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

<jats:p>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.</jats:p>

section_content: Antibodies (Abs) are the workhorses of biomedical research. Enhancing the research community's access to extensively validated Abs remains an important goal of antibody developers in both academia and industry (1). This has been the topic of a number of recent conferences and commentaries, led to changes in journal practices as related to transparent reporting of antibody-based research including the use of RRIDs, and resulted in large-scale NIH and EU Ab development initiatives (e. g., the NIH Common Fund Protein Capture Reagent Program, the EU Affinomics Program). It is widely recognized that production of Abs in a renewable form, such as monoclonal antibodies (mAbs), represents a substantial advance over polyclonal Abs from antisera that are available in finite quantity, and that comprise a heterogeneous and less definable population of Abs (2) (3) (4). Having mAbs available in recombinant form as recombinant mAbs (R-mAbs) offers numerous additional advantages (5). Recombinant expression systems allow for the reliable production of an R-mAb not prone to loss by genetic instability of tetra-or hexa-ploid hybridoma cells or other factors (6, 7). Moreover, unlike hybridoma cell lines that can express multiple functional heavy and light immunoglobulin chains (8) (9) (10), recombinant expression ensures production of a single, molecularly defined R-mAb. 

Recombinant expression can also yield production levels 100s or 1000s of times higher than is possible with endogenous expressions of mAbs from hybridoma cells [e. g., (11) (12) (13), etc. ]. 

Furthermore, the cloning of R-mAbs provides for permanent, dependable and inexpensive archiving of the R-mAb as plasmid DNA and nucleic acid sequences versus an archiving system relying on expensive cryopreservation of hybridoma cell lines in liquid nitrogen, and their subsequent recovery as viable cell cultures. The conversion of existing mAbs to R-mAbs also allows for more effective dissemination of R-mAbs as plasmids, bacterial stocks or as DNA sequences. However, in spite of these advantages, it remains that most mAbs used for research are produced by hybridomas in culture. 

Modern techniques employing Abs as immunolabels [e. g., immunoblotting (IB), immunocytochemistry (ICC) and immunohistochemistry (IHC)] utilize simultaneous multiplexing of numerous Abs to simultaneously detect multiple targets within a single cell or tissue sample. This allows for direct comparison of the relative amounts and respective characteristics of multiple target molecules within the same sample, while reducing the number of samples needed to accomplish a comprehensive analysis. Typically, the individual primary Abs bound to the sample are detected with secondary antibodies conjugated to distinct reporters, most commonly organic fluorescent dyes, although enzymes, gold particles, etc. are also routinely employed as detection modalities. Multiplex labeling is often accomplished using Abs raised in distinct species, with their subsequent individual detection accomplished using species-specific secondary antibodies. However, mouse mAbs offer an important advantage for multiplex labeling procedures. Each mouse mAb is a single immunoglobulin (Ig) isotype, generally of the IgG class and if so specifically of a single IgG subclass, most commonly IgG1, IgG2a or IgG2b. Mouse mAbs of distinct IgG subclasses can be robustly, reliably and specifically detected with commercial subclass-specific secondary antibodies, and, as such, can be multiplexed in a manner analogous to Abs from different species [e. g., (14) (15) (16) (17) (18) ]. One limitation to greater adoption of this approach is that mouse mAb collections generally have an extremely high representation (≈70%) of IgG1 mAbs (18), which limits the flexibility of multiplex labeling. The conversion of mAbs into R-mAbs allows for their subsequent engineering into forms with properties distinct from their parent mAb, as is routinely done to impact diverse aspects, including target binding affinity, of therapeutic R-mAbs (19). Such engineering could also include switching the heavy chain constant region to impact subclass-specific secondary Ab binding specificity, an approach similar to that used to successfully modify subclass-specific in vivo effector functions (20). 

There are numerous routes to obtaining validated R-mAbs, including their de novo generation from high complexity immune repertoire libraries produced from naïve or immunized animals, combined with selection of target-specific R-mAbs by in vitro display (21). Alternatively, R-mAbs can be generated from existing hybridoma cell lines, which express well-characterized mAbs [e. g., (22) ]. Here we undertook conversion of a widely used collection of hybridomagenerated mAbs extensively validated for neuroscience research applications (14, 23, 24) into recombinant form. We developed a coherent pipeline of protocols for effective cloning of intact R-mAbs from cryopreserved hybridoma cells and their subsequent validation compared to their parent mAbs. Further, we developed a process to engineer these R-mAbs to IgG subclassswitched forms that provide additional utility for multiplex labeling employing mouse IgG subclassspecific secondary Abs. This approach is feasible and relatively inexpensive for any laboratory that uses standard molecular biology and mammalian cell culture techniques. This approach also represents a reliable method to convert valuable cryopreserved hybridoma collections to the immortalized form of a DNA sequence archive, including hybridomas that are no longer viable in cell culture.
section_name: Introduction

section_name: Results

section_content: We previously generated a large library of mouse mAbs that have been extensively validated for efficacy and specificity for immunolabeling endogenous target proteins in mammalian brain samples in immunoblotting (IB) and immunohistochemistry (IHC) applications (14, 23, 24). Here, we undertook the systematic conversion of a sizable subset of this existing mAb collection to R-mAbs. We developed an innovative pipeline for R-mAb cloning, expression and validation. For the cloning steps, we built upon a previously described method (22, 25) to clone IgG VH and VL region sequences, but with the modification that we cloned directly from cryopreserved hybridomas, without the need for their labor-intensive recovery into cell culture. Our overall cloning strategy (Figure 1A ) employs PCR-mediated amplification to generate IgG VH and VL region sequences from hybridoma-derived cDNA, followed by PCR-based fusion of these VH and VL regions with a joining fragment that contains a subset of the elements needed for high level expression of R-mAbs from transfected mammalian cell lines. The product of this fusion PCR reaction is then inserted into a plasmid containing the remainder of the elements for propagation in bacteria and expression of intact heavy and light chains in, and secretion of R-mAbs from, transfected mammalian cells (Figure 1B ). 

We extracted RNA from cryopreserved hybridoma cell vials, followed by first strand cDNA synthesis and RT-PCR of the IgG VH and VL region sequences (Figure 1A ). To enhance throughput, we performed the first three steps of this process in a 96 well plate format. We obtained hybridoma cells directly from cryovials and added an aliquot of 1-3 x 10 6 cells to individual wells of a commercially available 96 well RNA extraction plate. After performing the RNA isolation procedure in the 96 well plate, we replica plated 1/16 of the extract volume to a second 96 well plate for first strand cDNA synthesis. Following the cDNA synthesis reaction, we replica plated ≈5% of the volume of the cDNA synthesis product to a third 96 well plate to serve as the template for PCR amplification of the IgG VH and VL domain sequences. As such, the templates for the VH and VL domain PCR amplification represented the cDNA yield from ≈3,000-7,500 hybridoma cells. We amplified the IgG VL kappa (κ) and VH domain sequences from the hybridoma cDNA templates using a degenerate mouse Ig variable region primer set developed by Gavin Wright and colleagues (22) expanding on a previously used set (33) that at the time of their design recognized 97% and 98% of known functional heavy and kappa (κ) light chain sequences, respectively. Lambda light chains were not targeted for amplification because they constitute only a small percentage of light chains used in mouse immunoglobulins (34, 35). This PCR amplification reliably gave products of the expected 360 base pairs (bp) for both the VL and VH domain sequences, examples of which are shown in Figure 2A for representative mAbs N59/36 ("N59", anti-NR2B/GRIN2B glutamate receptor) and K39/25 ("K39", anti-Kv2. 1/KCNB1 potassium channel). 

