Journal: bioRxiv

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

doi: 10.1101/483537

Figure Lengend Snippet: A. Schematic of cloning, expression and validation pipeline. Orange steps involve V H and V L 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 V L and V H domain PCR products are fused to a joining fragment comprising a κ light chain constant domain (C L ) and the κ light chain polyA tail sequences ( κ 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 (C H ) that is either γ 1 or γ 2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA).

Article Snippet: In preparation for fusion of the V L and V H 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: Clone Assay, Expressing, Plasmid Preparation, Derivative Assay, Sequencing, Translocation Assay

Journal: bioRxiv

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

doi: 10.1101/483537

Figure Lengend Snippet: A. Agarose gel analysis of V L and V H 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 V L and V H domains is ≈ 360 bp. B. Agarose gel analysis of V H and digested V L 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 7 kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. E. Agarose gel analysis of PCR products of V L 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 V L domains are ≈ 360 bp, and the digested aberrant light chains ≈ 180 bp.

Article Snippet: In preparation for fusion of the V L and V H 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: Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

Journal: bioRxiv

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

doi: 10.1101/483537

Figure Lengend Snippet: A. Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈ 360 bp, and of the cleaved aberrant V L domain is ≈ 180 bp. B. Agarose gel analysis of D3/71 V H and digested V L 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 7 kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp.

Article Snippet: In preparation for fusion of the V L and V H 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: Agarose Gel Electrophoresis, Amplification, Synthesized, Derivative Assay, Plasmid Preparation