HHS Public Access Author manuscript Author Manuscript
N Biotechnol. Author manuscript; available in PMC 2017 September 25. Published in final edited form as: N Biotechnol. 2016 September 25; 33(5 Pt A): 551–564. doi:10.1016/j.nbt.2015.11.007.
Developing High-Quality Mouse Monoclonal Antibodies for Neuroscience Research – Approaches, Perspectives and Opportunities Belvin Gong1, Karl D. Murray1,2, and James S. Trimmer1,3 1Department
Author Manuscript
of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, California 95616 2Center
for Neuroscience, University of California, Davis, Davis, California 95616
3Department
of Physiology and Membrane Biology, University of California Davis School of Medicine, Davis, California 95616
Abstract
Author Manuscript Author Manuscript
High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research
Corresponding Author: Dr. James S. Trimmer, Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Ave, Davis, CA, 95616-8519. [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gong et al.
Page 2
Author Manuscript
will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.
Keywords Brain; immunoblot; immunofluorescence; immunohistochemistry; neuroscience; validation
Introduction
Author Manuscript Author Manuscript
Antibodies (Abs) are valuable and essential reagents for many proteomic level applications that are key to the effective pursuit of molecular and cellular neuroscience research. While Abs are only one class of the diverse binder types used in neuroscience research, they remain the primary tool for the labeling and capture of molecular targets in cells and tissues from the nervous system. They provide neuroscientists with familiar, stable and high-affinity reagents for which there are a wide array of readily available reagents for their subsequent detection and/or capture. Antibodies can be developed and validated for a broad range of labeling and capture applications, and compared to many other classes of binders, can generally be used under a broad range of assay conditions (although, as discussed below, sample preparation and assay conditions can fundamentally impact the nature of Ab-antigen interaction). High-quality and well-characterized polyclonal or monoclonal Abs (pAbs or mAbs, respectively) provide enormous benefits to neuroscience research due to their wide availability, familiarity and ease of use, such that the same Ab can be used globally across many independent laboratories. Antibodies have been crucial to the expansion of knowledge regarding the expression, localization, structure, function, and molecular interactions of a wide variety of proteins expressed in the nervous system, leading to significant advances in the field. However, in spite of the importance of Abs in neuroscience research, and the large number of commercial and public suppliers of Abs, neuroscientists are often faced with challenges and frustrations when applying Abs in their experiments [1–3]. Lack of reliable results using Abs, and their reproducibility, whether due to the poor quality of the Abs themselves, or their application in experiments under conditions for which the Ab was not validated for use, or insufficient reporting of the details of the Abs themselves, has caused considerable frustration among researchers in many fields of research [4]. In the neuroscience arena this has resulted in documentation of numerous incorrect and irreproducible results using Abs, for examples see [5–11]. This is coupled with frustration with the high cost of many commercial Abs (francesscientist.wordpress.com/2010/10/12/ scamming-the-antibody/). The estimates of the resulting cost of “bad” Abs to global research budgets in the hundreds of millions of US dollars annually [12].
Author Manuscript
The UC Davis/NIH NeuroMab Facility was created in 2005, in large part as a response to inquiries from neuroscientists interested in having the same systematic approach to mAb generation and characterization we were using to generate high quality anti-ion channel mAbs in our research laboratory, for examples see [13–16], applied more broadly to targets of general importance to the neuroscience research community. This approach is similar to that used in many other laboratories for stringent in-house validation of Abs, and similar to that described in a prominent publication on Ab quality and validation [17], but with a
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 3
Author Manuscript
specific focus on validation in brain samples. NeuroMab aims to provide mAbs (i.e., NeuroMabs), validated for the critical applications of immunoblotting (IB) and immunohistochemistry (IHC) against mammalian brain samples, and to distribute them on a non-profit low cost basis. Over the last ten years, the NeuroMab Facility has established a strong track record towards this mission by both following a proven neuroscience-based approach to screening that is briefly described here, and by its subsequent involvement in controlling quality of the final mAb product distributed to end users. Here we describe the approaches used at the NeuroMab Facility to generate and validate mAbs for neuroscience research applications, provide examples of validation data from such efforts, and use this as a platform to discuss some aspects of the general state of Abs for neuroscience research.
