METHOD OF CULTURING SINGLE B-CELL Russian patent published in 2019 - IPC G01N33/50 G01N33/53 

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, specifically to a method of producing B-cells secreting antigen-specific antibodies. Following steps are taken to obtain B-cells: obtaining B cells from rabbit blood; labelling of IgG + -B-cells and/or CD138 + -B-cells; incubation of B-cells at temperature of 37 °C for one hour in a medium for co-cultivation before placing labelled B-cells in form of single cells with subsequent combined cultivation with feeder cells in a medium for co-cultivation; selection of B-cell, proliferating at this stage; obtaining B-cells. Method may also include a step for centrifugation of separately placed B-cells before combined cultivation. Feeder cells are murine EL-4 B5 cells, and the feeder mixture contains interleukin-1-beta, tumour necrosis factor alpha, Staphylococcus aureus cells Cowan, BAFF, interleukin-2, and/or interleukin-10, and/or interleukin-6, and/or interleukin-4. To obtain antibodies, a mature B-cell population derived from rabbit blood is taken; marking of IgG + -B-cells and/or CD138 + -B-cells with at least one fluorescent label; B cells are incubated at temperature of 37 °C for one hour in a medium for co-cultivation before placing single cells from the population of labelled B-cells into separate containers; cultivating separately placed B-cells in the presence of feeder cells and a feeder mixture; specificity of antibody binding and amino acid sequence of variable domains of light and heavy chains of specifically binding antibodies by PCR with reverse transcription; introducing a nucleic acid encoding the variable domains of the light and heavy chains of the monoclonal antibody for antibody expression; introducing nucleic acid into a cell; culturing the cell and isolating the antibody from a cell or cell supernatant.

EFFECT: group of inventions enables fast characterization of binding of monoclonal antibodies prepared from individual B-cell clones obtained from rabbit blood.

7 cl, 13 tbl, 22 ex