To permit cloning of both the VH and VL regions into a single expression vector, fusion PCR (F-PCR) was performed using as templates the VH and VL PCR products, as well as a joining fragment amplified from the P1316 expression plasmid (22, 25) to produce a 2. 4 kbp amplicon (Figure 2B ). The joining fragment (Figure 1B ) contains kappa light chain constant region sequences and an associated polyadenylation signal, followed by a CMV promoter to drive VH expression, and a VH leader sequence (22, 25). The F-PCR reaction products were then treated with NotI and AscI restriction enzymes and purified in preparation for cloning into a NotI/AscI restriction enzyme-digested fragment of the P1316 expression plasmid (Figure 1B ). Upstream of the NotI cloning site, the digested P1316 fragment contains a CMV promoter to drive light chain expression and a VL leader sequence, and downstream of the AscI cloning site a mouse IgG1 CH sequence, and a polyadenylation signal (Figure 1B ). Standard methods were used for DNA ligation and subsequent transformation of E. coli. Clones expressing the full-length IgG expression cassette were identified by colony PCR (Figure 2C ). Following NotI/AscI restriction digestion to verify the correct insert size (Figure 2D ), these plasmid clones were subjected to further analysis (Figure 1A ), including expression in mammalian cells, and R-mAb validation and sequencing as detailed in the subsequent sections. 

While a number of bona fide R-mAbs were isolated using this approach, we found a high degree of variability in the number of colony PCR-and restriction enzyme digest-verified positive plasmids that yielded functional expression. A major obstacle in cloning functionally rearranged IgG sequences from many mouse hybridomas is the presence of an aberrant kappa IgG light chain transcript expressed by the Sp2/0-Ag14 (Sp2/0) hybridoma (36) that frequently serves as a "myeloma" partner for fusion with mouse splenocytes to generate mAb-producing hybridomas (37), and that was used as the fusion partner in all of our mAb generation efforts (14, 24). The source of this non-productive IgG light chain is the MOPC-21 myeloma cell line used to generate the Sp2/0 hybridoma (37). As we experienced, and as previously reported by others (36), aberrant chain mRNA expression varies greatly among distinct hybridoma cell lines but in certain cases can exceed the levels of functional light chain transcripts. For certain of our projects, this resulted in >90% of the colony PCR positive clones failing to produce detectable levels of functional R-mAbs, thus necessitating a high volume of screening. 

As such, we sought to eliminate this aberrant light chain during the cloning process. We treated the VL PCR products with the restriction enzyme BciVI, as the restriction site for this enzyme is present in the VL region of the aberrant Sp2/0-derived transcript, but is predicted to occur at a low frequency in functional mouse VL kappa sequences (38). We used VL PCR products derived from the Sp2/0 cell line and from pooled BALB/c mouse splenocytes as positive and negative controls, respectively, for sensitivity to BciVI digestion. Due to the exclusive presence of aberrant light chain in Sp2/0 cells, VL PCR products from these cells were completely digested, as shown by the decreased size of the VL PCR products from ≈360 bp typical of VL PCR products (see Figure 2A for examples) to the ≈180 bp fragment that results from BciVI digestion (Figure 2E ). In contrast, the sample prepared from the pooled mouse splenocytes was not detectably affected by BciVI digestion (Figure 2E ). Treatment of VL PCR products from various Sp2/0-derived hybridomas with BciVI resulted in varying degrees of digestion, yielding different proportions of the bands representing the intact VL PCR product of ≈360 bp and the cleaved aberrant SP2/0derived VL fragment of ≈180 bp (Figure 2E ). After BciVI digestion was incorporated into the protocol, DNA sequencing of 149 colony PCR-positive clones from 26 different hybridomas revealed that only 12 (8%) still contained the aberrant Vk light chain (Table 1 ). In certain cases, digestion of hybridoma VL PCR products resulted in fragments of unexpected sizes (see the K58/35 lane in Figure 2E ), indicating, as predicted by an earlier bioinformatics analysis (38), that in rare cases (in our hands, 3/248 clones pursued to this step) the BciVI restriction site was also present in these functionally rearranged splenocyte-derived VL genes. As such, we attempted to clone these VL PCR products without BciVI treatment. As one example, the splenocyte-derived Vk light chain PCR products from the K58/35 hybridoma were sensitive to BciVI digestion, which necessitated their cloning in the absence of the BciVI digestion step. This project yielded somewhat lower frequency of clones that produced functional R-mAbs able to detect target antigen (≈37%) than, on average, those containing splenocyte-derived VL PCR products refractory to BciVI digestion (≈48%; Table 2 ). For the bulk of mAbs encoded by splenocyte-derived VL PCR products resistant to BciVI digestion, ligations were performed following BciVI digestion, and 10-14 candidate clones that were colony PCR-positive were selected. Plasmid DNA was digested with AscI/NotI restriction enzymes to confirm the correct insert (Figure 2D ). On average, ≈93% of all clones subjected to restriction analysis passed this screening step. 

We next transfected the colony PCR and restriction digest-validated plasmids into COS-1 cells cultured in 12 well or 96 well tissue culture plates. After a 3 to 6 day incubation, we tested the conditioned culture media for production of target-specific R-mAbs. While the parent mAbs had previously been validated for efficacy and specificity in a variety of applications (IF-ICC, IB and IHC on brain samples), we selected the IF-ICC assay in transiently transfected heterologous cells for R-mAb validation. This method was chosen because it is high-throughput, employing 96 well microtiter plates, it requires only a small amount of R-mAb sample, and the robust difference between target-expressing and non-expressing cells in the same sample allows for sensitivity and clarity of results. Importantly, each of the parent mAbs had been previously validated in this procedure. We took advantage of the fact that in most cases we switched the IgG subclass during conversion of the hybridoma-derived mAb into the corresponding R-mAb, allowing for their separate detection by subclass-specific secondary Abs (18). This assay involved expressing the full-length target protein in transiently transfected COS-1 cells cultured in individual wells of a black polystyrene 96 well clear bottom plate that allows for microscopic visual analysis and imaging using indirect immunofluorescence. For each of 1 to 15 R-mAb candidate clones to be assayed from a given project, a set of replicate wells were prepared expressing the given target protein. After fixation and permeabilization, the individual wells were then immunolabeled with either the hybridoma-generated mAb alone, the R-mAb alone, or the mAb and R-mAb together. 

