Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 1. Schematic representation of the R-mAb pipeline. (A) Schematic of cloning, expression and validation pipeline. Orange steps involve VH and VL regions of individual hybridomas, blue steps involve steps involving backbone components, and green step involves expression of target for R-mAb validation. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters (orange). Hybridoma-derived VL and VH domain PCR products are fused to a joining fragment comprising a k light chain constant domain (CL) and the k light chain polyA tail sequences (k pA), a CMV promoter for heavy chain expression, and an ER signal/leader sequence (L) for translocation of the heavy chain across the ER membrane. PCR-mediated fusion of these three elements is followed by their insertion into the p1316 plasmid that contains an upstream CMV promoter for light chain expression, and an ER signal/leader sequence (L) for translocation of the light chain across the ER membrane. Downstream of the insert is a heavy chain constant domain (CH) that is either g1 or g2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA). DOI: https://doi.org/10.7554/eLife.43322.003

Article Snippet: In preparation for fusion of the VL and VH PCR products, a joining fragment was produced using the mouse Ig expression plasmid P1316 (a gift of Dr. Gavin Wright, Sanger Institute, Cambridge, UK, now available from Addgene as plasmid #28217).

Techniques: Cloning, Expressing, Biomarker Discovery, Plasmid Preparation, Derivative Assay, Sequencing, Translocation Assay, Membrane

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 2. Cloning of VL and VH domain sequences from hybridomas into the R-mAb expression plasmid. (A) Agarose gel analysis of VL and VH domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG VL and VH domains is » 360 bp. (B) Agarose gel analysis of VH and digested VL fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. (E) Agarose gel analysis of PCR products of VL domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact VL domains are » 360 bp, and the digested aberrant light chains » 180 bp. DOI: https://doi.org/10.7554/eLife.43322.004

Article Snippet: In preparation for fusion of the VL and VH PCR products, a joining fragment was produced using the mouse Ig expression plasmid P1316 (a gift of Dr. Gavin Wright, Sanger Institute, Cambridge, UK, now available from Addgene as plasmid #28217).

Techniques: Cloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/elife.43322

Figure Lengend Snippet: Figure 5. Cloning of anti-Kv2.1 D3/71 VL and VH domain cDNAs from a nonviable hybridoma. (A) Agarose gel analysis of PCR amplified VL and VH domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the VL after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG VL and VH domains is » 360 bp, and of the cleaved aberrant VL domain is » 180 bp. (B) Agarose gel analysis of D3/71 VH and digested VL fragments joined by fusion PCR (F- PCR) to the P1316 joining fragment to create a dual IgG chain cassette. (C) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. (D) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the VL and VH domains and the intervening joining fragment is 2.4 kbp. DOI: https://doi.org/10.7554/eLife.43322.008

Article Snippet: In preparation for fusion of the VL and VH PCR products, a joining fragment was produced using the mouse Ig expression plasmid P1316 (a gift of Dr. Gavin Wright, Sanger Institute, Cambridge, UK, now available from Addgene as plasmid #28217).

Techniques: Cloning, Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation