Antibody validation using ip-mass spectrometry
AssigneePierce Biotechnology, Inc.
InventorsJohn ROGERS, Bhavinkumar PATEL, Greg POTTS, Leigh FOSTER
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Abstract
The invention relates, in part, to compositions and methods for validating antibodies utilizing immunoprecipitation and mass spectrometry.
Claims63
A method for identifying proteins that specifically bind to an antibody comprising:
- i) Selecting a test antibody;
- ii) Preparing a cell lysate from a biological sample;
- iii) Contacting the cell lysate with the test antibody, and immunoprecipitating the antibody and its protein binding partner(s);
- iv) Analyzing the immunoprecipitated antibody and its protein binding partner(s) by mass spectrometry;
- v) Determining the fold enrichment of the protein(s) bound to the test antibody as compared to the proteins in the cell lysate;
- and
- vi) Identifying the proteins that specifically bind to the antibody, wherein proteins that specifically bind to the antibody are enriched as compared to proteins in the cell lysate.
A method for identifying proteins that specifically bind to an antibody comprising:
- i) Selecting a test antibody;
- ii) Preparing a first and second preparation of cell lysate from a biological sample, wherein the first and second preparations are nearly identical;
- iii) Contacting the first cell lysate with the test antibody, and the second cell lysate with a second antibody, and immunoprecipitating the antibodies and their protein binding partner(s);
- iv) Analyzing the immunoprecipitated test and second antibody and their protein binding partner(s) by mass spectrometry;
- v) Plotting the intensity and/or fold enrichment of each protein identified by mass spectrometry as being bound to the test antibody on an x- or y-axis, and plotting the intensity or fold enrichment of each protein identified by mass spectrometry as being bound to the second antibody on the opposite axis;
- and
- vi) Identifying the proteins that specifically bind to the test and second antibodies, wherein
- a. proteins that specifically bind to the test and second antibody are those that do not display equal or nearly equal binding to both the test and second antibodies (those that fall along the diagonal when plotted);
- b. proteins that specifically bind to the test antibody fall above the diagonal if plotted along the y-axis, or below the diagonal if plotted along the x-axis;
- and
- c. proteins that specifically bind to the second antibody fall above the diagonal if plotted along the y-axis, or below the diagonal if plotted along the x-axis.
The method of any of the preceding claims, wherein the mass spectrometry is selected from tandem mass spectrometry using data dependent acquisition and data independent acquisition.
The method of any of the preceding claims, wherein the identified interaction partners, isoforms, or modifications may indicate distinguishable epitopes for different antibodies.
The method of any of the preceding claims, wherein the immunoprecipitated antibody-target protein is digested prior to mass spectrometry.
The method of any of the preceding claims, further comprising desalting after immunoprecipitation or after digestion and prior to mass spectrometry.
A method for characterizing an antibody, the method comprising:
- (a) determining the affinity of the antibody to an antigen, and
- (b) determining the selectivity of the antibody for the antigen,
- wherein the affinity of the antibody and/or the selectivity of the antibody are determined using immunoprecipitation-mass spectrometry (IP-MS), and
- wherein the immunoprecipitate is generated by contacting the antigen with the antibody under conditions that allow for the formation on the immunoprecipitate between the antibody and the antigen.
A method for preparing a matched set of antibodies, the method comprising:
- (a) determining the affinity of each antibody for its respective antigen in a cell lysate,
- (b) determining the selectivity of each antibody for its respective antigen in the cell lysate, and
- (c) selecting antibodies to form the matched set,
- wherein the affinity of the antibody and/or the selectivity of the antibody are determined using immunoprecipitation-mass spectrometry (IP-MS), and
- wherein the matched set is composed of two or more antibodies that each have selectivity of at least 100 fold enrichment of for its respective antigen present in the cell lysate.
A method for determining the selectivity of an antibody, the method comprising:
- (a) contacting the antibody with a cell extract under conditions that allow for the formation on an immunoprecipitate between the antibody and one or more antigen in the cell extract,
- (b) collecting the immunprecipitate formed in step (a), and
- (c) indentifying one or more non-antibody molecules present in the immunprecipitate by mass spectrometry,
- wherein the cell extract contains cell components from two or more cell types or one or more cell types from two or more species.
A method for determining the selectivity of an antibody, the method comprising:
- (a) contacting the antibody with two or more proteins under conditions that allow for the formation on an immunoprecipitate between the antibody and one or more of the two or more proteins,
- (b) collecting the immunprecipitate formed in step (a), and
- (c) quantifying the amount of individual proteins present in the immunprecipitate by mass spectrometry.
A method for identifying an antibody that selectively binds to target molecules of cells obtained from different species, the method comprising:
- (a) contacting the antibody with two or more cell lysates generated from cell of different species under conditions that allow for the formation on two or more immunoprecipitates between the antibody and one or more target molecule present in each cell lysate,
- (b) collecting the immunoprecipitate from each cell lysate, and
- (c) determining the fold purification for the target molecules in each immunoprecipitate by mass spectrometry.
The method of claim 65, wherein the species are selected from the group consisting of:
- (a) Homo sapiens,
- (b) Oryctolagus cuniculus,
- (c) Mus musculus, and
- (d) Rattus norvegicus.
The method of claim 65, wherein the antibody is generated in response to an epitope or a protein that is conserved across the different species from which the cell lysates are obtained.
The method of claim 67, wherein the epitope is from a protein or the protein is in a category selected from the group consisting of:
- (a) heat shock proteins,
- (b) polymerases,
- (c) cell surface receptors,
- (d) transcription factors,
- (e) kinases,
- (f) dephosphorylases,
- (g) membrane associated transporters, and
- (h) zinc finger proteins.
Dependencies are inferred from the claim text.