NeuroMab generation, characterization and validation Author Manuscript Author Manuscript
NeuroMab projects begin with input from the neuroscience community regarding potential targets that are important for neuroscience research and for which no reliable, widely available, and/or renewable binder currently exists. A Scientific Advisory Board and NIH program official prioritize target suggestions based on information regarding the scientific justification and biological importance of the target, and the availability and suitability of existing Abs or other binders. The suggesting lab is also asked to provide a list of available reagents for the project, including plasmids for expression of full-length recombinant target protein in mammalian cells, and samples from knockout (KO) mice if available. Suggesting labs are also asked to provide any other information relevant to the target that may facilitate the screening and validation (e.g., the spatial and temporal expression pattern of the target in brain, any unusual aspects of the target protein as far as instability during sample preparation, behavior on SDS gels, etc.). Through this process, the NeuroMab Facility has taken on projects for a broad array of targets, ranging from novel proteins for which little information and few reagents were available, to well-characterized proteins for which the quality of, or accessibility to, existing Abs was not reliable, or for which there is a dwindling supply of a non-renewable reagent (e.g., a pAb). The array of targets we have pursued include cytoskeletal proteins, enzymes, receptors, ion channels, transcription factors and other proteins of diverse and/or unknown function, as well as specific epigenetic marks on DNA and proteins, specific phosphorylation sites, splice junctions of alternatively spliced proteins, newly generated ends of proteolytic cleavage products, disease-associated mutations, adducts arising from bioterrorism/chemical warfare agents, and many other targets for which no reliable or renewable reagent existed.
Immunogen design and production Author Manuscript
A critical step in Ab production is the design and production of an appropriate immunogen. While many sophisticated approaches can be used in immunogen design, guided by intensive efforts in the area of vaccine development, for example see [18], our proximate goal is to generate research reagents for labeling and capturing targets from brain tissue, so our immunogen design is guided by only a few simple considerations. The design of our immunogens is similar to that used by the Human Protein Atlas project except we do not limit the length of our immunogens to 150 amino acids [19]. One key feature is that the immunogen be hydrophilic and, in a best-case scenario, have extensive representation of
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 4
Author Manuscript Author Manuscript
charged residues to increase the probability that these regions will be surface-accessible in aqueous solutions. The immunogen should also contain extensive regions not present in other proteins, including other highly related members of the same family. It should also provide a reagent that is likely to yield mAbs that recognize the specific target across species (i.e., mAbs that recognize mammalian orthologs, but not paralogs within a given species). For complex polypeptide targets such as ion channel subunits and other integral membrane proteins, we have commonly used fragments of intracellular or extracellular loops, or N- or C-terminal domains. For smaller targets, we have used full-length protein immunogens to successfully generate target-specific NeuroMabs. We prefer that our immunogens are at least 5 kDa in size, and do not have any known requirements for establishing or maintaining proper folding. In cases where the only attractive options for immunogens were a few separate short stretches of less than 50 amino acids each, we generated fusions of these fragments. We have a focus on human targets, as many of our neuroscience end users study human samples and the mechanisms underlying human neurological disease. However, much of this research also involves animal models, especially rodents, such that specific immunoreactivity against both human and rodent orthologs is desirable. Selecting human sequences that share substantial identity with rodent sequences allows us to use rat and mouse brain tissue in screens, which offers advantages as discussed below.
Author Manuscript
Our most common immunogens are recombinant protein fragments, which typically generate a more robust and varied immune response, and yield a high number of mAbs that work well in different applications. Smaller synthetic peptides are used almost exclusively for highly focused projects such as disease-associated mutations, posttranslational modifications (PTMs), splice junctions, etc. Because of their size and complexity, designing recombinant protein immunogens that produce maximal target specificity and minimal cross-reactivity can be challenging. Expression, solubility, and affinity purification of recombinant mammalian proteins in E coli can be difficult compared with the ease of outsourcing chemical synthesis of a peptide immunogen. However, in our experience, the time, effort and cost put towards generating or obtaining recombinant protein immunogens has been worthwhile. From a retrospective analysis of 414 NeuroMab projects, we find a much higher project success rate for generating at least one validated NeuroMab when using recombinant protein immunogens (211 out of 290 projects or a 73% success rate) than when using synthetic peptide immunogens (47 out of 124 projects or a 38% success rate). One step we have incorporated into our workflow is to validate each affinity-purified immunogen by tandem mass spectrometry (MS) analysis, which has substantially reduced the number of failed projects due to an incorrect immunogen.
