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US 12,590,120 B2Granted
Materials and methods for isolating self-antigen polypeptides
AssigneesImpact Proteomics, University of Pittsburgh—Of the Commonwealth System of Higher Education
InventorsJonathan Minden, Dana Ascherman
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Abstract
Materials and methods for isolating self-antigen polypeptides are provided.
Claims19
A method of isolating self-antigen polypeptides, the method comprising:
- a) and a lysis buffer comprising leupeptin and pepstatin, wherein the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides, thereby forming a mixed sample comprising compound bonded polypeptides,
- wherein the cell sample comprises self-antigen polypeptides;
- wherein the compound comprises
- a first moiety comprising a methyltetrazine (mTet);
- a second moiety comprising a carboxyl dimethyl maleic anhydride (CDM);
- and
- a linker linking the first moiety and the second moiety;
- b) adding the compound bonded polypeptides in the mixed sample to an antibody coupled substrate, wherein the antibody coupled substrate comprises antibodies from serum of a patient with an autoimmune disorder bound to a bead or solid surface and wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate;
- c) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
- d) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface;
- and
- e) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
9 more elements
A method of isolating self-antigen polypeptides, the method comprising:
- a) adding compound bonded polypeptides in a mixed sample to an antibody coupled substrate, wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate,
- wherein the antibody coupled substrate comprises antibodies from serum of a patient with an autoimmune disorder bound to a bead or solid surface, and
- wherein the mixed sample comprising compound bonded polypeptides is a and a lysis buffer comprising leupeptin and pepstatin, and the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides,
- wherein the cell sample comprises self-antigen polypeptides;
- wherein the compound comprises
- a first moiety comprising a methyltetrazine (mTet);
- a second moiety comprising a carboxyl dimethylmaleic anhydride (CDM);
- and
- a linker linking the first moiety and the second moiety;
- b) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
- c) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface;
- and
- d) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
10 more elements
A method of isolating self-antigen polypeptides, the method comprising:
- a) adding compound bonded polypeptides in a mixed sample to an antibody coupled substrate, wherein the compound bonded polypeptides bind to the antibody coupled substrate when the compound bonded polypeptides have an affinity to antibodies on the antibody coupled substrate, thereby forming compound bonded polypeptides bound to the antibody coupled substrate,
- wherein the antibody coupled substrate comprises antibodies from a biological source of a patient with an autoimmune disorder bound to a bead or solid surface, and
- wherein the mixed sample comprising compound bonded polypeptides is a and a lysis buffer comprising leupeptin and pepstatin, and the compound is bonded to polypeptides in the cell sample by formation of an acid-labile amide bond between a dicarboxylic anhydride of the compound and primary amines of the polypeptides,
- wherein the cell sample comprises self-antigen polypeptides;
- wherein the compound comprises
- a first moiety comprising a methyltetrazine (mTet);
- a second moiety comprising a carboxyl dimethyl maleic anhydride (CDM);
- and
- a linker linking the first moiety and the second moiety;
- b) eluting the compound bonded polypeptides bound to the antibody coupled substrate by adding a solution comprising sodium dodecyl sulfate (SDS), thereby forming compound bonded self-antigen polypeptides in an eluted sample;
- c) adding the compound bonded self-antigen polypeptides in the eluted sample to a trans-cyclooctene (TCO), wherein the TCO is linked to a second bead or solid surface, thereby forming self-antigen polypeptides bound to the second bead or solid surface;
- and
- d) eluting the self-antigen polypeptides bound to the second bead or solid surface in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the acid-labile amide bond between the primary amines of the self-antigen polypeptides and the second moiety comprising a carboxyl dimethyl maleic anhydride (CDM).
10 more elements
Dependencies are inferred from the claim text.