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Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain-only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification. Key features • Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment. • Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells., Competing Interests: Competing interestsThe authors declare that they have no competing interests with respect to the work described., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Background: The World Health Organization targeted Trypanosoma brucei gambiense human African trypanosomiasis (gHAT) for elimination as a public health problem and for elimination of transmission. To measure gHAT elimination success with prevalences close to zero, highly specific diagnostics are necessary. Such a test exists in the form of an antibody-mediated complement lysis test, the trypanolysis test, but biosafety issues and technological requirements prevent its large-scale use. We developed an inhibition ELISA with high specificity and sensitivity that is applicable in regional laboratories in gHAT endemic countries., Methods: The T. b. gambiense inhibition ELISA (g-iELISA) is based on the principle that binding of monoclonal antibodies to specific epitopes of T. b. gambiense surface glycoproteins can be inhibited by circulating antibodies of gHAT patients directed against the same epitopes. Using trypanolysis as reference test, the diagnostic accuracy of the g-iELISA was evaluated on plasma samples from 739 gHAT patients and 619 endemic controls and on dried blood spots prepared with plasma of 95 gHAT and 37 endemic controls., Results: Overall sensitivity and specificity on plasma were, respectively, 98.0% (95% CI 96.7-98.9) and 99.5% (95% CI 98.6-99.9). With dried blood spots, sensitivity was 92.6% (95% CI 85.4-97.0), and specificity was 100% (95% CI 90.5-100.0). The g-iELISA is stable for at least 8 months when stored at 2-8°C., Conclusion: The g-iELISA might largely replace trypanolysis for monitoring gHAT elimination and for postelimination surveillance. The g-iELISA kit is available for evaluation in reference laboratories in endemic countries., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Metacaspases (MCAs) are ideal drug and diagnostic targets for animal and human African trypanosomiasis, as these cysteine peptidases are absent from the metazoan kingdom and have been implicated in the parasite cell cycle and cell death. Tsetse fly-transmitted trypanosomes that live free in the bloodstream and/or cerebrospinal fluid of the mammalian host cause animal and human African trypanosomiasis (nagana or sleeping sickness respectively). Chemotherapy and chemoprophylaxis are the main forms of control, but in contrast to human trypanocides, the veterinary drugs are old and drug resistance is on the increase. A peptidomimetic library targeting the MCA2 from Trypanosoma brucei brucei has ligands with low IC 50 values, some of which were antiparasitic. This study validates the inhibitory activity of these ligands using the protein structure solved by X-ray diffraction after the ligand library was published. Water molecules were shown to be important in substrate binding and strategies to improve the efficacy of these ligands are highlighted. These ligands appear to be pan-specific as they were docked into the active site of the homology modelled MCA5 of animal infective Trypanosoma congolense with similar binding energies and conformations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we term nickel-induced over autoprocessing. When both the catalytic His and Cys residues were mutated (TcoMCA5 H147A/C202G ), no nickel-induced over autoprocessing was observed and was enzymatically active, suggesting the existence of a secondary catalytic Cys residue, Cys81. Immunoaffinity purification of native TcoMCA5 from the total parasite proteins was achieved using chicken anti-TcoMCA5 IgY antibodies. The full length native TcoMCA5 and the autoprocessed products of recombinant TcoMCA5 H147A/C202G were shown to possess gelatinolytic activity, the first report for that of a MCA. Both the native and recombinant enzyme were calcium independent, had a preference for Arg over Lys at the P 1 site and were active over a pH range between 6.5 and 9. Partial inhibition (23%) of enzymatic activity was only achieved with leupeptin and antipain. These findings are the first step in the biochemical characterisation of the single copy MCAs from animal infective trypanosomes towards the design of novel trypanocides., (Copyright © 2019 Elsevier Inc. All rights reserved.)

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATL cat , was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C 1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S 2 pocket. Leucine was preferred in P 2 and basic and non-bulky, hydrophobic residues accepted in P 1 and P 3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P 1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P 2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA., (Copyright © 2018 Elsevier B.V. All rights reserved.)