Ligand-binding assays have long been a staple for researchers who need to know when and how strongly two species bind. Examples include agonists binding to receptors, molecules interacting in an intracellular signaling cascade and G-protein-coupled receptor membrane proteins binding to ligands extracellularly or signaling molecules intracellularly. The traditional way to keep track of the components of a ligand-binding assay is to label with radioactive isotopes. Radiolabels give strong, clean signals that are unmistakable and easy to detect.
However, anyone who uses radioactive reagents in the lab, for ligand-binding or other assays, will tell you that it entails work above and beyond normal experimental protocols. There are extra preparatory steps, additional cleanup steps, protective gear that hinders your movements and special handling and disposal rules. The hazards involved in using radioactive reagents won’t help you remain calm, cool and collected throughout the process, either. And the list of reasons to nix radiolabels in the lab doesn’t stop there. “The shelf life of nonradioactive labels is a key advantage,” says Roger Bosse, global product leader of Alpha Technologies at PerkinElmer. “For example, the shelf life of [PerkinElmer’s] DELFIA-labeled molecules is generally in the range of one [to] two years when stored correctly. This is in contrast to radioactively labeled ligands—125I-labeled ligands typically have shelf lives of two [to] three weeks. Even tritiated ligands can have shelf lives of only a few months, due to degradation of bonds of the ligand from the energy resulting from radioactive decay.” Today, ligand-binding assays come in more nonradioactive varieties than ever before.
One of the best-studied ligand-binding systems is the movement of membrane proteins whose extracellular domains act as ligand receptors for extracellular signals. This event can be studied biochemically, with purified proteins in solution, or it can be examined in a more physiological, whole-cell context using cell cultures. Cisbio’s Tag-lite® system of homogeneous time-resolved fluorescence (HTRF) tools is a cell-based system for studying receptor-ligand binding that uses time resolved-fluorescence resonance energy transfer (TR-FRET) as a readout for binding activity. “The technology is based on the encoding of a tagged receptor,” says François Degorce, director of marketing and communication at Cisbio Bioassays. “Once the receptor expressed at the membrane, it can be very specifically labeled via the SNAP-tag with one of the HTRF dyes. The pre-labeled cells can then be combined with a ligand labeled with the other HTRF acceptor dye (either red or green). This combination will generate a TR-FRET signal upon binding of the ligand to the receptor.” The Cisbio platform has grown recently to incorporate labeling technology called SNAP-tag and CLIP-tag (originally offered by New England Biolabs) and HaloTag® (from Promega).
Tag-lite is also valuable as a cell-based screening platform for studying the binding of antibodies to membrane proteins such as G-protein-coupled receptors and receptor tyrosine kinases. After expressing the membrane protein with a SNAP-tag, the tag is labeled covalently with an HTRF dye via a fluorophore-bearing substrate. The cells are then ready to use in screening applications in one of three formats: ligand inhibition, biobetter selection and sandwich assay. “The technology works equally well or even better for the screening of biotherapeutics,” says Degorce. “In those cases, monoclonal antibodies can be even more easily labeled with acceptors to look for direct binders, biosimilars or biobetters, or for antibody characterization. Some of our users are also starting to use Tag-lite to determine ligand affinity constants such as Kon and Koff.” The availability of a labeled ligand is crucial for small molecules and peptides, because it is necessary to ensure that the label will not affect the binding ability and characteristics of ligands. “For biotherapeutics, the labeling is quite straightforward, and binding tests work in almost all situations,” Degorce says. “Other than this, these binding assays are very easy to implement, as they just involve the binding between a prelabeled cell and a fluorescent ligand.”
Using similar signaling mechanisms, Life Technologies also uses TR-FRET signals to indicate ligand binding. In its LanthaScreen™ kinase activity assays, Life Technologies typically uses two types of FRET donor-acceptor pairs—terbium/fluorescein and europium/AlexaFluor® 647—but researchers can order custom donor-acceptor pairs according to their needs. For example, the company offers europium or terbium donors, additional Life Technologies acceptor fluorophores and fluorophores such as GE Healthcare’s CyDyes and PerkinElmer’s ULight dyes.
Life Technologies also incorporates LanthaScreen into kits for different screening purposes. The LanthaScreen™ TR-FRET PPAR gamma Competitive Binding Assay is designed for high-throughput screening of ligands for peroxisome proliferator-activated receptor-gamma (PPAR gamma). The kit includes the ligand-binding domain of human PPAR gamma (tagged with glutathione S-transferase (GST)), a terbium-labeled anti-GST antibody and a fluorescent small-molecule pan-PPAR ligand called Fluormone™ Pan-PPAR Green. Another assay, the LanthaScreen™ PXR (SXR) Competitive Binding Assay, is designed for testing the affinity of compounds. It includes purified PXR (SXR)-LBD protein, a terbium-labeled anti-GST antibody and a proprietary fluorescent Fluormone™ ligand.
Enzyme-linked immunoadsorbent assays (ELISAs) are commonly used ligand-binding assays in which a ligand-binding species (usually an antibody) is attached to a multiwell ELISA plate. PerkinElmer puts a new spin this, combining the convenience of the ELISA with the precision of time-resolved fluorescence assays. The company’s dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA®) uses a lanthanide-based fluorescent label. “Lanthanide fluorescence provides long signal decay times, allowing a delayed signal measurement to remove background interference,” says Bosse. “Due to the time-resolved nature of the assay, background is very low and sensitivity increased compared to traditional fluorescence assays. Biochemical ligand-binding assays, whole-cell or fixed-cell ligand-binding assays and cell membrane-based assays can be performed.” In the place of a traditional radiolabeled ligand is a ligand labeled with a lanthanide chelator, which is often europium, but could instead be samarium, terbium or dysprosium. Lanthanide chelates are used because their unique fluorescence properties make them sensitive labels. Europium is the most sensitive and gives the best fluorescent signal. However, other chelates, such as samarium and terbium, are important as second labels in dual-labeling assays to detect the analyte requiring less sensitivity.
DELFIA ligand-binding assays can take the form of coated plates or filtration assays, and they have been used to characterize antibodies and to study the binding of therapeutic antibodies. In addition, says Bosse, “the enhanced sensitivity [of the DELFIA assay] allows working with endogenous or transfected receptors.” Researchers can label their own ligands with europium (PerkinElmer supplies materials and protocols for this) or use PerkinElmer’s custom labeling services. DELFIA assays can detect down to attogram levels of ligands, and they have a 4- to 5-log dynamic range, which is wider than traditional ELISAs. In addition, DELFIA assays can be used in a 384-well format and multiplexed with up to three analytes per well.
Another readout for ligand binding is DiscoveRx's proprietary enzyme fragment complementation (EFC) system, which uses the enzyme β-galactosidase as a detection tool. The company splits β-galactosidase into two fragments that are inactive when separated but form an active enzyme when combined in a process called complementation. The subsequent enzymatic activity in the presence of substrate is then detected by chemiluminescence. In the EFC system, the two inactive fragments of β-galactosidase are attached to the interacting proteins of interest. When binding interactions occur between the two labeled proteins, the β-galactosidase fragments are in close enough proximity to combine and form an active β-galactosidase enzyme.
DiscoveRx incorporates its EFC technology into both biochemical and cell-based formats. The biochemical HitHunter® assays are competitive immunoassays in which one of the β-galactosidase fragments is conjugated to a ligand of interest (such as cAMP, cGMP or cortisol). The labeled ligand competes with free ligand for binding to the target—and the subsequent signal when the other β-galactosidase fragment is introduced indicates the proportion of labeled and free ligands bound. For studying protein trafficking and protein-protein interactions in whole cells, the PathHunter® assays use EFC technology in cell cultures expressing β-galactosidase fragments as tagged fusion proteins.
With so many new and varied tools to study ligand binding, do we need radioactive labels anymore? Occasionally, yes—for example, in assays that require very small ligands, such as ion channel studies. In some situations, nonradioactive labels are simply too bulky. “Radioactive isotopes are very small and generally do not cause steric hindrance,” says Bosse. “Radioisotopes such as 3H, 35S, 33P and 14C have the same size as their naturally occurring and stable counterparts. 125I, a bulky radioisotope, is by far less bulky than any fluor. Chemistries for labeling based on primary amines on sulfhydryl group modifications with radioactive isotopes are well established.” This sentiment is echoed by Degorce, who says that the limit of Cisbio’s labeling technology thus far is very small ligands (such as ions) and lipidic ligands. In addition, complex ligands can sometimes lose their activity when coupled to a dye. Nevertheless, complex peptides such as chemokines have been successfully labeled with small HTRF dyes without loss of binding capabilities. For instance, the combination of CXCR4 pre-labeled cells and labeled SDF1-alpha was shown to provide a much more robust and reliable binding assay system compared to those involving iodinated ligands. As new detection tools emerge, perhaps steric hindrance will no longer hinder our ability to be radioactivity-free.
The image at the top of the page is PerkinElmer's AlphaScreen® SureFire® Assay.