Roy Matkovic is studying epigenetic repression of HIV. After the virus integrates in the host genome, it can be inactive for a long time, and Matkovic, a molecular virologist at Institut Cochin in Paris, is learning how proteins keep the virus latent. Among other methods, he uses immunohistochemistry to confirm that the proteins of interest are present together in the same parts of the cell.
Matkovic ran into a problem in distinguishing co-localized proteins using immunofluorescence. “I need to make sure the primary antibodies are from two different species,” he says. If they’re from the same species, the secondary antibodies can’t distinguish them. Many of the antibodies that Matkovic has to use are from rabbit, so to really push his research forward, he needed a different way to visualize the proteins.
Antibodies are a staple of proteomic research, because they can specifically detect unique proteins in a range of applications, such as western blotting, immunohistochemistry or flow cytometry. But to visualize the primary antibodies, many applications require a labelled secondary antibody. These can distinguish primary antibodies that were generated in rabbit from those generated in mouse or other species, but can’t tell primary antibodies from the same species apart. This was an issue for Matkovic and his multiple rabbit antibodies. Researchers working with larger screens that detect many different proteins have even greater problems.
“There are more than 100 different antibodies that we routinely use in our screens,” says a senior biotech researcher, who asks to remain anonymous as his company is deep in stealth mode. In the search for new drugs for a range of diseases, he uses high-throughput and high-content screens to determine the mechanisms of action of novel compounds. Several of his primary antibodies are from the same species, and if he can’t detect those together, the combinations of proteins he can monitor is limited.
“We want to increase our multiplexing, so we’ve been trying different methods,” the researcher says. One method he is currently optimizing is to strip and reprobe his samples so he can stain the same cells with different sets of antibodies. That process proved useful, but wasn’t sufficient alone. The breakthrough came in the form of an innovative new kit, which gave him the flexibility to directly label primary antibodies from the same host species.
Matkovic also tried the new kit to detect his many rabbit antibodies. The only alternative he had considered was to overexpress one of the proteins with a fluorescent tag, but this leaves the cell with an unnaturally high amount of protein. Antibody detection of endogenous proteins is more likely to show colocalization that is functionally relevant, and that only became possible with labelling of the primary antibodies.
Primary antibody labelling hits the sweet spot
With this kit, called FlexAble from Proteintech, primary antibodies can be labelled with one of several fluorophores in about 10 minutes. This not only saves time, but also means that researchers don’t have to worry about using primary antibodies from the same species in the same experiment.
“The people who want to do these conjugations tend to be doing multiplexing,” says Deepa Shankar, Proteintech’s chief scientific officer. “We wanted to make sure multiplexing was easy.”
There are other methods of primary antibody labelling that allow multiplexing without a secondary antibody. However, those usually entail a complex process that often requires custom formulations of antibodies to create covalent chemical bonds. FlexAble uses an affinity linker that binds tightly, but not covalently, to the antibody, similar to a biotin-streptavidin interaction. The reaction only takes a few minutes.
“It should work in any buffer,” says Shankar. “You can use it with any antibody from the refrigerator or freezer.” Small volumes of antibody, as low as 0.5 micrograms, can be labelled. They can also be used in existing workflows alongside secondary antibody detection in the same experiment.
Matkovic found the signal and protein distribution were the same as he would get using secondary antibodies. And most importantly, he was able to visualize both targets at once. “I could get proper signals for the two anti-rabbit antibodies at the same time on the same coverslip,” he says.
A flexible future
For the biotech researcher, primary antibody labelling has made setting up high-content screens more manageable.
“If we want to stain 40 antibodies using one cell line and a couple of different conditions, there will be a lot of different plates and different wells,” he points out. FlexAble has saved time in his high-throughput workflow because there is no secondary antibody incubation step.
The biotech researcher is now limited only by the different wavelengths that his machines can pick up. To further increase the readings he can get, he is planning to use the strip and reprobe method in conjunction with primary antibody labelling. “Anything we can do to increase the data we get from a single plate is useful,” he says.
Now that Matkovic can finally see whether the proteins he studies are indeed both present in the right location to play a role in HIV repression, he can follow up with further characterization studies to confirm protein function.
Multiplex immunohistochemistry is far from the only application for the FlexAble kits. They can also be used in western blotting to detect proteins of the same size without stripping the blot, or to label antibodies for flow cytometry.
Shankar is keeping future applications and current research trends in mind. “People are doing spatial biology, cyclic immunofluorescence, or combining multiple readouts within a single cell.” All these methods rely on having antibodies that can detect a clear signal. “We're trying to make it simpler and easier,” she says. “The idea is to keep it as transparent as possible and leave the choice with the scientist.”