WO1998015572A1 - Composition comprenant un peptide acyle et immobilise - Google Patents

Composition comprenant un peptide acyle et immobilise Download PDF

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Publication number
WO1998015572A1
WO1998015572A1 PCT/EP1997/005572 EP9705572W WO9815572A1 WO 1998015572 A1 WO1998015572 A1 WO 1998015572A1 EP 9705572 W EP9705572 W EP 9705572W WO 9815572 A1 WO9815572 A1 WO 9815572A1
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Prior art keywords
peptide
peptides
lower alkyl
aralkyl
group
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PCT/EP1997/005572
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English (en)
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Elma Elisabeth Maria Gerarda Loomans
Wilhelmus Joseph Gerardus Schielen
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Akzo Nobel N.V.
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Priority to AU49462/97A priority Critical patent/AU4946297A/en
Publication of WO1998015572A1 publication Critical patent/WO1998015572A1/fr

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    • 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
    • C07K17/06—Peptides being 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates to a composition, a process for the preparation of said composition, the use of said composition in a diagnostic assay, and a process for immobilising an antigenic peptide onto solid supports
  • SPPS solid-phase peptide synthesis
  • immobilised protein antigens In contrast to immobilised protein antigens, immuno-assays (like i.e. ELISA) using immobilised peptide antigens are often not satisfactory, since simple adso ⁇ tion of a peptide onto a solid support frequently results in low coating efficiency. Alteration of the conformation upon immobilisation may result in a lowering of the accessibility of the peptide for antibody binding. Inefficient adso ⁇ tion as a result of inappropriate hypophilic or hydrophilic properties of the peptides is another possibility. Most authors point out that relatively short peptides (e.g., less than 20 residues in length), lack sufficient secondary and tertiary structural features or do not have enough side-chains to bind well to plastic surfaces (Briand et al., 1985, J.
  • composition comprising an antigenic peptide immobilised on the surface of a solid support, wherein said peptide is linked to an acyl group of formula I:
  • Rl is lower alkyl, aryl or aralkyl
  • R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-.
  • Coating efficiency is defined as the peptide concentration in the coating solution, that is required to achieve 50% of the maximum EIA signal (ECso).
  • the improvement of the coating efficiency which is realised with the introduction of said acyl group is found to be between 2 and 5 orders of magnitude, as compared with the parent peptide.
  • This improvement substantially reduces the amount of peptide needed to develop a sensitive peptide-based immunoassay.
  • lower alkyl refers to a branched or unbranched alkyl group having from 1-6 carbon atoms, like hexyl, isobutyl, propyl, isopropyl, ethyl, and preferably, methyl.
  • aryl refers to an aromatic group like phenyl and naphthyl. Also included in the definition of aryl are heteroaromatic groups like pyridinyl, pyrimidyl and thienyl.
  • the aryl groups may be substituted by lower alkyl groups, O-lower alkyl, wherein lower alkyl is as previously defined, and by halogen.
  • Preferred aryl groups are phenyl and 2-pyridinyl.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • aralkyl refers to a lower alkyl group, as previously defined, which is substituted with at least one aryl group, as previously defined.
  • Preferred divalent radicals derived from lower alkyl, aryl or aralkyl, terms which have each been previously defined, are methylene (-CH2-) and 1,2-ethanediyl (- CH2-CH2-).
  • a preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by Rl being a lower alkyl, R2 being derived from a lower alkyl, and A being -CO-S-.
  • Another preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by Rl being methyl, R2 being methylene, and A being -CO-S- (this group is also known as acetyl-thio-acetyl-group (Ata)).
  • a further preferred embodiment of the present invention is directed to a composition wherein said acyl group is represented by A is -CO-S- and: Rl being methyl and R2 being 1,2-ethanediyl (acetyl-thio-propionyl (Atp)), by Rl being phenyl and R2 being methylene (benzoyl-thio-acetyl (Bta)), or by Rl being phenyl and R2 being 1,2-ethanediyl (benzoyl-thio-propionyl (Btp)).
  • Another preferred embodiment of the present invention is directed to a composition wherein said acyl group is linked at the N-terminus of the peptide.
  • a further preferred embodiment of the present invention is directed to a composition wherein said solid support is a polystyrene support.
  • peptide refers to a molecular chain of amino acids with a biological (immunological) activity, and does not refer to a specific length of the product.
  • proteins, fusion-proteins or -peptides, oligopeptides, nucleopeptides and polypeptides are included.