Each well was subsequently incubated with a cocktail of the two distinct subclass-specific secondary Abs, one specific for the respective mAb and one specific for the subclass-switched R-mAb mouse IgG subclasses, and both conjugated to spectrally distinct Alexa Fluors. The "mAb only" well was used to demonstrate that the target protein was expressed in a subset of the transiently transfected cells, and that the only detectable secondary Ab labeling was for the IgG subclass of the parent hybridoma-generated mAb. Similarly, the R-mAb only wells were used to show that the R-mAb labeled a comparable number of cells, and that the only detectable secondary Ab labeling was for the IgG subclass of the subclass-switched R-mAb. The wells containing both the parent mAb and candidate R-mAb were used to show that the mAb and R-mAb gave indistinguishable labeling patterns at both the cellular and subcellular level and could be detected separately using subclass-specific secondary Abs. Numerous examples of this assay are shown in the following sections focusing on specific R-mAb projects. We note that only in rare cases was the labeling for one or both secondaries noticeably depressed in the well containing both mAb and R-mAb relative to the wells with these primary antibodies alone, as would occur due to competitive binding to the same epitope. However, this was generally not apparent, suggesting that in this assay system, in which the target protein was overexpressed, we were typically operating under conditions of antigen excess. 

In comparing the overall results from 180 recent projects that had BciVI-resistant splenocyte-derived VL PCR products, and that were taken through this entire pipeline one time (Table 2 ), we found that a range of 1-15 colony PCR-and restriction digest-positive candidate R-mAbs per project were evaluated in the COS-ICC assay (919 total, mean/project = 5. 11 ± 0. 23 S. E. M. ). For these 180 projects, cloning was performed after BciVI digestion, and all restriction digest-validated candidates were tested for functional mAb production. Of these 180 projects, 72% (129 projects) yielded at least one positive R-mAb on their first pass through the pipeline. A retrospective analysis of these 129 successful projects revealed a range of 1 to 13 restriction digest-validated candidates were evaluated (mean/project = 5. 74 ± 0. 26 S. E. M. ), with an overall success rate of 48. 4% for all 741 restriction digest-validated candidate R-mAbs tested. A parallel analysis of the 51 projects that did not yield a positive R-mAb on their first pass through the pipeline revealed a similar range of 1 to 15 restriction digest-validated candidates per project evaluated. However, an overall lower number of colony PCR-and restriction digest-positive candidates were evaluated in the COS-ICC assay (178 total, mean/project = 3. 5 ± 0. 37 S. E. M. ) in these projects than for the successful projects. A statistically significant difference (two-tailed P value = 2. 48 x 10 -6 ) existed between the number of candidates evaluated in successful versus unsuccessful projects. The per-project COS-ICC rate success was impacted by number of clones tested. For 1-3 clones tested (66 projects), the project success rate was 50%, for 4-6 (62 projects) it was ≈ 77%, and for ³ 7 (52 projects) it was ≈92%. However, the per clone success rate was similar between the three bins (≈ 45% vs. ≈39% vs. ≈ 45%). 

Following validation in the COS-ICC assay, a subset of positive clones for each project was subjected to DNA sequencing. We employed a set of sequencing primers that allowed for determination of sense and antisense strands of the VH and VL domain-encoding cDNA inserts that were unique to each R-mAb. The sequences were searched against the NCBI database, and against a custom database that contained the VH and VL domain sequences of the parent P1316 plasmid, the VL domain of the aberrant Sp2/0 cell line, and the VH and VL domain sequences of all of the R-mAbs we had cloned to date. The set of COS-ICC validated R-mAb plasmids derived from a single hybridoma that had matching sequences unique from any sequences in the custom database were subsequently archived as frozen plasmid DNA and bacterial glycerol stocks, and their sequence used as the archival sequence of that particular R-mAb.
section_name: Effective Cloning of Immunoglobulin VH and VL Regions from Cryopreserved Hybridomas and Generation of R-mAbs

section_content: We initiated our R-mAb cloning efforts with the K28/43 mAb, a mouse mAb specific for the neural scaffolding protein PSD-95. This mAb is widely used as a marker of excitatory synapses (e. g., as of 7/1/18 ≈500 publications have cited the use of the K28/43 mAb as obtained from the UC Davis/NIH NeuroMab Facility alone). While the K28/43 mAb is already of a less common IgG subclass (IgG2a), generating a subclass switched version with an alternate IgG subclass would provide greater flexibility in its use in multiplex labeling experiments, and also provide proof of concept that we could use our process to effectively generate subclass switched R-mAbs with efficacy and specificity comparable to the parent mAb. We generated VL and VH domain cDNA fragments from the cryopreserved K28/43 hybridoma and cloned them into the original P1316 plasmid that contains a mouse IgG1 CH domain (22). COS-1 cells were transiently transfected with the resultant plasmids. We evaluated the conditioned medium from the transfected cells for the presence of the subclass-switched K28/43R IgG1 R-mAb by ICC against fixed and permeabilized transiently transfected COS-1 cells expressing full-length PSD-95 (Figure 3A ). We note that we have designated the recombinant R-mAb versions of each of our mAbs by a capital "R" after the clone designation, in this case the R-mAb cloned from the K28/43 hybridoma is designated K28/43R. We screened the COS-1 cell-generated R-mAbs for functional R-mAb immunoreactivity in the 96-well IF-ICC assay detailed in the previous section. After primary antibody labeling, all wells received both IgG1-and IgG2a-subclass-specific, fluorescently labeled secondary antibodies. As shown in Figure 3A, as expected, the sample receiving only the native K28/43 hybridoma-generated mAb exhibited a signal corresponding to the IgG2a subclassspecific secondary Ab (red) with no detectable signal for the IgG1 subclass-specific secondary Ab (green). Conversely, labeling with the K28/43R R-mAb alone produced only an IgG1 subclassspecific green signal demonstrating a successful IgG subclass switch for the R-mAb (Figure 3A ). 

Simultaneous multiplex labeling with the hybridoma-generated mAb and a positive R-mAb resulted in an identical pattern of immunolabeling at the cellular level in the specific cells recognized, and at the subcellular level as to the pattern of immunolabeling within the labeled cells, as shown by the uniform hue of the signal in the merged panel, indicating that both the mAb and R-mAb were recognizing the same target (Figure 3A ). We next performed multiplex immunofluorescent IHC on adult mouse brain sections with the K28/43 mAb and the K28/43R R-mAb. As shown in Figure 3B, as detected with secondary Abs specific for their respective mouse IgG subclasses, the signal from these two primary antibodies was indistinguishable in its laminar and subcellular pattern in cerebellar cortex, consistent with previous studies of PSD-95 [e. g., (39) ]. Both signals were especially intense in the terminal pinceau of basket cells located adjacent to the Purkinje cell layer, and both signals were also present in the molecular layer, and for the most part lacking in the granule cell layer (Figure 3B ). This demonstrates that consistent with the validation in heterologous COS-1 cells expressing exogenous PSD-95, the K28/43 R-mAb can be used reliably for multiplex immunolabeling of endogenous PSD-95 in brain sections. 