Author Manuscript
Mice are immunized using a short (30 day) but immunogen-intensive immunization protocol [13] that is a modified version of protocols developed to generate a strong IgG response in a much shorter time period than conventional immunization protocols [20; 21]. For each project, two naïve female BALB/c mice (6–8 weeks old) are immunized intraperitoneally using immunogen mixed with Sigma Adjuvant System adjuvant weekly for four weeks. Antiserum from the mice is analyzed for immunoreactivity against the immunogen by enzyme-linked immunosorbent assay (ELISA), by immunocytochemistry (ICC) against the target protein heterologously overexpressed in mammalian COS-1 cells, and by immunoblot against the target protein endogenously expressed in brain tissue. For three days preceding N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 5
Author Manuscript Author Manuscript
splenocyte isolation and fusion, mice receive intravenous tail vein injections of immunogen in physiological saline. Splenocytes from both mice are then harvested and pooled to maximize the population of mAb specificities, and used in a fusion with Sp2/0-Ag14 (ATCC CRL-1581) B-lymphocyte hybridomas. A small fraction (1
>1
1.3
2.7
>1
>1
Fold Δ
32%
14%
8%
2%
15%
16%
PEG
74%
100%
24%
46%
83%
94%
EF
2.3
7.1
3.0
23.0
5.5
5.9
Fold Δ
% of wells with hybridomas @ 1 × 106 splenocytes/plate
2%
2%
1%
ND
2%
0%
PEG
9%
66%
3%
ND
40%
18%
EF
4.5
33.0
3.0
ND
20.0
NA
Fold Δ
% of wells with hybridomas @ 1 × 105 splenocytes/plate
Using larger capacity chamber: 100%, 100% and 50% of wells with hybridomas @ 1 × 107, 1 × 106 and 1 × 105 splenocytes/plate, resulting in >1, >3 and 25.0 fold increases, respectively.
*
NA = not applicable; ND = not done
PEG
Project
% of wells with hybridomas @ 1 × 107 splenocytes/plate
Author Manuscript
Fusion efficiency with electrofusion (EF) and polyethylene glycol (PEG)
Author Manuscript
Table 1 Gong et al. Page 29
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Author Manuscript
Author Manuscript
Author Manuscript O00591 P19490 Q9JKA9 NP_599190
GABA(A)R, Pi
GluR1 glutamate receptor
HCN2
KCC2
O08773 Q8R464 Q62666
Pan-MAGUK
Pan-Nav1 Na+ channel
RGS14
SynCAM4
VAChT
Navbeta4
Q7M730
Q56A07
-
channel
Na+
Navbeta2
P38647
Pan-KChIP K+ channel
channel
Na+
Mortalin/GRP75
Currently not available
*
P31644
GABA(A)R, Alpha5
channel
P23576
GABA(A)R, Alpha2
KChIP4
P16157
Ankyrin-R
Q3YAB7
E9PE32
Ankyrin-G (staining)
K+
Accession #
Target
N425/45
N244/5
N133/21
N419/40
K28/86
K55/82
N168/6
N395/68
N52A/42
N423/75
N1/12
N71/37
N355/1
N431/64
N415/24
N399/19
N388A/10
N106/36
Clone
IgG1
IgG1
IgG2a
IgG2a
IgG1
IgG2a
IgG1
IgG2b
IgG1
IgG2a
IgG2a
IgG1
IgG1
IgG2a
IgG1
IgG1
IgG2b
IgG2a
IgG Subclass
RRID:AB_2532047
RRID:AB_10673109
RRID:AB_10698026
RRID:AB_2491079
RRID:AB_10698179
RRID:AB_2132595
RRID:AB_10673578
RRID:AB_2315895
RRID:AB_10674108
RRID:AB_2491080
RRID:AB_10697875
RRID:AB_10672304
RRID:AB_2315839
RRID:AB_2493092
RRID:AB_2491075
RRID:AB_2336904
RRID:AB_2336901
RRID:AB_10697718
RRID TCS
NA*
RRID:AB_10676101
RRID:AB_2179931
RRID:AB_2491098
RRID:AB_10673115
RRID:AB_10673406
RRID:AB_2301402
RRID:AB_2315896
RRID:AB_2120479
RRID:AB_2493100
RRID:AB_10672851
RRID:AB_2279449
RRID:AB_2315840
RRID:AB_2532051
RRID:AB_2491187
NA*
RRID:AB_2491109
RRID:AB_10673030
RRID Pure
NeuroMabs highlighted in current manuscript and their associated Research Resource Identifiers (RRIDs)
Author Manuscript
Table 2 Gong et al. Page 30
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
N Biotechnol. Author manuscript; available in PMC 2017 September 25. Published in final edited form as: N Biotechnol. 2016 September 25; 33(5 Pt A): 551–564. doi:10.1016/j.nbt.2015.11.007.
