Method for introduction of reporter groups into bacterial lipopolysaccharide-derived carbohydrates and the subsequent coupling of such derivatives onto solid surfaces

• US 20020128381A1
• Filed: 04/25/2002
• Published: 09/12/2002
• Est. Priority Date: 12/15/1998
• Status: Active Grant

• Current Assignee: Exiqon A/S (Qiagen NV)
• Original Assignee: Exiqon A/S (Qiagen NV)
• Inventors: Boas, Ulrik, Heegaard, Peter M. H., Jakobsen, Mogens Havsteen, Jauho, Eva Irene Stenbaek
• Granted Patent: US 6,881,549 B2
• Time in Patent Office: Days
• US Class Current: 525/54.2
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: G01N 2333/255 Salmonella (G)
• CPC Class Codes: G01N 2400/50 Lipopolysaccharides; LPS
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56916 Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

Abstract

The present invention provides a method for immobilising a polysaccharide (PS) to a solid surface, said polysaccharide having a keto-carboxy group (—C(═O)—COOH) or a ketal or hemiketal group corresponding thereto, e.g. derived from KDO (2-keto-3-deoxy-D-manno-octonic acid)), the method comprising the steps of: (a) forming a covalent bond between the carboxy group of the polysaccharide and a reporter molecule (RM), thereby forming a polysaccharide-reporter molecule conjugate (PS-RM), said reporter molecule comprising a recognition/substrate site (e.g. biotin or an anthraquinone); and (b) immobilising the polysaccharide-report molecule conjugate by forming a specific bond (e.g. by photocoupling or formation of an affinity pair) between the recognition/substrate site of said reporter molecule and a reception/reagent site of the solid surface. The present invention also provides a solid surface thus obtainable and the use of such solid surfaces for diagnostic purposes, e.g. for the detection of bacterial infections from Gram-negative bacteria that are human or veterinary pathogens, e.g. enterobacteria, respiratory bacteria, urogenitial bacteria, and neuropathogenic bacteria such as Salmonella sp., Actinobacillus sp.

Claims

1. A method for immobilising a polysaccharide (PS) to a solid surface, said polysaccharide having a keto-carboxy group (—

C(═

O)—

COOH) or a ketal or hemiketal group corresponding thereto), the method comprising the steps of;

a) forming a covalent bond between the carboxy group of the polysaccharide and a reporter molecule (RM), thereby forming a polysaccharide-reporter molecule conjugate (PS-RM), said reporter molecule comprising a recognition/substrate site; and

b) immobilising the polysaccharide-reporter molecule conjugate (PS-RM) by forming a specific bond between the recognition/substrate site of said reporter molecule and a reception/reagent site of the solid surface.

3. A method according to claim 1 or 2, wherein the keto-carboxy group of the polysaccharide is a part of a KDO (2-keto-3-deoxy-D-manno-octonic acid) monosaccharide unit of the polysaccharide.

4. A method according to any of the preceding claims, wherein the polysaccharide (PS) is substantially identical to the carbohydrate part of a Gram-negative bacterial lipopolysaccharide (LPS).

5. A method according to claim 4, wherein the polysaccharide is obtained by selective hydrolysis of the ketal bond between the inner core part and the lipid A part of a Gram-negative bacterial lipopolysaccharide.

6. A method according to claim 1 or 3, wherein the polysaccharide further comprises the lipid part of a Gram-negative bacterial lipopolysaccharide, thereby constituting a bacterial lipopolysaccharide.

7. A method according to any of the claims 4-6, wherein the bacterial lipopolysaccharide is derived from a bacteria selected from Gram-negative bacteria that are human or veterinary pathogens, e.g. enterobacteria, respiratory bacteria, urogenitial bacteria, and neuropathogenic bacteria.

8. A method according to claim 7, wherein the bacteria is a veterinary pathogen or zoonotic bacteria including enterobacteria selected from Escherichia coli, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella enterica, and all serotypes hereof, especially Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Choleraesuis, Salmonella Manhattan, Salmonella Dublin, Salmonella Infantis, Escherichia coli spp. including O157, oedema-disease causing Escherichia coli, Yersinia enterocolitica, and Campylobacter ejuni.

9. A method according to claim 7, wherein the bacteria is a respiratory bacteria selected from the HAP group of bacteria especially Actinobacillus sp, in particular the HAP bacteria Actinobacillus pleuropneumoniae, Haemophilus somnus, Pasteurella haemolytica, Pasteurella multocida, Haemophilus parasuis and Mannheimia sp.

10. A method according to claim 1 or 2, wherein the polysaccharide has a molecular weight of at least 1000.

11. A method according to any of the claims 1 and 3-10, wherein formation of the covalent bond between the polysaccharide and the reporter molecule is mediated by using a coupling reagent.

12. A method according to any of the claims 2-10, wherein formation the covalent bond between the polysaccharide and the chemical functionality of the solid surface is mediated by using a coupling agent.

13. A method according to any of the claims 1 and 3-11, wherein the reporter molecule comprises a reporter part for presenting the recognition/substrate site and a linker part for linking the reporter part to the polysaccharide.

14. A method according to claim 13, wherein the polysaccharide-reporter molecule conjugate (PS-RM) has the general formula PS′

—

C(═

O)—

N(R^N)—

F—

L—

R, where PS′

—

C(═

O) is the polysaccharide, N(R^N)—

F is the group directly involved in the covalent link between the polysaccharide and the reporter molecule, RN designates hydrogen or C_1-4-alkyl, L is the linker part of the reporter molecule, and R is the reporter part of the reporter molecule.

