A one-step reverse transcription recombinase polymerase amplification assay for lateral flow-based visual detection of PVY.

Potato virus Y (PVY) is an abundant and damaging virus which reduces crop yield and marketability. Accurate detection of this economically important virus both in-field and in seed potatoes is considered essential in the control of PVY spread. Current detection methods are focused on immunodetection and PCR-based methods, however, identification of PVY through isothermal amplification is a promising avenue for developing accessible, on-site diagnostics with quick turnaround times. In this work, a rapid recombinase polymerase amplification assay was developed which could readily amplify PVY nucleic acids with good sensitivity and specificity. Additionally, this assay was shown to be capable of amplification directly from RNA in a one-step amplification process, without the need for prior reverse transcription. The assay was coupled with lateral flow technology to provide a rapid visual confirmation of amplification. This nucleic-acid lateral flow immunoassay could feasibly be employed in-field, or at any location where testing is required, to aid in the detection and control of PVY.

Antibody Purification Using Affinity Chromatography.

Antibodies are an integral part of many biological assays and biotherapeutics. However, the sources from which antibodies are derived frequently contain other contaminants which may interfere with assays or cause adverse reactions if administered in vivo. Therefore, a means of isolating these antibodies from their source at high levels of purity is critical. Affinity chromatography is currently one of the most widely applied methods for the purification of antibodies. This method relies on specific and reversible, interactions between antibody structures, or recombinant tags fused to these structures, and ligands immobilized on solid support matrices, generally within a column. Herein, common chromatographic methods applied to antibody purification are described. These include the purification of IgG, and its recombinant forms, through protein A, protein G and immobilized metal affinity chromatography.

Generation, selection and modification of anti-cardiac troponin I antibodies with high specificity and affinity.

Current diagnosis of acute myocardial infarction involves quantification of circulating cTn levels. This work endeavoured to generate and enhance recombinant antibody fragments targeting various epitopes on the N- and C-terminals of the cTnI molecule, thereby facilitating highly sensitive detection of the troponin molecule. From this approach, two anti-cTnI scFv antibodies were successfully selected using either phage display or structural reformatting of full length anti-cTnI IgG. Their antibody binding affinity was further optimised via chain shuffling and/or site directed mutagenesis, resulting in scFv with heightened sensitivity when compared to the wild-type scFv. If used in conjunction with existing anti-mid fragment cTnI antibodies, these N- and C- terminal-targeting scFvs show high potential for the enhancement of current cTnI detection assays by limiting the effects from cTnI degradation or troponin complex formation.

The role of antibody-based troponin detection in cardiovascular disease: A critical assessment.

Cardiovascular disease has remained the world's biggest killer for 30 years. To aid in the diagnosis and prognosis of patients suffering cardiovascular-related disease accurate detection methods are essential. For over 20 years, the cardiac-specific troponins, I (cTnI) and T (cTnT), have acted as sensitive and specific biomarkers to assist in the diagnosis of various types of heart diseases. Various cardiovascular complications were commonly detected in patients with COVID-19, where cTn elevation is detectable, which suggested potential prognostic value of cTn in COVID-19-infected patients. Detection of these biomarkers circulating in the bloodstream is generally facilitated by immunoassays employing cTnI- and/or cTnT-specific antibodies. While several anti-troponin assays are commercially available, there are still obstacles to overcome to achieve optimal troponin detection. Such obstacles include the proteolytic degradation of N and C terminals on cTnI, epitope occlusion of troponin binding-sites by the cTnI/cTnT complex, cross reactivity of antibodies with skeletal troponins or assay interference caused by human anti-species antibodies. Therefore, further research into multi-antibody based platforms, multi-epitope targeting and rigorous validation of immunoassays is required to ensure accurate measurements. Moreover, in combination with various technical advances (e.g. microfluidics), antibody-based troponin detection systems can be more sensitive and rapid for incorporation into portable biosensor systems to be used at point-of care.

Sowing seeds for the future: The need for on-site plant diagnostics.

Point-of-care technology is a term used to describe any treatment given at the "site-of-need". The span of point-of-care has broadened, growing from its current dominant usage in clinical settings to encompass many sectors, such as water and food testing. This review focuses on applications of point-of-care/use technology in phytodiagnostics, a field which requires accurate diagnostic systems with increasing urgency due to the demands of global food needs in conjunction with persistent crop loss due to pathogens and increased awareness of the environmental impacts of extensive agrochemical usage.