The specificity of recombinant K28/43R was also assessed on immunoblots of samples from COS-1 cells exogenously expressing various representatives of the MAGUK superfamily of scaffolding proteins, of which PSD-95 is one member (Figure 3C-D ). Immunoblots were probed with a rabbit polyclonal antibody raised against PSD-95 and that also cross-reacts with SAP97 as a positive control, and recombinant K28/43R in either the IgG1 or IgG2a mouse IgG subclass form (Figure 3C-D ). Generation of the IgG2a expression plasmid is described below. Both the IgG1 and IgG2a subclass isoforms of the K28/43R R-mAb gave identical immunolabeling patterns against samples from rat brain and COS-1 cells overexpressing PSD-95. Moreover, the pattern of R-mAb immunolabeling was similar to that obtained with the rabbit polyclonal antibody, and absent against samples of COS-1 cells exogenously expressing other MAGUK superfamily members (Figure 3C-D ). To confirm expression of these MAGUK proteins, immunoblots were probed with rabbit polyclonal anti-PSD-95 and with the hybridoma-generated K28/43 mAb (IgG2a) or a mAb that recognizes all mammalian MAGUK proteins mAb (K28/86; IgG1) (27) (Figure 3E ). 

These initial results demonstrated that we could use this cloning and expression process to generate and validate subclass-switched R-mAbs that recapitulate the immunolabeling characteristics of the native mAbs.
section_name: Effective Cloning and IgG Subclass Switching of a Widely Used Monoclonal Antibody

section_content: Most (≈70%) mouse IgG mAbs are of the IgG1 subclass (18). Generating R-mAbs employing the efficient VH and VL cloning approach developed by Gavin Wright and colleagues includes their subsequent insertion into an expression plasmid that yields R-mAbs of the mouse IgG1 subclass due to the presence of the mouse g1 CH domain in the plasmid backbone (22). To generate R-mAbs of less common mouse IgG subclasses, we set out to modify this plasmid by replacing this g1 CH domain with a mouse g2a CH domain. We developed PCR primers to amplify the g2a CH domain sequence from cDNA generated from the hybridomas producing the K28/43 IgG2a mAb. 

We then replaced the g1 CH domain present in the K28/43R plasmid with this g2a CH domain using restriction digestion, gel isolation and ligation. This plasmid was sequenced verified and validated for expression of a K28/43R R-mAb of the IgG2a subclass (Figure 3C ). 

We subsequently used this plasmid as the target cloning vector for generation and expression of numerous R-mAb plasmids encoding functional IgG2a R-mAbs. Our strategy entailed cloning all native IgG1 and IgG2b mAbs into this plasmid to generate forms distinct from the parent native mAbs. Toward this end, we have successfully cloned and validated as functional R-mAbs a total of 178 mAbs (Table 3 ). This includes 148 mAbs of distinct subclasses converted to IgG2a R-mAbs (125 IgG1, 21 IgG2b, and 3 IgG3), and 29 IgG2a mAbs that were retained in their native IgG subclass (Table 3 ). We also converted the IgG2a mAb K28/43 to an IgG1 R-mAb. 

Each of these R-mAbs have been assigned unique RRID numbers in the Antibody Registry (Table 3 ) and all will be deposited in plasmid form at Addgene, a subset of which are already available (https://www. addgene. org/James_Trimmer/).
section_name: Generation of an R-mAb Expression Plasmid with an IgG2a CH Backbone Allows for Effective Subclass Switching of mAbs from Prevalent IgG1 Subclass

section_content: One benefit of subclass switching R-mAbs is the ability to perform multiplex immunolabeling previously not possible due to subclass conflicts. Examples of such enrichment in cellular protein localization are shown in Figure 4. Figure 4A shows labeling with the subclass switched IgG2a R-mAb derived from the widely used pan-voltage-gated sodium channel or "pan-Nav channel" IgG1 mAb K58/35 (40). Like the corresponding mAb, K58/35 R-mAb gives robust labeling of Nav channels concentrated on the axon initial segment (AIS, arrows in Figure 4A main panel), and at nodes of Ranvier (arrows in Figure 4A insets). Importantly, subclass switching allowed Nav channel labeling at nodes to be verified by co-labeling with K65/35 an IgG1 subclass antibody directed against CASPR, a protein concentrated at paranodes (41, 42). Similarly, simultaneous labeling for the highly-related GABA-A receptor b1 and b3 subunits (43) with their widely used respective mAbs N96/55 and N87/25 could not be performed as both are IgG1 subclass. 

Switching N96/55 to IgG2a allowed simultaneous detection of these two highly-related but distinct GABA-A receptor subunits. In Figure 4B localization of GABA-A receptor b1 and b3 subunits appeared completely non-overlapping in separate layers of cerebellum. Figure 4C illustrates localization of protein Kv2. 1 and AnkyrinG in separate subcellular neuronal compartments. 

Labeling for the K89/34R R-mAb (IgG2a, red), specific for the Kv2. 1 channel, highly expressed in the plasma membrane of the cell body and proximal dendrites (arrows in panel C1) is shown together with labeling for N106/65 (green), an IgG1 mAb specific for AnkyrinG, a scaffolding protein highly expressed in the AIS (arrows in panel C2) and at nodes of Ranvier. Subclass switching N229A/32 (IgG1, GABA-AR a6) to IgG2a, allows comparison with Kv4. 2 potassium channel (K57/1, IgG1) in the cerebellum where both are highly expressed in the granule cell layer (Figure 4D ). While both are prominently found in the glomerular synapses present on the dendrites of these cells, simultaneous labeling reveals that some cells express both (Figure 4D, magenta) while others appear to predominantly express Kv4. 2 (Figure 4D, blue). Labeling for both proteins is in contrast to that for mAb N147/6 (IgG2b) which recognizes all isoforms of the QKI transcription factor and labels oligodendrocytes within the granule cell layer and throughout the Purkinje cell layer (PCL, green). In Figure 4E, localization of pan-QKI (N147/6, IgG2b, blue) is compared with GFAP (N206A/8, IgG1, green) predominantly thought to be in astrocytes. Surprisingly many (but not all) cells co-label both proteins. We also labeled cortical neurons with these two mAbs (pan-QKI in blue, GFAP in green). Multiplex labeling for the neuron-specific Kv2. 1 channel, using subclass-switched K89/34R (IgG2a, red) confirms non-neuronal localization of both proteins. Lastly, we labeled for the postsynaptic scaffold protein PSD-93 using R-mAb N18/30R (IgG2a, red), which in the cerebellum is prominently localized to Purkinje cell somata and dendrites (44). As shown in Figure 4F, the R-mAb labeling is consistent with the established localization of PSD-93. Because of subclass switching the N18/30R R-mAb, this labeling can now be contrasted with labeling for the excitatory presynaptic terminal marker VGluT1, labeled with mAb N28/9 (IgG1, blue), which exhibits robust labeling of parallel fiber synapses in the molecular layer, and glomerular synapses in the granule cell layer. Together these results demonstrate the utility of employing subclass-switched R-mAbs to obtain labeling combinations not possible with native mAbs.
section_name: Multiplex brain immunofluorescent labeling with subclass switched R-mAbs

section_content: While not a common event, mAb-producing B cell hybridomas can lose their mAb production due to genetic instability (45) (46) (47), mycoplasma contamination leading to amino acid depletion and cytopathic effects (48), or other factors. Non-optimal cryopreservation due to problems during the freezing process itself or inadequate storage conditions can result in non-recoverable frozen seeds. As the method described here for cloning of VH and VL domain sequences from cryopreserved hybridomas does not require expansion of cells in culture prior to RNA extraction, we speculated that it may be possible to use this approach to generate functional R-mAbs from even those hybridoma cell lines that are no longer viable in cell culture. 

