General overview of ELISA
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.
Overview of antibodies
Antibodies are part of the adaptive immune response of the body. After an initial defense against a pathogen from immune cells such as neutrophils, our body launches B and T cells to create antibodies to fight a pathogen. The structure of an antibody looks like the letter Y of the latin alphabet. The central part, which goes from the stalk up to the arms of the antibody, is called the “heavy chain”. A “light chain” is attached to the upper arms of the heavy chain. The stalk is also called the “Fc” region and the arms on top are called the “Fab” region. The Fab region contains the part of the antibody that binds pathogens. This region that binds pathogens is called the paratope, and it binds an epitope on a pathogen. This interaction is specific and is based on the tertiary structure and amino acid sequence. There is one epitope per paratope.
For a successful procedure for the magnetic bead conjugation, there are three important aspects to take in consideration when designing the assay: i) the planning of the conjugation protocol, ii) the density of the functional groups on the surface of the magnetic beads, and iii) the controlled magnetic separation of the beads.
The main reason for changing from latex to magnetic latex particles is the need for a change from an homogeneous to an heterogeneous immunoassay. Latex beads are micrometer sized polystyrene beads. They are made of polymer chains that form a sphere with a hydrophobic exterior that can participate in hydrophobic interaction. The beads bind proteins through passive adsorption or can be functionalized with chemical groups which better bind amine groups on proteins to bind them. Magnetic latex particles have an easy protocol that allows you to do washing steps quickly and efficiently, which helps to improve analytical sensitivity and to reduce interference from sample components. The main consideration in shifting from a process that utilizes latex beads to one that uses magnetic latex beads will be the physical separation process itself. Applications that utilize latex beads traditionally make use of a centrifuge or, alternatively, tangential filtration. In contrast, processes that use magnetic latex beads are carried out in a biomagnetic separator. As such, it is necessary to acquire an adequate separator for the process. Ideally, this would be a homogeneous separator.