Developing High-Quality Mouse Monoclonal Antibodies for Neuroscience Research – Approaches, Perspectives and Opportunities Belvin Gong1, Karl D. Murray1,2, and James S. Trimmer1,3 1Department
Author Manuscript
of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, California 95616 2Center
for Neuroscience, University of California, Davis, Davis, California 95616
3Department
of Physiology and Membrane Biology, University of California Davis School of Medicine, Davis, California 95616
Abstract
Author Manuscript Author Manuscript
High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research
Corresponding Author: Dr. James S. Trimmer, Department of Neurobiology, Physiology and Behavior, University of California Davis, One Shields Ave, Davis, CA, 95616-8519. [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gong et al.
Page 2
Author Manuscript
will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.
Keywords Brain; immunoblot; immunofluorescence; immunohistochemistry; neuroscience; validation
Introduction
Author Manuscript Author Manuscript
Antibodies (Abs) are valuable and essential reagents for many proteomic level applications that are key to the effective pursuit of molecular and cellular neuroscience research. While Abs are only one class of the diverse binder types used in neuroscience research, they remain the primary tool for the labeling and capture of molecular targets in cells and tissues from the nervous system. They provide neuroscientists with familiar, stable and high-affinity reagents for which there are a wide array of readily available reagents for their subsequent detection and/or capture. Antibodies can be developed and validated for a broad range of labeling and capture applications, and compared to many other classes of binders, can generally be used under a broad range of assay conditions (although, as discussed below, sample preparation and assay conditions can fundamentally impact the nature of Ab-antigen interaction). High-quality and well-characterized polyclonal or monoclonal Abs (pAbs or mAbs, respectively) provide enormous benefits to neuroscience research due to their wide availability, familiarity and ease of use, such that the same Ab can be used globally across many independent laboratories. Antibodies have been crucial to the expansion of knowledge regarding the expression, localization, structure, function, and molecular interactions of a wide variety of proteins expressed in the nervous system, leading to significant advances in the field. However, in spite of the importance of Abs in neuroscience research, and the large number of commercial and public suppliers of Abs, neuroscientists are often faced with challenges and frustrations when applying Abs in their experiments [1–3]. Lack of reliable results using Abs, and their reproducibility, whether due to the poor quality of the Abs themselves, or their application in experiments under conditions for which the Ab was not validated for use, or insufficient reporting of the details of the Abs themselves, has caused considerable frustration among researchers in many fields of research [4]. In the neuroscience arena this has resulted in documentation of numerous incorrect and irreproducible results using Abs, for examples see [5–11]. This is coupled with frustration with the high cost of many commercial Abs (francesscientist.wordpress.com/2010/10/12/ scamming-the-antibody/). The estimates of the resulting cost of “bad” Abs to global research budgets in the hundreds of millions of US dollars annually [12].
Author Manuscript
The UC Davis/NIH NeuroMab Facility was created in 2005, in large part as a response to inquiries from neuroscientists interested in having the same systematic approach to mAb generation and characterization we were using to generate high quality anti-ion channel mAbs in our research laboratory, for examples see [13–16], applied more broadly to targets of general importance to the neuroscience research community. This approach is similar to that used in many other laboratories for stringent in-house validation of Abs, and similar to that described in a prominent publication on Ab quality and validation [17], but with a
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 3
Author Manuscript
specific focus on validation in brain samples. NeuroMab aims to provide mAbs (i.e., NeuroMabs), validated for the critical applications of immunoblotting (IB) and immunohistochemistry (IHC) against mammalian brain samples, and to distribute them on a non-profit low cost basis. Over the last ten years, the NeuroMab Facility has established a strong track record towards this mission by both following a proven neuroscience-based approach to screening that is briefly described here, and by its subsequent involvement in controlling quality of the final mAb product distributed to end users. Here we describe the approaches used at the NeuroMab Facility to generate and validate mAbs for neuroscience research applications, provide examples of validation data from such efforts, and use this as a platform to discuss some aspects of the general state of Abs for neuroscience research.
