Column chromatography is a method used in many areas of science to isolate a single compound from a mixture. The basic principle of column chromatography is the adsorption of target to the column by designing a column with specific affinity to the target. The target compound adsorbs to the column resin while the remaining mixture easily flow through the column and out the other end. A similar method is used to purify protein and nucleic acids, and it is generally referred to as affinity chromatography. Affinity chromatography requires a solid support (typically a magnetic bead or a resin column) on which to covalently attach a capture molecule which has affinity to the target protein or nucleic acid.
Cell lysis is the act of breaking the cell membrane to enable the study of specific proteins, nucleic acids, and other molecules inside of cells. When cell lysis is successful, the undamaged contents of the cell escape through the damaged cell membrane. These contents are then separated out of the mixed sample and used for further study. The methods used for separation of the lysed cell contents are dependent on the goal of the study. Careful investigation of these inner workings can reveal disease patterns, improve our understanding of normal cellular function, and elucidate biochemical pathways and therapeutic targets. Protein isolation is different from nucleic acid separation, and the reagents used vary drastically. There are a few ways to lyse the cell membrane; these include mechanical disruption, liquid homogenization, freeze/thaw cycles, manual griding, and the use of detergents. Sonication cell lysis is an example of mechanical disruption used for releasing the contents of cells.
An antigen is a molecule that is part of an object that is foreign to the body. The body uses antibodies to recognize the foreign object by its antigens and stimulates an immune response, activating white blood cells to produce more antibodies and other immune pathways. Antigens can be proteins or sugars that are located on the outer surfaces of pathogenic cells. All cells have antigens including the ones inside the body, bacteria, and even viruses. The antibodies produced by the immune system are custom-fitted to the antigen that initially stimulated the immune response. The antibodies have an antigen recognition site (paratope) that is highly specific affinity for a region on the antigen called the epitope.
There are a few more considerations for optimizing the CLIA assay that in this chapter will be discussed. The following considerations are related to the performance of your assay.
An isolation kit helps you isolate a material of interest. When we talk about an isolation kit, we are likely talking about a kit that helps you isolate nucleic acid (RNA or DNA) or protein. These kits often contain all the buffers and hardware you need for your isolation. Let’s do a review of the types of isolation kits and how they work, then we’ll give you a general protocol to understand how the process works!
The tracer, the antigen or antibody labelled with a chemiluminescent tag for CLIA, is the next vital optimization step of a chemiluminescent immunoassay. As mentioned earlier, chemiluminescent labels generate light from a chemical reaction. Widely used CLIA labels are based on luminol derivatives or acridinium esters.
Introduction to dNTP’s
dNTP stands for deoxynulceoside triphosphate. dNTP’s are what make up one of the four macromolecules of life, nucleic acids. A nucleoside is a molecule that consists of a ribose (sugar) bound to a nitrogenous base. On dNTP’s the ribose is actually a deoxyribose because it lacks an oxygen atom on the second carbon position. There are four dominant types of nitrogenous bases that define the type of dNTP it is, they are A,T,C,G. The triphosphate is the three phosphate groups that bind the ribose as well. Our DNA is made up of these dNTPS, A binding favorably to T and C binding favorably to G. In addition to their role in the genetics of nature, dNTP’s are also used as a tool for polymerase chain reactions (PCR). Let’s discuss how PCR works and how dTNP’s are used for it.
There are several types of CLIA formats that can be used depending on the target analyte of your assay. The choice of assay format will impact four major aspects of development. The first will be the choice of magnetic bead coated with antigen/s or antibody/ies for binding the target analyte. The tracer will then be required to match the target analyte using a conjugated antibody/ies or antigen/s conjugatedith a CLIA label. The assay buffer will need to be optimized to improve the specificity and sensitivity for each step. Lastly there will be components such as blockers, other linking molecules or stabilizing molecules. These aspects can be optimized once an assay format is chosen.