Brianna White, a research scientist at Emory Pharma, walks through ligand binding assays and how they work in drug development, immunology, and biomarker discovery. She breaks down sandwich ELISA step by step: coating wells with capture antibodies, adding samples and detection antibodies, then reading results against a standard curve. ELISA handles single targets well, while mass spectrometry works better for analyzing multiple targets or structures.
Chapters
Hello everyone. My name is Briana White and I am a research scientist here at Emory Pharma. I have a master's degree with a focus in molecular biology and biotechnology and have five years of experience in GMP quality control and assay development. Today we will be diving into ligand binding and their importance. What are ligand binding assays? Ligand binding assays or LBAs are analytical techniques designed to measure the interaction between a ligand and its binding partner. Specifically, they measure the binding affinity between the reactants. The binding partners normally are receptors, antibodies or other macromolecules. Why are they important? LBAs help in characterizing the strength, specificity and kinetics of interactions in drug development and pharmacology. They are essential for understanding drug receptor interactions, facilitate the identification of leading compounds and aid in optimizing drug concentrations and dosages. For biomarker discovery, they enable the identification and validation of biomarkers for diseases and are crucial in diagnosing and monitoring disease progression. For pharmacokinetics, they help in determining how a drug is absorbed, distributed, metabolized and excreted and provide critical data for dosage regimen optimization. In immunology, they determine the antibody concentrations for vaccine development and immune responses and can be used to quantify cytokines. In the food industry, LBAs help to identify and quantify proteins and allergens in food projects. Finally, in research and development, LBAs are used to study protein interactions, to understand cellular processes and signaling pathways, and characterize ligand receptor binding kinetics and affinities. The type of LBAs employed in your analysis is highly dependent on a variety of factors such as the nature of molecule of interest, the matrix sensitivity and the suitability of the assay, and even the cost of implementation. There are numerous types of LBAs that can be tailored to fit your analytical needs. Here we will discuss one of the most common types of lba. Enzyme linked immunosorbent assay or ELISA relies on the specificity of antibodies to capture and detect the target molecule. Here is the workflow for a common sandwich elisa. First, the capture antibody is added to the multi well plate, incubated, then washed. During incubation, the capture antibody binds to the surface of the wells. Next, any unbound sites present on the surface of the wells are blocked by the addition of the blocking buffer. This is a key step to help reduce any non specific binding and the background signal. After incubation and washing of excess blocking buffer blanks, standards and diluted samples that contain the target antigen are added to the wells. The blank is the formulation buffer or diluent used for the standards and samples. The standards will have known concentrations of the target antigen. After another incubation and wash step, the enzyme conjugated detection antibody is added to the plate. This antibody will bind to the available target antigens present. Next, following the incubation and wash, an imaging substrate is added to the plate and incubated. During this step, the enzyme bound to the detection antibody converts the substrate to to a colored product creating a detectable signal. Next, the stop solution is added to the plate which terminates the enzymatic reaction and the signal is measured. Most commonly this is absorbance, but luminescence and fluorescence are also common readouts. Then, finally, the measured signal of the samples is compared to the standard curve generated from the known concentrations of the target protein. This is an example of an ELISA calibration curve used to quantitate the unknown analytes present. It was generated using the absorbance of the standards with curve plotting software. There are many advantages of using ELISA. ELISAs are generally very rapid and easy to use. They have a relatively low cost of supplies and equipment, usually requiring a plate reader. ELISA kits are commercially available and can be a more cost effective option for routine and large scale studies. Alases are normally used for single target analysis, but if multiple target analysis is needed, mass spectrometry can be preferable. Mass spectrometry can provide very detailed structural information including composition, modifications and interactions of the molecule of interest, as well as identification of unknown molecules and quantitation. Thank you for watching and I hope that this video was informative. If you would like to discuss your project with one of our scientists, please reach out via our website or email. Thank you.
