The Enzyme Linked ImmunoSorbant Assay (ELISA) is the gold standard immunoassay for clinical diagnosis of disease. The basis of any immunoassay is the specific molecular recognition between antibody and antigen. This is something that the immune system does naturally. The production of monoclonal antibodies in a laboratory has become commonplace and standardized, which makes it possible to use monoclonal antibodies in immunoassays such as an IgG ELISA. The antibodies are easy to purchase from commercial vendors, and they come with quality control reports ensuring that they will recognize the target antigen.
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 hierarchal structures in order to perform specialized functions within the cells and tissues of all living organisms. These higher structures are imperative to the proper function of the protein within it biochemical pathway. The tertiary structure creates the chemical and morphological landscape that imparts the biorecognition abilities to ligand, receptors, antibodies, and all of the other workhorse proteins in the organism. The hydrophobicity or electrosatic nature of the binding pockets are responsible for the specific affinities between proteins. It is no wonder that the ability to analyze proteins with a protein assay is fundamental to biological research and clinical diagnosis.
Purpose of Protein Assays
The purpose of the protein assay is to determine the amount or concentration of a specific protein or an array of different proteins a sample. This can be a primary step before further manipulation in a research and development process, an initial capture of protein before structural analysis, or it can be a final detection step in a clinical laboratory as part of a disease diagnosis.
Molecular diagnostics entails the analysis of biomarkers to help diagnose, track the progression of, or determine risk factors and prognosis of disease. Biomarkers have been identified within the realm of genomics, epigenomics, transcriptomics, proetomics, metabolomics, and lipidomics:
Molecularly imprinted polymers (MIP) are relatively new diagnostic and therapeutic tool. MIPs are highly specific three dimensional polymer imprints of molecules. The power of this tool may not be immediately obvious, but it is indeed a very useful technology. The power of MIPs lies in their specificity of binding to target molecules. Before the invention of MIPs, the only way to achieve this type of specificity was through antibody-antigen binding, surface receptor-ligand interactions, or protein affinities such as streptavidin and biotin. The problem with all of these systems is that they need to already exist. But what about a target that doesn't have a known molecule with a natural affinity? Herein enters molecularly imprinted polymers.
For the indirect and direct elisa, the antigen is applied to the surface of the elisa plate, but for a capture antibody is attached directly to the surface of the plate for sandwich elisa. Aside from this major difference the indirect and elisa protocol is very similar to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly. The indirect elisa requires two antibodies—a primary antibody to bind to the antigen, and a secondary antibody conjugated to an enzyme or fluorophore. The direct elisa uses a primary antibody that is directly conjugated to an enzyme or fluorophore. Either way, both of these methods—and indeed every elisa protocol, is a labeled assay. The antibody-antigen binding event cannot be quantified without the presence of the enzyme or fluorophore.
Faster and more efficient methods of pathogen detection are in high demand. The traditional methods involve collection of patient blood or swab samples for multi-day cultures. These methods are time-consuming and require full laboratories with skilled technicians and sterile equipment. As such, they are not ideal for low-income areas or for rapid pathogen detection. There is a need for rapid pathogen technology and point-of-care diagnostic tools. Ideally, these technologies will come with a built-in validation protocol. Magnetic nanoparticles and molecularly imprinted polymers are good candidates for improved pathogen detection systems. An additional benefit to using magnetic nanoparticles is that the separation process is easy to quantitatively measure with a validation protocol.
The northern blot protocol is a technique used to study gene expression via mRNA transcripts. The northern blot was named after the southern blot, which was developed to study DNA. The two techniques are the same except that the northern blot is used to detect RNA while the southern blot is used to detect DNA. The northern blot protocol, in brief, involves gel electrophoresis to separate mRNA by size, a blotting step to transfer the separated mRNA to a membrane, and a probe hybridization step to identify the mRNA sequence of interest. Even with the advent of powerful RNA analysis techniques such as RT-qPCR and sequencing, the northern blot protocol is still useful for comparing gene expression between samples. The northern blot protocol is relatively inexpensive, and makes it easy to visualize the results on a single membrane.
Equipment and materials needed
A northern blot requires several pieces of equipment for its main steps which include, running the gel, transfering your sample onto a membrane, and immobilizing it. The process also requires many buffers for your gel to run smoothly and correctly, as well as buffers for your RNA samples and the subsequent steps.
For a northern blot you will need an apparatus for setting up an agarose gel, as well as a power supply for it. You will also need a transfer system, such as a vacuum and a nylon membrane on which to transfer your RNA. You will also need a device to produce UV rays onto the nylon membrane for immobilization of the RNA. For materials you will need to make a typical RNA separation buffer called MOPS, and a denaturing agarose gel. You will also need to create a buffer for your RNA. You will also need a saline-sodium citrate buffer (SSC) for wash steps. One of the time consuming aspects of a northern blot is preparing the buffers required for each step.
Enzymes are the catalysts for biochemical reactions. As such, they speed up the transition from reactants to products without being consumed in the process. Multiple enzymes can be found in every cell, from bacteria up through to humans. We as humans have found ways to exploit enzymes to produce food products, fuel, pharmaceutical products, biotechnological tools, sensors, and much more. The potential uses for enzymes are seemingly limitless. The creation of solid support structures with immobilized enzymes has improved our ability to reuse enzymes in a controlled manner for a variety of applications. Immobilized enzymes can be reused multiple times before their efficacy is lost. This allows them to be an affordable part of industrial processes.
Pharmaceutical validation is important to the manufacturing process to ensure product consistency and safety. It involves regulation of all raw materials and production procedures as well as testing of final product. The general rule of thumb is to follow good manufacturing practice (GMP). This demands that all protocols be up to date and followed by trained personnel. It also requires that equipment be well-maintained and inspected. In the case of clean-room usage the clean room needs to be verified.
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.
