is subjected to a working pressure of approximately 3.4 10 -1 mbar and a radioelectric power of 100 W.
- the support is then brought back to atmospheric pressure, under a neutral atmosphere.
- the support is deposited (for about 5 minutes and at room temperature) in a dilute aqueous solution of polyvinylpyrrolidone grade K30. The support is then dried completely under an air stream at about 40 ° C.
- the support is stored in a neutral atmosphere (N2), and protected from light in sealed packaging.
- N2 neutral atmosphere
- Plasma treatment alone O PS (-N 2, -CO 2 and -CF 4) o PC (-N 2, -CO 2 and -CF 4) o PP (-N 2, -CO 2, -CF 4, Ar and O 2)
- Plasma treatment + polymer deposition o PP-N2 (- PVP, -PVA, -CMC, -HPMC, PEG and PHEMA) GD PC-N2 (- PVP, -PVA, -CMC, -HPMC, PEG and PHEMA) I) Analysis of the media produced:
- the commercial supports used consist of: 95 to 100% of polymers (or plastics) such as polycarbonate, polyester terephthalate, polystyrene or polypropylene,
- additives liquid petrolatum, paraffin or stearate. These additives have the disadvantage of emerging from the support over the days and forming a polluting film covering the plastic surface.
- Wetting test This test is performed by the water drop test. During this, a drop of water is deposited on the surface of the support and the internal angle thereof is measured in order to estimate the hydrophobic (or hydrophilic) nature of the support.
- Plasma-treated support Contact angle of 20 to 30 °, therefore relatively hydrophilic and generally not stable over time
- Coating Buffer 0.05M NaHCCG - Na2CO3 O ; 05M - pH 9.4.
- PBS buffer 0.14M NaCl - 8mM Na2HPO4.2H2O - 1.5mM KH2PO4
- Saturation buffer PBS buffer + 3% BSA - pH 7.4.
- Dilution buffer A PBS buffer + BSA 1% + Tween 20 0.1%
- Dilution buffer B PBS buffer + 1% BSA - pH 7.4.
- BSA wash buffer 1/5% PBS + BSA buffer - pH 7.4.
- Tween wash buffer PBS buffer + 0.1% Tween 20 - pH
- Monoclonal capture antibodies SAF32 (Eurogentec) and 12F10 (Bio-Rad).
- TMB 3,3 ', 5,5' tetramethyl benzedine
- ELISA ELISA-type wells marketed by the German company Greiner (catalog number 655081) is used.
- the working volume is 50 ⁇ l / well.
- the steps of the protocol are as follows: Coat, overnight at 4 ° C., of the SAF32 capture monoclonal antibody diluted to a concentration of 1 ⁇ g / ml in coating buffer (50 ⁇ l / well).
- PrPrec bovine protein (Roboscreen) diluted at various concentrations in dilution buffer A (50 ⁇ l / well).
- Washings (300 ⁇ l / well): 2 in Tween wash buffer followed by 2 in BSA wash buffer.
- the capture monoclonal antibody 12F10 diluted to a concentration of 10 ⁇ g / ml in coating buffer (100 ⁇ l / well).
- the plasma modified support 02 is less effective than the Argon modified support.
- An improvement in the detection limit of a factor close to 4 is observed for the Argon-treated support compared to that treated with O 2.
- the PP Ar has a larger detection range than the control. untreated, resulting in better discrimination power. The latter therefore seems better suited to this type of application.
- All these polymer deposits improve the detection limit by a factor of at least 4 and the discrimination power by a factor of 2.5 minimum.
- PrPrec protein Robot
- dilution buffer B 50 ⁇ l / well
- 6- Washes 250 ⁇ l / well: 3 in Tween wash buffer followed by 3 in BSA wash buffer.
- the invention therefore proposes
- a transfer container, storage or detection Poui * entities in particular biological solubilized or suspended, characterized in that it is made of a material (plastic, glass, mineral or organic compound), preferably in polypropylene or polycarbonate, surface treated with a plasma followed by a polymer deposit.
