IDENTIFICATION OF AFFINELY-MATURE HUMAN ANTIBODIES Russian patent published in 2017 - IPC C07K16/00 C12N15/11 C12Q1/68 C07K16/18 C40B30/04 C40B50/06 

FIELD: biotechnology.

SUBSTANCE: inventions relate to methods for creation of a library of nucleotide sequences of human immunoglobulin for detection of nucleic acid sequences encoding somatic hypermutated variable immunoglobulin regions that bind to the antigen of interest and kits for use in the said methods. The method for library creation includes: a PCR step to amplify the nucleotide sequences of the leader and V regions of the heavy and light chains of human immunoglobulin from the cDNA library created from a sample of human peripheral blood mononuclear cells derived from a human donor who previously had antibodies, specific for the antigen of interest, where a set of primer-specific primer sequences is used for PCR amplification, including those, specific relative to 90% of all leader primer sequences. Isolation of amplification products obtained at the PCR step. Creation of a constructs set by embedding the isolated amplification products obtained at the PCR step into expression vectors. Introduction of a set of constructs into the set of host cells to create a library of human immunoglobulin nucleotide sequences. The method for sequences detection includes: obtaining of a library of nucleotide sequences from the previous method as the first step. The second step of the PCR to amplify the human immunoglobulin nucleotide sequences from the obtained library or a human tissue sample. Isolation of the second set of amplification products. Creation of the second set. Introduction of the second set of constructs into the second set of host cells. Screening of the second library. Preparation of a nucleotide sequence encoding a variable region that specifically binds to the target antigen. Creation of a reverse primer and its application at the third stage of PCR.

EFFECT: inventions provide detection of more complete V-region libraries encoding antibody sequences containing critical somatic mutations to produce high affinity antibodies.

6 cl, 5 dwg, 4 tbl, 3 ex