Detection of trypanosome‐specific antibodies in saliva, towards non‐invasive serological diagnosis of sleeping sickness

Lejon, Veerle; Jamonneau, Vincent; Solano, Philippe; Atchade, Pascal S.; Mumba, Dieudonné; Nkoy, N; Bebronne, Nicolas; Kibonja, T; Balharbi, Fatima; Wierckx, A; Boelaert, Marleen; Büscher, Philippe

doi:10.1111/j.1365-3156.2006.01620.x

Cited by 22 publications

(27 citation statements)

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“…gambiense , combining these three VSGs has been developed [22] and the ELISA/ T.b. gambiense with the same antigen combination has a proven high sensitivity and specificity on serum, plasma, CSF and even saliva [23], [24]. However, stability and/or logistical requirement issues prevented replacement of the CATT by these tests.…”

Section: Introductionmentioning

confidence: 99%

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

et al. 2014

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BackgroundScreening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.Methodology/Principal FindingsWe have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.Conclusions/SignificanceThe diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

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Section: Introductionmentioning

confidence: 99%

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

et al. 2014

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“…There are also several promising developments in disease biomarkers (Papadopoulos et al 2004 ;Agranoff et al 2005). Blood or CSF markers for staging are in the early stages of development (Lejon et al 2002 ;Courtioux et al 2005) as are non-invasive diagnostic tools (Lejon et al 2006).…”

Section: Diagnostics and Chemotherapymentioning

confidence: 99%

Controlling sleeping sickness – a review

2009

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SUMMARYFollowing a period characterized by severe epidemics of sleeping sickness, restoration of effective control and surveillance systems has raised the question of eliminating the disease from sub-Saharan Africa. Given sufficient political and financial support, elimination is now considered a reasonable aim in countries reporting zero or less than 100 cases per year. This success may lead health authorities across the affected region to downgrade the disease from ‘neglected’ to simply being ignored. In view of the significant levels of under-reporting of sleeping sickness mortality in rural communities, this could be a short-sighted policy. Loss of capacity to deal with new epidemics, which can arise as a consequence of loss of commitment or civil upheaval, would have serious consequences. The present period should be seen as a clear opportunity for public-private partnerships to develop simpler and more cost-effective tools and strategies for sustainable sleeping sickness control and surveillance, including diagnostics, treatment and vector control.

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“…Our prototype LAMPole assay is based on a published ELISA that detects host antibodies against T. b. gambiense LiTat 1.3, 1.5, and 1.6 variable surface glycoproteins (VSGs) (13). To create the solid-phase substrate for use in our assays, we conjugated 2.8-m tosylactivated beads (Invitrogen) with either purified LiTat 1.3 VSG (23) or purified mouse total IgG (Jackson Immunologicals) in 0.1 M borate (pH 9.5)-1 M (NH 4 ) 2 SO 4 -0.1 M NaCl for 16 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Various VSGs have traditionally been selected as antigenic targets in serological assays for HAT (13,22). For all these assays, we scavenged antiparasite IgG from sera using T. b. gambiense LiTat 1.3 VSG-coated magnetic beads and detected bound IgG by forming a sandwich with our anti-IgG LAMPole (Fig.…”

Section: Figmentioning

confidence: 99%

“…We further investigated the specificity of the LAMPole fusion for recognizing human IgG and, as a benchmark of this approach, applied it to measuring antiparasite antibodies in humans and mice. Our assay was inspired by an ELISA for detecting host antibodies to Trypanosoma brucei gambiense variable surface glycoproteins (VSGs), which detected titers of 1:30,000 and 1:40 from serum and saliva, respectively (13). This work indicated that detection of relatively low titers of antibodies in noninvasive specimen types is possible provided the method is sufficiently sensitive.…”

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confidence: 99%

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Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

Burbulis

Yamaguchi

Nikolskaia

et al. 2015

Clin. Vaccine Immunol.

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f Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a "LAMPole") that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratorybased quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections. N umerous applications require sensitive measurement of biological analytes to provide actionable information in real time. In considering biomarker measurement, no other molecule is as easily or sensitively quantified as nucleic acid because of facile means to enzymatically replicate sequence-specific templates in vitro. The detection limit of specific DNA templates has been reported to be at or below 10 molecules when measured by real-time PCR (quantitative PCR [qPCR]) (1, 2). This level of sensitivity is useful in the context of a laboratory, but there is need for diagnostics that can be used at the point of care (POC) without thermocycling equipment. An alternative method for amplifying DNA is loop-mediated isothermal amplification (LAMP), which requires four different oligonucleotides to promote Bst polymerase-catalyzed DNA synthesis at a constant 65°C. The end products of LAMP (pyrophosphate and high-molecular weight DNA) can be monitored by measuring changes in turbidity or color with the addition of Mg 2ϩ (3) or dyes (4), respectively. Highly sensitive measurement of non-DNA analytes (e.g., proteins, toxins, lipids, and carbohydrates) at the POC can be achieved by combining the molecular recognition of immunoassays with the signal amplification of LAMP. We previously developed means to label protein and small-molecule ligands with unique oligonucleotides measurable by PCR (5). We labeled molecular targets using a protein-DNA fusion known as a "Tadpole," which binds ligand with high specificity so as to attach an identifying oligonucleotide. When we labeled antibodies or protein antigens with oligonucleotides, we created toxin and biomarker assays that were hundreds of times more sensitive than matched enzyme-linked immunosorbe...

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Detection of trypanosome‐specific antibodies in saliva, towards non‐invasive serological diagnosis of sleeping sickness

Cited by 22 publications

References 13 publications

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Controlling sleeping sickness – a review

Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

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