Nano-Secondary Antibodies

A new way to think about secondary antibodies
Nano-Secondaries are a novel class of secondary “antibodies” based on Nanobodies (tiny binding elements from Alpaca antibodies*) that bind to primary antibodies in a species and subtype specific manner. These recombinant antibody fragments are conjugated to the popular Alexa Fluor® dyes. Because Nano-Secondaries are derived from Nanbodies they are about 10 times smaller than conventional secondary antibodies.   This small size enables better tissue penetration and decreases the distance between epitope and label.  Nano-Secondaries bind to its particular target primary antibody with ultra-high affinity and specificity making them faster to stain with much smaller point resolution and great for cleaner images with lower background.  Currently, Nano-Secondaries against rabbit polyclonal IgGs and three different subclasses of mouse IgG (1, 2b, and 3) are available.

*As compared to conventional antibodies, alpacas uniquely express a single domain antibody (sdAbs). sdAbs contain only heavy chains and are devoid of light chains. The recombinantly expressed antigen binding domain of a sdAb is called VHH or Nanobody. Nano-Secondaries are IgG-specific Nanobodies that are coupled to Alexa Fluor dyes.

Unrivaled binding leads to cleaner images
Nano-Secondaries are subclass-specific and do not cross-react with IgGs from other commonly used species. This high specificity in combination with incredibly low background enables challenging immuno-assays such as multiplexed western blots or multiplexed super resolution microscopy. It is universally common to see some cross- reactivity between mouse IgG subclasses even after pre-adsorption against mouse serum. Nano-Secondaries eliminates this expectation.

Faster protocols by combining primary and secondary incubations
One-step immunostaining is the simultaneous incubation of a primary antibody and a Nano-Secondary antibody. This is due to the ultra-fast and strong binding kinetics of the Nano-Secondary to its target primary IgG. There is no need for two serial incubation steps, simply add both the primary IgG and the Nano-Secondary together to reduce incubation time and “hands-on” time. Simultaneous incubation supports multiplexing, deeper tissue penetration, and cell staining for flow cytometry.

Multiplex your immuno-assays with confidence
Nano-Secondaries can be applied in parallel in multiplex fluorescent western blotting or immunofluorescence microscopy experiments. This allows multiple targets to be analyzed simultaneously on the same blot or slide at the same time. Results rival those of traditional, sequential protocols. The combined image to the right shows triple stained HeLa cells via immunofluorescence. This experiment used polyclonal primary antibody (rabbit anti-lamin), and 2 monoclonal primary antibodies (mouse IgG1 anti-COX4 and mouse IgG2b anti-Tubulin) simultaneously stained with Nano-Secondary antibodies anti-rabbit IgG Alexa 568 (yellow), anti-mouse IgG1 Alexa 488 (green), and anti-mouse IgG2b Alexa 647 (magenta).

With western blot multiplexing it is not necessary to strip and re-probe the western blot membrane. Of course, detection requires the use of a  fluorescence blot reader.

Achieve finer details with smaller secondary antibodies
Nano-Secondary antibodies are about 1/10th the size of conventional secondary antibodies. Even after contently attaching numerous Alexa Fluor molecules, its small size enables better tissue penetration and decreases the distance between epitope and label. No secondaries are as uniquely designed as Nano-Secondary antibodies for super-resolution microscopy (e.g. STED and STORM). Your lab will get more detailed images in a single-plex, 2-plex or even a 3-plex staining experiment.

Camelidae single-domain antibodies are like IgGs on steroids

The family of animals known as Camelidae (camels, llamas, and alpacas) produce functional antibodies devoid of light chains, so-called "heavy chain" antibodies. These heavy chain antibodies recognize and bind their antigens via a single variable domain. When cleaved from their carboxy tail, these barrel-shaped structures (2x3 nm) are extraordinarily small, naturally-occurring, and intact antigen binding fragments (MW of 13 kDa). These fragments, called Nanobodies, are characterized by high specificity, affinities in the low nanomolar range, and dissociation constants in the sub-nanomolar range (typically 10- to 100-fold better than mouse IgGs). The compact size of Nanobodies makes them extremely stable at temperatures up to 70°C, and functional even in 2M NaCl or 0.5% SDS. These small and powerful antibody fragments can be used in a variety of unique applications. They will open up your research possibilities.