Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
• • 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
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/545—Synthetic resin
• • 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
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran

Definitions

• micro-titre-plate is chemically modified with functional groups, whereafter non-adsorbing molecules can be bound covalently by conventional cross-linking technology (cf. e.g. Neurath, AR and Strick, N "Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates. J. Virol. Methods. 3: 155-65, 1981, and e.g. GlueTech Aps "A method for covalent fixation of molecules on a solid phase", W089/05329).
• conventional cross-linking technology cf. e.g. Neurath, AR and Strick, N "Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates. J. Virol. Methods. 3: 155-65, 1981, and e.g. GlueTech Aps "A method for covalent fixation of molecules on
• activated poly- saccharide such as activated dextran or activated agarose
• nucleophilic groups such as NH 2 - and SH- groups
• an " activated polysaccharide which is not a cellulose ether, and which does not have a “cloud-point”, and which acts as a kind of multifunctional cross-linking reagent which connects e.g. antigenes or antibodies to the solid phase, the polysaccharide being activated in such a way that the activated groups can form covalent bonds.
• polysaccharides are activated dextran and activated agarose.
• dextran and agarose use can be made of corresponding amounts of other polymers containing OH-groups, such as other natural or synthetic poly- or oligosaccharides, e.g. gum xanthan or poly- alcohols, such as polyvinyl alcohol or polyhydroxy methyl methacrylate.
• a micro-titre-plate containing primary amino groups may e.g. be treated with perjodate- or tresyl-activated dextran or agarose, whereby the surface obtains quite new and strongly improved binding properties.
• a micro-titre-plate in a particularly simple way can be imparted with the expedient physicochemical properties which are e.g. known from materials used for affinity chromatography.
• the excess hydrophilic groups e.g. ether- and alcohol-groups
• the surface is hydrophilic and in other words possessing the same non-binder properties. It is further achieved that the function and structure of the biomolecules are retained after binding to the surface.
• the method is designated "hydro-coating”.
• glutaraldehyde is associated with a number of working environmental disadvantages. Additionally, it is often seen that the methacrylate-monomer, which is toxic, does not diffuse into the patient. Alternatively it is therefore suggested that perjodate-activated dextran is used for pretreating the hydrofluoric acid treated dentin surface. Hereby a coating is performed with a well-known biocompatible material, and it must be expected that the aldehyde groups on the dextran will have the same initiating effect as glutaraldehyde on the succeeding methylmethacrylate-polymerization.
• Fig. 1 shows binding of biotin-MP7 to micro-titre-plates with (•) and without POD (°), respectively; binding of biotin-MP9 to micro-titre-plates with (T) and without POD (v), respectively, and binding of biotin-peptide T to micro-titre-plates with ( ⁇ ) and without POD ( ⁇ ), respectively.
• Fig. 2 shows binding of biotin-MP7 to micro-titre-plates with (•) and without TAD (°), respectively; binding of biotin-MP9 to micro-titre-plates with (T) and without TAD (v), respectively, and binding of biotin-peptide T to micro-titre-plates with ( ⁇ ) and without TAD ( ⁇ ), respectively.
• Fig. 3 shows the binding of peroxidase-labeled porcine immunoglobulin (PMS) to micro-titre-plate surfaces, which were and were not, respectively, coupled with POD.
• PMS added to a POD treated surface (•); PMS added to an untreated surface (° ).
• Fig. 6 shows the result of a sandwich-ELISA for detecting murine immunoglobulin, where the catching antibody (goat anti-mouse immunoglobulin) was immobilized to a POD- treated surface (°) and an untreated micro-titre-plate surface (•), respectively.
• the capacity for binding the catching antibody was examined by performing a titration thereof, and at the same time retaining the amount of the intermediate layer which contained murine IgG (0x6).
• Fig. 7 shows the binding of biotin-MP7 to a POD-treated surface in PBS ( ) and in carbonate buffer (• ), respectively.
• POD was oxidized to various degrees (0, , 1%, 2 and 2 ).
• the molar ratio between the OH-groups on dextran and perjodate during the oxidation was 0, %, 1%, 2 and 2%, respectively.
