An enzyme immunoassay is used to detect the presence of a target protein in a biological sample. A more common name for an enzyme immunoassay is the enzyme linked immunosorbent assay (ELISA). These assays are the cornerstone of most established clinical tests, and are typically the gold star diagnostic method. The ELISA is also used in many research environments because of its reliability and sensitivity. It is commonly able to detect protein down to concentrations as low as single picograms per milliliter. The sensitivity depends on the antibody-antigen pair and on the performance of the enzyme-substrate pair. Enzyme immunoassays are labeled assays because they don’t detect a protein binding event (between antibody and antigen) directly. Instead, these assays rely on an enzymatic reaction between an enzyme-conjugated label and a substrate to produce a colorimetric, chemiluminiscent, or fluorescent product. These products are then detected via a spectrophotometer or fluorometer and compared to a standard curve to produce quantitative concentration information about the target protein in the experimental sample.
Cell based assays are used to quantify cellular function, measure how stimuli affect cells, or to localize an effect within the cell. The cells are live and intact, and require the use of fluorescent tags and chemiluminescent or colorimetric enzymes. The quantification is performed by flow cytometry or microscopy. This is very different from studies of protein or nucleic acid which require destruction of the cell and isolation of those components from cell lysate. A cell based assay is conducted entirely within live, intact cells. The goal is to understand a cellular process, localization of a molecule or drug to a cellular compartment, or to measure how cells react to a substance. Cell based assays are usually performed in tightly controlled cell lines to test for a wide range of behaviors:
The enzyme linked immunosorbent assay (ELISA) is a gold standard method for protein and antibody detection. It is routinely used for clinical lab work and is often used in research and development. The assay is based on the lock-and-key specificity between an antibody-antigen pair. Antibodies are naturally made by the adaptive immune system to recognize a specific piece of an antigenic protein. This is what allows the body to identify and fight invading pathogens so effectively. Since antibodies have such a fundamental role in most biosensing and diagnostic systems it is very easy to purchase them or have them custom-made to recognize a protein of interest. The ELISA assay is performed in 96- or 384-well plates that are treated to favorably adsorb antigen or antibody depending on the type of ELISA being performed. The ELISA assay is built up from the bottom of each well; it doesn’t float freely in the well. As a result, the unbound proteins are easily washed away and only the target remains to be quantified.
What Does the Webinar Cover?
Biomagnetic separation has proven to be a quick, efficient and clean process in Life Sciences. However, most researchers and developers focus only on the magnetic beads or particles to optimize their separation process. The effectivity of the biomagnetic separation depending on the magnetic carrier is only half of the story. To have the complete picture we also need to pay attention to the role of the applied magnetic field on the play. Not understanding or controlling the parameters linked to the magnetic separator will result in failure when developing new applications, and also in industrializing lab-scale developments. The webinar will review the basic concepts of magnetic separation and help the attendees understand how advanced systems may enlight key aspects of the process. These concepts will be applied to parameterize, monitor and validate the magnetic beads behavior in controlled conditions. Afterwards, the discussion will focus on how to transfer the correctly characterized biomagnetic separation process from laboratory to production scale. Finally, the webinar will address how to use this knowledge to assure the quality of the magnetic-carriers based products.
Gold nanoparticles display unique optical properties. These properties make gold nanoparticles useful tools for biotechnology and medicine. Gold nanoparticles are also called nano gold or colloidal gold due to the fact that they are less than 100nm in size and are suspended in a liquid solution. The color of the colloidal gold is dependent upon the size and shape of the gold nanoparticles comprising it. Larger particles and aggregates of particles cause the absorbance spectrum to broaden and shift towards longer wavelengths and a red color. The metallic nature of the particles makes them very useful for imaging by electron microscopy, which was one of the first applications for them. The gold nanoparticles can be functionalized with antibodies, carbohydrates, and nucleic acids. This makes them very useful for scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal light microscopy as well as pathogen detection and other diagnostic assays. The ability of gold to absorb light via surface plasmon resonance (SPR) makes them useful tools for photothermal therapy in the treatment of cancer.
The ELISA, or enzyme linked immunosorbent assay, is the gold standard immunoassay for detection of small quantities of protein in samples as varied as serum, urine, saliva, and more. The ELISA is a labeled assay, which means that some type of label is needed to detect protein binding events. These labels are typically fluorescent, chromatic, or chemiluminiscent, and require the use of a plate reader to quantify the amount of protein in the sample. The major benefit of the ELISA is that low concentrations (often down to pg/mL) of protein are easily quantified. One disadvantage to ELISA is that many steps and reagents are required throughout the protocol. However, this can be mitigated by purchasing an ELISA kit that is pre-bound with capture antibodies and contains a detailed protocol for using all of the included buffers in a clear, easy to follow format. The use of an ELISA kit can improve diagnostic results from assay to assay because the kits are all validated between lots and come with protein standards. This means that a standard curve (detected signal vs. protein concentration) is generated during each assay and this standard curve can be checked against the expected values to ensure that the kit is still functioning as expected. The kit streamlines the process and takes the guesswork out of protocol design.
Antibodies are produced by the adaptive immune system in response to invading pathogens. The antibody has specific lock and key recognition for the offending bacteria, virus, or other molecule, which are collectively called antigens. Antibodies are proteins, which are folded polypeptides, or strands of amino acids which have antigen recognition sites that specifically recognize a binding site of its specific antigen. They are produced by B-cells of the adaptive immune system.
As with any nanoparticle, the properties of silver nanoparticles are dependent on size. Many synthesis strategies produce nanoparticles with a wide size distribution. One way to separate these particles based on size is to use density gradient centrifugation. A sucrose gradient is an easy way to perform this separation because the sucrose gradient is simple to create using common laboratory equipment.
Proteins are fundamental building blocks for life. All tissues and organisms are made up of protein, and all of the work performed inside and outside of cells is mediated by protein signaling cascades. Proteins are polymers of amino acids with three or four layers of organized structure. Primary structure is defined as the linear order of amino acids. This is dictated by the genome: the code is transcribed from DNA and translated into the string of amino acids. Secondary structure is thought of as two basic forms: a beta sheet or alpha helix. The string of amino acids adopts the conformation that allows the lowest energy state. Beyond the sheets and helices, the chain can take other twists and turns to fold into a shape known as its tertiary structure. Some proteins are actually made up of two or more subunits of individually folded amino acids strands. The complexing of protein subunits to form one functional protein is called quaternary structure. All of this folding is extremely important to the character and function of each individual protein because it results in certain side chains of amino acids being located on the exterior or interior of the protein. Importantly, the folds create binding pockets where key amino acids are located to create a unique chemical landscape that allows the protein to bind to other proteins and carry out its job in a signaling cascade.