  • Preferred are peptides with a length of 2-30 amino acids. Also modifications by replacement of amino acids by non- natural amino acids and other building blocks to obtain a biologically active molecule are included.
  • solid support refers to polymeric substances such as polystyrene, polyvinylchloride (PVC), nylon, vinypolymers and nitrocellulose, or to glass.
  • the surface of the solid phase may have the form of the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or die surface of a particle such as, for example, a latex particle, a dye sol, a metal sol or metal compound as sol particle.
  • the preparation of the peptides according to the invention is effected by means of one of the known organic chemical methods for peptide synthesis or with the aid of recombinant DNA techniques.
  • the condensation reaction can be carried out as follows: a) condensation of a compound (amino acid, peptide) with a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) with a free amino group and protected other reactive groups, in the presence of a condensation agent; b) condensation of a compound (amino acid, peptide) with an activated carboxyl group and free or protected other reaction groups with a compound (amino acid, peptide) with a free amino group and free or protected other reactive groups.
  • Activation of the carboxyl group can take place, inter alia, by converting the carboxyl group to an acid halide, azide, anhydride, imidazolide or an activated ester, such as the N-hydroxy-succinimide, N-hydroxy-benzotriazole or p- nitrophenyl ester.
  • a particular suitable solid phase is, for example, the p-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystrene-l % divinyl-benzene resin), described by Wang (1974; J. Am. Chem. Soc. 95, 1328).
  • the peptides can be split from this solid phase under mild conditions.
  • detaching of the peptide from the resin follows, for example, with trifluoroacetic acid (Fmoc), or with trifluoromethanesulphonic acid (t-boc).
  • the peptide can also be removed from the carrier by transesterification with a lower alcohol, preferably methanol or ethanol, in which case a lower alkyl ester of the peptide is formed directly.
  • a lower alcohol preferably methanol or ethanol
  • a lower alkyl ester of the peptide is formed directly.
  • splitting with the aid of ammonia gives the amide of a peptide according to the invention.
  • the reactive groups which may not participate in the condensation reaction are, as stated, effectively protected by groups which can be removed again very easily by hydrolysis with the aid of acid, base or reduction.
  • a carboxyl group can be effectively protected by, for example, esterification with methanol, ethanol, tertiary butanol, benzyl alcohol or p-nitrobenzyl alcohol and amines linked to solid support.
  • Groups which can effectively protect an amino group are the ethoxycarbonyl, benzyloxycarbonyl, t-butoxy-carbonyl (t-boc) or p-methoxy-benzyloxycarbonyl group, or an acid group derived from a sulphonic acid, such as the benzene- sulphonyl or p-toluene-sulphonyl group, but other groups can also be used, such as substituted or unsubstituted aryl or aralkyl groups, for example benzyl and triphenylmethyl, or groups such as ortho-nitrophenyl-sulphenyl and 2-benzoyl-l- methyl-vinyl.
  • a particularly suitable ⁇ -amino-protective group is, for example, the base-sensitive 9-fluorenyl-methoxycarbonyl (Fmoc) group [Ca ⁇ ino & Han, 1970, J. Amer. Chem. Soc. 92, 5748].
  • Fmoc base-sensitive 9-fluorenyl-methoxycarbonyl
  • Customary protective groups in this connection are a Boc-group for lysine and a Pmc- or Pms- or Mbs-group or Mtr-group for arginine.
  • the protective groups can be split off by various conventional methods, depending on the nature of the particular group, for example with the aid of trifluoroacetic acid or by mild reduction, for example with hydrogen and a catalyst, such as palladium, or with HBr in glacial acetic acid.
  • Covalent linkage of two or more peptides in a hybrid- or combi-peptide can for instance be carried out through solid phase peptide synthesis, using the methods described above, of a peptide sequence wherein the amino acid sequences of the individual peptides are aligned. It is understood that a linker sequence may be inserted between the individual peptides sequences. Such a linker sequence may for instance be a stretch of 2-5 residues of glycine.
  • a hybrid- or combi-peptide can also be prepared through solid phase synthesis using the fragment condensation approach.
  • the peptides are derivatized to contain an additional residue of cysteine at either the carboxyl- or the amino-terminal end.
  • One of the peptides is subsequently activated at the single cysteine thiol function with 2,2'-dithiodipyridine.
  • the resulting pyridyl-dithio-peptide derivative is then reacted with the second peptide containing the cysteine thiol group to yield a hybrid peptide in which the individual peptides are linked through a disulfide bond.
