EP0669942A1 - Anticorps artificiels, procede de production desdits anticorps et utilisation desdits anticorps - Google Patents

Anticorps artificiels, procede de production desdits anticorps et utilisation desdits anticorps

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Publication number
EP0669942A1
EP0669942A1 EP93924895A EP93924895A EP0669942A1 EP 0669942 A1 EP0669942 A1 EP 0669942A1 EP 93924895 A EP93924895 A EP 93924895A EP 93924895 A EP93924895 A EP 93924895A EP 0669942 A1 EP0669942 A1 EP 0669942A1
Authority
EP
European Patent Office
Prior art keywords
antibodies
artificial antibodies
monomers
artificial
binding sites
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Withdrawn
Application number
EP93924895A
Other languages
German (de)
English (en)
Inventor
Klaus Mosbach
Georg Foundation for Research & Techn. VLATAKIS
Lars I. Andersson
Ralf MÜLLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP0669942A1 publication Critical patent/EP0669942A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • the present invention concerns artificial antibodies, a method for producing the artificial antibodies, a method for determination of an organic molecule in a fluid sample, a method for separation or isolation of an organic molecule and use of the latter methods in immunoassays as well as a method of therapy or diagnostics.
  • Antibodies are used in several areas, such as thera ⁇ py, immunoaffinity, purification and in particular in immunoassays. As to the latter aspect the corresponding antigens can either be small or large molecules.
  • Antibodies are normally produced by immunising ani- mals with the corresponding antigen leading to polyclonal antibodies, or by using fused cells (B cells) allowing the obtained cell lines to produce monoclonal antibodies.
  • Antibodies can be raised against most compounds; they are versatile reagents employed in numerous applica- tions 1-5, ranging from basic research to clinical analy ⁇ sis. However, being bio-macromolecules they require care-
  • a potentially useful alternative would be the produc ⁇ tion of non-biologically derived antibody mimics or arti ⁇ ficial antibodies, such as polymer structures that are similar to biological antibodies in binding and recogni- sing antigens.
  • the technique has been applied to make synt-whet.i-c enzymes9,10
  • the technique of molecular imprinting and its special form of non-covalent imprinting as developed by the inven ⁇ tors makes it possible to achieve the above objects.
  • the technique involves polymerisation of functional monomers in the presence of a print molecule (see Scheme 1). Subsequent removal of the print molecule from the rigid polymer results in sites within the polymer that are complementary to and have an affinity for the original print molecule.
  • artifi ⁇ cial antibodies which consist of polymers that carry specific binding sites mimicking the properties of anti- bodies.
  • a method for producing artificial anti ⁇ bodies in which poly erisable monomers carrying functiono ⁇ nal groups and crosslinking monomers are polymerised in the presence of a print molecule and subsequently the print molecule is removed leaving specific binding sites complementary to the print molecule.
  • the invention also provides for a method for determi ⁇ nation of an organic molecule in a fluid sample.
  • a known amount of the organic molecule provided with a label is added to the sample, the sample is contacted with artificial antibodies having specific binding sites for the organic molecule, whereby the labelled and unlabelled organic molecules are competi- tively bound to the binding sites, and the labelled orga ⁇ nic molecule is determined either unbound in the superna ⁇ tant or bound by the polymer.
  • the invention also provides a method for separation or isolation of an organic molecule from a fluid sample, in which the sample, labelled or not, is contacted with an excess of artificial antibodies consisting of a polymer having specific sites for the organic molecule, whereby the organic molecule is bound to the binding sites, and optionally the organic molecule is measured bound to the artificial antibodies or eluted from the antibodies.
