WO1996037778A1 - Complexe contenant un mediateur polymere et un peptide, pour fixation par adsorption sur des surfaces de substrat - Google Patents

Complexe contenant un mediateur polymere et un peptide, pour fixation par adsorption sur des surfaces de substrat Download PDF

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Publication number
WO1996037778A1
WO1996037778A1 PCT/DE1996/000936 DE9600936W WO9637778A1 WO 1996037778 A1 WO1996037778 A1 WO 1996037778A1 DE 9600936 W DE9600936 W DE 9600936W WO 9637778 A1 WO9637778 A1 WO 9637778A1
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WO
WIPO (PCT)
Prior art keywords
peptide
complex according
complex
peptides
terminal
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Application number
PCT/DE1996/000936
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German (de)
English (en)
Inventor
Wieland Keilholz
Hans Georg Rammensee
Stefan Stevanovic
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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.)
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Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Publication of WO1996037778A1 publication Critical patent/WO1996037778A1/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
    • G01N33/53—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 complex containing a polymeric mediator and a peptide for adsorptive binding to support surfaces.
  • the peptides In the ELISA, the peptides must bind to a polystyrene support surface and not all peptides have this ability to the same extent, so that small peptides in particular do not have the necessary sites to bind tightly enough for detection.
  • the current state of research in the field of C-terminal sequencing of proteins is such that proteins in the range from 500 pmol to 1 nmol can be sequenced by 3 cycles as a standard C-terminal.
  • the only commercially available device for C-terminal sequencing from Hewlett-Packard is designed for samples with a molecular weight above 5kD, ie peptides with a length of approx. 50 amino acids can only be sequenced.
  • the sample is applied to a Teflon membrane, to which it is bound by adsorption and on which the degradation cycles take place in the device. Small peptides are washed from the membrane in the course of the degradation cycles and therefore cannot be sequenced.
  • the present invention is therefore based on the object of providing a means with which peptides, in particular small peptides, can be bound to support surfaces in a simple and rapid manner. Furthermore, the detection limit for such peptides should be improved.
  • peptides adhere better to support surfaces, for example made of polyethylene, Teflon or glass, when the peptides are bound to a polymeric mediator. If a C-terminal peptide sequencing is to take place subsequently, the peptide is bound N-terminally to the polymeric mediator.
  • a polymer with many N-termini is preferably used as mediator, to which the peptide is coupled at the C-terminal and then has a free N-terminus.
  • mediators are polycarboxylic acids and their derivatives, preferably polyacrylic acid, and their lower esters and amides.
  • the polyacrylic polymethyl ester or polyacrylic acid polyethyl ester formed by reacting polyacrylic acid with methanol or ethanol is particularly suitable. It is possible to allow the esterification to take place on the support surface after the polycarboxylic acid has bonded to it, or to form the ester beforehand and only then to form a complex according to the invention, which will later be bound to the support surface.
  • the esterification of the polycarboxylic acid has the advantage that an inventive appropriate complex adheres better to the support surface by increasing the hydrophobicity.
  • the amidation ie the reaction of the polycarboxylic acid with amines, leads to compounds which can be used as mediators.
  • Other compounds which are suitable as polymeric mediators are polyglutamate, polyaspartate or polylysine, polyvinyl sulfate and polyvinyl alcohols.
  • the polymeric mediator in particular the polycarboxylic acid, is preferably bound to the peptide via a coupling agent, e.g. N-ethyl-N '- (3'-dimethyl-laminopropyl) carbodiimide hydrochloride (EDC), dicycloheylylcarbodiimide (DCC) or hydroxybenzotriazole / 2- (1 -H-benzotriazol-1 -yl) - 1, 1, 3, 3-tetramethyluronium hexafluorophosphate (HBTU / HOBT).
  • a coupling agent e.g. N-ethyl-N '- (3'-dimethyl-laminopropyl) carbodiimide hydrochloride (EDC), dicycloheylylcarbodiimide (DCC) or hydroxybenzotriazole / 2- (1 -H-benzotriazol-1 -yl) - 1, 1, 3, 3-tetramethyluronium hex
  • a customary peptide detection method e.g. an ELISA.
  • C-terminal sequencing of the peptide can also be carried out.
  • sequencing devices available on the market, e.g. the C-terminal sequencer G 1009 from Hewlett-Packard can be used.
  • Teflon membranes are used, to which a complex according to the invention can be bound by adsorption.
  • each peptide can be sequenced.
  • the amounts of the peptide are preferably above 50 pmol in order to be able to sequence enough cycles.
  • Native, synthetic or biologically processed peptides can be used as peptides.
  • a complex according to the invention can be produced in the simplest way. It can be used to bind the smallest peptides to support surfaces, which means that the peptides are used in detection methods, eg ELISA, or sequencing methods 4 driving can be subjected.
  • a complex according to the invention is particularly suitable for routine use. Its use in the C-terminal sequence analysis of peptides makes it possible for the first time to sequence peptides in a quantity range that is not far from that of the N-terminal Edman degradation.
  • FIG. 1 shows a chromatogram of the synthetic peptide HDTWHLM
  • FIG. 2 shows a C-terminal "repetitive yield" of the peptide HDTWHLM
  • FIG. 3 shows a C-terminal histogram of the peptide HDTWHLM
  • FIG. 4 shows a C-terminal "carryover "of the HDTWHLM peptide
  • Figure 5 shows a chromatogram of a peptide pool
  • Figure 6 shows a C-terminal histogram of the peptide pool.
  • PAS polyacrylic acid
  • HDTWHLM peptide was synthesized, purified by HPLC, and its mass was checked. An aqueous solution with a concentration of 3 nmol / ⁇ l was prepared and the concentration was confirmed by N-terminal sequence analysis.
  • This peptide was adsorptively bound according to Example 1 on a support surface and subjected to a C-terminal sequence analysis.
  • the sequencing was carried out with 3 nmol of the peptide over 4 degradation cycles. The result of the sequencing is shown in FIGS. 1-4.
  • the "initial yield” is 1 35 pmol
  • the “repetitive yield” is 40%
  • the “carryover” for methionine is 1 8%
  • leucine 51% histidine 1 1 5% and tryptophan 1 10%.
  • a peptide in an amount of 150 pmol was successfully sequenced over 2 cycles.
  • (b) A peptide pool from a cell supernatant was used and a complex with polyacrylic acid was formed in accordance with Example 1. The C-terminal sequencing was carried out over 9 cycles and all amino acids could be detected. In cycle 1, a dominant signal can be seen for arginine, which pulls into cycle 2 with a "Carryover" of 100%, but then disappears.
  • the hydrophobic amino acids A, Y, M, V, F, I and L have a high carryover of up to 100%. The result of the sequencing is given in FIGS. 5 and 6.
  • This peptide was adsorptively bound to a support surface in accordance with Example 2 (b) and subjected to a C-terminal sequence analysis. Sequencing was carried out with 1,50 pmol of the peptide over 3 cycles of degradation. The initial yield for methionine is 4.3 pmol. The signal rises slightly in cycle 2, but is no longer visible in cycle 3. Cycle 2 clearly shows leucine, which increases by 3 pmol from 4.4 to 7.4 and then drops again in cycle 3 to 3.6 pmol. Histidine is only recognized in cycle 3 and, at 3.7 pmol, is a comparatively high signal for this late cycle. The "repetitive yield" is surprisingly 93%.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un complexe contenant un médiateur polymère et un peptide, pour fixation par adsorption sur des surfaces de substrat. L'invention concerne en outre l'utilisation de ce complexe.
PCT/DE1996/000936 1995-05-22 1996-05-22 Complexe contenant un mediateur polymere et un peptide, pour fixation par adsorption sur des surfaces de substrat WO1996037778A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1995118771 DE19518771C2 (de) 1995-05-22 1995-05-22 Mittel enthaltend einen Komplex aus einem polymeren Mediator und einem Peptid, sowie eine Trägeroberflächen zur adsorptiven Bindung des Komplexes
DE19518771.7 1995-05-22