  • the peptides according to the invention can likewise be prepared with the aid of recombinant DNA techniques. This possibility is of importance particularly when the peptide is incorporated in a repeating sequence ("in tandem") or when the peptide can be prepared as a constituent of a (much larger) protein or polypeptide or as a fusion protein with, for example, (part of) ⁇ - galactosidase. This type of peptides therefore likewise falls within the scope of the invention.
  • a nucleic acid sequence is used which codes for a peptide according to the invention and which, furthermore, is substantially free from nucleic acid segments.
  • This latter method involves the preparation of the desired peptide by means of bringing to expression a recombinant polynucleotide with a nucleic acid sequence which is coding for one or more of the peptides in question in a suitable microorganism as host.
  • the peptides expressed by the host can be purified, if necessary, and coupled to acyl-groups according to the present invention moieties by known methods (use can be made of the chemical methodology that has been developed in the field of protein-protein conjugation. An overview is given by Means and Feeney (Bioconj. Chem. 1, 2-12, 1990)).
  • oligonucleotide molecules can be linked to an acyl group according to the present invention.
  • Another embodiment of the present invention is directed to a process for the preparation of a composition
  • a composition comprising an antigenic peptide immobilised on the surface of a solid support, wherein an aqueous solution of said peptide which is linked to an acyl group of the formula:
  • Rl is lower alkyl, aryl or aralkyl
  • R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-, is brought into contact with a solid support at a pH range of 8.0-10.0.
  • Another embodiment of the present invention is directed to the use of a composition according to the present invention, in a diagnostic assay.
  • Another embodiment of the present invention is directed to a process for immobilising an antigenic peptide onto solid supports, wherein said peptide is linked to an acyl group of the formula:
  • Rl is lower alkyl, aryl or aralkyl
  • R2 is a divalent radical derived from lower alkyl, aryl or aralkyl; and A is a sulfur containing functional group selected from -CO-S-, -S-, -SO-, -SO2- and -S-S-, after which the modified peptide is brought into contact to said solid support.
  • Another embodiment of the present invention is directed to a test kit wherein a composition according to the present invention is used.
  • Ata acetyl-thio-acetyl: represented by A is - CO-S-, Rl is methyl and R2 is methylene
  • Atp acetyl-thio-propionyl: A is -CO-S- , Rl is methyl and R2 is 1,2-ethanediyl
  • Bta benzoyl-thio-acetyl: A is -CO-S-, Rl is phenyl and R2 is methylene
  • Btp benzoyl-thio-propionyl: A is -CO-S-, Rl is phenyl and R2 is 1,2-ethanediyl).
  • Monoclonal antibodies Five mouse monoclonal antibodies of which the epitope was previously determined were selected, and were all of the IgGl subclass.
  • the anti-hCG Mabs were further purified by protein A affinity chromatography as described by Van Sommeren et al. (1992, Prep. Biochem.
  • Murine monoclonal antibody HB.OT95A was produced by injecting Balb/c mice with E. coli derived recombinant HBeAg in Freund's complete adjuvans. The best responding mouse received an intravenous dose of the recombinant antigen dissolved in PBS. Fusion and selection were performed according to standard methods. Reactive clones were recloned to 100% clonality.
  • Murine monoclonal antibody HB.OT95A was deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No.95090611.
  • the synthesis of the peptides was carried out by an automated procedure on a Perkin Elmer/Applied Biosystems Inc. 433A peptide synthesizer, using standard FastMoc 0.25 mmol procedures with UV-monitoring and feedback option. Some of the peptides were synthesized in a semi-automated manner on an in-house built multiple peptide synthesizer using standard Fmoc/tBu-chemistry with in situ activation by carbodiimide. An overview of techniques, protecting groups, linkers, and solid supports in the SPPS 'Fmoc chemistry' is given by Fields & Noble (Int. J. Peptide Protein Res., 35, 161, 1990).
  • Fmoc amino acid derivatives were obtained from Bachem (Bubendorf, Switzerland). Desaminophenylalanine was purchased from Merck (M ⁇ nchen, Germany). The peptides were synthesized on a TentaGel S RAM Fmoc resin (RAPP Polymere, Tubingen, Germany) via the Fmoc/tBu chemistry.
  • the linker is of a Rink-amide type, which automatically yields a C-terminally amidated peptide.
