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What VHH ligands are

Camelid’s sera, in addition to conventional Ig antibodies, contain a unique class of functional heavy chain–only antibodies (HCAbs), lacking light chains. Antigen recognition is mediated by a single variable domain, originally referred to as VHH but also known as Nanobody®, a designation meant to point out its nanometer-scale dimensions. Due to their small size – ∼15 kDa instead of 150 kDa- and more compact structure, VHHs can contact target regions that are not accessible to conventional antibodies.

Their biochemical properties are outstanding:

Long shelf life (months at 4°C and years at -20°).
Can stand harsh conditions, which can be used for panning, including a marked resistance to chemical and thermal denaturation
NOT immunogenic due to their small size, stability, rapid blood clearance, and high identity with human VH
High solubility
High affinity and selectivity of only a single cognate target.

In addition, the unique convex structure of the paratope makes them capable of allosterically inhibiting the catalytic activity of specific target enzymes and/or stabilizing the enzyme in a particular conformation.

These properties coupled with a fast VHH selection technology (based on phage display) allow a vast range of applications such as:

Manufacturing of well-characterized bio-reagents directed against important cellular targets such as intracellular signaling proteins and cancer biomarkers
Production of chromophoric derivatives, for tracing specific antigens in living cells, allowing high-resolution, single-molecule localization/imaging in vivo (look at the NIRFP section for further details on this type of application).
Functional abrogation of specific intracellular antigens/proteins –a gene product-based knockdown approach complementary to CRISPR and RNAi
Investigation of specific protein-protein interactions, including disease-relevant interactions commonly considered undruggable
Deliver specific cargos to hard-to-access tissues
Generation of dedicated affinity adsorbents by covalently linking to solid, inert or magnetic supports.

Small is more!

Bioluminescence, our Bonfire probes

Bioluminescence is the production of light (photons) in living organisms. Bioluminescent assays usually rely on luciferase enzymes, which oxidize specific substrates (luciferins) that emit photons upon ground-state relaxation. Luciferase does not require light excitation, thus allowing for a sensitive, low-background assay, less prone to interference by surrounding fluorescent compounds and fluorophore photobleaching.

Bonfire 1 - Fluc

Fluc is a luciferase with a molecular mass of 62kDa, produced by the firefly Photinus pyralis. This enzyme catalyzes a two-step reaction that uses as substrates D-luciferin, adenosine triphosphate (ATP) and oxygen to yield oxyluciferin in an electronically excited state. Due to its ATP dependence, this luciferase is typically employed in biochemical assays that measure ATP and ADP concentrations. Because of very low-level cellular background luminescence, it is also commonly employed as a reporter for ex-vivo and in-vivo gene expression studies.

Bonfire 2 - YY5luc

YY5 is a Fluc mutant with improved thermostability and catalytic activity at low D-luciferin concentrations that displays enhanced bioluminescence and a superior animal tissue penetration capacity due to its longer wavelength light emission. These features make YY5 an excellent tool for in vivo imaging.

Bonfire 3 - Rluc

Rluc is a luciferase variant from the soft coral Renilla reniformis with a molecular mass of 36 kDa. It differs in sequence from Fluc and does not require ATP as a co-substrate for light production. Rluc catalyzes the oxidation of its luciferin coelenterazine substrate to coelenteramide, with the emission of light. Rluc is also used as a reporter gene, often in combination with Fluc in dual-luciferase assays. In such assays, one luciferase (typically Fluc) is used to monitor target gene expression, while the other (Rluc) serves as an internal reference for assay normalization as well as to inform on cytotoxicity, differences in cell number and transfection efficiency in the case of transiently transfected cell lines.

Peroxidase Tag

The Peroxidase Tag can catalyze a colorimetric reaction or emit light (chemiluminescence) depending on the substrate that is used for detection. Both reactions are used for a variety of immunoassays measuring different biochemical and clinical analytes as well as for multiple detection and assay techniques such as enzyme-linked immunosorbent assays (ELISA) or Western blotting (with conjugated antibodies as key reagents). Chemiluminescence, which is also widely employed for imaging purposes and biosensor construction, does not require external excitation and thus affords a deeper tissue penetration, a superior bio-imaging sensitivity and a generally higher signal-to-background ratio.

