Identification of antigen-specific adaptive immune responses using ARM-PCR and high-throughput sequencing
Disclosed is a method for correlating at least one amino acid sequence from an antibody isolated from human or animal blood with at least one DNA sequence corresponding to the antibody in the immunorepertoire of the human or animal. The method also provides a means for pairing heavy and light chains to produce synthesized monoclonal antibodies.
1. A method for identifying antigen-specific T cells, the method comprising:
a) obtaining a sample of blood or tissue from a human subject that has been previously challenged with a disease-associated-antigen in vivo;
b) isolating peripheral blood mononuclear cells, wherein the peripheral blood mononuclear cells comprise T cells, from the sample and culturing the peripheral blood mononuclear cells in vitro;
c) adding an effective amount of the antigen to a portion of the peripheral blood mononuclear cells in vitro;
d) harvesting RNA from the peripheral blood mononuclear cells at an empirically-determined time after adding the antigen;
e) amplifying the harvested RNA using arm-PCR and sequencing the resulting amplicons to generate an immunorepertoire for T cells stimulated with the antigen in vitro and for T cells not stimulated with the antigen in vitro; and
f) diagnosing the human subject with disease associated with the antigen where an increase in one or more T cell clones is detected in the T cells stimulated with the antigen in vitro but is not detected in the T cells not stimulated with the antigen in vitro.
2. The method of claim 1 , further comprising diagnosing the human subject with influenza where an increase in one or more T cell clones is detected in the T cells stimulated with the antigen in vitro but is not detected in the T cells not stimulated with the antigen in vitro, wherein the antigen is hemagglutinin.