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.

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins.

Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an "active" enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme-inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-ß-lactamase and its inhibitor protein, such that target binding disrupts the enzyme-inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics.

Detection and circulation of hepatitis B virus immune escape mutants among asymptomatic community dwellers in Ibadan, southwestern Nigeria.

BACKGROUND: In 2012, the first Nigerian Hepatitis B Virus (HBV) immune escape mutant (IEM) case was detected in a pregnant woman in southwestern Nigeria. Consequently, this study was designed to investigate the presence and possible circulation of IEMs amongst asymptomatic community dwellers in southwestern Nigeria. METHODS: Blood specimens collected from 438 asymptomatic community dwellers were screened for HBsAg using ELISA technique. Subsequently, the S-gene was amplified in HBsAg positive samples by a nested PCR protocol, and amplicons sequenced. Isolates were then subtyped by amino acid residues at positions 122, 127, 134 and 160, and genotyped by phylogenetic analysis. RESULTS: Of the 31 (7.08%) samples positive for HBsAg, the ∼ 408 bp Sgene fragment was successfully amplified and sequenced in 27. Samples obtained from 4 patients could not be amplified due to low titres. Sequence data from only 15 of the isolates could be analysed further as eight of the remaining 12 had multiple peaks while the rest three showed no similarity to any HBV gene when subjected to BLAST analysis. Thirteen of the 15 isolates were identified as genotype E. Eleven of which were subtyped as ayw4 while the remaining two could not be subtyped due to sR122Q/P substitutions. The last two isolates that could not be genotyped and subtyped had other mutations in the "a" determinant associated with IEMs. CONCLUSIONS: This study confirmed presence and circulation of HBV IEM in Nigeria, the country's inclusion in the genotype E crescent, and the value of phylogenetic analysis in HBV identification.