Horseradish Peroxidase - HRP

HRP is a 44 kDa, heme- and Ca2+-containing enzyme from horseradish (Armoracia rusticana) belonging to the family of plant peroxidases. It catalyses the hydrogen peroxide-mediated, one electron oxidation of various organic and inorganic substrates, yielding a characteristic colour change easily detectable by spectrophotometry. Due to its wide substrate specificity and high catalytic activity and stability, HRP is often employed as a building block for the construction of chemiluminescent bio- and immuno-sensors. Because of its versatility, HRP is often used as an antibody conjugate for target detection and quantification by western blot, ELISA and immunochemistry.

Fluorescence, our Pharos probes

Fluorescence is the emission of light by a compound, called fluorophore, which becomes excited upon light absorption and emits longer wavelength light when returning to its relaxed, ground state. A clear example of fluorescence occurs when the absorbed radiation is in the UV region, while the emitted light is in the visible range of the electromagnetic spectrum. Different fluorophores emit different light colors based on their emission light wavelengths, thus allowing multiplex assays and the measurement of multiple targets in the same microplate.

Pharos 1 - NIRFP (Near Infrared Fluorescent Proteins) (670nm-720nm)

NIRFPs are composed of fluorophores, derived from natural photoreceptors incorporating biliverdin Ixα autocatalytically, that emit in the NIR part of the electromagnetic spectrum (650-900 nm). These innovative fluorescent proteins allow overcoming some of the main drawbacks of fluorescence-based assays such as the low signal-to-noise ratio caused by naturally occurring cellular fluorophores and interference by intrinsically fluorescent test compounds. The wavelength range of 650–900 nm enables high-resolution imaging through deep tissue penetration and the presence of biliverdin in eukaryotic cells as an intermediate of heme metabolism allows to use NIRFP in a manner similar to GFP-like probes. NIRPs can thus be exploited for the setting-up of multiplex assays and can be imaged from subcellular to whole-body scales in small animals, such as mice. NIR fluorophores have also been employed to pinpoint new cellular events as well as to explore cell architecture with the use of fluorescence nanoscopy techniques. We offer miRFPs (monomeric infrared FPs), which are enhanced monomeric NIRFPs derived from cyanobacteriochrome photoreceptors with a higher apparent cellular brightness compared to previously described Infrared FPs. We have a whole set of miRFPs (miRFP670, miRFP680, miRFP703, miRFP713 and miRFP720) that can be combined in different assortments for two-colors or multicolor NIR imaging.

Pharos 2 - NIR-Fbs (Near-infrared Fluorescent-bodies) (ex 645nm-em 670nm)

NIR-tracks are small, 32 kDa fluorescent VHHs internally tagged with NIRFPs. VHHs bind their predefined targets with high affinity and specificity, plus VHH conjugation increases the photostability of the fluorophore, producing a higher molecular brightness in mammalian cells without the addition of biliverdin, as well as its chemical stability over a wide range of pH values (from pH 4 to 11). The most innovative feature of NIR-track is their enhanced fluorescence in cells co-expressing the cognate antigen and their nearly complete degradation, and concomitant background abrogation, in the absence of the target antigen.

Pharos 3 - GFP

GFP from the jellyfish Aequorea victoria is the most studied FP and multiple, optimized fluorescence as well as folding/stability variants are available. What made GFP so popular and easy to use is its internal (i.e., synthesized from resident, polypeptide chain amino acid residues) p-hydroxybenzylidene-imidazolidone chromophore that only needs O2 to fluoresce. These features have made GFP an optimal reporter gene, and a first-choice tag for multiple cellular components and applications.

Pharos 4 - mCherry

mCherry is a small (26.7 kDa) monomeric red fluorescent protein (mRFPs) derived from sea anemones belonging to the genus Discosoma. As GFP, it is used as a reporter gene and protein tag. Its low molecular weight and ensuing reduced impact on the targeting system, along with its strong brightness, folding efficiency and tagging tolerance, as well as chemical (especially pH) stability, have made it almost as popular as GFP and one of the most widely-used red fluorescent proteins.

Fragment-crystallizable

In recent years there has been a surge of monoclonal antibody (mAb) applications for research, diagnosis and therapy. Due to their large (150 kDa) size, however, IgG1 mAbs often exhibit poor tissue penetration and a limited ability to access their target epitopes. Some of these limitations can be overcome through the design of smaller-size scaffolds based on the IgG Fc region. Conjugation of a VHH to an IgG-derived Fc not only will allow its detection with the use of standard secondary antibodies but will also lead to an extended in vivo half-life, close to that of conventional IgG antibodies. We offer human and mouse recombinant Fcs as well as Fcs from other species on a customer request basis, to render VHH easily detectable with secondary antibodies and to increase their in vivo half-life.