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

Abstract

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.

Keywords: antibodies; brain; immunohistochemistry; mouse; neuroscience; rat.

Figure 1.. Schematic representation of the R-mAb pipeline.

(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 γ1 or γ2a depending on the plasmid, followed by the SV40 polyA tail (SV40 pA).

Figure 2.. Cloning of V_L and V_H domain sequences from hybridomas into the R-mAb expression plasmid.

(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 seven 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.

Figure 3.. Validation of subclass-switched anti-PSD-95 K28/43R R-mAb.

(A) Validation of the K28/43R R-mAb in heterologous cells. COS-1 cells transiently transfected to express human PSD-95 in a subset of cells were immunolabeled with K28/43 mAb (IgG2a) alone (top row), K28/43R R-mAb (IgG1) alone (middle row), or K28/43 mAb plus K28/43R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG2a (red, for the K28/43 mAb) and anti-mouse IgG1 (green, for the K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 µm and holds for all panels in A. (B) Validation of the K28/43R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K28/43 mAb plus K28/43R R-mAb and immunolabeling detected with a cocktail of anti-mouse IgG2a (red, for K28/43 mAb) and anti-mouse IgG1 (green, for K28/43R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). The region of interest shown is from cerebellar cortex. Scale bar in the left panel = 100 µm, and in the right merged panel = 30 µm. (C) Immunoblots against brain membranes and COS cell lysates over-expressing various members of the MAGUK superfamily of scaffolding proteins. To confirm expression of MAGUK proteins, immunoblots were probed with rabbit anti-PSD-95 (red). K28/86 is an anti-MAGUK mAb. Primary antibodies were detected with the appropriate combinations of fluorescently labeled species-specific anti-rabbit and subclass-specific anti-mouse IgG secondary Abs as indicated. Control indicates COS cells transfected with an empty vector.

Figure 4.. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain.

(A) A section from neocortex labeled with anti-pan-Nav R-mAb K58/35R (IgG2a, red) at nodes of Ranvier and AIS (arrows), anti-CASPR mAb K65/35 (IgG1, green) at paranodes, and anti-Kv2.1 rabbit polyclonal (KC) antibody (blue) on somata and proximal dendrites. Scale bar = 150 μm. Insets (dashed box) show details of labeling for pan-Nav (red) and CASPR (green) at the node and paranodes (arrows), respectively, at a single node of Ranvier as indicated by box in main panel. (B) A section through cerebellum showing labeling with anti-GABA-AR β1 R-mAb N96/55R (IgG2a, magenta) in the molecular layer (ML), and anti-GABA-AR β3 mAb N87/25 (IgG1, green) in the granule cell layer (GL). PCL = Purkinje cell layer. Scale bar = 150 μm. (C) A section through hippocampus labeled with anti-Kv2.1 R-mAb 89/34R (IgG2a, red) on somata and proximal dendrites, anti-AnkyrinG mAb N106/65 (IgG2b, green) on AIS, and nuclear stain Hoechst 33258 (blue). Scale bar = 150 μm. Panels C1-C3 show magnified details of labeling for pan-Kv2.1 (red) on somata and proximal dendrites (arrows in C1), and anti-AnkyrinG (green) on AIS (arrows in C2). Scale bar = 50 μm (C1–C3). (D) A section through cerebellum labelled with anti-GABA-AR α6 R-mAb K229A/32R (IgG2a, red) in the granule cell layer (GL), anti-pan-QKI mAb N147/6 (IgG2b, green) labeling glial cells in/near the Purkinje cell layer (PCL), and anti-Kv4.2 mAb K57/1 (IgG1, blue) labeling the granule cell layer (GL). Scale bar = 30 μm. (E) A section from neocortex labelled with anti-Kv2.1 R-mAb 89/34R (IgG2a, red) on somata and proximal dendrites of neurons, and anti-GFAP mAb N206A/8 (IgG1, green) and anti-pan-QKI mAb N147/6 (IgG2b, blue) labeling glial cell processes and cell bodies respectively. Scale bar = 15 μm. (F) A section through cerebellum showing labeling with anti-PSD-93 R-mAb N18/30R (IgG2a, red) in the cell bodies and dendrites of Purkinje cells, the nuclear stain Hoechst 33258 (green) and anti-VGluT1 mAb N28/9 (IgG1, blue) in the molecular layer (ML). PCL = Purkinje cell layer. Scale bar = 10 μm.

Figure 5.. Cloning of anti-Kv2.1 D3/71 V_L and V_H domain cDNAs from a nonviable hybridoma.

(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 seven kbp, and the intact insert comprising the V_L and V_H domains and the intervening joining fragment is 2.4 kbp.

Figure 6.. Recovery of a functional anti-Kv2.1 D3/71R R-mAb from nonviable hybridomas.

(A) Validation of the D3/71R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), D3/71R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus D3/71R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. (B) Validation of the subclass-switched K89/34R R-mAb in heterologous cells. COS-1 cells transiently transfected to express rat Kv2.1 in a subset of cells were immunolabeled with K89/34 mAb (IgG1) alone (top row), K89/34R R-mAb (IgG2a) alone (middle row), or K89/34 mAb plus K89/34R R-mAb (bottom row). Immunolabeling in all samples was detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the K89/34R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Labeling in blue in panels A and B is for the DNA-specific dye Hoechst 33258 and shows nuclei of both transfected and untransfected cells. Scale bar in the lower right merged panel = 30 µm and holds for all panels in A and B. (C) Validation of the D3/71R R-mAb in brain sections. A brain section from an adult rat was immunolabeled with K89/34 mAb plus D3/71 R-mAb and the immunolabeling detected with a cocktail of anti-mouse IgG1 (green, for the K89/34 mAb) and anti-mouse IgG2a (red, for the D3/71R R-mAb) subclass-specific Alexa Fluor conjugated secondary antibodies. Cell nuclei are labeled with the DNA-specific dye Hoechst 33258 (blue). Region of interest shown is from neocortex. Scale bar = 30 µm. (D) Strip immunoblots on a crude rat brain membrane fraction immunolabeled with the K89/34 mAb, the K89/34R R-mAb, and the D3/71 R-mAb as indicated. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and ECL.