What You always Wanted to Know about Ligand Binding Assays
These kind of assays are quite common in biological research. In general, a ligand binding assay is a tool to investigate the binding activity of a biological or chemical component to another biological or chemical component. Either both components bind to each other in a liquid phase (like buffer), or one of the binding partners is previously immobilized to a solid phase (like beads, column resins, multiplates, filters, etc.). This is why the huge amount of different ligand binding assays currently available can be separated into the liquid phase binding assays, and the solid phase ligand binding assays, respectively.
Putative ligands can principally comprise the following: 1. chemicals, e.g. like reporter fluorophores, or other small molecules; 2. biologicals, e.g. proteins, peptides, nucleic acids, lipids, or charbohydrates; 3. complex organisms, e.g. like human pathogens of viral or bacterial origin. By definition the ligand is the molecule which binding properties are to be analyzed. This ligand can be bound to one or more binding partners. Ideally one of the binding partners emit a signal upon binding, which afterwards can be detected by an appropriate apparatus or device. If the binding event proceeds in a quantitative manner, the detected signal should also increase with the binding. This opens the possibility to create a pretty good signal-to-analyte ligand binding assay plot, which in most cases can be described by a mathematical model or function.
Liquid phase ligand binding assays can include: 1. intercalation of a fluorescence dye into a RNA or DNA ligand, which emits fluorescence through secondary structure conversion; 2. hybridisation of a molecular beacon to an nucleic acid ligand of pathogen origin, the emitted fluoroescence can be detected in a fluorometer; 3. Real-Time RT-qPCR, which is a powerful method to amplify DNA ligands for more sensitive detection, mediated again by a beacon or fluorophore intercalator.
The main problem you have to tackle with is the specificity and selectivity of ligand binding assays. In terms of the liquid phase ligand binding assays mentioned above, the fluorescent intercalator is the most insensitive option, combined with low selectivity, because the dye will intercalate into virtually any nucleic acid present in the liquid phase. The molecular beacon only hybridises to complementary nucleic acid sequences, which increases dramatically the ligand binding assay selectivity, but might reduce the assay sensitivity as well, because only one fluorophor per binding event is detected. Finally the Real-Time RT-qPCR in combination with a molecular beacon implies both accurate selectivity and satisfying sensitivity, as the target ligand DNA is amplified through PCR.
If you can not overcome the low ligand binding assay selectivity, it is a good idea to introduce another molecule, which is also capable to bind to the ligand. In other words, these kind of assays imply two molecules, which bind to the same target ligand, but exhibit distinct binding sites, and ideally do not interfere with each other. In the scientific literature, this kind of ligand binding assays are often referred to as sandwich assays, because the ligand is usually captured by one molecule, and detected by another molecule.
Sandwich ligand binding assays are often performed as solid phase assays, because this opens the possibility to capture the ligand from a sample by immobilization of the first binding partner, followed by (almost) complete removal of all unbound traces (or ligand) from the formed complex. Immobilization can occur on any material or surface, e.g. like multiwell plates, beads, filters, or column resins. Immobilized ligands in this way can be detected by another molecule, which often bears a reporter molecule or catalytically active enzyme capable to convert a substrate into the active (= detectable) form.
The following liquid phase ligand binding assays are discussed in more detail:
- Real-Time RT-qPCR as a powerful tool to quantify nucleic acid therapeutics like microRNA, siRNA, shRNA, aptamers, etc in biological samples,
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- immunoprecipitation, which can be used to selectively capture a compound from a biological sample, followed by precipitation of the capture complex, and removing of all nonrelevant trash: an underestimated tool to increase the ligand binding assay sensitivity dramatically, Continue Reading ?