  • the amino acid side-chains were protected with acid-labile protecting groups: the ⁇ - aminogroup of lysine with Boc, the ⁇ -guanidino group of arginine with 2,2,5,7,8- pentamethylchroman-6-sulphonyl (Pmc), the ⁇ -carboxyl group of glutamic acid and the ⁇ -carboxyl group of aspartic acid with OtBu, the ⁇ -amide group of glutamine and the ⁇ -amide group of asparagine with trityl (Trt), histidine and cysteine with Trt, the ⁇ -hydroxyl group of serine and threonine with tBu, and tyrosine with tBu.
  • acid-labile protecting groups the ⁇ - aminogroup of lysine with Boc, the ⁇ -guanidino group of arginine with 2,2,5,7,8- pentamethylchroman-6-sulphonyl (Pmc), the ⁇ -carboxyl group of gluta
  • the N-acetyl-peptide was obtained by acetylation of the N-terminus with acetic anhydride.
  • the fully protected peptides were cleaved from the resin during a 2-hr reaction at room temperature under nitrogen with 5% thioanisole (vol/vol), 3% ethanedithiol (vol/vol), 2.5% water (vol/vol), and 2% anisole (vol/vol) in trifluoroacetic acid (87.5% vol/vol) followed by precipitation in diethylether.
  • the crude peptides were washed twice with diethylether, dried at the air, dissolved in water/acetonitrile (3:1) and lyophilized. The composition of the peptides were confirmed by mass and amino acid analysis.
  • HPLC analysis and purification were carried out on a Beckman Gold HPLC system.
  • HPLC analyses were performed on a RP-C2/C18 column (Supe ⁇ ack prepS, 4x250 mm, Pharmacia) at a flow rate of 1 ml/min, using a 3 min isocratic elution with 0.1 % trifluoroacetic acid in acetonitrile followed by a 30 min linear gradient from in water (100%) to 75 % 0.1 % trifluoroacetic acid in acetonitrile.
  • Peptides were detected by UV measurement at 206 nm.
  • the synthesised peptides (summarised in table II) were of more than 75% purity (obtained by purification when necessary).
  • the molecular weight, HPLC -purity and retention times of these peptides are listed in table II.
  • Polystyrene microtitre plates (Greiner, Frickenhausen, Germany) were sealed during every incubation (to prevent evaporation) and washed (Washer Microelisa system 400, Organon Teknika, Boxtel, The Netherlands) four times after each incubation with PBS-Tween (6.7 mmol/1 phosphate buffer pH 7.2; 0.13 mol/1 NaCl; 0.05% (v/v) Tween-20). During every experiment blanks were included to check for non-specific binding of the monoclonal antibody and the sheep anti-mouse IgG conjugate. The plates were freshly coated for each experiment.
  • the synthetic peptides (parent or N-acylated), serial diluted (135 ⁇ l) in 0.05 mol/1 bicarbonate coating buffer (pH 9.6) in concentrations of 1 mg/ml to 1 ng/ml, were allowed to passively adsorb to a well of the microtiter plate during overnight incubation at room temperature with constant shaking (600 rpm, TPM-2 shaker; Sarstedt, N ⁇ mbrecht, Gemany).
  • Non-specific adso ⁇ tion of the monoclonal antibody and/or conjugate was prevented by performing a subsequent incubation with aliquots of 135 ⁇ l of bovine serum albumin (2.0 g/1; Organon Teknika, Boxtel, The Netherlands) in Tris-buffer (pH 7.4) for 2 h at room temperature, after the first washing procedure.
  • 100 ⁇ l of the appropriate monoclonal antibody solution diluted in sample diluent (20% v/v normal goat serum in 1% (v/v) triton in 7.6 mmol/1 sodium phosphate buffer (pH 7.4) with 120 mmol/1 NaCl), was added to each well for 1 h at room temperature with constant shaking.
  • the Ala-scanning method is frequently used to determine the contribution of individual amino acids to the binding interaction between peptide and antibody. Therefore every amino acid of the peptide sequentially is replaced by the amino acid alanine. When the natural peptide sequence contains an alanine itself, it is replaced by aspartic acid.
  • residues are found which can be replaced by alanine without impairing ELISA reactivity. Presumably, they do not correspond to critical residues that contribute to the energy of interaction. For example, this pattern of replaceability is important for modelling peptide-antibody interaction.
  • N-acylated peptide 3A The effects of the antigenic properties of various N-acyl groups according to the present invention were tested by comparing the coating efficiency (the term surface reactivity or binding capacity is also used in the same context) of N-acylated peptide 3A to the parent peptide 3A in the following ELISA-format.
