1. An expression library comprising a repertoire of nucleic acid sequences, which sequences are not cloned from an immunised source, each nucleic acid sequence encoding at least part of a variable domain of a heavy chain derived from an immunoglobulin naturally devoid of light chains (VHH) wherein the extent of sequence variability in said library is enhanced compared to the corresponding naive expression library repertoire by the introduction of mutations in one or more of the complementarity determining regions (CDRs) of said nucleic acid sequences or by generating alternative combinations of CDR and framework sequences not naturally present in the naive library repertoire.
2. A library according to claim 1, wherein the nucleic acid sequences are modified by random or partially random mutation of one or more of the CDR sequences.
3. A library according to claim 1 or claim 2, wherein at least part of one or more of the CDR sequences is replaced by synthetic nucleic acid sequences.
4. A library according to any one of claims 1 to 3, wherein at least part of each of the CDR sequences is replaced by synthetic nucleic acid sequences.
5. A library according to claim 1, wherein VHH sequences comprising alternative CDR and framework sequence combinations are generated by random recombination of fragments of VHH sequences derived from a naive library or a library according to any one of claims 2 or 4.
6. A library according to any one of claims 1 to 5, wherein conserved residues within the CDR sequences are retained in the modified nucleic acid sequences.
7. A library according to any one of claims 1 to 6 wherein sequence variability present in the naive library repertoire in those parts of the VHH domain other than the CDRs is conserved.
8. An expression library according to any one of claims 1 to 7, wherein the variable domain of a heavy chain derived from an immunoglobulin naturally devoid of light chains is derived from a camelid immunoglobulin.
9. Use of a naive VHH expression library to prepare an expression library according to any one of claims 1 to 8.
10. A method for preparing a library according to any one of claims 1 to 8 comprising the steps:
(i) taking sequence data obtained from a number of cDNA clones randomly selected from a naive VHH library;
(ii) identifying a series anchor regions which show substantial conserved homology within said sequence data and on which basis framework primers with a capacity to amplify framework regions of the naive library target DNA can be constructed;
(iii) amplification from a non-immunised source of a maximal number of different framework regions using primers from step (ii);
(iv) combining the DNA sequences encoding each CDR present in the naive library, optionally modified by mutation or replacement, at least in part, by synthetic sequences, with framework primers to form a range of CDR primers;
(v) assembling nucleic acid sequences to create a VHH repertoire by random recombination of the range of CDR primers with the amplified framework regions using a technique of splicing by overlap extension.
11. Method for the preparation of antibody fragments derived from a non-immunised source having specificity for a target antigen comprising screening an expression library according to any of claims 1 to 9 for antigen binding activity and recovering antibody fragments having the desired specificity.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A blind rivet washer, comprising:
a washer body with an outer surface, an inner surface, and a rivet aperture; and
a watertight or airtight membrane comprising at least one raised pressure point skirt and at least one recessed troughs on each side of the at least one raised pressure point skirt, wherein the membrane covers the outer surface of the washer body and is sealably watertight or airtight and continuously connected to the perimeter of the washer body, and optionally to a circumferential portion of the outer surface,
wherein the watertight or airtight membrane encapsulates the washer body and the rivet aperture to create a lumen which provides a space between a portion of the outer surface of the washer body and the watertight or airtight membrane,
wherein the raised pressure point skirt and the at least one recessed trough on each side of the at least one raised pressure point skirt are oriented to face a substrate on which the blind rivet washer is sealed against.
2. The blind rivet washer of claim 1, wherein the watertight or airtight membrane is sealably watertight or airtight connected to the perimeter of the washer body, and optionally a circumferential portion of the outer surface, by a fixing means.
3. The blind rivet washer of claim 2, wherein the fixing means is a bonding agent.
4. The blind rivet washer of claim 1, wherein the watertight or airtight membrane is sealably watertight or airtight connected to the perimeter of the washer body by a protrusion extending radially and circumferentially from the outer surface perimeter of the washer body.
5. The blind rivet washer of claim 4, wherein the watertight or airtight membrane is sealably watertight or airtight connected by friction pressure, a bonding agent, or any combination thereof.
6. The blind rivet washer of claim 1, wherein the watertight or airtight membrane is a material selected from the group consisting of silicone, silicone rubber, natural rubber, and synthetic rubber.
7. The blind rivet washer of claim 1, wherein the watertight or airtight membrane is silicone rubber.
8. A blind rivet assembly, comprising:
a blind rivet washer body with an outer surface, an inner surface, and a rivet aperture;
a watertight or airtight membrane comprising at least one raised pressure point skirt and at least one recessed troughs on each side of the at least one raised pressure point skirt, wherein the membrane covers the outer surface of the washer body and is sealably watertight or airtight and continuously connected to the perimeter of the washer body, and optionally to a circumferential portion of the outer surface; and
a rivet configured to be installed with the washer body,
wherein the watertight or airtight membrane encapsulates the washer body, a head of the blind rivet and the rivet aperture to create a lumen which provides a space between a portion of the outer surface of the washer body and the watertight or airtight membrane,
wherein the raised pressure point skirt and the at least one recessed trough on each side of the at least one raised pressure point skirt are oriented to face a substrate on which the blind rivet washer is sealed against.
9. The blind rivet assembly of claim 8, wherein the watertight or airtight membrane is sealably watertight or airtight connected to the perimeter, and optionally a circumferential portion of the outer surface, of the washer body by a fixing means.
10. The blind rivet assembly of claim 9, wherein the fixing means is a bonding agent.
11. The blind rivet assembly of claim 8, wherein the watertight or airtight membrane is sealably watertight or airtight connected to the perimeter of the washer body by a protrusion extending radially and circumferentially from the outer surface perimeter of the washer body.
12. The blind rivet assembly of claim 11, wherein the watertight or airtight membrane is sealably watertight or airtight connected by friction, a bonding agent, mechanical methods or any combination thereof.
13. The blind rivet assembly of claim 8, wherein the watertight or airtight membrane is a material selected from the group consisting of silicone, silicone rubber, natural rubber, and synthetic rubber.
14. The blind rivet assembly of claim 8, wherein the watertight or airtight membrane is silicone rubber.
15. A blind rivet washer, comprising:
a washer body with an outer surface, an inner surface, and a rivet aperture; and
a watertight or airtight silicone rubber membrane comprising at least one raised pressure point skirt and at least one recessed trough on each side of the at least one raised pressure point skirt, wherein the silicone rubber membrane covers the outer surface of the washer body and is sealably watertight or airtight and continuously connected to the perimeter of the washer body, and optionally to a circumferential portion of the outer surface, by a fixing means,
wherein the silicone rubber membrane encapsulates washer body and rivet aperture to create a lumen which provides a space between a portion of the outer surface of the washer body and the silicone rubber membrane,
wherein the raised pressure point skirt and the at least one recessed trough are oriented to face a substrate on which the blind rivet washer is sealed against.
16. The blind rivet washer of claim 15, wherein the fixing means is a bonding agent.
17. The blind rivet washer of claim 15, wherein the fixing means is mechanical.