A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
- University of California, Davis, United States
Abstract
Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.
Data availability
Plasmids and R-mAb sequences will be made available via Addgene (https://www.addgene.org/James_Trimmer/).
Article and author information
Author details
Funding
National Institute of Neurological Disorders and Stroke (U24 NS050606)
- James S Trimmer
National Institute of Neurological Disorders and Stroke (R24 NS092991)
- James S Trimmer
National Institute of Neurological Disorders and Stroke (U24 NS109113)
- James S Trimmer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#20485) of the University of California Davis. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California Davis (Animal Welfare Assurance Number A-3433-01). All procedures were performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.
Copyright
© 2019, Andrews et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,990
- views
-
- 1,066
- downloads
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
eLife 8:e43322.
https://doi.org/10.7554/eLife.43322
Further reading
-
- Neuroscience
Unipolar brush cells (UBCs) are excitatory interneurons in the cerebellar cortex that receive mossy fiber (MF) inputs and excite granule cells. The UBC population responds to brief burst activation of MFs with a continuum of temporal transformations, but it is not known how UBCs transform the diverse range of MF input patterns that occur in vivo. Here, we use cell-attached recordings from UBCs in acute cerebellar slices to examine responses to MF firing patterns that are based on in vivo recordings. We find that MFs evoke a continuum of responses in the UBC population, mediated by three different types of glutamate receptors that each convey a specialized component. AMPARs transmit timing information for single stimuli at up to 5 spikes/s, and for very brief bursts. A combination of mGluR2/3s (inhibitory) and mGluR1s (excitatory) mediates a continuum of delayed, and broadened responses to longer bursts, and to sustained high frequency activation. Variability in the mGluR2/3 component controls the time course of the onset of firing, and variability in the mGluR1 component controls the duration of prolonged firing. We conclude that the combination of glutamate receptor types allows each UBC to simultaneously convey different aspects of MF firing. These findings establish that UBCs are highly flexible circuit elements that provide diverse temporal transformations that are well suited to contribute to specialized processing in different regions of the cerebellar cortex.
-
- Neuroscience
The specific role that prolactin plays in lactational infertility, as distinct from other suckling or metabolic cues, remains unresolved. Here, deletion of the prolactin receptor (Prlr) from forebrain neurons or arcuate kisspeptin neurons resulted in failure to maintain normal lactation-induced suppression of estrous cycles. Kisspeptin immunoreactivity and pulsatile LH secretion were increased in these mice, even in the presence of ongoing suckling stimulation and lactation. GCaMP fibre photometry of arcuate kisspeptin neurons revealed that the normal episodic activity of these neurons is rapidly suppressed in pregnancy and this was maintained throughout early lactation. Deletion of Prlr from arcuate kisspeptin neurons resulted in early reactivation of episodic activity of kisspeptin neurons prior to a premature return of reproductive cycles in early lactation. These observations show dynamic variation in arcuate kisspeptin neuronal activity associated with the hormonal changes of pregnancy and lactation, and provide direct evidence that prolactin action on arcuate kisspeptin neurons is necessary for suppressing fertility during lactation in mice.