We had in our cryopreserved hybridoma archive a hybridoma cell line that produced the D3/71 mAb. This hybridoma cell line had been generated in 1994 among a series of projects targeting the Kv2. 1 voltage-gated potassium channel α subunit, projects that also yielded the D4/11 mAb (14). The D3/71 mAb has particular value in binding at a site distinct from other anti-Kv2. 1 mAbs. In particular, the binding site for the D3/71 mAb, but not most other available anti-Kv2. 1 mAbs (and pAbs), remains intact in truncated isoforms of Kv2. 1 found in patients with severe neurodevelopmental disorders linked to de novo frameshift or nonsense mutations in the KCNB1 gene (49, 50). Unlike our experience with other cryopreserved hybridomas in our collection, when we attempted to resuscitate this cryopreserved hybridoma cell line over 20 years after it was cryopreserved, the D3/71 hybridoma cells were no longer viable in cell culture. We extracted RNA from a cryopreserved vial of D3/71 hybridoma cells in an attempt to generate a functional D3/71R R-mAb from these non-viable hybridoma cells. Cloning was performed as described above including insertion of D3/71 VH and VL domain cDNA into the mouse IgG2a expression plasmid (Figs. 5A-B ). After bacterial transformation, most of the colonies tested (20/24) were positive for the insert by PCR (Figure 5C ). Restriction analysis of 12 PCR positive clones confirmed that they each contained the full-length VL-joining fragment-VH cassette (Figure 5D ). We next tested whether these plasmids encoded functional D3/71R R-mAb in double immunolabeling experiments in Kv2. 1-expressing transiently transfected COS-1 cells using a distinct native anti-Kv2. 1 IgG1 mAb K89/34 (51) as a control. We also included a comparison of D3/71R to the subclass-switched K89/34R R-mAb (31, 52) as an additional control. Two out of 12 clones exhibited specific immunolabeling of Kv2. 1-expressing cells as detected with an anti-IgG2a secondary antibody. An example for one of these is shown in Figure 6A. As shown for one of the positive clones, the recovered D3/71R R-mAbs exhibited immunolabeling that when detected with an IgG2a-specific secondary antibody (red) was indistinguishable at both the cellular level in the specific cells recognized, and at the subcellular level as to the pattern of immunolabeling within the labeled cells, to that obtained with the K89/34 IgG1 mAb detected with an IgG1-specific secondary antibody (green). Parallel experiments demonstrated successful subclass-switching of the K89/34 IgG1 mAb when converted into the K89/34R R-mAb (Figure 6B ). Moreover, the D3/71R R-mAb exhibits immunolabeling of endogenous Kv2. 1 in neurons in mouse neocortex that recapitulates the previously established pattern of Kv2. 1 immunolabeling in these neurons [e. g., (31, 53, 54) ] and that overlaps precisely with simultaneous immunolabeling obtained with the native K89/34 mAb (Figure 6C ). The D3/71R R-mAb also yields an immunoreactive band on immunoblots similar to that obtained with K89/34 and K89/34R (Figure 6D ). These results suggest that the mAb cloning approach described here can be used to effectively recover functional R-mAbs from non-viable hybridomas and could be applied by researchers that have such hybridomas in their collections.
section_name: Recovery of Functional R-mAbs from a Non-Viable Hybridoma

section_content: In this study, we developed a novel R-mAb generation and validation procedure that we used to generate a valuable R-mAb resource by converting a library of widely used mouse mAbs into recombinant reagents. Our approach was adapted from that of Gavin Wright and colleagues (22) with modifications to facilitate higher throughput cloning and validation steps. The efficiency of the process was further enhanced by restriction digest of VL PCR products to eliminate the MOPC-21 derived aberrant light chain. R-mAbs were produced in the culture medium from transiently transfected cells under standard mammalian cell culture conditions in sufficient quantities that they did not require purification for effective use in IB, ICC, and IHC assays. We were also able to recover functional R-mAbs from a cryopreserved hybridoma cell line that was no longer viable in cell culture via RNA extraction, cloning and expression. Moreover, in the process we generated subclass-switched R-mAbs that are more amenable to multiplex labeling in IB, ICC, and IHC applications. We then employed these mAbs in multiplex IHC experiments in combinations that were not possible prior to their cloning and expression as subclass-switched R-mAbs. Using the method described here, most researchers with standard molecular biology expertise can generate functional R-mAb expression plasmids with the advantages associated with recombinant reagents from their own cryopreserved hybridomas, whether viable or non-viable. Moreover, producing R-mAbs from cells transfected with any of the large collection of expression plasmids whose generation is described here is within the reach of anyone with standard mammalian cell culture capabilities. 

Building on our long standing and established practice of utilizing mAbs of different IgG subclasses in simultaneous multiplex immunolabeling experiments (14-17), we previously undertook a systematic analysis of anti-mouse IgG subclass-specific secondary antibodies. We demonstrated that the use of mouse mAbs of different IgG subclasses in combination with antimouse IgG subclass-specific secondary antibodies for simultaneous multiplex immunolabeling allows for robust and specific labeling in several immunolabeling applications, and also provides enhanced signal-to-noise (background) in immunolabeling of brain samples when compared to generic anti-mouse IgG (H+L) secondary Abs (18). Simultaneous multiplex labeling of multiple targets within a single sample allows for better comparison of their relative levels, co-localization, and overall tissue architecture. Multiplex labeling also conserves valuable samples, as multiple targets can be interrogated in the same sample instead of across multiple samples, which can be a particular concern in studies using human tissue samples that may be available in limited quantity. However, multiplex labeling using the classical approach of employing primary Abs raised in different species and their subsequent detection with species-specific secondary Abs is highly limited by the lack of available Abs from numerous species. As one example, interrogating the Abs registered with the Antibody Registry (2,381,068 as of 9/9/18) shows a preponderance (84% in total) of Abs raised in only three species: rabbits (44. 6%), mice (31. 2%) and goats (8. 2%), with all other species together accounting for the remaining 16%. Among the predominant species, goat Abs are exclusively polyclonal and as such cannot be used with one another in simultaneous multiplex labeling. Moreover, the bulk of secondary Abs are raised in goats, constraining facile detection of primary Abs in multiplex labeling experiments employing goat primary Abs. Rabbit Abs (whether poly-or mono-clonal) have limited utility for multiplex labeling, as unlike most other mammals, rabbits do not make IgGs of distinct subclasses but only a single generic IgG (55). As such employing multiple goat or rabbit primary Abs for multiplex immunolabeling involves costly and time-consuming procedures such as direct labeling with different fluorophores or sequential multiplexing technologies such as Opal TM (56). 

While the utility of mouse mAbs (and less common rat mAbs) for simultaneous multiple immunolabeling is enhanced by the availability of subclass-specific secondary antibodies, it remains that mouse mAbs against any given target may be available in only a single IgG subclass. 

In general, mouse mAb collections reflect the representation of these subclasses in the circulating serum IgGs in immunized BALB/c mice, which is ≈70% IgG1, ≈20% IgG2a and ≈10% IgG2b (57). 

