An extended immunization procedure over a total of 78 days, involving 4 injections (1 priming dose of immunogen in Freunds Complete Adjuvant and 3 boosts with immunogen in Freunds Incomplete Adjuvant) of immunogen preparation was performed. also known as African sleeping sickness. These include detection of anti-trypanosome antibodies [1], [2], amplification of DNA sequences [3], [4] and direct observation of parasites by microscopic examination of patient blood or cerebrospinal fluid (CSF), usually preceded by parasite enrichment techniques [5]. Although each of these methods has problems that hinder reliable, high throughput and cost effective disease diagnosis, together, they do help disease control efforts [6]. Currently, the only way to definitively diagnose HAT in the field is usually to microscopically observe trypanosomes in the blood (early stage disease) and in the CSF (late stage disease). Using parasite enrichment techniques, the current limit of microscopic detection is usually 100 trypanosomes/mL of blood [5], thus between parasitemic waves, parasite numbers lower than this make microscopic detection unreliable. Due to low sensitivity and low throughput, microscopic diagnosis is only used to confirm suspected infections and is not an effective MBX-2982 tool for mass screening campaigns. The card agglutination test for trypanosomiasis (CATT, [1]) is the most commonly used assay for mass screening in the field as it is usually relatively easily performed and requires minimal instrumentation [5]. It is not ideal, since it is only useful for detecting antibodies generated during infections with that often, but not usually, express a particular, defined variant surface glycoprotein (VSG) during waves of parasitemias. Infections with parasites in west and central Africa may be missed if that VSG type is not expressed. In addition, the CATT is not useful for detecting infections with infections are available, MBX-2982 diagnostic assays for African sleeping sickness based on parasite antigen detection are deemed to be more desirable. Previous studies have shown that trypanosome antigens are detectable by immunoassay in the sera of infected cattle [7], [8], [9], rodents [10], vervet monkeys [11] and humans [12], [13]. Previous work from our lab showed that trypanosome antigens (with both and infections) appeared in the blood soon after contamination [10], [11] were present at detectable levels throughout the contamination (regardless of the oscillating parasite populace associated with antigenic variation) and were reduced to undetectable levels within weeks of the infections being cured [12], [13]. Despite reports of antigen detection assays for animal and human trypanosomiasis [7], [8], [9], [10], [11], [12], [13], [14], [15] only one (for animal trypanosomiasis; [9]) described the identity of the analyte. No reliable antigen detection assessments have been developed and implemented for wide-scale use in the field. The collective data PIK3CG suggest that antigen detection assays have potential for diagnosis and monitoring of HAT although it is usually clear that more effort is required to identify parasite antigens of best utility. Several strategies towards antigen identification have been publically suggested [16], [17]. However these are based on examination of the parasites themselves and are not aimed directly at identification of the most relevant molecules that are found circulating MBX-2982 in a patients bloodstream. Identification of parasite proteins in the blood or plasma of an infected host is the most direct approach for discovery of candidate biomarkers for diagnosis and monitoring of trypanosomiasis. This strategy, however, is made technically difficult by the high abundance of human plasma proteins [18], [19], almost certainly explaining the failure over the past 30 years to identify trypanosome antigens in the blood of infected patients. Here we describe a deep mining protein discovery methodology using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify low abundance trypanosome proteins in human plasma. The approach used involved immunodepletion of the most abundant human plasma proteins,.
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