NOVEL SYNTHETIC BLOCKING REAGENTS

• US 20100317048A1
• Filed: 06/10/2010
• Published: 12/16/2010
• Est. Priority Date: 06/10/2009
• Status: Active Grant

• Current Assignee: Borstel Forschungszentrum
• Original Assignee: Borstel Forschungszentrum
• Inventors: Frey, Andreas, Ramaker, Katrin, Röckendorf, Niels, Fujimoto, Naho, Bade, Steffen
• Granted Patent: US 8,501,199 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention concerns novel synthetic blocking reagents for the reduction of non-specific bindings in solid phase assays that rely on biological and specific recognition, e.g., in enzyme-linked immunosorbent assays (ELISAs). The invention provides the use of compounds as blocking reagents as well as kits comprising these compounds. The compounds comprise one or more poly(ethylene glycol) moieties, one or more alkyl- or aminoalkyl groups and another unit bridging the aforementioned groups, wherein the compound comprises at least one amino group.

Claims

1. A test system for the qualitative and/or quantitative determination of an analyte in a sample comprising a compound as a blocking reagent, wherein the test system is based on specific binding between the analyte and a ligand thereof, wherein the analyte and/or the ligand is bound to a solid support, wherein the compound is a compound of Formula I:

R¹-PEG-Bridge-Alkyl-R²

wherein R¹ is a residue selected from the group consisting of —

H, —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃ and —

CH(CH₃)₂,

PEG is poly(ethylene glycol) having an average molecular weight of at least 300 Da,

Bridge is a non-cyclical group comprising an amino group N¹ and/or an amide and/or an —

O—

ether,

Alkyl is an alkylene moiety having an average alkyl chain length of at least 10 carbon atoms, and

R² is a residue selected from the group consisting of —

H or an amino group N²,

wherein the compound comprises at least one amino group, so that, if Bridge comprises no amino group, R² is the amino group N²;

or a salt of a compound of Formula I.

2. The test system of claim 1, wherein Bridge is a group comprising an amino group N¹.

3. The test system of claim 1, wherein Bridge is selected from the group consisting of —

NH—

, —

N(PEG-R³)—

, —

N(Alkyl-R⁴)—

, —

NHCH₂CH(Alkyl-R⁴)—

, —

NHCH₂CH[CH₂NH(PEG-R³)]—

, —

NHCH₂C[CH₂NH(PEG-R³)](Alkyl-R⁴)—

, —

O—

, —

OCH₂CH(Alkyl-R⁴)—

, —

OCH₂CH[CH₂O(PEG-R³)]—

, —

OCH₂C[CH₂O(PEG-R³)](Alkyl-R⁴)—

,

and wherein R³ is a residue selected from the group consisting of —

H, —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃ and —

CH(CH₃)₂, and R⁴ is a residue selected from the group consisting of —

H and the amino group N², wherein preferably R³ is the same as R¹.

4. The test system of claim 1, wherein Bridge is selected from the group consisting of:

5. The test system of claim 1, wherein PEG is a linear or branched poly(ethylene glycol) having an average molecular weight of 300-3,500 Da, preferably, 400-2,500 Da, and/or

wherein Alkyl is a linear or branched, saturated or unsaturated alkylene moiety preferably having an average alkyl chain length of 10-26 carbon atoms, preferably of 12-20 carbon atoms.

6. The test system of claim 1, wherein the amino group N² is selected from the group consisting of —

NH₂, and a mono-, di- or trisubstituted secondary, tertiary or quaternary amino or ammmonium group, wherein the substituent(s) may be identical or non-identical and selected from the group consisting of —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃, —

CH(CH₃)₂, —

CH₂CH₂CH₂CH₃, —

CH₂CH(CH₃)(CH₂CH₃) and —

C(CH₃)₃.

7. The test system of claim 1, wherein the compound is selected from the group comprising

8. The test system of claim 1, wherein the critical micelle concentration (n/v) of the compound is 0.02-1 mmol/L.

9. The test system of any of claim 1, wherein the solid support and/or its surface is made from a material selected from the group comprising polystyrene, polypropylene, PVDF, nylon, nitrocellulose, glass, ceramics, metals or semi-metals and combinations or composite materials thereof.

10. The test system of claim 1, wherein the test system is selected from the group comprising FLISA, FELISA, ELISA, ELISPOT, FIA, EIA, RIA, Western Blot, Southern Blot and Northern Blot.

11. The test system of claim 1, wherein the ligand and/or the analyte are cells, oligo- or polynucleotides, nucleic acids, small molecules, lipids, oligo- or polysaccharides, oligo- or polypeptides, proteins, or derivatives or combinations of any of the aforementioned substances, wherein peptides and/or proteins may be glycosylated.

12. A blocking reagent comprising a compound of Formula I as defined in claim 1, wherein the compound is a compound of Formula II or a compound of Formula III:

13. The blocking reagent of claim 12, wherein

Alkyl is a linear or branched, saturated or unsaturated alkylene group with an average chain length of 10-26 carbon atoms, preferably of 12-20 carbon atoms, and/or

PEG is a linear or branched poly(ethylene glycol) having an average molecular weight of 300-3,500 Da, preferably, 400-2,500 Da, and/or

R¹ is selected from the group comprising —

H, —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃ and —

CH(CH₃)₂ and/or

R² is —

H.

14. A blocking reagent according to claim 12, wherein the compound is selected from a group comprising:

a compound of Formula II, wherein R¹ is —

CH₃, R² is —

H, PEG is linear poly(ethylene glycol) with an average molecular weight of 400-700 Da and Alkyl is —

(CH₂)₁₂—

or —

(CH₂)₂₀—

,

a compound of Formula III, wherein R¹ is —

CH₃, R² is —

H, PEG is linear poly(ethylene glycol) with an average molecular weight of 400-700 Da and Alkyl is —

(CH₂)₁₂—

,

and a compound of Formula III, wherein R¹ is —

CH₃, R² is —

H, PEG is linear poly(ethylene glycol) with an average molecular weight of 1,500-2,500 Da and Alkyl is —

(CH₂)₂₀—

.

15. A kit comprising (a) a blocking reagent, which is a compound of Formula I as defined in claim 1 or a salt thereof, and (b) a reagent selected from the group consisting of cells, oligo- or polynucleotides, nucleic acids, small molecules, lipids, oligo- or polysaccharides, oligo- or polypeptides, proteins, or derivatives or combinations of any of the aforementioned substances, wherein peptides and/or proteins may be glycosylated.