As a prominent example, the large mouse mAb catalog of ThermoFisher Scientific (10, 992 independent IgG mouse mAbs as of 6/28/18) follows this remarkably closely, comprising 69% IgG1, 18% IgG2a and 12% IgG2b (Matt Baker, ThermoFisher Scientific, personal communication). As such, the flexibility of using mouse mAbs in multiplex labeling is restricted by the preponderance of those of the IgG1 subclass. It is possible to selectively screen for mAbs of the rarer IgG2a and IgG2b subclasses of mouse IgGs in the process of their development and screening (24, 58). It is also possible, although labor-intensive, to manipulate hybridomas in culture and select and/or screen for subclass switched hybridoma-generated mAbs (59). 

However, it remains that the optimal utility of mouse mAbs in research and diagnostics is limited by the preponderance of IgG1 mAbs. Here, we have enhanced the flexibility of a substantial fraction of an existing library of widely used mouse mAbs by converting them to subclass-switched R-mAbs without altering target binding specificity. We show using mixtures of mAbs and R-mAb that we can obtain effective simultaneous multiplex labeling for combinations that were previously unattainable due to conflicting IgG subclass. 

Antibody reformatting in our system is easily accomplished by the choice of plasmid backbone used for the original cloning, or by subcloning the VL-joining fragment-VH cassette (Figure 1B ) between plasmids with distinct CH domains. Ultimately, one can envision using this approach to convert any mouse mAb into the corresponding set of mouse IgG1, IgG2a, and IgG2b subclass R-mAb expression plasmids. Multiplex labeling could be further expanded by constructing IgG expression vectors containing CH regions of other species that have IgG subclasses such as rat and human (but not rabbit, in which all IgG Abs are of a generic IgG class), for which subclass-specific secondary antibodies are also widely available. For example, we interrogated the ThermoFisher secondary Ab catalog, which contains 52 different IgG subclassspecific secondary antibodies for mouse IgG subclasses, 47 for rat IgG subclasses, and 43 for human IgG subclasses. Together, the wide availability of such a broad range of reliable secondary antibodies with distinct conjugates provides ample flexibility for specific detection of multiple R-mAbs with distinct CH regions engineered for separate detection in multiplex labeling experiments. 

Previous attempts by others to generate R-mAbs have been complicated by the MOPC-21 derived aberrant kappa light chain that is present in many widely used myeloma fusion partners (22, (60) (61) (62). For example, Crosnier et al., (22) showed that depending on the hybridoma, 26% to 70% of R-mAb plasmids they generated contained this aberrant sequence. We have incorporated restriction enzyme digest of this transcript into our process to eliminate the aberrant kappa light chain (38). For most of our hybridomas, the majority of the VL PCR product was cleaved by BciVI, indicating that the MOPC-21 aberrant light chain comprised the greatest fraction of total light chain amplicons. By eliminating this transcript, the overall screening and cloning burden for each mAb was reduced substantially. However, depending on the particular hybridoma, the efficiency of generating clones that produced functional R-mAbs even after BciVI digestion of the VL domain PCR products was still highly variable. This was evidenced by the overall efficiency of slightly below 50% for the successful projects tabulated in Table 2, and also by the projects that were unsuccessful on the first pass in which clones passed the colony PCR and restriction digest screening steps but failed in the COS-ICC screening assay for functional R-mAbs. Moreover, a lack of allelic exclusion at heavy and light chain IgG loci has been documented in hybridomas, resulting in expression of multiple IgG heavy and/or light chains at the mRNA and protein levels (8) (9) (10) 63). In these previous studies, while multiple combinations of heavy and light IgG chains were produced by monoclonal hybridoma cells, only one combination of heavy and light IgG chains present in the hybridoma produced an R-mAb capable of recognizing the target antigen. 

Varying degrees of allelic exclusion in the hybridomas that served as the source of our R-mAbs could also contribute to the variable efficiency of whether colony PCR and restriction enzyme assay-positive clones would produce an R-mAb that recapitulated immunolabeling obtained with the native mAb. Technical issues, such as PCR-induced mutations may have also contributed to the generation of R-mAb clones that failed in the COS-IF-ICC assay. We did not systematically analyze these negative clones, so we do not know whether these plasmids contain residual aberrant light chains from the BciVI digestion step, plasmids containing other heavy and/or light chains, or mutated forms of bona fide R-mAb chains with impaired/lost functionality. That the vast majority (84%) of the 114 projects for which ³4 clones were evaluated yielded at least one positive R-mAb suggests that the presence of a small and variable number of negative clones does not impact the overall success of the approach described here, given the simple but effective nature of microplate screening assays, such as the ICC assay employed here, to rapidly and easily screen candidate R-mAbs. 

There is a growing impetus to enhance research reproducibility by both improving the quality of Abs used in basic research and in diagnostics, and the transparency of their reporting as related to the exact molecularly-defined Ab that was used (1, 5, 64). Using mAbs in their recombinant form and defining them unambiguously at the molecular level by publishing their VL and VH domain sequences is a step towards achieving this goal and elevate reproducibility worldwide. Among the different forms of research and diagnostic Abs, polyclonal Ab preparations have unique benefits (65), due to the presence of Abs recognizing distinct epitopes on the target protein. However, in any of the diverse forms in which they are available (antiserum, IgG fractions, affinity-purified) polyclonal Abs are inherently limited by their intrinsic lack of precise molecular definition. The inability to rigorously define the clonal composition of specific preparations can contribute to batch-to-batch variation in the efficacy and specificity of the polyclonal collection for any specific application, negatively impacting the reproducibility of research performed with such Abs. Moreover, unlike mAbs, polyclonal Abs are a finite resource whose depletion can lead to batch-to-batch variability or complete lack of availability, stymieing independent reproduction of research using the same reagent. Conventional mAbs are advantageous in lacking the molecular complexity of polyclonal preparations, and in being renewable reagents. However, the conversion of conventional hybridoma-generated mAbs into R-mAbs offers numerous additional advantages. This includes ensuring their broad availability by generating a publicly accessible DNA sequence archive that can be used to synthesize the functional VL and VH domain sequences. R-mAbs are also more easily distributed as plasmids or DNA sequence, a feature that should enhance dissemination of this resource, relative to cryopreserved and living hybridomas that are comparatively fragile and may not survive long-distance transport. Cloning also enhances research reproducibility by the unambiguous definition of mAbs at the level of their cDNA sequence, as well as the enhanced control of molecular composition afforded by expressing a single light and heavy chain combination from transfected cells and their high-level animal-free production in cell culture. Cloning of mAbs also allows for increased utility as offered by their subsequent engineering into alternate forms, such as the subclass-switched R-mAbs generated here. The method described here represents a straightforward approach to producing functional R-mAbs from existing murine hybridomas that could be applied to other valuable mAb collections to ensure their permanent archiving which cannot be assured when they exist solely in the form of hybridomas cryopreserved in liquid nitrogen. In particular, there are many cases of important hybridomas, including entire collections, that have been discarded upon the closure of the laboratory that formerly housed and financially supported the maintenance of the cryopreserved collection. The enhanced utility, permanence, and cost-effectiveness of R-mAbs as generated by the approach described here is one route to circumvent the negative impact to research effectiveness and reproducibility that such losses represent. Simple conversion of mAbs to R-mAbs, as described here, also paves the way for higher throughput approaches, for example those employing high throughput "next generation" sequencing to simultaneously obtain light and heavy chain sequences from large pools of hybridomas (66). In theory this should allow for larger scale "en masse" (as opposed to the mAb-by-mAb approach used here) conversion efforts more suitable to large hybridoma collections. For example, our larger collection of ≈40,000 targetspecific mAbs archived in our cryopreserved hybridoma collection, and other widely used collections, such as that housed at the Developmental Studies Hybridoma Bank (http://dshb. biology. uiowa. edu/; comprising 3,672 mAbs as of 9/9/18) might represent appropriate targets for such next generation sequencing approaches. Future efforts to generate R-mAbs from cryopreserved hybridoma collections will further enhance the rigor, reproducibility and overall effectiveness of Ab-based research.
section_name: Discussion

