The development and optimisation of Nanobody based electrochemical immunosensors for IgG

Biosensors are increasingly heralded for their potential to create inexpensive diagnostic devices which are sensitive, selective and easy to use. One of the key categories of biosensor are immunosensors, which have historically used antibodies as bioreceptors. Though widely used, antibodies bring inherent limitations such as variability, limited stability and their reliance on animal sources. This has led to the development of alternative immuno-reagents such as non-antibody binding proteins (NABPs). These are low molecular weight proteins which largely avoid the aforementioned advantages of antibodies. They are commonly produced by bacteria enabling the use of DNA technology to manipulate bioreceptors at the molecular level. Single chain VHHs (commonly known as nanobodies) are an antibody derived NABP adapted from camelid heavy chain antibodies which are the isolated binding domain. Whilst nanobodies have been used for diagnostic and therapeutic applications, they have limited demonstration in biosensors. In this study, both antibodies and nanobodies were used to construct a biosensor. In addition nanobody performance was optimised by introducing a novel peptide spacer. The role of nanobody orientation and spacing was thus investigated and spacer length was optimised, leading to an increase in the sensitivity of the biosensor

Proximity assays for sensitive quantification of proteins

Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression

Immunodiagnostics and immunosensor design

This work compiles information on the principles of diagnostic immunochemical methods and the recent advances in this field. It presents an overview of modern techniques for the production of diag- nostic antibodies, their modification with the aim of improving their diagnostic potency, the different types of immunochemical detection systems, and the increasing diagnostic applications for human health that include specific disease markers, individualized diagnosis of cancer subtypes, therapeutic and addictive drugs, food residues, and environmental contaminants. A special focus lies in novel developments of immu- nosensor techniques, promising approaches to miniaturized detection units and the associated microfluidic systems. The trends towards high-throughput systems, multiplexed analysis, and miniaturization of the diag- nostic tools are discussed. It is also made evident that progress in the last few years has largely relied on novel chemical approaches

Coorie in to beat the winter darkness

No abstract available

A strange thing happened when we started to sign: an unexpected learning experience for all

No abstract available

Gravissimum Educationis (1965–2015) 50 years on

No abstract available

New approaches for the production of antibodies to chemical contaminants in food

The production of antibodies to a veterinary drug requires conjugation of the low molecular weight compound to a larger molecular weight substance (carrier protein) to enable immune recognition. The resulting immunogen is usually mixed with an adjuvant to create a prepnration thnt will elicit a strong and lasting inullune response to the complex when administered to the host animal. Fretmd's adjuvant has been previously used but can produce adverse side effects in the host. A replacement adjuvant for Fretllld's was sought: Montanide ISA 50V was found to be suitable for the production of antibodies to three veterinary clmg haptens without the adverse effects associated with Freund·s. Antibodies were required for the development of inummoassays for the coccidiostats diclazuril and . robenidine. TIle lack of a suitable fimctional group for coupling to the protein made immtlllogen preparation difficult for both these compounds. lvlimics possessing part of the relevant structures and more open to chemical manipulation were used as haptens to prepare inummogens and subsequently specific antibodies for diclazuril and robenidine were obtained. Polyclonal antisera to tlle antibiotic chloramphenicol were successfully raised in three different species. All of the antisera were affected to a similar e;-,.1ent when sanlple matrix was introduced to the assays. It was found that a heterologous format of both the ELISA and biosensor assay increased tlle crossreactivity of donkey and goat antibodies but not emile!. A camel antibody was separated into its three IgG subclasses. The conventional four chain molecule displayed affinity to chloramphenicol and the carrier protein \\rule the two heavy chain IgGs were only able to bind the carrier. TIle use of a suitable adjunult. antigen mimics and appropriate chemical manipulation techniques can enable the researcher to generate specific and sensitive antibodies to a range of small molecular weight compounds.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A strange thing happened when we started to sign: an unexpected learning experience for all

No abstract available