Methods of selecting binding reagents
Methods and systems are provided herein for selecting an affinity reagent which binds a desired peptide epitope in a plurality of sequence contexts. The method relies on obtaining a peptide library, each peptide having the sequence αXβ, wherein X is the desired peptide epitope, wherein each of α and β comprise an amino acid, using the peptide library to select an affinity reagent.
1. A method for selecting an affinity reagent which binds a desired peptide epitope in a plurality of sequence contexts, the method comprising:
obtaining a peptide library comprising a plurality of at least 400 sequence contexts, each peptide of the peptide library comprising the sequence αXβ, wherein X is the desired peptide epitope, wherein X is a sequence of 2, 3 or 4 amino acids, wherein each of a and comprises at least one amino acid, and wherein each of α and β varies amongst the plurality of sequence contexts within the peptide library; and
screening one or more affinity reagents against the peptide library to select an affinity reagent from among the one or more affinity reagents which binds the desired peptide epitope X in at least 10% of the plurality of sequence contexts, wherein the selected affinity reagent is selected from the group consisting of an antibody, an aptamer, a peptamer, and an antibody fragment.
2. The method of claim 1 , wherein each of α and β consists of one amino acid.
3. The method of claim 1 , wherein at least one of α and β further comprises a linker.
4. The method of claim 1 , wherein at least one of α and β further comprises a modification selected from the group consisting of a N-terminal modification, a C-terminal modification, a positively-charged group, a negatively-charged group, a hydrophobic group, and a sugar.
5. The method of claim 1 , wherein each of α and β consists of between one and three amino acids.
6. The method of claim 1 , wherein the selected affinity reagent is the antibody.
7. The method of claim 1 , further comprising:
Characterizing the binding of the selected affinity reagent by screening the selected affinity reagent against a plurality of peptides of the same length of the desired epitope.
8. The method of claim 7 , wherein the plurality of peptides of the same length of the desired epitope is representative of at least 90% of all possible variations of peptides of the same length of the desired epitope.
9. The method of claim 1 , further comprising:
characterizing the binding of the selected affinity reagent by screening the selected affinity reagent against a plurality of peptides, wherein each peptide of the plurality of peptides comprises the desired epitope and one or more flanking residues.
10. The method of claim 7 , further comprising:
characterizing the binding of the selected affinity reagent by screening the selected affinity reagent against a second plurality of peptides, wherein each peptide of the plurality of peptides comprises a secondary epitope and one or more flanking residues.
11. The method of claim 1 , further comprising:
characterizing the binding of the selected affinity reagent at least in part by screening the selected affinity reagent against a plurality of subsets of a plurality of peptides shorter than the desired peptide epitope.
12. The method of claim 1 , further comprising characterizing the selected affinity reagent by screening the selected affinity reagent against a panel of proteins or peptides which contain the desired epitope.
13. The method of claim 1 , wherein the selected affinity reagent binds to at least 20% of the plurality of sequence contexts.
14. The method of claim 1 , further comprising characterizing the selected affinity reagent by screening the selected affinity reagent against a panel of proteins which do not contain the X epitope.
15. The method of claim 1 , wherein the selected affinity reagent binds to at least 30% of the plurality of sequence contexts.
16. The method of claim 1 , wherein the selected affinity reagent is screened against a library of random peptides having a length between 2 and 10 amino acids, and further comprising:
identifying the peptides bound by the selected affinity reagent; and
retaining the selected affinity reagent if 1) it binds less than twenty different epitopes and 2) it binds at least 10% of all peptides longer than 5 amino acids containing that epitope.
17. The method of claim 16 , wherein the selected affinity reagent binds at least 15% of all peptides longer than 5 amino acids containing the epitope.
18. The method of claim 1 , further comprising:
characterizing the binding of the affinity reagent by screening the affinity reagent against a library of peptides, the library of peptides comprising peptides shorter than the desired epitope, peptides of the same length of the desired epitope, and peptides longer than the desired epitope; and
identifying the sequences of bound peptides, thereby characterizing the binding of the affinity reagent.
19. The method of claim 1 , wherein the selected affinity reagent is the aptamer.
20. The method of claim 1 , wherein the selected affinity reagent is the peptamer.
21. The method of claim 1 , wherein the selected affinity reagent is the antibody fragment.
22. The method of claim 1 , wherein the peptide library is used in a denatured state to select said affinity reagent.
23. The method of claim 1 , further comprising retaining the selected affinity reagent for further screening.