section_name: Materials and Methods

section_content: Primer sets for mouse Ig V region amplification and fusion PCR (F-PCR) were used as described previously (22, 25). The following primers were used for other PCR steps. Amplification of the Joining Fragment ( 22
section_name: Primers

section_content: The Ambion RNAqueous 96 Extraction kit (Thermo Fisher Cat# AM1920) was used for high throughput RNA extraction. Frozen vials containing 0. 5-1 x 10 7 hybridoma cells per vial were thawed in a 37ºC water bath for 5 minutes, in batches of 20 vials for high through-put purposes.
section_name: RNA preparation from cryopreserved hybridomas and RT-PCR

section_name: Cloning of Immunoglobulin Variable Domain Regions into a Dual Promoter Expression

section_content: F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37ºC for 20 min, followed by inactivation at 80ºC for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106). The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706). P1316 is a derivative of the pTT3 expression vector (26) and consists of (5' to 3'): a CMV promoter, the mouse V kappa leader sequence, a NotI restriction site, an insert consisting of VL/joining fragment/VH, and the mouse IgG1 CH sequence amplified from mouse genomic DNA, flanked by AscI and XbaI restriction sites (22, 25). Ligation was performed overnight at 16ºC with T4 DNA ligase (Thermo Fisher Cat# 15224017) using 20 ng insert and 20 ng vector, a 3:1 molar ratio. Half of each ligation reaction was used to transform 25 µL of Mach I chemically competent E. coli. 

(Thermo Fisher Cat# C862003). Cells and DNA were incubated on ice for 30 min, heat shocked at 42ºC for 30 s, incubated on ice for 2 min, and allowed to recover for 1. 0 hr in 250 µL SOC medium in a 37ºC shaking incubator. The cells were spun in a centrifuge at 3000 rpm (≈950 x g) for 2 min and the supernatant was removed until 150 µL remained. Cells were resuspended, and the entire volume was plated on LB plates containing 100 µg/mL ampicillin and incubated overnight at 37ºC.
section_name: Plasmid

section_content: Colony PCR was performed to identify E. coli colonies that contained the full-length, 2. 4 kb Ig cassette. Colonies were diluted in 96-well plates containing 50 µL water and patch plates were made for later recovery of positive clones. 2 µL of diluted colony was used in each PCR. 

Conditions were 94°C for 5 min; 23 cycles of 94°C for 20 s, 58°C for 30 s, 72°C for 2. 5 min; 72°C for 10 min. For additional confirmation of the presence of the full-length Ig cassette, plasmid DNA was isolated from PCR positive clones and subjected to restriction enzyme digestion with NotI/AscI at 37ºC for 20 min followed by agarose gel electrophoresis. The VL and VH regions of functional R-mAbs were subjected to sequencing in both orientations to generate a permanent archive. The primers "UpNotI" and "Seq_VL_Rev_IgG2a" were used for VL domain sequencing, and the "VH seq forward", and either "DownAscI" or "Rev IgG2a" for sequencing of VH regions in the IgG1 or IgG2a expression plasmids, respectively.
section_name: Colony PCR, Restriction Analysis and Sequencing of R-mAb Clones

section_content: The mouse g2a CH domain was amplified from the cDNA preparation that was obtained from the K28/43 (RRID: AB_2292909) hybridoma (27, 28) that was used for cloning of the K28/43

VL and VH domains. PCR conditions were: 94ºC for 5 min; 29 cycles of 94ºC for 30 s, 65ºC for 30 s, 72ºC for 30 s. The forward primer included an AscI restriction site and the reverse primer included an XbaI restriction site to facilitate cloning into the K28/43 IgG1 recombinant R-mAb plasmid. The K28/43 IgG1 recombinant R-mAb plasmid was derived from the P1316 plasmid (22) by restriction enzyme-based cloning of K28/43 variable region sequences as described above. 

The IgG2a CH PCR product and the K28/43 IgG1 recombinant plasmid were both digested with AscI and XbaI restriction enzymes (New England BioLabs Cat# R0558 and R0145, respectively) and column purified (Qiagen/QiaQuick PCR Purification Cat# 28106) or agarose gel purified, respectively (Qiagen/QiaQuick Gel Extraction Cat# 28706). Because XbaI is methylation sensitive, the K28/43 IgG1 plasmid was sourced from dam -/dcm -E. coli (New England Biolabs Cat# C2925). T4 DNA ligase (New England Biolabs Cat# M0202) was used to insert the IgG2a CH fragment into the digested K28/43R plasmid to generate a K28/43 IgG2a R-mAb plasmid which was confirmed by DNA sequencing.
section_name: Generation of a mouse IgG2a expression vector

section_content: For production of recombinant mAbs in mammalian cell culture, 3 x 10 5 COS-1 cells were plated on 35 mm tissue culture dishes and cultured overnight in DMEM (high glucose/pyruvate, Thermo Fisher Cat# 11995065) with 10% Fetal Clone III (HyClone Cat# SH30109. 03) and 100 µg/ml penicillin/streptomycin (Thermo Fisher Cat# 15140122). Cells were then transfected with a 1:1 ratio of plasmid (1 µg):Lipofectamine 2000 (1 µL) (Thermo Fisher Cat# 11668019) diluted in Opti-MEM reduced serum medium (Thermo Fisher Cat# 31985070). Following an overnight incubation, the transfection solution was replaced with culture medium, and the cells incubated for an additional six days, at which time the conditioned medium was collected as R-mAb tissue culture supernatant (TC supe) for analysis.
section_name: R-mAb Expression in Mammalian Cells

section_content: R-mAb TC supes were screened for immunoreactivity in an immunofluorescence assay against transiently transfected COS-1 cells cultured in 96-well plates. COS-1 cells were plated in black, clear bottom 96-well plates (Greiner Cat# 655090) at a density of 4,700 cells/well. After overnight incubation, each well received 50 ng plasmid DNA encoding the R-mAb target protein plus Lipofectamine 2000 at a 1:1 ratio as described above. On day 3 post-transfection, cells were washed 3 times with DPBS (138 mM NaCl, 2. 67 mM KCl, 1. 47 mM KH2PO4, 8. 1 mM Na2HPO4, 1 mM CaCl2 and 1 mM MgCl2), pH 7. 4 and then fixed using 3. 0% formaldehyde (prepared fresh from paraformaldehyde) in in DPBS plus 0. 1% Triton X-100 on ice for 20 min. Cells were washed 3 times with DPBS/0. 1% Triton X-100, blocked with Blotto/0. 1% Triton X-100 for 1 hr, and stored in DPBS/0. 02% sodium azide. For primary antibody labeling, R-mAb TC supes were used without dilution and hybridoma-generated mAb TC supe controls (see Table 4 for details of non-R-mAb

