Methods for identification of an antibody or a target

摘要

This disclosure relates to methods for identifying an antibody, a target molecule, or an agent by analyzing the immunoglobulin repertoire sequence data in a sample and by determining the most dominant VH and VL chains present in said sample, as well as materials used therewith.

主权项

1. A method of identifying an antibody, or a target, comprising the steps of:
a) obtaining a sample of at least 106 B cells from an immunized mammal, extracting mRNAs encoding immunoglobulins from the B cells, and reverse transcribing cDNAs of the mRNAs, thereby obtaining a mixture of the cDNAs; b) using single, massive parallel sequencing technology to directly parallel sequence within 24 hours, without any prior cloning steps, the cDNAs encoding the variable heavy (VH) and variable light (VL) chains of the immunoglobulins from the sample, thereby obtaining sequence data of the VH and VL chains of the immunoglobulins present in the sample; c) determining the frequency of the VH and VL chain sequences present in the sample by counting or statistical analysis, d) identifying the most abundant VH and VL chain sequences present in the sample according to their frequency; e) synthesizing polynucleotides encoding the most abundant VH and VL chain sequences present in the sample and producing antibodies comprising said VH and VL chains by cloning the polynucleotides into an expression vector, introducing the expression vector into a host cell and expressing the antibodies; and f) identifying the antibody(ies) specific for a target of interest or identifying the target molecule to which the antibody(ies) is specific.