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ELISA steps, principle, and how it works

The ELISA (Enzyme Linked ImmunoSorbent Assay) is the gold star immunoassay, which means that it is the standard procedure that all new assay technology is compared to during research and development. The ELISA is also fundamental to most clinical tests for diagnosis of disease because it is currently the most characterized and standardized method. The ELISA is an immunoassay, the principle of which relies on the specific recognition between an antibody and antigen. This specificity comes from the unique three dimensional structure of the antibody paratope and the antigen epitope. These two regions fit like a lock and key via non-covalent, charge-based, and/or hydrophobic interactions. The clinical purpose of the ELISA is to detect either antibody or antigen from a biological fluid such as blood (serum), urine, or saliva. When the ELISA is used to antibody, the assay is being used to assess whether or not the patient has been exposed to a certain antigen at some point. It is difficult to assess current infection with this method because the body retains antibodies forever after the first introduction. However, elevated amounts of antibody can be indicative of active immune response to the pathogen. One major benefit of the ELISA is that it is quantitative, meaning that an actually number of protein can be evaluated. When the ELISA is used to detect antigen it provides a better understanding of current infection since the antigen would be cleared if it was no longer active in the body. 

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ELISA reader

ELISA stands for Enzyme Linked Immunosorbent Assay. The immunoassay utilizes the specific lock-and-key recognition between antibodies and antigens. This recognition occurs naturally in the adaptive immune system; antibodies are created by the immune system when an antigen such as a virus or bacteria invades the body. The immune system recognizes the foreign invader and creates antibodies that specifically recognize surface proteins on the virus or bacteria. The antibodies are either attached to the surface of an immune cell or move freely through the body to tag the invader and begin a cascade of destruction and elimination. The most useful part of this process from a biotechnology and engineering perspective is the specificity of the antibody-antigen recognition.

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Biodetection and biosensing

Biodetection is a general term that encompasses the global strategies in place for the detection of biological threats. Biological threats are pathogens, infectious disease, and biological weapons that can infect significant populations of humans and to which we have little innate immunity or defense against. Wee must improve our ability to detect the infectious pathogens at the earliest sign of an outbreak. This will be accomplished by improving our methods of biodetection by developing more sensitive and portable biosensing devices. The use of bioassays in clinical laboratories are standardized and validated to improve the accuracy and speed of pathogen detection and disease diagnosis. New technologies are being developed to integrate biodetection platforms with smartphone devices and extend the sensing range to the hands of ordinary individuals.

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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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Transferring magnetic bead coating from R&D to Production

The key issue when transferring a bead coating process from the Research and Development department to the manufacturing department is scalability. It is essential to ensure that the system being utilized for a particular protocol is adaptable to larger volumes. Ideally, any scale-up would be carried out with the use of a homogeneous biomagnetic separator, as this would ensure that the conditions of the protocol are well-defined and able to be reproduced for a larger volume.

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Scaling up magnetic bead coating protocols

Once a protocol for coating beads is developed and put in place, it will need to be scaled up in order to meet demand. Scaling up a process, however, requires careful attention to ensure that the details of the protocol are replicated for larger volumes.

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Pros and Cons of Classical Biolinks (Streptavidin, Biotin, Protein A, Protein G)

This is the last of a series of posts on pros and cons of classical covalent links, which we have been publishing during the last few weeks. This one is about 3 well-known classical covalent linksstreptavidin beads, biotin, protein A and protein G.

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Pros and Cons of Classical Covalent Links (Tosyl, Epoxy, Chloromethyl)

This is the second of a series of posts on pros and cons of classical covalent links, which we will publish during the next few weeks. This one is about 3 well-known classical covalent links: tosyl, epoxy and chloromethyl groups. In the next posts we are going to review other examples, such biotin or streptavidin beads.

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Pros and Cons of Classical Covalent Links (-COOH, -OH, -NH2)

This is the first of a series of posts on pros and cons of classical covalent links, which we will publish during the next few weeks. The very first one is about 3 well-known classical covalent links: carboxyl, hydroxyl and amino groups. In the next posts we are going to review other examples, such as tosyl or streptavidin beads.

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Is There any Advantage of Using Beads with Plain Coatings?

Attaching a protein to a bead can be a detailed process that requires forethought and careful planning. Generally, a molecule is attached to a particle through a surface group available on the coating of that particle, for example in the case of streptavidin beads. In cases where the attachment is covalent, it is essential to choose a binding site on the molecule that will allow for proper orientation, maximally presenting the desired site to the sample while still retaining a strong attachment between the molecule and the bead.

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