ABSTRACT
Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.
ABSTRACT
Background: Haematuria often requires investigation with an imaging test and flexible cystoscopy to rule out urinary tract cancers. With a reduction in diagnostic services due to the COVID-19 pandemic there is a risk of compromise in the care of patients referred with haematuria. We aimed to provide a pragmatic strategy that optimises the use of scarce resources by reducing patient visits to hospital and allocating the appropriate diagnostic tests according to risk of bladder cancer. Methods: The IDENTIFY study was an international, prospective, multicentre cohort study of over 11,000 patients referred to secondary care for investigation of newly suspected urinary tract cancer. Patients underwent cystoscopy, imaging tests, urine cytology and transurethral resection of bladder tumour (TURBT), where indicated. We developed strategies using combinations of imaging and cytology as triage tests to flexible cystoscopy. These strategies aimed to maximise cancer detection within a pragmatic pathway in a resource-limited environment. Findings: 8112 patients (74 4%) received an ultrasound or a CT urogram, with or without cytology. 5737 (70 7%) patients had visible haematuria (VH) and 2375 (29 3%) had non-visible haematuria (NVH). Amongst all patients, 1474 (18 2%) had bladder cancer;1333 (23 2%) in VH group and 141 (5 94%) in NVH group. Diagnostic test performance was used to determine optimal age cut-offs for each proposed strategy. We recommended proceeding directly to TURBT for patients of any age with positive triage tests for cancer. Patients with negative triage tests under 35-years-old with VH, or under 50-years-old with NVH can safely be discharged without undergoing flexible cystoscopy. The remaining patients may undergo flexible cystoscopy, with a greater priority for older patients (threshold of 60-years-old with VH, or 70-years-old with NVH) to capture high risk bladder cancer. Interpretation: We suggest diagnostic strategies in patients with haematuria, which focus on detection of bladder cancer, whilst reducing the burden to healthcare services in a resource-limited setting.
ABSTRACT
The COVID-19 pandemic has changed the world;people are living differently in all aspects: the ways they are socializing, buying, or even thinking before doing something, have led to dramatic transformations in different types of business. Some companies are thriving while others are struggling to stay alive, restructuring their products management and services. Many workers fear for their livelihoods and still working even not being paid. Consumers, in general, tend to retract non-essential purchases but on the other hand they have learned to adapt quickly to overcome the constraints imposed by governments. They also started working from home at the same time their younger children started taking online classes. Suddenly our home became our world, the place where we study, build, eat, do sports, and share our fears and our hopes. So, if we all stay there longer, what does your wardrobe look like? The purpose of this paper is to gather as much information as possible to understand how the enclosure experiences have transformed people’s ways of dressing. © 2021, The Author(s), under exclusive license to Springer Nature Switzerland AG.
ABSTRACT
Introduction: Diagnostic haematuria services have been reduced due to the COVID-19 pandemic, compromising patient care, and necessitating a more pragmatic pathway. Method: The IDENTIFY study was an international, prospective, multicentre cohort study of over 11,000 patients referred to secondary care for investigation of haematuria. Using this data, we developed strategies using combinations of imaging and cytology as triage tests to maximise cancer detection within a pragmatic pathway. Results: 8112 patients (74 4%) received an ultrasound or a CT urogram, with or without cytology. 5737 (70 7%) patients had visible haematuria (VH) and 2375 (29 3%) had non-visible haematuria (NVH). Diagnostic test performance was used to determine optimal age cut-offs for four proposed strategies. We recommended proceeding directly to transurethral resection of bladder tumour for patients of any age with positive triage tests for cancer. Patients with negative triage tests under 35-years-old with VH, or under 50-years-old with NVH can safely be discharged without undergoing flexible cystoscopy. The remaining patients may undergo flexible cystoscopy, with a greater priority for older patients to capture high risk bladder cancer. Conclusions: We suggest diagnostic strategies in patients with haematuria, which focus on detection of bladder cancer, whilst reducing the burden to healthcare services in a resource-limited setting.