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

Abstract

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

eLife digest

The immune system fights off disease-causing microbes using antibodies: Y-shaped proteins that each bind to a specific foreign molecule. Indeed, these proteins bind so tightly and so specifically that they can pick out a single target in a complex mixture of different molecules. This property also makes them useful in research. For example, neurobiologists can use antibodies to mark target proteins in thin sections of brain tissue. This reveals their position inside brain cells, helping to link the structure of the brain to the roles the different parts of this structure perform.

To use antibodies in this way, scientists need to be able to produce them in large quantities without losing their target specificity. The most common way to do this is with cells called hybridomas. A hybridoma is a hybrid of an antibody-producing immune cell and a cancer cell, and it has properties of both. From the immune cell, it inherits the genes to make a specific type of antibody. From the cancer cell, it inherits the ability to go on dividing forever. In theory, hybridomas should be immortal antibody factories, but they have some limitations. They are expensive to keep alive, hard to transport between labs, and their genes can be unstable. Problems can creep into their genetic code, halting their growth or changing the targets their antibodies recognize. When this happens, scientists can lose vital research tools.

Instead of keeping the immune cells alive, an alternative approach is to make recombinant antibodies. Rather than store the whole cell, this approach just stores the parts of the genes that encode antibody target-specificity. Andrews et al. set out to convert a valuable toolbox of neuroscience antibodies into recombinant form. This involved copying the antibody genes from a large library of preserved hybridoma cells. However, many hybridomas also carry genes that produce non-functional antibodies. A step in the process removed these DNA sequences, ensuring that only working antibodies made it into the final library. Using frozen cells made it possible to recover antibody genes from hybridoma cells that could no longer grow.

The recombinant DNA sequences provide a permanent record of useful antibodies. Not only does this prevent the loss of research tools, it is also much more shareable than living cells. Modifications to the DNA sequences in the library allow for the use of many antibodies at once. This could help when studying the interactions between different molecules in the brain. Toolkits like these could also make it easier to collaborate, and to reproduce data gathered by different researchers around the world.