  • the invention also provides fo a method of therapy or diagnosis, in which artificial antibodies are administra ⁇ ted to a mammal body, which artificial antibodies consist of a biocompatible polymer carrying specific binding sites mimicking the properties of antibodies towards an organic molecule.
  • the polymers are prepared by non-covalent polymerisation.
  • the polymers constituting the artificial antibodies are preferably built up of polymerisable monomers carrying functional groups and crosslinking monomers.
  • the polymerisable monomers carrying functional groups are chosen among negatively charged monomers such as methacry- lic acid, itaconic acid, basic monomers such as vinylpyri- dine, vinylimidazole, hydrophobic monomers carrying alkyl chains, monomers allowing ⁇ - ⁇ -interactions, van der Waals forces.
  • polymers are built up of methacrylic acid crosslinked by ethylene glycol dimethacrylate.
  • the polymers must be bio ⁇ compatible. Preferably they must be of the size not more than 5 ⁇ m or the size'of normal biological antibodies, most preferred 10-100 nm.
  • the polymer is ground to a particle size of normally ⁇ 25 ⁇ m for use in so-called heterogenous assays.
  • the fines that is particles with a size of 10-100 or 1000 nm, resulting from the grinding, can be kept in solu ⁇ tion or suspension and used for instance in so-called homogenous immunoassays.
  • Such assays are extremely sensi ⁇ tive and can be performed involving e.g. two different antibodies.
  • Another advantage with the fine particles is that they are more suitable for use in therapy or diagnostics.
  • binding sites are specific for a com ⁇ pound chosen from the group consisting of drugs, metabo ⁇ lites, nucleotides, nucleic acids, carbohydrates, pro ⁇ teins, hormones, toxins, steroids, prostaglandins and leukotrienes.
  • binding sites are specific for theofylline or diazepam.
  • Suitable labels for use in the methods according to the invention are radioligands, enzymes, biotin, steroids, fluorochromes, gold.
  • the methods according to the invention are preferably used in immunoassays, especially in radioimmunoassays.
  • the method of therapy or diagnosis according to the invention comprises several different modes of action. For example, it can be used to withdraw an undesired organic molecule from a mammal body, such as a toxin.
  • the artificial antibodies assemble around a cancer cell to indicate the presence of such a cell.
  • the artificial antibodies are bringing a drug to specific targets, for instance cancer cells.
  • an extra corporal device containing the artificial antibodies is coupled to the body via a shunt in the bloodstream, and the bloodstream is passed through the device.
  • Theophylline a commonly used drug in the prevention and treatment of asthma, apnea and obstructive lung diseases, has a narrow therapeutic index (56-112 ⁇ mol L ⁇ serum) requiring careful monitoring of serum concent- rations 17.
  • Diazepam e.g. valium
  • Benzodiazepines are one of the most commonly implicated substances in drug overdose situations and their detection in body fluids is very useful in clinical and forensic toxicology.
  • Current methods for measuring theophylline and benzodiazepines are based on high-performance liquid chromatography and, on lmmunol,ogical, t.ec,hni.ques22-26
  • the polymers were prepared using methacrylic acid
  • the solvent compositions giving optimal binding and selectivity were determined for each polymer (see Example 2 and Fig. 1 below).
  • a general guide 14'27 i) in a more apolar solvent the substrate binds more strongly to the polymer than in polar solvents, and ii) small amounts of acetic acid can be added to the solvent in order to supress non-specific binding.
  • the eqilibrium dissociation constants (K ) for binding of the drugs to the correspon ⁇ ding polymers were estimated by Scatchard plot analysis using radio-labelled ligands. In both cases, the Scatchard plots were nonlinear and fitted well with two K values, for high and low affinity binding sites.
