Background
Multiplex immunostaining has become an increasingly important approach in current biomedical research. However, the availability of directly labeled primary antibodies often poses a bottleneck for multiplex methods. Here we aimed to develop an antibody-labeling process that enables fast and easy conjugation of primary antibodies to fluorophores at a microgram to milligram scale, e.g. for the amount needed for one immunostaining experiment1 (figure 1).
Methods
For the development of this labeling procedure, we employed a broad range of methods including in silico protein design, molecular cloning, protein expression, protein purification, site-directed conjugation, BLI, DLS, cell culture, IF, IHC, flow cytometry and X-ray crystallography. Successful application of the developed labeling technique takes around 10 minutes and does not require any special equipment or proficiency in chemical conjugation or antibody purification.
Results
We designed peptide-based multivalent linkers that bind to primary IgGs from rabbit, mouse, rat or human species with an apparent dissociation rate koff (s-1) below 1.0 x 10-4 s-1 and covalently conjugated them to different fluorophores (CL+405/488/555/647/750). We validated the specificity of the binding between the linkers and their target IgGs and confirmed the proposed molecular arrangement with X-ray crystallography. Since these linkers are fluorescent, they offer a simple and convenient tool to functionalize primary IgGs from the aforementioned species with the desired fluorophores. We called this labeling approach FlexAble labeling because it embodies the simple steps of mixing and briefly incubating linkers and primaries. This procedure can be performed virtually in any laboratory for any amount of primaries right before applying the primaries in the desired assay. Moreover, primary antibodies can be in any buffer, since glycerol or other additives do not interfere with FlexAble labeling.
Here, we show that FlexAble labeled antibodies are suitable for immunofluorescence stainings, especially when multiplexing of primaries from the same species is required. Also, we present examples of FlexAble applied to flow cytometry, tissue IF, live time-lapse imaging, cyclic IF, and other more advanced experiments employing immunofluorescence detection of the target molecules.
Conclusions
We have established FlexAble, a novel antibody labeling process that enables rapid conjugation of primary antibodies to fluorophores and facilitates multiplex immunofluorescence experiments. We are expanding this method further to expedite labeling of primary antibodies with reporter proteins (HRP, PE, APC), biotin, DNA oligonucleotides, rare-earth metals etc. to support multiplex flow cytometry, nucleotide barcoding (e.g. 10x Genomics) and IMC (e.g. CyTOF).
Reference
www.nature.com/articles/d42473-023-00120-w
Abstract 76 Figure 1
FlexAble Antibody Labeling