  • the (N-acylated) peptide was coated in a serial dilution in the range of 1 mM to 1 nM and a fixed concentration of antibody was presented to the immobilised peptide.
  • Comparison of coating efficiency between parent and N-acylated equivalents is made in terms of the amount of peptide coat concentration required to achieve 50% of the maximum ELISA-signal (ECso).
  • the coating efficiencies of the parent peptide 3A, the N ⁇ acetyl-peptide 3A, and the N" Ata-peptide 3A were determined. The results are presented in Figure 1 :
  • Fig. 1A shows that the coating efficiency of peptide 3A can be substantially improved when N-acylated with an Ata-group.
  • the concentration of peptide required to produce the dose-response curve with an equal amount of antibody could be reduced from 390 ⁇ M to 18 nM which is a reduction of more than four orders of magnitude.
  • Fig. IB shows that the surface reactivity of the peptide 3 A coating cannot be improved by N-acylation with an acetyl-group.
  • peptide sequences were N-acylated with the Ata-group to further explore die general applicability of the Ata-group and analogues tiiereof. All peptides clearly differed in amino acid sequence, length, and hydrophobicity (see also table I and II).
  • the peptides are: - peptide 1A [amino acid sequence: DTPILPQ ( ⁇ -hCG)], - peptide 5-1-1 [amino acid sequence:
  • HBeAg [amino acid sequence: LEDPASRDLVVNYVNTN (HBeAg)].
  • Fig. 3A The adso ⁇ tion of the parent peptide 1A to the solid phase did not result in any ELISA-reactivity up to a coating concentration of 1 mM (Fig. 3A).
  • the binding of die specific antibody could be detected when an N-acylated group according to die invention was introduced to the peptide, indicating poor coating efficiency for the parent peptide 1A.
  • the parent peptide 5-1-1 did not exceed the background signal unambiguously in contrast to the N-acylated equivalents (Fig. 3B).
  • Fig. 3 (A t/m C) shows that the Ata-group and analogues tiiereof systematically increase the surface reactivity of all three peptides in comparison to their parent counte ⁇ arts but each to a different extent (see table III).
  • the presented results support the finding that the use of high molecular weight carriers (e.g., MAP or BSA) or covalent activation of polystyreen (PS) can be circumvented in the immobilisation procedure of small synthetic peptides.
  • Application of small N-acylgroups, such as the Ata-group or analogues thereof, to the peptide is sufficient to improve the coating efficiency at least two orders of magnitude. This substantially reduces the amount of peptide needed to develop a sensitive peptide-ELISA.
  • the use of an Ata-group or analogues thereof at the N-terminal end of the peptide has the additional advantage that the solubility of the peptides improves due to the addition of extra polarity.
  • a represents peptides from the C-terminal part of the ⁇ -chain of human chorionic gonadotropin.
  • HCV-NS4-C100 refers to the C100 part of the NS4 region of the Hepatitis-C virus genome.
  • c CMV-PP52 refers to the PP52 part of the Cytomegalovirus.
  • UL44 refers to the correct reading frame.
  • d represents a part of the non-particulate e-antigen (HBeAg) secreted by the hepatitis B-virus.
  • Retention time is determined by reverse phase column analysis
  • A ELISA was performed using dilution series of parent peptide 3A (- ⁇ - ) and N ⁇ Ata-peptide 3 A ( - - ). Binding was determined using an anti-hCG Mab OT-3A concentration of 1 ⁇ g/ml.
  • B ELISA was performed using dilution series of parent peptide 3A (-f-) and N" Acetyl-peptide 3A Hfc-). Binding was determined using an anti-hCG Mab OT-3A concentration of 10 ⁇ g/ml.
  • Fig. 2 Effect of coating time on coating efficiency of parent peptide 3 A and its N" Ata-peptide 3 A.
  • the coating efficiency decreased when the coating time was decreased from 88 h to 10 min via intermediate coating times for both parent peptide 3A from 19.5 h (-ft-), 4 h (-#-), 1 h (H-) to 10 min (-f-), and for N ⁇ Ata-peptide 3A from 19.5 h (-fr), 5 h (-A-), 1 h (-$ ⁇ -) to 10 min (-#-). Binding was assessed with an anti-hCG Mab OT-3A concentration of 1 ⁇ g/ml.
  • Fig. 4 Ala-scanning of the N-acylated HBeAg-peptide (sequence of HBeAg peptide in Table I). Coating efficiency was tested with parent peptides and N ⁇ Ata- peptides.
