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 Nanoparticle Exposure Can Awaken Dormant Viruses in the Lungs
16.01.2017 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt

nachricht Cholera bacteria infect more effectively with a simple twist of shape
13.01.2017 | Princeton University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Interfacial Superconductivity: Magnetic and superconducting order revealed simultaneously

Researchers from the University of Hamburg in Germany, in collaboration with colleagues from the University of Aarhus in Denmark, have synthesized a new superconducting material by growing a few layers of an antiferromagnetic transition-metal chalcogenide on a bismuth-based topological insulator, both being non-superconducting materials.

While superconductivity and magnetism are generally believed to be mutually exclusive, surprisingly, in this new material, superconducting correlations...

Im Focus: Studying fundamental particles in materials

Laser-driving of semimetals allows creating novel quasiparticle states within condensed matter systems and switching between different states on ultrafast time scales

Studying properties of fundamental particles in condensed matter systems is a promising approach to quantum field theory. Quasiparticles offer the opportunity...

Im Focus: Designing Architecture with Solar Building Envelopes

Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.

As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...

Im Focus: How to inflate a hardened concrete shell with a weight of 80 t

At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).

Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...

Im Focus: Bacterial Pac Man molecule snaps at sugar

Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.

The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

12V, 48V, high-voltage – trends in E/E automotive architecture

10.01.2017 | Event News

2nd Conference on Non-Textual Information on 10 and 11 May 2017 in Hannover

09.01.2017 | Event News

Nothing will happen without batteries making it happen!

05.01.2017 | Event News

 
Latest News

Water - as the underlying driver of the Earth’s carbon cycle

17.01.2017 | Earth Sciences

Interfacial Superconductivity: Magnetic and superconducting order revealed simultaneously

17.01.2017 | Materials Sciences

Smart homes will “LISTEN” to your voice

17.01.2017 | Architecture and Construction

VideoLinks
B2B-VideoLinks
More VideoLinks >>>