Array-based epitope-captured antibody display

• US 20050042658A1
• Filed: 07/27/2004
• Published: 02/24/2005
• Est. Priority Date: 04/17/2001
• Status: Active Grant

• Current Assignee: XenoPort, Inc. (Arbor Pharmaceuticals LLC)
• Original Assignee: XenoPort, Inc. (Arbor Pharmaceuticals LLC)
• Inventors: Dower, William J., Cwirla, Steven E.
• Granted Patent: US 7,192,720 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2600/00 Assays involving molecular imprinted polymers/polymers created around a molecular template
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

Reagents and methods for detecting target proteins in a sample are provided. The reagents include a replicable genetic package, a protein displayed on an exterior surface of the package that is expressed from a heterologous nucleic acid borne by the package, and one or more antibodies complexed with the expressed protein and which have an open binding site for a target protein. Thus, a segment of the nucleic acid encodes for an epitope that is shared by the expressed polypeptide and the target protein. The reagents can be utilized individually or as part of a library or an array to bind target proteins within protein samples to form one or more complexes. By determining the sequence of the segment of the heterologous nucleic acid of a package within a complex, one can identify the target protein since the segment encodes for an epitope that is shared by the expressed and target proteins.

Claims

1-18. -18. (cancelled)

19. A method of analyzing the amino acid sequence of polypeptides by determining whether they share an epitope, the method comprising:

(a) providing an array comprising a support and a population of replicable genetic package/antibody reagents immobilized to the support, each replicable genetic package/antibody reagent (package/antibody reagent) comprising (i) a replicable genetic package having a heterologous nucleic acid segment that encodes a first polypeptide displayed on the replicable genetic package and (ii) a captured antibody having a plurality of binding sites, each site having specific affinity for the first polypeptide, with at least one of the sites available for binding, the first polypeptide and the captured antibody complexed with it varying between at least some of the package/antibody reagents;

(b) contacting the population of package/antibody reagents with a sample containing a second polypeptide, whereby package/antibody reagents bearing captured antibodies having specific affinity for the second polypeptide bind to the second polypeptide;

(c) identifying at least one package/antibody reagent that binds to the second polypeptide; and

(d) determining the nucleotide sequence of the nucleic acid segment of the at least one package/antibody reagent, whereby the amino acid sequence corresponding to the nucleotide sequence can be deduced and is an indication of the amino acid sequence of an epitope shared by the first and second polypeptide.

20. The method of claim 19, wherein

(i) the second polypeptide is one of multiple different polypeptides within the sample;

(ii) the identifying step (c) comprises identifying package/antibody reagents that have formed complexes to the different polypeptides; and

(iii) the determining step (d) comprises determining the sequence of the segment of the nucleic acid for each of the package/antibody reagents that are complexed to the different polypeptides.

21. The method of claim 19, further comprising preparing the array, the preparing step comprising spotting the package/antibody reagents onto the support.

22. The method of claim 19, further comprising preparing the array, the preparing step comprising

(i) growing bacterial colonies that express the replicable genetic packages of the package/antibody reagents;

(ii) transferring colonies onto the support; and

(iii) contacting the support with the captured antibodies to form the array.

23. The method of claim 19, wherein the replicable genetic packages of the package/antibody reagents are lytic bacteriophage and the method further comprises preparing the array, the preparing step comprising

(i) growing the bacteriophage on a lawn of bacteria to produce a plurality of plaques;

(ii) transferring plaques to the support; and

(iii) contacting the support with the captured antibodies to form the array.

24. The method of claim 19, wherein the second polypeptide is labeled and the identifying step (c) comprises detecting labeled second polypeptide complexed to the at least one package/antibody reagent.

25. The method of claim 19, wherein

(i) the contacting step (b) further comprises contacting the array with one or more labeled detection reagents, whereby a labeled detection reagent with specific affinity for the second polypeptide binds to the second polypeptide of the at least one package/antibody reagent to form a tertiary complex comprising the at least one package/antibody reagent, the second polypeptide and the detection reagent;

and

(ii) the identifying step (c) comprises detecting the tertiary complex, thereby identifying the at least one package/antibody complex.

26. The method of claim 25, wherein the one or more detection reagents comprise a detection antibody with specific affinity for the second polypeptide.