  • the amino acid (one-letter code) directly at the bottem of the bar represents the amino acid in the HBeAg sequence which is substituted by alanine.

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Abstract

On décrit un nouveau procédé générique de revêtement destiné à améliorer l'efficacité de revêtement de petits antigènes peptidiques synthétiques dans la technique ELISA. Dans cette invention on a comparé les capacités de fixation de plusieurs peptides liés à diverses fractions, avec celles de leurs homologues parentaux dans ELISA. On a trouvé que l'allongement d'un épitope/séquence par un groupe acyle de la formule R1-A-R2-CO-, dans laquelle R1 représente alkyle inférieur, aryle, ou aralkyle, R2 représente un radical divalent dérivé à partir d'alkyle inférieur, aryle ou aralkyle, et A représente un soufre contenant un groupe fonctionnel choisi dans le groupe constitué par -CO-S-, -S-, -SO-, -SO2- et -S-S-, augmentait de manière spécifique la réactivité de fixation avec l'anticorps monoclonal, lorsque cet épitope/séquence était adsorbé sur un support solide, de préférence des puits de polystyrène. Cet allongement s'effectue préférablement de manière N-terminale. Plusieurs fractions ont effectué une énorme réduction de concentration du revêtement peptidique pour tous les peptides testés, réduction comprise entre deux et quatre ordres de grandeur. Le remplacement d'une extension Ata (où R1 représente méthyle, R2 représente méthylène et A représente -CO-S-) par des analogues (à savoir Bta, Atp, Btb) a été possible sans réduction trop importante des propriétés activatrices. En outre, une synthèse directe du groupe Ata sur l'extrémité N-terminale d'un peptide semble être une stratégie simple et générale pour une adsorption efficace de petits peptides sur du polystyrène.
PCT/EP1997/005572 1996-10-09 1997-10-07 Composition comprenant un peptide acyle et immobilise WO1998015572A1 (fr)

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AU49462/97A AU4946297A (en) 1996-10-09 1997-10-07 A composition comprising an immobilised, acylated peptide

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006113958A3 (fr) * 2005-04-26 2007-05-03 Sandoz Ag Ligands d'affinités
AU2011253661B2 (en) * 2005-04-26 2013-06-13 Boehringer Ingelheim Rcv Gmbh & Co Kg Production of recombinant proteins by autoproteolytic cleavage of a fusion protein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0339264A1 (fr) * 1988-03-24 1989-11-02 Roche Diagnostics GmbH Composés pour l'introduction d'un groupe mercapto dans la tyrosine
EP0479376A1 (fr) * 1990-10-05 1992-04-08 Akzo Nobel N.V. Peptides agissants immunochimiquemeur avec des anticorps contre le virus d'hépatite non-A, non-B
WO1996018645A1 (fr) * 1994-12-16 1996-06-20 Merck Patent Gmbh Peptides et membranes cellulaires synthetiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0339264A1 (fr) * 1988-03-24 1989-11-02 Roche Diagnostics GmbH Composés pour l'introduction d'un groupe mercapto dans la tyrosine
EP0479376A1 (fr) * 1990-10-05 1992-04-08 Akzo Nobel N.V. Peptides agissants immunochimiquemeur avec des anticorps contre le virus d'hépatite non-A, non-B
WO1996018645A1 (fr) * 1994-12-16 1996-06-20 Merck Patent Gmbh Peptides et membranes cellulaires synthetiques

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006113958A3 (fr) * 2005-04-26 2007-05-03 Sandoz Ag Ligands d'affinités
JP2008539170A (ja) * 2005-04-26 2008-11-13 サンド・アクチエンゲゼルシヤフト 親和性リガンド
EP2348053A3 (fr) * 2005-04-26 2011-11-02 Sandoz AG Ligands oligopeptidiques
US8058410B2 (en) 2005-04-26 2011-11-15 Sandoz Ag Affinity ligands
AU2006239722B2 (en) * 2005-04-26 2012-02-02 Boehringer Ingelheim Rcv Gmbh & Co Kg Affinity ligands
US8163890B2 (en) 2005-04-26 2012-04-24 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein
US8372959B2 (en) 2005-04-26 2013-02-12 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein
AU2011253661B2 (en) * 2005-04-26 2013-06-13 Boehringer Ingelheim Rcv Gmbh & Co Kg Production of recombinant proteins by autoproteolytic cleavage of a fusion protein

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