Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications

Reagentless Affimer- and antibody-based impedimetric biosensors for CEA-detection using a novel non-conducting polymer

Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1–100 fM vs. 1 fM – 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 μl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers

Do pediatric hospitalizations have a unique geography?

BACKGROUND: In the U.S. small-area health services research studies are often based on the hospital service areas (HSAs) defined by the Dartmouth Atlas of Healthcare project. These areas are based on the geographic origins of Medicare Part A hospital patients, the great majority of whom are seniors. It is reasonable to question whether the geographic system so defined is appropriate for health services research for all ages, particularly for children, who have a very different system of healthcare financing and provision in the U.S. METHODS: This article assesses the need for a unique system of HSAs to support pediatric small-area analyses. It is a cross-sectional analysis of California hospital discharges for two age groups – non-newborns 0–17 years old, and seniors. The measure of interest was index of localization, which is the percentage of HSA residents hospitalized in their home HSA. Indices were computed separately for each age group, and index agreement was assessed for 219 of the state's HSAs. We examined the effect of local pediatric inpatient volume and pediatric inpatient resources on the divergence of the age group indices. We also created a new system of HSAs based solely on pediatric patient origins, and visually compared maps of the traditional and the new system. RESULTS: The mean localization index for pediatric discharges was 20 percentage points lower than for Medicare cases, indicating a poorer fit of the traditional geographic system for children. The volume of pediatric cases did not appear to be associated with the magnitude of index divergence between the two age groups. Pediatric medical and surgical case subgroups gave very similar results, and both groups differed substantially from seniors. Location of children's hospitals and local pediatric bed supply were associated with Medicare-pediatric divergence. There was little visual correspondence between the maps of traditional and pediatric-specific HSAs. CONCLUSION: Children and seniors have significantly different geographic patterns of hospitalization in California. Medicare-based HSAs may not be appropriate for all age groups and service types throughout the U.S

Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

Cancer is frequently characterised by dysregulation of the cellular signalling processes that govern proliferation, survival and attachment. Understanding such dysregulation continues to present a challenge given the importance of protein-protein interactions in intracellular processes. Exploring this protein-protein interactome requires novel tools capable of discriminating between highly homologous proteins, individual domains and post-translational modifications. This review examines the potential of scaffold-based binding proteins to fulfil these requirements. It also explores protein-protein interactions in the context of intracellular signalling pathways and cancer, and demonstrates the uses of scaffold proteins as functional moderators, biosensors and imaging reagents. This review also highlights the timeliness and potential to develop international consortia to develop and validate highly specific “proteome” scaffold-based binding protein reagents with the ultimate aim of developing screening tools for studying the interactome

Affinity purification of fibrinogen using an Affimer column

Background Fibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen. Methods Fibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis. Results Affimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII. Conclusions The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. General significance Our methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions

Analysis of patient flows for orthopedic procedures using small area analysis in Switzerland

BACKGROUND: In general cantons regulate and control the Swiss health service system; patient flows within and between cantons are thereby partially disregarded. This paper develops an alternative spatial model, based upon the construction of orthopedic hospital service areas (HSA(O)s), and introduces indices for the analysis of patient streams in order to identify areas, irrespective of canton, with diverse characteristics, importance, needs, or demands. METHODS: HSA(O)s were constructed using orthopedic discharge data. Patient streams between the HSA(O)s were analysed by calculating three indices: the localization index (% local residents discharged locally), the netindex (the ratio of discharges of nonlocal incoming residents to outgoing local residents), and the market share index (% of local resident discharges of all discharges in local hospitals). RESULTS: The 85 orthopedic HSA(O)s show a median localization index of 60.8%, a market share index of 75.1%, and 30% of HSA(O)s have a positive netindex. Insurance class of bed, admission type, and patient age are partially but significantly associated with those indicators. A trend to more centrally provided health services can be observed not only in large urban HSA(O)s such as Geneva, Bern, Basel, and Zurich, but also in HSA(O)s in mountain sport areas such as Sion, Davos, or St.Moritz. Furthermore, elderly and emergency patients are more frequently treated locally than younger people or those having elective procedures. CONCLUSION: The division of Switzerland into HSA(O)s provides an alternative spatial model for analysing and describing patient streams for health service utilization. Because this small area model allows more in-depth analysis of patient streams both within and between cantons, it may improve support and planning of resource allocation of in-patient care in the Swiss healthcare system

Estimating Design Effect and Calculating Sample Size for Respondent-Driven Sampling Studies of Injection Drug Users in the United States

Respondent-driven sampling (RDS) has become increasingly popular for sampling hidden populations, including injecting drug users (IDU). However, RDS data are unique and require specialized analysis techniques, many of which remain underdeveloped. RDS sample size estimation requires knowing design effect (DE), which can only be calculated post hoc. Few studies have analyzed RDS DE using real world empirical data. We analyze estimated DE from 43 samples of IDU collected using a standardized protocol. We find the previous recommendation that sample size be at least doubled, consistent with DE = 2, underestimates true DE and recommend researchers use DE = 4 as an alternate estimate when calculating sample size. A formula for calculating sample size for RDS studies among IDU is presented. Researchers faced with limited resources may wish to accept slightly higher standard errors to keep sample size requirements low. Our results highlight dangers of ignoring sampling design in analysis

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

Hospital service areas – a new tool for health care planning in Switzerland

BACKGROUND: The description of patient travel patterns and variations in health care utilization may guide a sound health care planning process. In order to accurately describe these differences across regions with homogeneous populations, small area analysis (SAA) has proved as a valuable tool to create appropriate area models. This paper presents the methodology to create and characterize population-based hospital service areas (HSAs) for Switzerland. METHODS: We employed federal hospital discharge data to perform a patient origin study using small area analysis. Each of 605 residential regions was assigned to one of 215 hospital provider regions where the most frequent number of discharges took place. HSAs were characterized geographically, demographically, and through health utilization indices and rates that describe hospital use. We introduced novel planning variables extracted from the patient origin study and investigated relationships among health utilization indices and rates to understand patient travel patterns for hospital use. Results were visualized as maps in a geographic information system (GIS). RESULTS: We obtained 100 HSAs using a patient origin matrix containing over four million discharges. HSAs had diverse demographic and geographic characteristics. Urban HSAs had above average population sizes, while mountainous HSAs were scarcely populated but larger in size. We found higher localization of care in urban HSAs and in mountainous HSAs. Half of the Swiss population lives in service areas where 65% of hospital care is provided by local hospitals. CONCLUSION: Health utilization indices and rates demonstrated patient travel patterns that merit more detailed analyses in light of political, infrastructural and developmental determinants. HSAs and health utilization indices provide valuable information for health care planning. They will be used to study variation phenomena in Swiss health care