Claims
- 1 A method for isolating a purification target comprising a protein which specifically binds sialic acid, said method comprising the step of:
allowing the purification target to contact a molecule comprising sialic acid which has been linked to a solid support, whereby the purification target having a binding site for sialic acid is selectively bound by the molecule, wherein the purification target comprises a capsid protein selected from the group consisting of an AAV serotype 4 capsid protein, an AAV serotype 5 capsid protein, and a capsid comprising a fragment of an AAV4 or AAV5 capsid which contains the binding site for sialic acid.
- 2 The method according to claim 1, wherein the molecule comprising sialic acid is mucin.
- 3. canceled.
- 4 The method according to claim 3, wherein the solid support is sepharose activated with a compound selected from the group consisting of CnBr, carbonyldiimidazole, glutaraldehyde, hydroxysuccinimide, and tosyl chloride.
- 5-8. canceled.
- 9 The method according to claim 8, further comprising the step of concentrating the separated purification target.
- 10 A method for isolating a viral vector having a protein with a binding site for sialic acid from a mixture, said method comprising the steps of:
(a) allowing a mixture which contains the viral vector to contact mucin which has been linked to a solid support, whereby the virus is selectively bound by the mucin-linked support, and (b) washing the solid support to remove material from the mixture which is non-specifically bound to the mucin-linked support.
- 11 The method according to claim 10 further comprising, following the washing step (b), the step of separating the viral vector from the solid support.
- 12 The method according to claim 11, wherein the separating is performed by a further washing step using a concentrated salt solution.
- 13 The method according to claim 12, wherein the concentrated salt solution is phosphate buffered saline and 0.4 M sodium chloride.
- 14 The method according to claim 11, further comprising the step of concentrating the separated viral vector.
- 15. canceled.
- 16 A kit useful for detecting a purification target in a mixture, said kit comprising a sialic acid linked to a solid support and a reagent which permits visual detection of binding of the purification target to the molecule, wherein the purification target comprises a protein selected from the group consisting of an AAV serotype 4 capsid protein, an AAV serotype 5 capsid protein, or a fragment of an AAV4 or AAV5 capsid protein having a binding site for sialic acid.
- 17 The kit according to claim 16, wherein the solid support is activated agarose with mucin linked thereto.
- 18 The kit according to claim 16, wherein the solid support is loaded in an affinity column.
- 19 The kit according to claim 18, wherein the affinity column is a liquid chromatography column.
- 20-21. canceled.
- 22 A composition useful in binding a purification target from a mixture comprising at least one 2-3,O-sialic acid moiety and at least one 2,2-N-sialic acid moiety linked to a solid support.
- 23 A method of binding an adeno-associated viral (AAV) vector having an AAV serotype 4 protein or an AAV serotype 5 capsid protein from a culture, said method comprising the step of incubating the composition of claim 22 on lysate or media from the culture.
- 24. The method according to claim 1, wherein the solid support is selected from the group consisting of sepharose, agarose, and a polyacrylamide resin.
- 25 The method according to claim 1, wherein the purification target and the molecule-linked solid support are contacted by incubating a solution comprising the AAV and cellular materials and the support.
- 26 The method according to claim 1, wherein the solid support is packed in a liquid chromatography column.
- 27 The method according to claim 1, further comprising the step of washing the solid support to remove material from the mixture which is non-specifically bound to the molecule-linked support.
- 28 The method according to claim 1, further comprising the step of separating the purification target from the solid support.
- 29 The method according to claim 10, wherein the capsid protein is selected from the group consisting of an AAV serotype 4 capsid protein, an AAV serotype 5 capsid protein, and a capsid comprising a fragment of an AAV4 or AAV5 capsid which contains the binding site for sialic acid.
- 30 The kit according to claim 16, wherein the kit further comprises wash, elution, and concentration reagents.
- 31 A method of detecting in a sample the presence of a virus with a surface protein selected from an adeno-associated virus (AAV) serotype 4 capsid protein or AAV serotype 5 capsid protein, or fragment thereof, said method comprising the step of utilizing the kit of claim 16 on a sample to visually detect binding of the virus.
Government Interests
[0001] This work was supported, in part, by funding by the National Institutes of Health, P30 DK47757. The US Government may have certain rights in this invention.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US02/25096 |
8/6/2002 |
WO |
|
Provisional Applications (1)
|
Number |
Date |
Country |
|
60310772 |
Aug 2001 |
US |