Polyclonal libraries

A method of preparing a diagnostic kit containing a polypeptide library having affinity for a target, comprising:

• providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, and wherein the library of viral particles has been enriched by affinity selection;
• subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules;
• introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and
• incorporating the library of polypeptides into a diagnostic kit.

A method of preparing a diagnostic reagent comprising a polypeptide library having affinity for a target, comprising:

• providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, and wherein the library of viral particles has been enriched by affinity selection;
• subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules;
• introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and
• formulating the library of polypeptides as a diagnostic reagent.

A method of preparing a therapeutic composition comprising a polypeptide library having affinity for a target, comprising:

• providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides vary between members,
• subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector;
• introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and
• formulating the library of polypeptides with a pharmaceutically acceptable carrier to form a therapeutic composition.

A method of producing a polypeptide library having affinity for a target, comprising:

• providing a library of viral particles, wherein a member comprises a viral particle capable of displaying from its outersurface a fusion protein comprising a viral coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members,
• contacting the library members with a receptor that specific affinity for a peptide sequence of the fusion protein that is the same in each library member and isolating library members bound to the receptor in immobilized form;
• contacting the isolated library members with a target molecule, and isolating library members bound to the target molecule in immobilized form;
• subcloning a mixture of DNA molecules encoding at least ten different polypeptides from the isolated library members that bind to the same target molecule;
• into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules;
• and
• introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms.

A method of producing an antibody library having affinity for a target, comprising:

• providing a library of phage, wherein a member of the library comprises a phage capable of displaying from its outersurface an antibody comprising an antibody heavy chain variable domain complexed with an antibody light chain variable domain, wherein either the heavy or light chain variable domain is expressed as a fusion protein with a coat protein of the phage and either the heavy or light chain variable domain or both is/are encoded by the genome of the phage, and the heavy and/or light chain varies between members;
• contacting the library members with a receptor that has specific affinity for a peptide sequence of the fusion protein that is the same in each library member and isolating library members that bind to the receptor in immobilized form;
• contacting the isolated library members with a target molecule, and isolating library members that bind to the target molecule in immobilized form;
• subcloning a mixture of DNA molecules encoding the heavy and/or light chain variable domains from the isolated phage library members that bind to the same target molecule into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules;
• introducing the modified forms of the expression vector into a host and expressing antibodies formed by the heavy and light chain variable domains of the phage library in the host, the antibodies being released from the host to form an antibody library of at least ten antibodies wherein at least 90% of modified forms of the expression vector encode antibodies with specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms.