When developing a CLIA it is helpful to understand what is available commercially. CLIA kits are available from many different companies that formulate reagents or components of the assay specifics for the analyte to detect and tailor made for the company analyser (platform). The solid phase can be based on superparamagnetic beads or polystyrene beads usually kept in liquid formulation. In both cases these beads require a CLIA-label reagent, discussed in more detail earlier in this ebook.
HRP stands for horseradish peroxidase, an enzyme derived from horseradish. Streptavidin is a protein derived from a species of bacteria in the genus streptomyces. Streptavidin has a high affinity for the molecule biotin. Streptavidin HRP is a streptavidin protein conjugated to HRP. HRP is used for detection/read out signals in an assay such as ELISA. Depending on which substrate you give the HRP, the enzyme will produce a different signal that you can read out with whatever hardware/technology you have in your laboratory. Let’s discuss the general protocol for using streptavidin HRP and the various substrates you can use to get an output.
Designing CLIA assays requires the consideration of different aspects, encompassing the raw materials for the reagent development & methods selection, together with the choice of the assay format. Material suppliers are a key factor for a successful design and development of an assay. An ideal supplier should be able to provide required raw materials not only at a reliable cost but also available to provide the required bulk quantities for scaling up the reagent. Moreover, suppliers should provide different lots to assess the lot to lot variability to check the impact in the assay to be developed.
Proteins are one of the four macromolecule building blocks of life. The other three are carbohydrates, lipids, and nucleic acids. Proteins are long strings of amino acids that fold together into what are called “hierarchical structures” in order to perform specialized functions within the cells and tissues of all living organisms. Let’s describe what the hierarchical structures for proteins are. First is the chain of amino acids that make up a protein, called its primary structure. Each amino acid has a “side chain” composed of elements that make that amino acid unique. The side chains interact with one another to create the next part of the hierarchical structure, the secondary structure. The secondary structure takes the form of an alpha helix or beta sheet. The next structure is the tertiary structure in which the sheets and helices fold in an physically and chemically favorable way to create a “folded” structure. When multiple folded proteins come together to form a bigger structure, that is called the quaternary structure.
Advantages of the CLIA
There are many types of assays that can be performed for detection of a molecule of interest, all with their own advantages and disadvantages. Many scientists choose to perform chemiluminescent immunoassays over the enzyme-linked immunosorbent assays (ELISA), fluorescence or radioimmunoassays. This is because the CLIA has been shown to have an improve detection at lower concentration and a wide dynamic range.
Today we are going to talk about a piece of technology found in laboratories around the world, the spin column. It is used for “solid phase extraction.” In simple terms, the spin column has a solid material that can be used to retain or bind certain molecules while letting other molecules pass through it. When referring to spin columns, this usually refers to nucleic acid purification/isolation (DNA or RNA) or proteins. The starting material can vary from blood to tissue.
General introduction to RIPA
RIPA stands for Radio immunoprecipitation assay. Let’s start out by talking about what a radio immunoprecipitation assay is and why it is used. In general, an immunoprecipitation assay uses antibodies to pull a protein of interest. The “immuno” part refers to the antibody (a molecule of the immune system) and the “precipitation” refers to a substance coming out of solution. You can read about the immunoprecipitation protocol in our other article "immunoprecipitation protocol". The radio part of this refers to using isotope labeling to track molecules. In a RIPA assay you radiolabel an antigen so you can track it when it binds an antibody, while the antibody is used to precipitate the antigen.
How can magnetic beads improve CLIA tests?
The combination of CLIA and magnetic beads brings together all the advantages of both parts. CLIA is known for its high sensitivity which allows the detection of analytes at very low concentrations, and thus providing an excellent limits of detection in a wide dynamic range.
Luminescence is the emission of light, and it can occur in many ways. In research and biomedical industry fluorescence and chemiluminescence are often used. Fluorescence is when light is absorbed then emitted by a substance. A photon of a higher energy state is absorbed, then a lower energy photon is emitted in another range of the electromagnetic spectrum.