NeuroMab generation, characterization and validation Author Manuscript Author Manuscript
NeuroMab projects begin with input from the neuroscience community regarding potential targets that are important for neuroscience research and for which no reliable, widely available, and/or renewable binder currently exists. A Scientific Advisory Board and NIH program official prioritize target suggestions based on information regarding the scientific justification and biological importance of the target, and the availability and suitability of existing Abs or other binders. The suggesting lab is also asked to provide a list of available reagents for the project, including plasmids for expression of full-length recombinant target protein in mammalian cells, and samples from knockout (KO) mice if available. Suggesting labs are also asked to provide any other information relevant to the target that may facilitate the screening and validation (e.g., the spatial and temporal expression pattern of the target in brain, any unusual aspects of the target protein as far as instability during sample preparation, behavior on SDS gels, etc.). Through this process, the NeuroMab Facility has taken on projects for a broad array of targets, ranging from novel proteins for which little information and few reagents were available, to well-characterized proteins for which the quality of, or accessibility to, existing Abs was not reliable, or for which there is a dwindling supply of a non-renewable reagent (e.g., a pAb). The array of targets we have pursued include cytoskeletal proteins, enzymes, receptors, ion channels, transcription factors and other proteins of diverse and/or unknown function, as well as specific epigenetic marks on DNA and proteins, specific phosphorylation sites, splice junctions of alternatively spliced proteins, newly generated ends of proteolytic cleavage products, disease-associated mutations, adducts arising from bioterrorism/chemical warfare agents, and many other targets for which no reliable or renewable reagent existed.
Immunogen design and production Author Manuscript
A critical step in Ab production is the design and production of an appropriate immunogen. While many sophisticated approaches can be used in immunogen design, guided by intensive efforts in the area of vaccine development, for example see [18], our proximate goal is to generate research reagents for labeling and capturing targets from brain tissue, so our immunogen design is guided by only a few simple considerations. The design of our immunogens is similar to that used by the Human Protein Atlas project except we do not limit the length of our immunogens to 150 amino acids [19]. One key feature is that the immunogen be hydrophilic and, in a best-case scenario, have extensive representation of
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 4
Author Manuscript Author Manuscript
charged residues to increase the probability that these regions will be surface-accessible in aqueous solutions. The immunogen should also contain extensive regions not present in other proteins, including other highly related members of the same family. It should also provide a reagent that is likely to yield mAbs that recognize the specific target across species (i.e., mAbs that recognize mammalian orthologs, but not paralogs within a given species). For complex polypeptide targets such as ion channel subunits and other integral membrane proteins, we have commonly used fragments of intracellular or extracellular loops, or N- or C-terminal domains. For smaller targets, we have used full-length protein immunogens to successfully generate target-specific NeuroMabs. We prefer that our immunogens are at least 5 kDa in size, and do not have any known requirements for establishing or maintaining proper folding. In cases where the only attractive options for immunogens were a few separate short stretches of less than 50 amino acids each, we generated fusions of these fragments. We have a focus on human targets, as many of our neuroscience end users study human samples and the mechanisms underlying human neurological disease. However, much of this research also involves animal models, especially rodents, such that specific immunoreactivity against both human and rodent orthologs is desirable. Selecting human sequences that share substantial identity with rodent sequences allows us to use rat and mouse brain tissue in screens, which offers advantages as discussed below.
Author Manuscript
Our most common immunogens are recombinant protein fragments, which typically generate a more robust and varied immune response, and yield a high number of mAbs that work well in different applications. Smaller synthetic peptides are used almost exclusively for highly focused projects such as disease-associated mutations, posttranslational modifications (PTMs), splice junctions, etc. Because of their size and complexity, designing recombinant protein immunogens that produce maximal target specificity and minimal cross-reactivity can be challenging. Expression, solubility, and affinity purification of recombinant mammalian proteins in E coli can be difficult compared with the ease of outsourcing chemical synthesis of a peptide immunogen. However, in our experience, the time, effort and cost put towards generating or obtaining recombinant protein immunogens has been worthwhile. From a retrospective analysis of 414 NeuroMab projects, we find a much higher project success rate for generating at least one validated NeuroMab when using recombinant protein immunogens (211 out of 290 projects or a 73% success rate) than when using synthetic peptide immunogens (47 out of 124 projects or a 38% success rate). One step we have incorporated into our workflow is to validate each affinity-purified immunogen by tandem mass spectrometry (MS) analysis, which has substantially reduced the number of failed projects due to an incorrect immunogen.
