The ELISA or Enzyme-Linked Immunosorbent Assay is the most established method of protein detection and quantification. The technique is commonly used in a wide variety of applications spanning from the basic research bench to clinical laboratories. The ELISA is a labeled assay, which means that it requires the use of a label to detect an antibody-antigen binding event. The label is commonly a fluorescent probe, chemiluminescent system, or enzymatic colorimetric reaction.
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. These polymers are crosslinked and formed around the target molecule. Once the polymerization process is complete, the target molecule template is destroyed. What remains is a three-dimensional polymer with binding sites in the exact physical and chemical configuration as the target molecule. These binding sites are specific with regard to hydrophobicity, hydrophilicity, chemical groups, hydrogen bonding, and charge. When these MIPs are added to solution they will bind the target molecule with exceptional specificity.
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 earliest chemists were on the hunt for new elements to add to the periodic table. Most of the chemistry that they were interested in doing was purification with the end goal of reaching a pure elemental substance. These chemists relied on a litany of methods—filtration, evaporation, distillation, and crystallization were some of the most used purification techniques for these discoveries. As the chemists were defining the elements, the biologists were trying to understand the human body, the cell, cellular organelles, and microbes. The point here is that in order to develop anything new we must first understand what everything is made of at the most basic and pure level. In modern science this means that we are trying to define matter beyond subatomic particles and we are attempting to map out every molecular pathway of disease. Our efforts to define complex systems by their purest constituents are rewarded by deep understanding and an ability to mimic, to engineer, develop, and create.
If you look closely at the product information for many commercial antibodies, you will see that they are protein A purified. Protein A is a surface protein that was originally found in the cell wall of staphyloccoccus aureus bacteria. On the surface of bacteria it serves as a defense against the host immune system and allows the bacteria to survive longer and be more virulent. Protein A binds the Fc portion of IgG antibodies.
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use (ICH) was founded in 1990 as a way to standardize the introduction of new drug substances to the worldwide market. The council wrote and maintains guidelines for how new pharmaceutical products must be tested for stability and quality before they can be approved for worldwide distribution. The guidelines protect consumers and allow new therapeutic drugs to reach patients across international borders more quickly. The ICH has written guidelines for the stability testing of new drug substances and products. There is a general document known as Q1A(R2) that outlines the details of every stability test that a new drug substance must undergo before being registered. These tests examine how the drug degrades in high temperature or high humidity over time. It outlines methods for defining the mechanism of degradation for the new drug, and how to test proposed protective packaging for efficacy. A supplementary document (Q1B) contains additional details specifically about photostability testing.
The northern blot 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 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.
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.
Until this point we have been thinking about antibodies as one of the five classes, IgG, IgE, IgD, IgA, or IgM. The basic unit of each antibody class is a Y structure, where the base of the Y is known as the Fc region and the arms are the Fab region. The entire IgG antibody is composed of four polypeptide chains (two heavy and two light). The Fc region is composed only of heavy chain, and the variable Fab region is built with heavy chain and light chain. The Fab region is where all of the antigen-binding occurs because the paratope, or antigen recognition site is located at the tip of each of the two arms of the Y. A single domain antibody paratope is made solely of a single heavy chain.