SCIENCE and SOCIETY Antibodies: they are proteins too... by Dr Michael Fiebig
J
ohnny stared in disbelief. This was the last Western Blot he needed…one last clear band at the right size…but what was THAT?! The lanes on his blot, even his previously blank negative control, had more bands than Glastonbury. What had happened? Johnny had done everything exactly the same, everything apart from opening a new vial of antibody. It looked the same; same target, species, isotype, supplier, catalogue number and concentration, and yet it dawned on Johnny that he had not the slightest idea what was in that vial.
Usually we biologists want to know. You wouldn’t work with a plasmid you haven’t sequenced and you want to know the sequence of the protein you’re working with. But somehow we don’t seem to care about the composition of antibodies we use. Superficially, there’s not much to it. There’s a constant region, which differs between species and comes in different isotypes, and there are variable domains that recognise the antigen. It’s so simple you could get a kid to draw it.
But there is more to it. The sequence that determines binding is extremely important and characteristic of an antibody, but usually it’s completely unknown. In a recent article in Nature (co-signed by 110 other leading scientists), Bradbury and Plückthun (1) address the issue of antibodies and reproducibility. They believe “poorly characterized and ill-defined antibodies were in large part to blame for a study (...) being able to replicate the scientific results of only 6 of 53 landmark preclinical studies.” Forget poor Johnny and his Western – think of the wasted time and funding (the authors estimate US$350 million per annum in the USA alone) and think about all those ‘fantastic’ papers you’ve always been a bit sceptical about. Of course antibodies are superb reagents, and the contribution readily raised polyclonal antibodies have made to science is undeniable. However, they are inherently only ever available in finite amounts. Hybridoma-based production can also bear problems. Long-term producing hybridomas can ‘drift’ and the antibody may not be as monoclonal as expected (e.g. multiple light chains (2) or aberrant chains (3)). Bradbury and Plückthun (1) propose that polyclonals should be “phased out of research entirely” and that for hybridoma-produced monoclonal antibodies, sequences should be determined and all antibodies produced recombinantly. This would revolutionize the way antibodies are made, ensuring that antibodies are not just defined by a label on a tube, but by their amino-acid sequence.
This technology exists and has been used extensively by the pharmaceutical sector, but has been inaccessible for most academic scientists – that is, until very recently. The Oxford based start-up, Absolute Antibody, provides antibody sequencing, engineering and entirely recombinant expression services. All production is performed in a serumfree mammalian cell system ensuring ultra-pure, absolutely defined products. Another advantage of producing antibodies starting from their sequence is that they can be reformatted – your favourite mouse IgG1 can now be a rat
IgG2B. Likewise, constant domains can be engineered like the Fc-Silent™ format from Absolute Antibody that doesn’t bind to complement and Fc-receptors.
Whilst Absolute Antibody are busy generating new antibody formats to suit the needs of our customers, we hope that the next time you pick up a tube of antibody you ask yourself, “do I really know what’s in this?” Maybe it’s time for a change; maybe it’s time for Absolute Antibody. If any of this interests you and you would like to find out more, email sales@absoluteantibody.com or visit www. absoluteantibody.com. References 1. Bradbury, A. & Plückthun, A. (2015) Reproducibility: Standardize antibodies used in research. Nature. 518(7537):27-29. 2. Yang, J. et al. (2013) Comparison of two functional kappa lightchain transcripts amplified from a hybridoma. Biotechnol. Appl. Biochem. 60(3):289-297. 3. Ding, G., et al.(2010) Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes. Cell. Mol. Immunol. 7(5):349-354.
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Dr Michael Fiebig recently completed his DPhil at the Sir William Dunn School of Pathology and now works for Absolute Antibody as Product Development Manager
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