Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

New technique efficiently turns antibodies into highly tuned 'nanobodies'

03.11.2014

Antibodies, in charge of recognizing and homing in on molecular targets, are among the most useful tools in biology and medicine. Nanobodies – antibodies' tiny cousins – can do the same tasks, for example marking molecules for research or flagging diseased cells for destruction. But, thanks to their comparative simplicity nanobodies offer the tantalizing prospect of being much easier to produce.

Unfortunately, their promise hasn't been fully realized, because scientists have lacked an efficient way of identifying the nanobodies most closely tuned to their targets. However, a new system, developed by researchers at Rockefeller University and their collaborators and described today in Nature Methods, promises to make nanobodies dramatically more accessible for all kinds of research.

Antibodies are defensive proteins deployed by the immune system to identify and neutralize invaders. But their power can be harnessed in other ways as well, and they are used in biology and medicine for visualizing cellular processes, attacking diseased cells and delivering specific molecules to specific places. Like their larger cousins, nanobodies can also be used for these tasks but their small size makes nanobodies much easier to grow in bacterial factories. They can also access hard to reach places that may be off limits to larger molecules.

"Nanobodies have tremendous potential as versatile and accessible alternatives to conventional antibodies, but unfortunately current techniques present a bottleneck to meeting the demand for them," says study author Michael Rout, head of the Laboratory of Cellular and Structural Biology at Rockefeller. "We hope that our system will make high-affinity nanobodies more available, and open up many new possible uses for them."

... more about:
»GFP »Laboratory »Rockefeller »immune »proteins »sequence

In their first studies, the team generated high-affinity antibodies, those that are capable of most precisely binding to their targets, directed against two fluorescent proteins that biologists often use as markers to visualize activity within cells: GFP and mCherry. Their new system, like existing ones for generating antibodies, begins with an animal, in this case llamas housed in a facility in Connecticut.

Llamas were chosen because the antibody variants they produce are easily modified to generate nanobodies, which are only one-tenth the weight of a regular antibody. The llamas were immunized with GFP and mCherry, prompting their immune systems to generate antibodies against these foreign proteins.

"The key was to figure out a relatively fast way of determining the genetic sequences of the antibodies that bind to the targets with the greatest affinity. Up until now obtaining these high-affinity sequences has been something of a holy grail," says Brian Chait, Camille and Henry Dreyfus Professor and head of the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry at Rockefeller. "Once those sequences are obtained, it's easy to engineer bacteria to mass produce the antibodies."

The researchers, led by graduate student Peter Fridy and postdoc Yinyin Li, started by making antibody sequence databases from RNA isolated out of antibody-producing cells in the llamas' bone marrow. Next, they picked out the tightest binding GFP and mCherry antibodies from blood samples from the same llamas, and chemically cut these into smaller pieces, keeping only the antigen-binding section to create nanobodies.

They then determined partial sequences of the amino acids that made up the protein of the nanobodies with a technique known as mass spectrometry. Using a computer algorithm called "llama magic," developed by David Fenyö and Sarah Keegan of New York University School of Medicine, they matched up the composition of the highest affinity nanobodies with the original RNA sequence. With this sequence, they could engineer bacteria to mass produce the nanobodies before putting them to use in experiments.

Antibodies are often used to isolate a particular structure within a cell so scientists can remove and examine it, and the team did just that with their new nanobodies. They purified various cellular structures tagged with GFP or mCherry, and also visualized these structures in situ.

All in all, their procedure generated 25 types of nanobodies capable of precisely targeting GFP and six for mCherry, a far more diverse set of high affinity nanobodies than is typically possible with conventional techniques.

This abundance opens up new options. Scientists can select only the best ones, eliminating nanobodies that by chance cross-react with other molecules, or string together two nanobodies that attach to different spots on the same target molecule to generate a super-high-affinity dimer, exactly as the researchers demonstrated for the GFP nanobodies. This super-high-affinity could be a powerful feature when delivering therapeutic or diagnostic molecules because it would lower the required dosage, and so reduce unwanted side effects.

"Given that we can now readily identify suites of high affinity nanobodies, the future for them as research tools, diagnostics and therapeutics looks bright," says Rout.

Zach Veilleux | EurekAlert!
Further information:
http://www.rockefeller.edu

Further reports about: GFP Laboratory Rockefeller immune proteins sequence

More articles from Life Sciences:

nachricht BigH1 -- The key histone for male fertility
14.12.2017 | Institute for Research in Biomedicine (IRB Barcelona)

nachricht Guardians of the Gate
14.12.2017 | Max-Planck-Institut für Biochemie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Long-lived storage of a photonic qubit for worldwide teleportation

MPQ scientists achieve long storage times for photonic quantum bits which break the lower bound for direct teleportation in a global quantum network.

Concerning the development of quantum memories for the realization of global quantum networks, scientists of the Quantum Dynamics Division led by Professor...

Im Focus: Electromagnetic water cloak eliminates drag and wake

Detailed calculations show water cloaks are feasible with today's technology

Researchers have developed a water cloaking concept based on electromagnetic forces that could eliminate an object's wake, greatly reducing its drag while...

Im Focus: Scientists channel graphene to understand filtration and ion transport into cells

Tiny pores at a cell's entryway act as miniature bouncers, letting in some electrically charged atoms--ions--but blocking others. Operating as exquisitely sensitive filters, these "ion channels" play a critical role in biological functions such as muscle contraction and the firing of brain cells.

To rapidly transport the right ions through the cell membrane, the tiny channels rely on a complex interplay between the ions and surrounding molecules,...

Im Focus: Towards data storage at the single molecule level

The miniaturization of the current technology of storage media is hindered by fundamental limits of quantum mechanics. A new approach consists in using so-called spin-crossover molecules as the smallest possible storage unit. Similar to normal hard drives, these special molecules can save information via their magnetic state. A research team from Kiel University has now managed to successfully place a new class of spin-crossover molecules onto a surface and to improve the molecule’s storage capacity. The storage density of conventional hard drives could therefore theoretically be increased by more than one hundred fold. The study has been published in the scientific journal Nano Letters.

Over the past few years, the building blocks of storage media have gotten ever smaller. But further miniaturization of the current technology is hindered by...

Im Focus: Successful Mechanical Testing of Nanowires

With innovative experiments, researchers at the Helmholtz-Zentrums Geesthacht and the Technical University Hamburg unravel why tiny metallic structures are extremely strong

Light-weight and simultaneously strong – porous metallic nanomaterials promise interesting applications as, for instance, for future aeroplanes with enhanced...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

See, understand and experience the work of the future

11.12.2017 | Event News

Innovative strategies to tackle parasitic worms

08.12.2017 | Event News

AKL’18: The opportunities and challenges of digitalization in the laser industry

07.12.2017 | Event News

 
Latest News

Plasmonic biosensors enable development of new easy-to-use health tests

14.12.2017 | Health and Medicine

New type of smart windows use liquid to switch from clear to reflective

14.12.2017 | Physics and Astronomy

BigH1 -- The key histone for male fertility

14.12.2017 | Life Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>