27. The method of claim 25, wherein

(i) the one or more detection reagents comprise a replicable genetic package which has a copy of the same nucleic acid as the package/antibody reagent that complexes with the second polypeptide, and which is complexed to a detection antibody having specific affinity for the second polypeptide;

and

(ii) the determining step (d) comprises determining the sequence of the segment of the nucleic acid of the replicable genetic package of the detection reagent, thereby providing an indication of the sequence of the segment of the nucleic acid of the at least one package/antibody reagent.

28. The method of claim 27, wherein the label is attached to the replicable genetic package of the detection reagent.

29. The method of claim 27, wherein the determining step (d) further comprises retrieving the replicable genetic package of the detection reagent from the tertiary complex, amplifying the segment of the nucleic acid of the detection reagent and sequencing the amplified segment, the sequence of the amplified segment corresponding to the sequence of the segment of the at least one package/antibody reagent.

30. The method of claim 29, wherein retrieval of the package/antibody reagent from the detection reagent is performed with a microdissection instrument.

31. The method of claim 30, wherein the microdissection instrument is a laser capture microdissection instrument.

32. The method of claim 19, wherein the determining step (d) further comprises retrieving the at least one package/antibody reagent from the array, amplifying the segment of the nucleic acid of the at least one package/antibody reagent and sequencing the amplified segment.

33. The method of claim 24, the method further comprising preparing an archival replica of the array, and wherein the determining step (d) further comprises isolating a replicable genetic package from the archival replica that corresponds to the at least one package/antibody reagent, and determining the sequence of the segment of the nucleic acid in the isolated replicable genetic package, thereby determining the sequence of the segment of the nucleic acid in the at least one package/antibody reagent.

34. The method of claim 25, the method further comprising preparing an archival replica of the array, and wherein the determining step (d) further comprises isolating a replicable genetic package from the archival replica that corresponds to the at least one package/antibody reagent, and determining the sequence of the segment of the nucleic acid in the isolated replicable genetic package, thereby determining the sequence of the segment of the nucleic acid in the at least one package/antibody reagent.

35-53. -53. (canceled)

54. A method of analyzing a sample containing a mixture of an polypeptides, the method comprising:

(a) providing an array comprising a support and a plurality of replicable genetic package/antibody reagents immobilized to the support, wherein the package/antibody reagents comprise a replicable genetic package that displays a polypeptide encoded by a segment of a nucleic acid of the package, and the polypeptide is complexed with a captured antibody having specific affinity for the polypeptide, the polypeptide and the captured antibody complexed with it varying between at least some of the package/antibody reagents;

(b) contacting the array with the sample containing the mixture of polypeptides, whereby package/antibody reagents bearing captured antibodies having specific affinity for a polypeptide in the mixture capture the polypeptide from the mixture to form a complex; and

(c) detecting at least one of the complexes, the sequence of the segment of the nucleic acid of the package/antibody reagent within the at least one complex and the corresponding amino acid sequence providing an indication of an amino acid sequence of an epitope on the captured polypeptide.

55. The method of claim 54, further comprising repeating steps (a)-(c) with a second array and a second sample and comparing which of the package/antibody reagents of the two arrays form complexes with the polypeptides in the samples.

56. The method of claim 54, further comprising quantifying the amount of polypeptide in the complex(es) detected in step (c).

57. The method of claim 54, further comprising differentially labeling polypeptides in two samples of proteins, such that polypeptides in one sample bear one label and polypeptides in the other sample bear a different label, and wherein

(i) the contacting step (b) comprises contacting the array with the two samples, whereby package/antibody reagents bearing captured antibodies having specific affinity for a polypeptide in the samples capture the polypeptide to form a complex;

and

(ii) the detecting step (c) comprises detecting differentially labeled complexes on the array.

58-94. -94. (canceled)

95. A method for preparing an array of polypeptides, the method comprising:

(a) immobilizing replicable genetic packages displaying a polypeptide to a support; and

(b) contacting the displayed polypeptides with a population of antibodies under conditions such that the antibodies form complexes with displayed polypeptides for which the antibodies have specific binding affinity, whereby the array of polypeptides is formed.

96. The method of claim 95, wherein the replicable genetic packages are phage and immobilizing comprises

plating the phage on a layer of cells to form bacterial microcolonies or an array of micro-plaques;

and

replicating the microcolonies or micro-plaques onto the support, whereby phage displaying polypeptides become immobilized to the support.

97. The method of claim 95, further comprising contacting the array with a sample containing a plurality of proteins under conditions such that proteins in the sample and antibodies on the array that have specific binding affinity for one another form complexes, thereby forming an array of captured proteins.

98. The method of claim 97, wherein the plurality of proteins in the sample are functional proteins.