Our discussions of various bioanalytical methodologies over the past few weeks has focused on chromatography and small molecule analysis. Today we are going to discuss a collection of methods that is commonly used for large molecules, such as peptides, peptides and macro-molecules. These molecules are often called “biologics” because they are generally derived from endogenous biological molecules. However, in most cases, they are modified in such a way that the body no longer recognizes them as an endogenous compound (although some are identical to actual endogenous molecules, eg, insulin). For some of these biologics, standard chromatographic methods may be adequate; however, in most cases, these molecules are too large, too hydrophilic, and too difficult to detect using HPLC/UV or even LC/MS/MS. To solve this problem, a new type of assay was developed, ligand binding assays.
They say that a picture is worth 1,000 words … but in this case, it is worth 3 pages of writing! The picture of a sandwich assay illustrates how ligand binding assays work (in general). I will describe the steps from left to right in the picture. (1) The plastic well of a 96-well plate has a ligand attached to it. This ligand has specific affinity for the molecule of interest. (2) A solution that contains the molecule of interest is added to the well and the target protein is captured by the ligand. (3) A detection ligand that also binds to the target protein is added to the well and creates a “sandwich” with two ligands surrounding the target protein. (4) In this example, a streptavidin protein with a horse radish peroxidase (HRP) is added to the well. The streptavidin binds to the biotin end of the detection ligand. (5) Finally the HRP substrate is added to the well and a colored product is produced that can be detected with a spectrophotometer.
The essence of a ligand binding assay is that a ligand (usually an antibody) is used to capture and/or detect the target molecule. Antibodies are designed to have high specificity and can therefore differentiate between target molecules much better than chromatography in many situations. Detection is achieved through a variety of means by attaching signaling tools to the antibodies. The example in the image is an enzyme that converts an uncolored substrate to a colored substrate. The variety of detection systems, and their abbreviations are listed below:
- ELISA – enzyme-linked immunosorbent assay: uses an enzyme to convert a substrate to a product which can be detected, usually with quantitative colorimetric methods.
- RIA – radioimmuno assay: links a radioactive particle to a detection antibody and uses liquid scintillation counting for quantitation.
- FLBA – fluorescent ligand binding assay: uses a fluorescent label in place of either a radioactive label, or as a product of an enzyme reaction
When evaluating ligand binding assays, there are several items that you should keep in mind:
- How specific are the ligands? Do they interact with molecules other than the target protein (this is called cross-reactivity)? How sensitive is the ligand?
- Are washing steps removing non-specific binding? After each capture step, a wash step should be performed to remove any molecules that are not binding to the ligand.
- How much amplification is needed? The choice of the detection method is dependent on the amplification requirements of the assay. If more amplification (i.e., lower detection levels) is necessary, often a radiometric assay provides better resolution. If less amplification is needed, colorimetric detection is usually adequate.
Ligand binding assays are very useful and efficient, particularly for bioanalysis of biologics. Yet they are not fool-proof and require the same level of validation as chromatographic methods.
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