  • the MIA for theophylline was linear over the range 14-224 ⁇ mol L ⁇ which is satisfactory for therapeutic monitoring of the drug.
  • the results for diazepam were linear over the range which is normally used in standard immunoassay techniques for benzodiazepines
  • H-theophylline bronchodilator
  • various benzodiazepines for binding of H-diazepam (tranqilizer) to artificial antibodies (ArtAb's) and natural antibodies (Ab's).
  • ligands were added to drug free serum and assayed as described in Fig. 1.
  • Cross- -reactivities are expressed as the molar ratio of theophylline and diazepam, respectively, to ligand giving 50% inhibition of radioligand binding to polymer.
  • the MIA method for theophylline (1,3-dimethylxanthine) appears to be highly specific since from all the compounds tested only 3-methylxanthine showed some cross-reactivity.
  • the ability of the MIA method for accurate measure ⁇ ment of theophylline was evaluated by analysing 32 patient serum samples. The sample were also analysed with the
  • Molecular imprints may be obtained against functiona- lity complementary to the monomer 14' 27. There is a poten ⁇ tial for molecularly imprinted artificial antibodies in the analysis of many other drugs, metabolites, hormones, toxins, etc.
  • molecularly imprinted polymers provide a potential alternative to the use of laboratory animals for the production of antibodies.
  • EMIT Enzyme-Multiplied Immunoassay Technique
  • the preparation follows the reaction of Scheme 1.
  • MAA is selected for its ability to form hydrogen bonds with a variety of chemical functionalities of the print molecule.
  • B) The polymerisation reaction is started with the addi ⁇ tion of initiator (AIBN) and a rigid insoluble polymer is formed. "Imprints", which are complementary in both shape and chemical functionality to the print molecule, are now present within the polymeric network.
  • Diazepam (1.27 g) was mixed with MAA (2.26 g), EDMA (26.1 g) and AIBN (0.5 g) in chloroform (39 ml).
  • the poly ⁇ merisation mixture was degassed under vacuum in a sonica- ting water-bath, sparged with nitrogen and then polymeri ⁇ sed under UV (366 nm) at 4°C for 16 h.
  • the resulting poly ⁇ mer was then treated as described above.
  • EMIT Enzyme-Multiplied Immunoassay Technique
  • Kurstak, E. in Enzyme Immunodiagnosis (ed Kurstak, E. ) 5-11 (Academic Press, London, 1986). 6. Ekberg, B. & Mosbach, K. Trends Biotechnol. 7, 92-96 (1989).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Manufacturing & Machinery (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Materials Engineering (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Anticorps artificiels ou mimes d'anticorps constitués de polymères qui portent des sites de liaison spécifiques imitant les propriétés des anticorps. Un procédé de production d'anticorps artificiels est également décrit. Selon ce procédé, des monomères polymérisables portant des groupes fonctionnels et des monomères de réticulation sont polymérisés en présence d'une molécule d'impression. Ladite molécule d'impression est ensuite enlevée, laissant des sites de liaison spécifiques complémentaires aux molécules d'impression. Des procédés permettant de déterminer et d'isoler des molécules organiques utilisant lesdits anticorps artificiels, ainsi que des procédés thérapeutiques ou diagnostiques utilisant ces anticorps sont en outre décrits.
EP93924895A 1992-11-11 1993-11-11 Anticorps artificiels, procede de production desdits anticorps et utilisation desdits anticorps Withdrawn EP0669942A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9203435 1992-11-11
SE9203435A SE9203435D0 (sv) 1992-11-11 1992-11-11 Framstaellande av artificiella antikroppar med molekylavtrycksmetoden samt deras anvaendning vid analys
PCT/SE1993/000960 WO1994011403A1 (fr) 1992-11-11 1993-11-11 Anticorps artificiels, procede de production desdits anticorps et utilisation desdits anticorps

