A Toolbox of IgG Subclass-Switched Recombinant Monoclonal Antibodies for Enhanced Multiplex Immunolabeling of Brain

Abstract

24Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers 25 numerous advantages that increase the effectiveness, reproducibility, and transparent reporting 26 of research. We report here the generation of a novel resource in the form of a library of 27 recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) 28 variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline 29 for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, 30 we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction 31 enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the 32 IgG subclasses without altering target binding specificity to generate R-mAbs useful in 33 simultaneous multiplex labeling experiments not previously possible. The method was also 34 employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable 35 cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated 36 from open source suppliers. 37Modern techniques employing Abs as immunolabels [e.g., immunoblotting (IB), 63 immunocytochemistry (ICC) and immunohistochemistry (IHC)] utilize simultaneous multiplexing 64 of numerous Abs to simultaneously detect multiple targets within a single cell or tissue sample. 65This allows for direct comparison of the relative amounts and respective characteristics of multiple 66 target molecules within the same sample, while reducing the number of samples needed to 67 accomplish a comprehensive analysis. Typically, the individual primary Abs bound to the sample 68 are detected with secondary antibodies conjugated to distinct reporters, most commonly organic 69 fluorescent dyes, although enzymes, gold particles, etc. are also routinely employed as detection 70 modalities. Multiplex labeling is often accomplished using Abs raised in distinct species, with their 71 subsequent individual detection accomplished using species-specific secondary antibodies. 72However, mouse mAbs offer an important advantage for multiplex labeling procedures. Each 73 mouse mAb is a single immunoglobulin (Ig) isotype, generally of the IgG class and if so specifically 74 of a single IgG subclass, most commonly IgG1, IgG2a or IgG2b. Mouse mAbs of distinct IgG 75 subclasses can be robustly, reliably and specifically detected with commercial subclass-specific 76 secondary antibodies, and, as such, can be multiplexed in a manner analogous to Abs from 77 different species [e.g., (14-18)]. One limitation to greater adoption of this approach is that mouse 78 mAb collections generally have an extremely high representation (≈70%) of IgG1 mAbs (18), 79 which limits the flexibility of multiplex labeling. The conversion of mAbs into R-mAbs allows for 80 their subsequent engineering into forms with properties distinct from their parent mAb, as is 81 routinely done to impact diverse aspects, including ...

Development, Screening, and Validation of Camelid‐Derived Nanobodies for Neuroscience Research

“…It is a common procedure to then extract the periplasmic proteins for recovery of the nAb prior to testing (Pardon et al., 2014). However, in an effort to work in a more high‐throughput manner, and parallel to the use of conditioned media from hybridomas or COS‐1 (i.e., tissue culture supernatants) for antibody screening (Andrews et al., 2019; Gong, Murray, & Trimmer, 2016), this protocol makes use of bacterial culture supernatants (BCS) for screening of the nAbs. The use of BCS eliminates the need to perform periplasmic protein extraction and nAb purification during the screening and validation phases, thus simplifying the workflow and increasing the number of nAbs that can be screened simultaneously.…”

An invariantTrypanosoma vivaxvaccine antigen inducing protective immunity

“…PCR products encoding the rearranged heavy and light chain regions were individually amplified using sets of degenerate oligonucleotides and then assembled in a subsequent fusion PCR using a linker fragment to create a single PCR product containing both the rearranged light and heavy chains, as previously described 45 . The fusion PCR product was ligated using the NotI and AscI restriction sites into an expression plasmid obtained from Addgene (plasmid # 114561) in frame with the mouse constant IgG2a heavy chain 46 . Competent E.coli were transformed and purified plasmids used in small-scale transfections of HEK293 cells to identify those plasmids encoding functional antibodies as described 47 .…”

Recombinant Antibodies in Basic Neuroscience Research

“…Example of an intact immunoglobulin G (IgG) form of recombinant monoclonal antibody (R‐mAb) expression plasmid. Schematic to the left shows the elements of the R‐mAb expression plasmid as developed by Gavin Wright and colleagues (Crosnier et al., 2010) that my group has used for our R‐mAb expression (Andrews et al., 2019). This plasmid allows for coexpression of H + L chains as driven by two CMV promoters (yellow).…”

“…This provides additional flexibility for pairing such R‐mAbs with other antibodies in simultaneous multiplex labeling experiments. My group has done this for a collection of mouse mAbs, allowing for their use in simultaneous multiplex labeling with subclass‐specific secondary antibodies (Manning, Bundros, & Trimmer, 2012) in combinations not possible with the progenitor conventional mAbs (Andrews et al., 2019). Rabbits do not have IgG subclasses (Knight, Burnett, & McNicholas, 1985), and thus engineering is required to distinguish one rabbit R‐mAb from another.…”

“…My group has used a dual promotor plasmid (Fig. 2) developed by Gavin Wright and colleagues (Crosnier, Staudt, & Wright, 2010) for our R‐mAb expression (Andrews et al., 2019).…”