An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. The assay can be used to identify the presence of pathogens in a clinical sample, or it can be used to measure the amount of a target biomolecule. If the goal of the immunoassay is to isolate a specific molecule then a separation system is needed. When the isolation is achieved by magnetic separation using a magnetic particle it is called a magneto-actuated immunoassay. The most common particle used in these assays is made of a core of magnetite that is coated with a biologically compatible material, and chemically modified by the attachment of antibodies. However, before designing a magnetic particle for an immunoassay one must decide which types of immunoassays best fits the goals of the experiment.
We have come a long way from the days of blood letting, trephination, and snake oil salesmen peddling cure-all tonics. The oversight and regulation of organizations such as the European Medicines Agency and the Federal Drug Administration (FDA) have significantly improved the quality and safety of our medical and pharmaceutical products. Of course, our medical understanding has deepened dramatically, our science has become more sophisticated, and we have developed tools to perform large scale drug discovery and screening. With this deeper understanding of chemistry and drug development we have realized the importance of preserving the chemical molecules via proper storage conditions.
The ICH guidelines for stability lay out the requirements for identifying and maintaining drug efficacy by understanding the pathways of degradation. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was founded in 1990. The European Commission, FDA from the USA, and the Ministry of Health, Labour, and Welfare (MHLW), which later became the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan are all founding members. Since that time, many other regulatory authorities from around the world have joined the ICH. The stated mission of the ICH is to “achieve greater harmonisation worldwide to ensure that safe, effective, and high quality medicines are developed and registered in the most resource-efficient manner.”
Superparamagnetism is a type of magnetism that lies between that of a permanent magnet and a paramagnet. Recall that a permanent magnet is always magnetic at temperatures below its Curie Temperature even in zero applied magnetic field, whereas a paramagnet is not magnetic at zero applied field but can become magnetic when an external magnetic field is applied. The potential for a paramagnet to be induced to have magnetization is called magnetic susceptibility. A superparamagnet behaves similarly to a paramagnet. The “super” means that it has a higher magnetic susceptibility than a regular paramagnet when a magnetic field is applied. Superparamagnets are typically made of iron oxide or other ferrous materials, and they are extremely small, on the order of 10-100 nanometers.
Proteins are constantly being created by the cellular machinery of living organisms. This article will first summarize the process as it occurs in a natural organism, and then discuss how protein expression and purification occurs in a laboratory setting for the generation of recombinant proteins.
In bacteria there are two main types of DNA—genomic and plasmid. Plasmid DNA is unique to bacteria. Eukaryotic cells don't typically have plasmid DNA unless it was put there by transfection for experimental purposes. The most important goal when isolating nucleic acids is to obtain the highest purity genetic material possible. When isolating genomic DNA it is important to remove plasmid DNA and RNA from the sample. Similarly, sometimes an experiment calls for the isolation of plasmid DNA, and the selective removal of genomic DNA is necessary. Also, some commercial RNA isolation kits include gDNA eliminator spin columns to remove genomic DNA from the isolate.
The concept of an antigen and antibody pair is central to modern medicine and biotechnology. These proteins match like a lock and key, with equisite specificity. The interactions are non-covalent, but have equilibrium constants ranging from 105 to 1012 M-1 Antibodies and antigens are proteins: polypeptide chains of amino acids. The IgG antibody is composed of four polypeptide chains, two heavy and two light, organized into a ‘Y’ shape. The base of the Y is called the Fc region, while the two tips are known as the Fab region.
Traditional immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are able to measure the presence or absence of only one analyte per reaction. Multiplex immunoassays measure dozens of different analytes in a single reaction. This is particularly beneficial for precious samples, and when only a small volume is collected for analysis. The multiplex immunoassay also saves working time since multiple assays can be completed simultaneously.
The ability to isolate and identify proteins from a biological solution is fundamental to basic research and clinical diagnosis. Proteins are the workhorses of the organism; they send and receive messages, they control the flow of information across the cell membrane, and they enact cascades of action within cells. It is rare that a single protein works alone, so it is imperative to understand how proteins interact with each other if we are to understand the nature of our bodies and to discover and treat disease.
Immunoprecipitation (IP) is a method used to purify target proteins from whole tissue or from cell culture. There are different types of IP: Single protein, Co-immunoprecipitation (co-ip), Chromatin immunoprecipitation(CHIP), RNA immunoprecipitation (RIP), and tagged protein immunoprecipitation. All of these techniques play an important role in modern medicine and research into the discovery of biochemical mechanisms, and they all are based on the basic principle of the specific lock-and-key interaction between antibody and antigen. Antibodies are naturally produced by the adaptive immune system, but they can also be engineered by modern techniques to specifically bind to any protein target desired.
Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogenous solution. It is column-based, which means that the solution is flowed through a column and eluted at the other end. The column is pre-functionalized with the capture antibody or antigen. The target protein is adsorbed onto the resin-bound capture protein and is retained in the column while the remaining solution is eluted. The fraction containing the target protein is later eluted and purified.