• Fig. 8 shows the binding of biotin-MP7 to a POD-treated surface in carbonate buffer ( ⁇ ) and in PBS ( a), respect- ively.
• the dextrans which were used for producing POD, had a MW of 10,000, 70,000 and 2000,000, respectively.
• Fig. 9 shows binding of biotin-N-hydroxy succinimide to a poly-L-lysine coated surface, which was not treated (°) and treated, respectively, with different amounts of POD having MW of 10,000 (•), 70,000 (v) and 2,000,000 (T), respectively.
• Fig. 11 shows binding of biotin-N-hydroxy succinimide to a poly-L-lysine coated surface which, respectively, was not treated with POD (0), and to surfaces which were treated with POD 70 000, and thereafter treated with 0.1 M NaOH (1); 1 M NaOH (2); 0.1 M HC1 (3); 1 M HC1 (4); PBS, pH 7.2 (5); 0.1 M carbonate buffer, pH 9.6 (6); washing buffer, pH 7.2 (7) and water (8), respectively, and
• hydro-coating is to couple a solid phase with an activated polysaccharide which hereby will act as a kind of "double adhering" tape.
• nucleophilic groups on the surface of the solid phase which it is desired to treat by the method according to the invention.
• This is achievable in various ways.
• Several micro-titre-plates are already on the market, where e.g. primary or secondary amino groups are associated covalently to the surface of the plate (e.g CovaLink from A/S Nunc, Denmark, or Costar, USA).
• coating can merely be by passive adsorption to the solid phase with e.g. protein, polyamine or poly-L- lysine, whereby amino groups are introduced non-covalently on the surface. It is well-known that this lastmentioned method leads to an especially stable modification, even through covalent forces are not involved.
• Other polymers containing the mentioned functional groups are similarly adsorbable to the surface, just as such groups can be introduced by various chemical methods (see e.g. "Radiation grafting", EP-A-155 252).
• dextran is used as the polysaccha ⁇ ride.
• other oligo- or polysaccharides such as e.g. agarose
• oligo- or polysaccharides such as e.g. agarose
• perjodate- activated dextran but examples are also shown where the dextran has been supplied with tresyl-groups.
• HMPTA hexamethyl phosphorous triamide
• N-methyl-pyrrolidinone N-methyl-pyrrolidinone
• hydro ⁇ philic polymer introduction may e.g. be made of aldehydes, ketones, vinyl sulphones, cyanogens, active esters, epoxides, disulfides and other active compounds of carboxylic acid, phosphoric acid and sulphonic acid.
• the mentioned activated groups can e.g. be attached separated by a "spacer-arm", whereby any sterical problems can be further alleviated.
• dextran instead of dextran use is made of corresponding amounts of agarose or other natural or synthetic oligo- or poly- saccharides, such as gum xanthan (Sigma, G 1253), or polyalcohols, such as polyvinyl alcohol or polyhydroxy methyl methacrylate.
• agarose or other natural or synthetic oligo- or poly- saccharides, such as gum xanthan (Sigma, G 1253), or polyalcohols, such as polyvinyl alcohol or polyhydroxy methyl methacrylate.
• micro-titre-plate used did not in advance contain primary amino groups and was therefore pretreated with poly-L-lysine.
• This pretreatment has also been made with other polyamino acids containing primary amino groups, such as polypeptides, just as it has been made with poly- amines, such as polimine on micro-titre-plates, micro- titre-strips, particles, membranes, test tubes, test strips or measuring pins consisting of materials, such as polyethylene glycol terephthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylo- nitrile and polymethacrylate.
• Dextran (100 mg MW: 70,000, Sigma D4751) was dissolved in water (10 ml) and was freeze-dried overnight to reduce the content of free water. The next day the dextran (10 mg) was dissolved in HMPTA (20 ml, Sigma H4006) at 120 °C in a 100 ml round-bottomed flask attached to a calcium-chloride tube with sicapent. After cooling freshly distilled pyridine (198 mg, 10.9 mmole, Aldrich 270407) and tresyl chloride (858 mg, 10.9. mmole, Aldrich 324787) were added. After two hours the dextran was precipitated with ethanol. Re-dissolution was in water, and again precipitation was with ethanol. Hereafter the active dextran was freeze- dried overnight.