Abs used in this study) were diluted 1:10 in COS-1 cell culture medium. Each R-mAb was tested alone and in combination with the corresponding hybridoma-generated mAb TC supe. Primary antibodies were incubated at room temperature for 1 hr and cells were washed 3 x 10 min with Blotto/0. 1% Triton X-100. Secondary labeling was performed at room temperature for 30 min using subclass-specific, anti-mouse secondary antibodies conjugated to Alexa Fluors (Thermo Fisher, Cat#/IgG subclass/Alexa Fluor dye conjugates: (A-21121/IgG1/488 and A-21241/IgG2a/647) and diluted to 1. 3 µg/mL in Blotto/0. 1% Triton X-100. Hoechst 33342 (Thermo Fisher Cat# H3570) was used at 0. 1 µg/mL in the secondary antibody cocktail to stain nuclear DNA. Cells were washed 3 x 10 min with DPBS/0. 1% Triton X-100. Imaging was performed using a Zeiss M2 AxioImager microscope. Images were processed using Zeiss Axiovision and ImageJ software. 

For higher resolution imaging, COS-1 cells were plated on poly-L-lysine coated #1. 5 glass cover slips and cultured overnight followed by transfection with plasmids encoding the target protein. Cells were fixed and immunolabeled as described in the previous section. Images were acquired on a Zeiss AxioImager M2 microscope using a 40x/0. 8 NA plan-Apochromat oilimmersion objective and an AxioCam MRm digital camera. Optical sections were acquired using an ApoTome 2 structured illumination system (Carl Zeiss MicroImaging). Imaging and post processing were performed in Axiovision (Carl Zeiss MicroImaging) and Photoshop (Adobe Systems).
section_name: COS Cell Immunofluorescence Immunocytochemistry Validation Assay

section_content: Multiplex immunofluorescence labeling of immunoblots using mouse IgG subclassspecific secondary antibodies was performed as described previously (18). In brief, samples were generated from COS-1 cells transiently transfected to express individual target proteins essentially as described above for the immunofluorescence experiments except that the cells were cultured in 35 mm tissue cultures dishes. Transfected COS-1 cells were washed once with ice-cold PBS and lysed with 150 µL of ice-cold lysis buffer containing 1% v/v Triton X-100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7. 4), 1 mM sodium orthovanadate, 5 mM NaF, 1 mM PMSF and a protease inhibitor cocktail for 10 min at 4ºC (29). The cell lysates were centrifuged at 12,000 x g at 4 ˚C for 10 min. The cell lysate supernatants were mixed with 150 µL of 2X RSB and size-fractionated by 7. 5% SDS-PAGE. Following SDS-PAGE, proteins were transferred to nitrocellulose membranes (Bio-Rad Cat# 1620115), which were blocked for 1 h with Blotto (3% w/v nonfat milk in Tris-buffered saline (TBS: 50 mM Tris, pH 7. 5, 150 mM NaCl) plus 0. 1% v/v Tween-20 followed by 2 h or overnight incubation with primary antibodies. Primary antibodies were mAb TC supes diluted 1:10, non-diluted R-mAb TC supes, and an in-house anti-PSD-95 rabbit polyclonal antibody raised against a GST fusion protein, GSTKAP1. 13, containing amino acids 77-299 of human PSD-95 [clone 2, (30) ] and that crossreacts with SAP97 (see Table 4 for details of non-R-mAb Abs used in this study). After 3 washes with Blotto, the membranes were incubated with the appropriate subclass-specific Alexa Fluor conjugated secondary antibodies (18) for 1 h. After 3 washes with TBS containing 0. 1% v/v Tween-20, the immunoblots were visualized directly on a FluorChem Q imager (Cell Biosciences Cat# DE500-FCQ). Alternatively, crude rat brain membranes (RBM) (29) were subjected to immunoblotting as described above except that a single RBM sample (3 mg protein) were size fractionated on a curtain gel, and after transfer to nitrocellulose the membrane was cut into 30 strips, each containing 100 µg of RBM protein. Immunolabeling was detected on autoradiography film after treatment of strip immunoblots with HRP-conjugated anti-mouse IgG-specific secondary antibody and enhanced chemiluminescence (ECL).
section_name: Multiplex immunofluorescence labeling of immunoblots

section_content: Multiplex immunofluorescence labeling of brain sections was performed essentially as described previously (18, 31). All experimental procedures were approved by the UC Davis Institutional Animal Care and Use Committee and conform to guidelines established by the National Institutes of Health (NIH). Rats were anesthetized with sodium pentobarbital (Fatal-Plus solution, 100 mg/kg sodium pentobarbital) and perfused transcardially with 100 mL of phosphate buffered saline (PBS), containing 10 units/mL heparin, pH 7. 4, followed by 400 mL of 4% formaldehyde (prepared fresh from paraformaldehyde) in 0. 1 M sodium phosphate buffer or PB (pH 7. 4). The brains were removed, cryoprotected for 48 hr in 30% sucrose, frozen in a bed of pulverized dry ice, and then cut into 30 μm sections on a freezing-stage sliding microtome. 

Sections were collected in 0. 1 M PB and processed immediately for immunohistochemistry. Freefloating brain sections were blocked with 10% goat serum in 0. 1 M PB containing 0. 3% Triton X-100 (vehicle) for 1 h at RT and then incubated overnight at 4°C in vehicle containing different combinations of primary antibodies (see Table 4 for details of non-R-mAb Abs used in this study). 

The following day sections were washed 4 X 5 min each with vehicle, and then incubated for 1 h at RT in mouse IgG subclass-specific or anti-rabbit Alexa-conjugated secondary antibodies as described previously (18, 32). Sections were then washed 2 X 5 min each with 0. 1 M PB, 2 X 5 min each with 0. 05 M PB and mounted on gelatin-coated microscope slides and air dried. Sections were cover slipped after adding ProLong Gold Antifade Mountant (Thermo Fisher Scientific catalog # P36930). Images were obtained on a Zeiss Axiovert 200 microscope with Apotome. 

Imaging and post-imaging processing was performed in Zeiss Axiovision and Adobe Photoshop software, taking care to maintain any linear differences in signal intensities present in the original samples. 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).
section_name: Multiplex immunofluorescence labeling of brain sections

section_content: This work was funded by NIH research grants U24 NS050606, R24 NS092991 and U24 NS109113 to J. S. Trimmer. We thank the current and former staff members of the UC Davis/NIH NeuroMab Facility and the Trimmer laboratory for their contributions and dedicated efforts, and Dr. Randall Stewart at the National Institute of Neurological Disorders and Stroke for support and helpful advice. We thank Dr. Gavin Wright of the Sanger Institute for his generous gift of the P1316 expression plasmid and helpful advice.

section_name: Clone