Firstly, suitable antigens are selected for animal immunization, and the sorting of bovine single B cells is an important step in preparing bovine polyclonal antibodies. Healthy cows are the preferred choice for experimental animals. By injecting the target antigen to stimulate an immune response in cattle, B cells are induced to produce antibodies, and then ELISA is used to confirm the effectiveness of the immune response. The peripheral blood of immunized cows was collected, and PBMCs were isolated by centrifugation and other methods. By using facs sorting to selectively sort enriched B cells, based on the expression patterns of cell surface markers, facs sorting can efficiently identify B cells. During sorting, fluorescently labeled antigens can be combined with antibodies on the surface of B cells to recognize and isolate B cells targeting specific antigens. The sorted individual B cells are lysed to release their nucleic acids. The mRNA inside the cell is reversed by RT-PCR, and primers are designed based on the genes of the variable heavy chain and variable light chain of the target antibody. The variable region sequence is then amplified by PCR. On this basis, single cell sequencing was used to sequence and analyze the antibody variable region genes encoded by these B cells, in order to obtain their gene sequence information. The variable region sequence was inserted into a vector plasmid containing a constant region gene, and a plasmid expressing monoclonal antibodies was obtained. An appropriate expression system is selected to obtain antibodies, and antibody validation and other operations can be performed on the obtained antibodies. Throughout the entire sorting process, the activity of cells needs to be maintained, avoiding the use of reagents and conditions that are harmful to cells, and ensuring that the entire sorting process is carried out under sterile conditions to avoid cell contamination. This ensures the efficiency of sorting and the activity of cells, providing a reliable foundation for subsequent antibody research and applications.

Single B cell sorting is not only suitable for experimental animals such as cows, but can also be used to prepare antibodies from PBMCs of recovered patients. Antibodies prepared through single B cell sorting technology can accurately target specific antigens of patients. Single B cell sorting can directly analyze individual B cells or their secreted antibodies without the need for cell fusion or antibody library establishment steps. The experimental cycle is greatly shortened and the screening efficiency is improved. With the continuous development of technology, single B cell technology has been used in more and more fields. Single B cell technology can be used to prepare antibodies against specific pathogens, such as HIV antibodies, tetanus antibodies, hepatitis B antibodies and influenza antibodies, for disease prevention and treatment. b cell antibody production technology can isolate specific B cells targeting tumor antigens and prepare efficient anti-tumor monoclonal antibodies, which can be used for tumor diagnosis and treatment. By isolating B cells targeting pathogen antigens, efficient and specific vaccine candidate antigens can also be prepared, which is helpful for vaccine development.