• micro-titre-plate used had in advance neither primary or secondary amino groups and was therefore pretreated with poly-L-lysine.
• This pretreatment has also been per ⁇ formed with other polyamino acids and polypeptides containing primary and/or secondary amino groups, just as it has been performed with polyamines, such as polimine, on micro-titre-plates, micro-titre-strips, particles, plates, test tubes, test strips or measuring pins, consisting of materials, such as polyethylene glycol terephthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile and polymeth ⁇ acrylate.
• the peptides biotin-MP7, biotin-MP9, and biotin-peptide-T were titrated 2-fold in double determination in 7 steps starting with a concentration of 100 ⁇ g/ml in phosphate buffered saline water (PBS, 0.1 M, 100 ⁇ l/well) and ending with a concentration of 1.6 ⁇ g/ml.
• PBS phosphate buffered saline water
• the peptides were titrated in double determination both in that half of the micro-titre-plate which was coated with poly-L-lysine and in that half which was not.
• washing buffer NaCl 29.2 g, KC1 0.2 g, KH 2 P0 4 H 2 0, Triton X-100 100 ml, distilled water to 1000 ml.
• biotin-MP7 biotin-PELFEALQKLFKHAY
• biotin-MP9 biotin-FAQKEPAFLKEYHLL
• biotin-peptide-T biotin-GGGASTTNYT
• Biotin-MP7/MP9 contain lysines and consequently free primary amino groups. This, however, is not so for biotin- peptide-T, which does not contain lysine.
• binding of the peptides to POD-treated surfaces is due to a specific covalent binding to the excess aldehyde- and ketone-groups on the POD-surface. If the surface does not contain amines, POD 70,000 does not bind, and consequently the peptides can only to a low degree or not at all be detected on the surface.
• Biotin-MP7, biotin-MP9, and biotin-peptide-T were added to surfaces both with and without poly-L-lysine, and any immobilization thereof was detected as described in example 3.
• biotin-MP7/MP9 bound strongly to TAD treated surfaces, but no binding of biotin-peptide- T was seen. Nor was any or only vague binding of the peptides to the surface seen without poly-L-lysine. Since biotin-MP7/MP9, but not biotin-peptide-T, contains free NH ? -groups, it can be concluded that the binding of peptide is due to a covalent immobilization via the tresyl-groups on the dextran. Tresyl-groups can, as opposed to aldehydes and ketones, form stable bonds with secondary amines.
• TAD additionally has the advantage that any excess tresyl-groups on the TAD-treated surface can be hydrolyzed off with base, whereby hydrophilic OH-groups are reestablished on the surface of the micro-titre-plate.
• these OH-groups can optionally be further activated with e.g. cyanogen bromide or tresyl chloride in e.g.
• TAD treated surfaces further possess the same properties as POD treated surfaces as regards the useful effect on binding and structure preservation of the peptides, which was described in example 3.
• a micro-titre-plate was used where the wells had been treated with POD, as described in example 3.
• Micro-titre-plates with poly-L-lysine were produced as described in example 3.

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• 1992
  • 1992-08-10 DK DK921005A patent/DK100592D0/da not_active Application Discontinuation
• 1993
  • 1993-08-10 CA CA002142011A patent/CA2142011A1/en not_active Abandoned
  • 1993-08-10 JP JP6504913A patent/JPH08503066A/ja active Pending
  • 1993-08-10 US US08/379,675 patent/US5874569A/en not_active Expired - Fee Related
  • 1993-08-10 EP EP93917569A patent/EP0654061A1/en not_active Withdrawn
  • 1993-08-10 WO PCT/DK1993/000259 patent/WO1994003530A1/en not_active Ceased
  • 1993-08-10 AU AU46986/93A patent/AU4698693A/en not_active Abandoned
• 1998
  • 1998-05-06 US US09/073,646 patent/US6262256B1/en not_active Expired - Fee Related

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