Sequencing and cloning of antigen-specific antibodies from mouse memory B cells

Abstract

Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico

“…Antibody heavy (IGH) and light (IGL) chain genes were amplified from single purified ZEDIII binding B cells by RT-PCR and sequenced (Scheid et al, 2009; von Boehmer et al, 2016). Overall, 290 antibodies were identified from the 6 individuals.…”

“…B cell purification, labeling and antibody discovery were performed as previously described in detail (Tiller et al, 2008; von Boehmer et al, 2016), with the following modifications. PBMCs were resuscitated and washed in 37°C RPMI.…”

“…RNA from single cells was reverse-transcribed using random primers (von Boehmer et al, 2016), followed by nested PCR amplifications and sequencing using the primers listed in (Tiller et al, 2008). V(D)J gene segment assignment and determination of the CDR3 sequences were with IgBlast (Ye et al, 2013).…”

Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site

“…Sequence analysis was performed to identify Ig gene usage, HC and LC CDR3s, and the number of V H -V L somatic hypermutations (IgBLAST, http://www.ncbi.nlm.nih.gov/igblast/ and IMGT, http://www.imgt.org/). For cloning by PCR, the primers contained 15–20 base pairs of 5′ and 3′ homology to restriction-digested expression vectors to allow for cloning by homologous recombination into DH5α, as previously described 49 .…”

Exploring Human Antimicrobial Antibody Responses on a Single B Cell Level

“…Murine antibodies were also shown to trigger immunogenic responses, provide less stability, and hamper downstream Fc effector functions. On the basis of all of these pitfalls, our laboratory and those of others use antigen-dependent B cell sorting for the isolation of a variety of different pathogen-specific molecules for a straightforward MAb isolation within a few weeks (4,30). For the detection of antigen-specific B cell populations, the antigen must be labeled either by fluorescently labeled streptavidin or by another fluorescent tag.…”