15. A method according to claim 14, wherein —

N—

F—

designates amino (—

N—

), anilino (—

N—

Ph), hydrazido (—

N—

C(═

O)—

), semicarbazido (—

N—

C(=O)—

NH—

), thiosemicarbazido (—

N—

C(=S)—

NH—

), or hydrazino (—

N—

NH—

), preferably amino.

16. A method according to claim 14, wherein L designates a biradical selected from C_1-20-alkylene optionally comprising aromatic or mono-/polyunsaturated hydrocarbons or cyclic hydrocarbons, oligo-oxyethylenes, oligo-amides such as oligo-glycine, oligo-alanine, oligo-lysine and oligopeptides in general, oligo-phospodiesters, oligo-phosphoamidates, oligo-phosphodiamides, oligo-sulfonesters, and oligo-sulfonamides.

17. A method according to any of the claims 1 and 3-16, wherein the reporter molecule has a molecular weight of at the most 10,000, preferably at the most 5,000, in particular at the most 2,500.

18. A method according to claim 14, wherein the reporter part is a photochemically reactive group.

19. A method according to claim 14, wherein the reporter part is a thermochemically reactive group.

20. A method according to claim 14, wherein the reporter part is a one part of an affinity pair.

21. A method according to any of the preceding claims, wherein the solid surface is a surface of an organic polymer, a glass, silicium, silicium oxide (silica) or a composite material thereof.

22. A method according to any of the preceding claims, wherein two or more types of polysaccharides are immobilised, where such types represent different bacterial serotypes.

23. A method according to any of the claims 4-22, wherein the LPS from which the PS is derived is LPS from a Salmonella bacteria and the reporter molecule comprises a reporter part selected from anthraquinones and biotin, preferably anthraquinone.

24. A method according to any of the claims 4-22, wherein the LPS from which the PS is derived is LPS from an Actinobacillus bacteria and the reporter molecule comprises a reporter part selected from anthraquinones and biotin, preferably anthraquinone.

25. A solid surface obtainable according to the method defined in any of the claims 1-24.

26. The use of a solid surface as defined in claim 25 in a diagnostic assay.

27. The use according to-claim 26, wherein the diagnostic assay is a solid phase immunoassay.

28. The use according to claim 26, wherein the diagnostic assay is a serological assay, e.g. a serological assay for detection of antibodies against microorganisms.

29. The use according to claim 26, wherein the diagnostic assay is a serological assay for detection of antibodies against Salmonella spp.

30. The use according to claim 26, wherein the diagnostic assay is a serological assay for detection of antibodies against Actinobacillus spp.

32. An assay device according to claim 31, wherein the polysaccharide is immobilised to the solid surface according to the method defined in any of the claims 1-24.

38. A compound according to claim 34, wherein L designates a biradical as defined in claim 16.

2. A method for immobilising a polysaccharide (PS) to a solid surface, said polysaccharide having a keto-carboxy group (—

C(═

O)—

COOH) or a ketal or hemiketal group corresponding thereto), by forming a covalent bond between the carboxy group of the polysaccharide and a chemical functionality linked to the solid surface.

31. An assay device for the detection of antibodies against one or more Gram-negative bacteria, comprising a solid surface having immobilised thereto a polysaccharide (PS), said polysaccharide (PS) corresponding to the carbohydrate part of the bacterial lipopolysaccharide (LPS) of the Gram-negative bacteria, via the carboxylic acid group of a KDO monosaccharide unit of said polysaccharide.

33. A method for estimating the number of bacteria or serotyping of bacterial antigen in a sample comprising whole bacteria, parts or lysates thereof, wherein an assay device according to claim 31 or 32 is used.

35. A compound according to claim 34, wherein R¹ is hydrogen.

36. A compound according to claim 34, wherein R¹ is L-R.

37. A compound according to claim 34, wherein —

N—

F—

designates amino (—

N—

), anilino (—

N—

Ph), hydrazido (—

N—

C(=O)—

), semicarbazido (—

N—

C(═

O)—

NH—

), thiosemicarbazido (—

N—

C(═

S)—

NH—

), or hydrazino (—

N—

NH—

), preferably amino.

39. A compound according to claim 36, wherein the reporter molecule (R) has a molecular weight of at the most 10,000, preferably at the most 5,000 and most preferred at the most 2,500.

40. A compound according to claim 34, wherein R⁴ is selected from KDO-2-yl, 4-phosphoethanolamine-KDO-2-yl, L-rhamnosyl-(1->

4)-KDO-2-yl, and KDO-(2->

4)-KDO-2-yl.

41. A compound according to claim 34, wherein the polysaccharide residue preferably comprises from 2-997 linked monosaccharide units, such as at least 7, e.g. 7-997, linked monosaccharide units, in particular at least 22, e.g. 22-497, linked monosaccharide units, such polysaccharide residue optionally being non-stoichiometrically substituted.

42. The use of a compound according to claim 34 for the preparation of an assay device for the detection of antibodies against Gram-negative bacteria.

43. The use of a compound according to claim 34 for the preparation of a solid surface carrying an immobilised polysaccharide.

44. A method for the preparation of a compound of the general formula I as defined in claim 34, comprising reacting a compound of the general formula III

45. A method according to claim 44, wherein the compound of the general formula III is used in its activated ester form.

46. A method according to claim 44, wherein the reaction between the carboxylic acid compound of the general formula III and the nitrogen compound IV is facilitated by using a coupling reagent.

34. A compound of the general formula I