Author Manuscript
Mice are immunized using a short (30 day) but immunogen-intensive immunization protocol [13] that is a modified version of protocols developed to generate a strong IgG response in a much shorter time period than conventional immunization protocols [20; 21]. For each project, two naïve female BALB/c mice (6–8 weeks old) are immunized intraperitoneally using immunogen mixed with Sigma Adjuvant System adjuvant weekly for four weeks. Antiserum from the mice is analyzed for immunoreactivity against the immunogen by enzyme-linked immunosorbent assay (ELISA), by immunocytochemistry (ICC) against the target protein heterologously overexpressed in mammalian COS-1 cells, and by immunoblot against the target protein endogenously expressed in brain tissue. For three days preceding N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Gong et al.
Page 5
Author Manuscript Author Manuscript
splenocyte isolation and fusion, mice receive intravenous tail vein injections of immunogen in physiological saline. Splenocytes from both mice are then harvested and pooled to maximize the population of mAb specificities, and used in a fusion with Sp2/0-Ag14 (ATCC CRL-1581) B-lymphocyte hybridomas. A small fraction (1
>1
1.3
2.7
>1
>1
Fold Δ
32%
14%
8%
2%
15%
16%
PEG
74%
100%
24%
46%
83%
94%
EF
2.3
7.1
3.0
23.0
5.5
5.9
Fold Δ
% of wells with hybridomas @ 1 × 106 splenocytes/plate
2%
2%
1%
ND
2%
0%
PEG
9%
66%
3%
ND
40%
18%
EF
4.5
33.0
3.0
ND
20.0
NA
Fold Δ
% of wells with hybridomas @ 1 × 105 splenocytes/plate
Using larger capacity chamber: 100%, 100% and 50% of wells with hybridomas @ 1 × 107, 1 × 106 and 1 × 105 splenocytes/plate, resulting in >1, >3 and 25.0 fold increases, respectively.
*
NA = not applicable; ND = not done
PEG
Project
% of wells with hybridomas @ 1 × 107 splenocytes/plate
Author Manuscript
Fusion efficiency with electrofusion (EF) and polyethylene glycol (PEG)
Author Manuscript
Table 1 Gong et al. Page 29
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
Author Manuscript
Author Manuscript
Author Manuscript O00591 P19490 Q9JKA9 NP_599190
GABA(A)R, Pi
GluR1 glutamate receptor
HCN2
KCC2
O08773 Q8R464 Q62666
Pan-MAGUK
Pan-Nav1 Na+ channel
RGS14
SynCAM4
VAChT
Navbeta4
Q7M730
Q56A07
-
channel
Na+
Navbeta2
P38647
Pan-KChIP K+ channel
channel
Na+
Mortalin/GRP75
Currently not available
*
P31644
GABA(A)R, Alpha5
channel
P23576
GABA(A)R, Alpha2
KChIP4
P16157
Ankyrin-R
Q3YAB7
E9PE32
Ankyrin-G (staining)
K+
Accession #
Target
N425/45
N244/5
N133/21
N419/40
K28/86
K55/82
N168/6
N395/68
N52A/42
N423/75
N1/12
N71/37
N355/1
N431/64
N415/24
N399/19
N388A/10
N106/36
Clone
IgG1
IgG1
IgG2a
IgG2a
IgG1
IgG2a
IgG1
IgG2b
IgG1
IgG2a
IgG2a
IgG1
IgG1
IgG2a
IgG1
IgG1
IgG2b
IgG2a
IgG Subclass
RRID:AB_2532047
RRID:AB_10673109
RRID:AB_10698026
RRID:AB_2491079
RRID:AB_10698179
RRID:AB_2132595
RRID:AB_10673578
RRID:AB_2315895
RRID:AB_10674108
RRID:AB_2491080
RRID:AB_10697875
RRID:AB_10672304
RRID:AB_2315839
RRID:AB_2493092
RRID:AB_2491075
RRID:AB_2336904
RRID:AB_2336901
RRID:AB_10697718
RRID TCS
NA*
RRID:AB_10676101
RRID:AB_2179931
RRID:AB_2491098
RRID:AB_10673115
RRID:AB_10673406
RRID:AB_2301402
RRID:AB_2315896
RRID:AB_2120479
RRID:AB_2493100
RRID:AB_10672851
RRID:AB_2279449
RRID:AB_2315840
RRID:AB_2532051
RRID:AB_2491187
NA*
RRID:AB_2491109
RRID:AB_10673030
RRID Pure
NeuroMabs highlighted in current manuscript and their associated Research Resource Identifiers (RRIDs)
Author Manuscript
Table 2 Gong et al. Page 30
N Biotechnol. Author manuscript; available in PMC 2017 September 25.