- the polymer deposit is obtained by dipping or placed in contact with a solution, preferably an aqueous solution, of an organic polymer, for example a polyethylene glycol, preferably at room temperature
- the polymer deposit is produced directly after the plasma treatment, p.e. 1 to 10 minutes, preferably within 5 minutes
- such a container may constitute a support for the assay of biological entities, making it possible to minimize the losses of these molecules by adsorption and / or by altering their native properties.
- Such a container can also be a support for the transfer and manipulation of biological or other molecules, reducing the loss of material on the walls thereof.
- It can be isolated tubes, strip or multi-well plates or consumables of different shapes (tips, cannulae, cups).
- the plasma used is a gas plasma (preferably N 2, Ar or CFA) of variable pressure, power and frequency,
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- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
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FR0503322 | 2005-04-04 | ||
PCT/BE2006/000030 WO2006105622A1 (en) | 2005-04-04 | 2006-04-04 | Novel supports, in particular for immunodetection of molecules of interest |
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EP1877238A1 true EP1877238A1 (en) | 2008-01-16 |
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EP06721551A Withdrawn EP1877238A1 (en) | 2005-04-04 | 2006-04-04 | Novel supports, in particular for immunodetection of molecules of interest |
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US (1) | US20080206795A1 (en) |
EP (1) | EP1877238A1 (en) |
WO (1) | WO2006105622A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5409696A (en) * | 1990-11-08 | 1995-04-25 | Cordis Corporation | Radiofrequency plasma treated polymeric surfaces having immobilized anti-thrombogenic agents |
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US4289748A (en) * | 1979-05-31 | 1981-09-15 | United States Of America | Ultrasensitive enzymatic radioimmunoassay method |
US4681782A (en) * | 1982-03-31 | 1987-07-21 | Biostar Medical Products, Inc. | Article for determining the presence of immune complexes |
US4806316A (en) * | 1987-03-17 | 1989-02-21 | Becton, Dickinson And Company | Disposable device for use in chemical, immunochemical and microorganism analysis |
US4967763A (en) * | 1989-03-13 | 1990-11-06 | Becton, Dickinson And Company | Platelet stable blood collection assembly |
US5628883A (en) * | 1991-06-18 | 1997-05-13 | Japan Vilene Co. Ltd. | Method for generating and activating plasma process of treatment using same, and apparatus therefor |
US5707624A (en) * | 1994-06-03 | 1998-01-13 | The Regents Of The University Of Michigan | Treatment of Kaposi's sarcoma by inhibition of scatter factor |
AU707484B2 (en) * | 1995-09-14 | 1999-07-08 | Regents Of The University Of California, The | Antibodies specific for native PrPsc |
GB9805938D0 (en) * | 1998-03-19 | 1998-05-13 | Glaxo Group Ltd | Valve for aerosol container |
US20040202579A1 (en) * | 1998-05-08 | 2004-10-14 | Anders Larsson | Microfluidic device |
SE9901100D0 (en) * | 1999-03-24 | 1999-03-24 | Amersham Pharm Biotech Ab | Surface and tis manufacture and uses |
US7456025B2 (en) * | 2001-08-28 | 2008-11-25 | Porex Corporation | Sintered polymer membrane for analyte detection device |
-
2006
- 2006-04-04 WO PCT/BE2006/000030 patent/WO2006105622A1/en active Application Filing
- 2006-04-04 US US11/910,554 patent/US20080206795A1/en not_active Abandoned
- 2006-04-04 EP EP06721551A patent/EP1877238A1/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5409696A (en) * | 1990-11-08 | 1995-04-25 | Cordis Corporation | Radiofrequency plasma treated polymeric surfaces having immobilized anti-thrombogenic agents |
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US20080206795A1 (en) | 2008-08-28 |
WO2006105622A1 (en) | 2006-10-12 |
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