These are two closely related proteins sharing 91% and 80% amino acid sequence similarity and identity, respectively. TvY486_0045500 lateral flow test prototype a sensitivity and specificity of 92.0% (95% CI, 83.4% to 97.0%) and 89.8% (95% CI, 77.8% to 96.6%), respectively. These data suggest that recombinant TvY486_0045500 shows promise for the development of a pen-side lateral flow test for the diagnosis of animal African trypanosomosis. Author Summary African Animal Trypanosomosis presents a significant problem for agricultural development in sub-Saharan Africa and leads to large economic losses. One of the main parasites responsible is usually proteins selectively recognized by infected cattle sera and developed two related proteins into ELISA assessments and one of these into a lateral flow test prototype. All three assessments performed well when tested against randomised calf sera, suggesting good potential for the development of a pen-side animal African trypanosomosis diagnostic device for use in endemic regions. Introduction is usually a protozoan parasite of the genus trypanosomatidae spread primarily by biting insects. Together with and Fst causes a severe version of AAT, often characterised by hemorrhagic fever as well as the more typical weight loss, fatigue and anaemia [1]. As does not require midgut gestation within the vector it can be can be transmitted mechanically by body fluid contamination and hematophagous flies [2,3]. This has allowed the spread of the disease in South America, an area previously free from RoTat1.2 and p64, as cross-reactive diagnostic antigens for cattle infections has been described [9] and these may lead to new diagnostic tools. Nevertheless, at the moment, farmers mostly rely upon symptom-based diagnosis, which is complicated by the numerous other diseases with comparable manifestations in the endemic regions. With this in mind, we set out to develop a low-cost pen-side diagnostic test for infections in cattle using lateral flow test (LFT) technology. We used the approach of identifying parasite antigens selectively recognised by cattle contamination sera by proteomics, followed by recombinant protein expression in and antigen assessment by ELISA to Ezatiostat hydrochloride select an antigen for LFT prototyping. This general approach has been successful for selecting diagnostic antigens for human and cattle infections [10C12]. One of these antigens, a recombinant invariant surface glycoprotein (rISG65-1), has been selected by the Foundation for Innovative New Diagnostics (FIND) for development of a next-generation all recombinant LFT for human African trypanosomiasis. Here, we report the identification, recombinant production and evaluation by ELISA of segments of two related invariant surface glycoprotein (ISG) diagnostic antigens for AAT caused by parasites to make the detergent lysates for immunoaffinity chromatography and proteomics. The animal procedures were carried out according the United Kingdom Animals (Scientific Procedures) Act 1986 and according to specific protocols approved by The University of Dundee Ethics Committee and as defined and approved in the UK Home Office Project License PPL 60/3836 held by MAJF. Cattle studies were approved by the ClinVet IACUC which complies with The South African National Standard: SANS 10386:2008: The care and use of animals Ezatiostat hydrochloride for scientific purposes. Sera All sera were provided by GALVmed. The sera used for the antigen identification by immunoprecipitation Ezatiostat hydrochloride and proteomics were from four animals obtained commercially in Burkina Faso and treated prophylactically for infections before experimental contamination with post-infection sera. The Mozambique samples (20 sera) were from 2 calves and consisted of 20 post-infection sera. The South Africa (ClinVet) samples (72 sera) were from 31 calves and consisted of 27 pre-infection and 32 post-infection sera and 13 post-infection sera. Strains used to infect cattle (one isolate per calf) were: In Mozambique, Y486 and IL700. In Burkina Faso, Sokoroni 18, Napie22, Komborodougou and Gondo Bengaly. At ClinVet, ILRAD560. IgG purification from pre- and post-infection sera Sera were collected in Burkina Faso from four calves before and 28 days after experimental contamination with parasite lysate Three BALB/c mice were injected with one stabilate of ILRAD V34. After five days, infected mouse blood was harvested with citrate anticoagulant, adjusted to 5104 parasites per ml with phosphate-buffered saline (PBS) and aliquots of 0.2 ml were injected into the peritoneal cavity of 45 NMR1 mice. The mouse blood Ezatiostat hydrochloride was harvested after 7 days and the parasites were purified by centrifugation, to yield a buffy coat enriched in trypanosomes, followed by DE52 ion exchange chromatography to remove white blood cells.