One of the critical factors underlysing the difficulty in generating human Mabs is the low frequency of antigen specific B cells in the peripheral blood. Typically, antigen specific B cells are present in periopheral blood of healthy volunteers at a frequency of less than 0.1% of total cells. Therefore, large numbers of PBLs are required from previously infected healthy individuals in order to obtain a significant number of antigne specific B cells.

Another technical difficulty in the isolation of virus specific B cells is the paucity of conformationally authentic viral antigens that are suitable for use in slection of epitope specific Mabs in human.

Selection of B cells by Fluorescence Activated Cell sorter (FACS)

Use of labeled antigen and FACS sorting: for the enrichment of these cells was demonstraed in 1972 (Dangl, J of Immunological Methods, 52 (1982) 1-14, citing Julius et al., 1972). These investigators sorted spleen cells from mice primed with keyhold limpet hemocyanin (KLH) stained with directly fluoresceinated KLH. After either 1 or 2 enrichment sorts, the percentage of cells binding the fluorescent antigen had increased from about 0.1% to 40%, an enrichment of 400 fold. 

Chang (US 5,213,960 and US 5,256,542) discloses that the selection for B cells expressing antibodies to specific antigens is increased by labeling B cells with at least two antigne problems that are labeled with a different fluorchrome. This increases the likelihood that antibodies expressed by a single B cell selected by FACS are specific for the antigen of interest.

Use of Labeled Antibody to identify B cells and FACS sorting

Allison (US 14/776,945, published as US 2014/0287952) discloses a method for identifying a B cell that expresses an antigen specific antibody which includes the steps of obtaining B cells for an immunized host or a host naturally exposed to an antigen of interest such as from a guinea pig, rabbit, mouse, rate or human, enriching a fraction of the B cell population such as by affinity purification to obtain a subpopulation of antigen specific B cells that produce an antibody that binds to an antigen of interest, culturing one or more fraction of said enriched subpopulation, detecting such as by ELISA a clonal B cell population that produces a single antibody that bind to the antigen of interest, staining the antigen specific B cells and then sorting the stained antigen specific B cells using fluoresesnce-activated cell sorting (FACS) or immunomagnetic cell sorting (MACS).  In one embodiment, the staining/sorting step is done using a negative selection method by staining B cells with a first label that stains irradiated EL4 cells, such as a frist label which is Thy1.2 (CD90) and a second label that stains dead cells such as propidium iodide (PI) and then sorting all viable non-EL4 using FACS or MACS. Cells that did not stain positive for Thy1.2 and PI were selected In a separate embodiment, the staining/sproting step facilitates a positive antigen specific B selection method by staining with a first label that stains species specific B cells such as an anti-rabbit IgG and a second label that stains dead cells such as propidium iodide (PI) and then sorting all viable species specific B cells using FACS or MACS. In one embodiment, the sorted opulation of cells can also be sorted directly into a RT-PCR reaction medium. 

Endl (WO2011/147903) discloses a method for obtaining a B cell which includes labeling the B cells for various markers such as IgG+ CD19+, IgG+CD38+, depositing the labeled B cells as single cells, co cultivating the single cell deposited B cells with feeder cells such as EL-4 B5 medium. The method can comprise the step of incubating the B cells with EL-4 B5 midum prior to the depositing step and can include centrifuging said single cell depositing B cells prior to the co-culivtation. . 

Meijer (WO 2008/104184) discloses a method for generating a vector encoding a chimeric antibody with human constant regions and non-human variable regions by providing a lymphoctye comprising cell fraction from a non-human animal, obtaining a population of isolated single cells by distributing cells from the cell fraction individually into a plurality of vessels and ampliyfing and effecting linkage of the V encoding nucleic acids of said isolated single cells. In one embodiment, plasma cells were isolated as CD43 high, CD138 high, PI (propdium iodide) positive or dead cells were exlucded. Then plasma cells were gated as CD43 high, CD138 high in bottom right panel. Finally, doublets were excluded in the SSC-H, SSC-W plot top right panel. Cells postive for all three gates were sorted into ELISPOT plastes. 

Tiller (J of Immunological Methods 329 (2008) 112-124) discloses starting with human samples, isolating the monocuclear cells from peripheral venous blood or bone marrow after enrichemnt with RosetteSep human B cell enrichment cocktail and purified by Ficoll-Paue (GE Healthcare) density gradeint centriguation. If necessary for example due to low B cell counts, B cells were enrched using anti-CD19 magnetic beads. Purified monocnulcear cells were stained on ice with anti-human antibodies directely coupled to FITC, PE and APC to distinguish amont individual B cell subpopulations. 

Urech (US 2010/0216170) discloses a FACS screening method for selecting B cells that bind to a target of interest. Specifically, lymphocytes suspension was prepared form spleen of rabbits immunized with the recombinant target. Cells were then incubated with PE and PerCP labeled scFv (single chain anti-VEGF antibody ESBA903) as well as antibody specific for IgG (APC labeled) or IgM (FITX labeded). ESBA903 positive B cells that expressed IgG but not IgM on their surface were sorted and selected in 96 well plates. 

B cell culture methods and Isolation

Carvalho Jensen (US 2012/0141982) discloses a method fo preparing a cell population that includes at least one antigen specific B cells by immunizing a host against an antigen, harvesting B cells from the host such as from the spleen, lymph nodes bone maarrow and PBMCs, enriching the harvested B cells to increase the frequency of antigen-specific cells, creating at least one single cell suspension, culturing a subpopulaiton form the single cell suspension and determining whether the B cell produces an antibody specific to the antigen. 

Use of Particular Compositions for Identifying B cells

Use of Virus-like Particles (VLPs) for selection: (see also “immunogenicity” on right hand panel under “vaccination”)

Weitkamp (J Immun. Methods 275, 2003, 223-237) disclose using structural virus proteins from rotavirus (RV) which self assemble into virus like particles (VLP) when coexpressed in the proper combinations to identify and isolate specific B cells from healthy or recently infected adults.

Antigen-Avidin Complexes:  

Diamond (US2005/0287607) discloses antigen-=avidin complexes for labeling a B cell reactive to a specific antigen, islating a B cell reactive to a specific antigen from a mixture of B cells. 

Warrington (WO 90/00625) discloses a pre-screening method for enrichment of specific B cells prior to immortilization by fusing with myeloma cells. In the depletion method, antigens are used to select for certin B cells in a population such that the non-reactive B cells in the supernatant are collection. In the enrichment procedure, B cells which one wishes to select can be boudn directly.