Fleet Bioprocessing Ltd. are experts in the development of immunoassays, widely-used tests for the diagnosis of diseases which rely on the well-known specificity of antibodies to detect specific molecules. Examples in common use include tests for detecting HIV and hepatitis, for diagnosing thyroid hormone abnormalities, or for differentiating heart attacks from other conditions such as angina which may display similar symptoms. New immunoassays are under development all the time, e.g. to improve the detection of cancer tumours or to monitor factors associated with the development of Alzheimer's disease. Immunoassays rely on the successful chemical "labelling" of antibodies and related proteins, so for example that they can be detected efficiently via the presence of a fluorescent dye - and Fleet are expert in the bioconjugation techniques required for this purpose. Fleet routinely use simple analytical techniques to characterise these labelled antibody conjugates, allowing us to determine basic information such as the antibody concentration and the mean number of dye molecules per antibody molecule. However these techniques tell us nothing about whether the labelled antibody conjugate has retained its ability to detect the molecule of interest, or has been damaged in the labelling process. For example it is possible to attach too many dye molecules to an antibody, with the result that its ability to bind the target molecule is compromised. It would be very useful to have access to a rapid, inexpensive analytical method allowing us to confirm that the conjugate has successfully retained its structure during the labelling procedure. Fleet have evaluated several techniques for this purpose, but to date all have failed to meet our requirements; simple techniques based on spectroscopy which would meet our needs of being rapid and inexpensive have not shown adequate sensitivity to differentiate between "good" and "bad" conjugates, while techniques capable of achieving the required sensitivity have proved prohibitively expensive and/or time-consuming. An initial project with LGC and NPL (completed in March 2019) identified techniques with exciting potential to fill this knowledge gap, and to better understand the mechanism of conjugate inactivation. This follow-up project aims to confirm the potential of these techniques. Fleet will prepare a range of antibody conjugates for evaluation and assess them in a model immunoassay, while LGC and NPL will characterise them using a range of candidate analytical techniques. This will hopefully allow us to confirm the capability of these techniques for routine use by Fleet Bioprocessing Ltd.

"**Fleet Bioprocessing Ltd.** are expert in the development of **immunoassays,** highly sensitive and specific antibody-based _in vitro_ diagnostic procedures widely used in the diagnosis of disease states. Immunoassay performance is critically dependent on **labelled antibody and antigen conjugates** which enable **immobilisation** or **detection** of these proteins: typical examples include **biotin-labelled antigens** which facilitate binding to a streptavidin-coated surface, and **fluorescent-labelled antibodies** which enable exquisitely sensitive detection _via_ **fluorometric** quantification. The performance of any given immunoassay is largely defined by the behaviour of these conjugates, which ideally display 100% retention of antibody/antigen binding activity, whilst incorporating an optimally high level of label to facilitate immobilisation or detection. A trade-off between these competing aims is required for optimal performance. The chemistry required to _create_ such conjugates is well-established; with Fleet's expertise, existing **bioconjugation** techniques are routinely exploited to yield conjugates with world-class immunoassay performance. By contrast, **the availability of** **rapid and inexpensive procedures for effective analysis and characterisation of these conjugates is extremely limited.** Simple cheap techniques can be used to determine basic conjugate characteristics such as antibody concentration or mean biotin or enzyme incorporation, but these provide no information on whether the antibody or antigen has retained immunological functionality; **without this information it is impossible to know whether the conjugate will perform effectively in the immunoassay**. This means that the only available additional approach is to ""try it in the assay"", which (see _Need or Challenge_ section) has serious weaknesses. The knowledge gap that this application aims to address, therefore, is t**o identify inexpensive and relatively fast techniques capable of confirming that antibody or antigen has retained adequate functionality during the conjugation process**. A technique capable of showing quickly and cheaply that the tertiary structure of the antigen or antibody remained intact after conjugation would be a massive step forward of great significance to our company and to the industry in general. Initial discussions with LGC and NPL have indicated that both parties have access to analytical techniques which may directly or indirectly help to address this knowledge gap, including (from LGC) **hydrogen/deuterium exchange mass spectrometry (HDX-MS)** and **ion mobility spectrometry mass spectrometry (IMS-MS)** and (from NPL) **circular dichroism spectroscopy (CD), Fourier transform infrared spectroscopy (FT-IR)** and **isothermal titration calorimetry (ITC).** This project comprises the preparation of suitable conjugate panels and determination of their immunoassay performance (Fleet), coupled with comparative evaluation of these conjugates using the techniques outlined above (LGC, NPL)."

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