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Pathogen detection using magnetic nanoparticles or molecularly imprinted polymers

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.

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Purification Techniques

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.

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Protein Purification with a protein A column or bead

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.

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Stability testing of pharmaceutical products

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.

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Northern Blot Protocol

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.  

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Immobilized enzymes

 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.

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Single Domain Antibody

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.

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Classes of Antibodies

The structure of the most commonly known antibody class (IgG) was a mystery until 1959, when it was elucidated by Edelman and Porter . The duo approached the question from two very different directions, but they were both awarded the 1972 Nobel Prize in Physiology or Medicine for their groundbreaking work. Gerald Edelman said that he was, “fascinated by the specificity of antigen recognition by antibodies,” and hoped that, “by doing the primary structure of antibody molecules, the basis of their specificity would be revealed.” Indeed, it was.

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Pharmaceutical validation

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.

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The step-by-step process of protein purification

Protein purification is the processes of isolating a protein of interest from its environment. In other words, from the other natural molecules surrounding the proteins in the natural niche in a host organism, or from a cell culture grown in a laboratory. Our protein purification handbook  explains that there are several available techniques and many options to consider, but the general procedure is the same. 

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