Publications (1)

Publication Number Publication Date
WO1996037778A1 true WO1996037778A1 (fr) 1996-11-28

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1996/000936 WO1996037778A1 (fr) 1995-05-22 1996-05-22 Complexe contenant un mediateur polymere et un peptide, pour fixation par adsorption sur des surfaces de substrat

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DE (1) DE19518771C2 (fr)
WO (1) WO1996037778A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2168789A5 (fr) * 1971-02-01 1973-08-31 Hoffmann La Roche
GB2005275A (en) * 1977-09-19 1979-04-19 Hoffmann La Roche Immunological diagnostic reagent
EP0038960A2 (fr) * 1980-04-04 1981-11-04 Toray Industries, Inc. Agent de diagnostic pour tests immunologiques et procédé pour sa préparation
EP0122969A2 (fr) * 1983-04-25 1984-10-31 GRUPPO LEPETIT S.p.A. Oligopeptides immobilisés
EP0410323A2 (fr) * 1989-07-26 1991-01-30 Perseptive Biosystems, Inc. Immobilisation de protéines et peptides sur un support insoluble

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4728639A (en) * 1982-07-27 1988-03-01 University Of Tennessee Research Corporation Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant
JPH05260991A (ja) * 1991-10-31 1993-10-12 Nalco Chem Co ポリアクリレートポリマーに対するモノクローナル抗体

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2168789A5 (fr) * 1971-02-01 1973-08-31 Hoffmann La Roche
GB2005275A (en) * 1977-09-19 1979-04-19 Hoffmann La Roche Immunological diagnostic reagent
EP0038960A2 (fr) * 1980-04-04 1981-11-04 Toray Industries, Inc. Agent de diagnostic pour tests immunologiques et procédé pour sa préparation
EP0122969A2 (fr) * 1983-04-25 1984-10-31 GRUPPO LEPETIT S.p.A. Oligopeptides immobilisés
EP0410323A2 (fr) * 1989-07-26 1991-01-30 Perseptive Biosystems, Inc. Immobilisation de protéines et peptides sur un support insoluble

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Handbook of fine chemicals", ALDRICH CATALOGUE, 1983 - 1984, pages 466, XP002014262 *

Also Published As

Publication number Publication date
DE19518771C2 (de) 1997-12-04
DE19518771A1 (de) 1997-06-12

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