Publications (1)

Publication Number Publication Date
EP0669942A1 true EP0669942A1 (fr) 1995-09-06

Family

ID=20387830

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93924895A Withdrawn EP0669942A1 (fr) 1992-11-11 1993-11-11 Anticorps artificiels, procede de production desdits anticorps et utilisation desdits anticorps

Country Status (6)

Country Link
EP (1) EP0669942A1 (fr)
JP (1) JPH08506320A (fr)
AU (2) AU5439794A (fr)
CA (1) CA2149043A1 (fr)
SE (1) SE9203435D0 (fr)
WO (1) WO1994011403A1 (fr)

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE9102622L (sv) 1991-09-06 1993-03-07 Klaus Mosbach Saett att aastadkomma specifikt adsorptionsmaterial laempligt foer biologiska makromolekyler genom prearrangemang av immobiliserbara till makromolekylen i fraaga bindande funktionella grupper
JPH09127116A (ja) * 1995-11-01 1997-05-16 Agency Of Ind Science & Technol タンパク質分子識別機能を有する物質
US7205162B1 (en) 1995-12-07 2007-04-17 Klaus Mosbach Artificial antibodies, method of producing the same and use thereof
US6255461B1 (en) * 1996-04-05 2001-07-03 Klaus Mosbach Artificial antibodies to corticosteroids prepared by molecular imprinting
US6884842B2 (en) 1997-10-14 2005-04-26 Alnis Biosciences, Inc. Molecular compounds having complementary surfaces to targets
JP2000254490A (ja) * 1999-03-15 2000-09-19 Toyo Ink Mfg Co Ltd 比較的低分子の化合物を選択的に捕捉する特性を有する高分子材料、被覆剤、分離方法、分離材、機能性化合物等の検知方法および検知材料
WO2000067899A1 (fr) * 1999-05-06 2000-11-16 Idemitsu Petrochemical Co., Ltd. Adsorbants issus de polymeres de styrenes
GB9925056D0 (en) * 1999-10-23 1999-12-22 Univ Cranfield Preparation of biologically-active molecules by template polymerization
GB0001513D0 (en) * 2000-01-25 2000-03-15 Univ Cranfield Rational design of mips using computational approach
CN1871515A (zh) * 2003-10-29 2006-11-29 冈田秀亲 包含互补肽的人工抗体
WO2005056613A2 (fr) * 2003-12-08 2005-06-23 The Research Foundation Of State University Of New York Polymeres a empreintes moleculaires a sites gabarits selectivement marques pour applications de capteurs
ES2384260T3 (es) 2004-05-24 2012-07-03 British American Tobacco (Investments) Limited Polímeros impresos molecularmente selectivos para nitrosaminas y procedimientos para usarlos
JP4547324B2 (ja) * 2005-11-24 2010-09-22 シャープ株式会社 タンパク質認識構造体、タンパク質認識基板、及びこれらの製造方法
JP2009527471A (ja) * 2006-02-21 2009-07-30 ミプサルス エーピーエス 分子インプリントポリマの改良した調製方法
GB201200878D0 (en) 2012-01-19 2012-02-29 British American Tobacco Co Polymer compositions
AR063873A1 (es) 2006-12-07 2009-02-25 British American Tobacco Co Polimeros impresos molecularmente selectivos para nitrosaminas especificas del tabaco y metodos para usarlos
EP2178540A4 (fr) * 2007-08-01 2012-01-04 Stellan Hjerten Composition pharmaceutique destinée à être adsorbée sur la surface cellulaire de microbes pathogènes
JP4724188B2 (ja) * 2008-01-24 2011-07-13 株式会社東芝 分子認識センサ
US9173943B2 (en) 2009-01-12 2015-11-03 The Regents Of The University Of California Imprinted polymer nanoparticles
DE102014008135A1 (de) 2014-06-06 2015-12-17 Gfl Gesellschaft Für Lebensmittel-Forschung Mbh Verfahren zur analytischen Authentizitätprüfung von Inhaltsstoffen in Lebensmitteln unter Verwendung künstlicher Antikörper

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE8900130L (sv) * 1989-01-16 1990-07-17 Klaus Mosbach Konceptet att med hjaelp av molekylavtrycksmetoden framstaella konstgjorda antikroppar genom imprinting av t ex antigener samt att framstaella konstgjorda entzymer genom imprintning med transition state analoger

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9411403A1 *

Also Published As

Publication number Publication date
AU5439794A (en) 1994-06-08
WO1994011403A1 (fr) 1994-05-26
SE9203435D0 (sv) 1992-11-11
AU5641798A (en) 1998-06-04
CA2149043A1 (fr) 1994-05-26
JPH08506320A (ja) 1996-07-09

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