Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device.
Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device.
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DOI：
10.1371/journal.pntd.0002087
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发表时间：
2013
影响因子：
3.8
通讯作者：
Ferguson MA
中科院分区：
文献类型：
--
作者：
Sullivan L;Wall SJ;Carrington M;Ferguson MA
人类非洲锥虫病的诊断主要依靠锥虫凝集卡试验（CATT）。虽然这一测试是成功的，但人们承认可能还有改进的余地。我们的目的是开发一个原型侧流试验的基础上检测锥虫抗原的抗体。我们采取了一种无偏见的方法来确定潜在的免疫诊断寄生虫蛋白抗原。用G蛋白亲和层析法从布氏冈比亚锥虫感染者和对照者血清中分离IgG组分，然后将其固定在琼脂糖凝胶珠上。将IgG珠与锥虫的去污剂裂解物一起孵育，并通过基于质谱的蛋白质组学方法鉴定结合的那些蛋白质。这种方法提供了24种锥虫蛋白质的列表，这些蛋白质选择性地与感染IgG组分结合，因此可能被认为是免疫诊断抗原。我们从该列表中选择了四种抗原（ISG 64、ISG 65、ISG 75和GRESAG 4），并在E. coli中构建了12个构建体。获得七种可溶性重组蛋白产物（三种用于ISG 64，两种用于ISG 65，以及ISG 75和GRESAG 4各一种），并使用个体和/或合并的患者血清通过ELISA评估其免疫诊断潜力。ISG 65和ISG 64构建体ELISA在检测T. B.冈比亚T. B. Rhodesiense感染，并且使用表现最好的ISG 65构建体来开发原型侧向流诊断装置。使用80个随机的T. B.冈比亚血吸虫感染血清和对照血清，原型显示出合理的敏感性（88%）和特异性（93%），使用视觉读数检测T。B.冈比亚感染。这些结果为进一步开发和优化侧流装置用于临床提供了鼓励。人非洲锥虫病是由布氏冈比亚锥虫或T. B.罗得西亚语初步诊断T. B.冈比亚锥虫感染主要依赖于锥虫卡凝集试验（CATT），该试验已被承认存在局限性。需要新的方法，首先是确定新的诊断抗原，其次是找到一个更合适的平台，以现场为基础的免疫诊断测试。我们采取了一种无偏见的方法来确定候选诊断抗原，通过询问哪些寄生虫蛋白质与感染患者的抗体结合，而不是与未感染患者的抗体结合。从这24种候选抗原中，我们选择了4种，并从中选择了在常规免疫诊断测试中效果最好的一种。这种抗原ISG 65用于制造侧流装置，其中将少量患者血清样品添加到垫中，并且30分钟后可以通过简单的光学读出来推断感染。这种简单的原型装置与CATT测试一样工作，并且可以被开发和优化用于该领域的临床使用。
The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens. We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device. Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use. Human African Trypanosomiasis is caused by infection with Trypanosoma brucei gambiense or T. b. rhodesiense. Preliminary diagnosis of T. b. gambiense infection relies mainly on a Card Agglutination Test for Trypanosomiasis (CATT), which has acknowledged limitations. New approaches are needed, first to identify new diagnostic antigens and, second, to find a more suitable platform for field-based immunodiagnostic tests. We took an unbiased approach to identify candidate diagnostic antigens by asking which parasite proteins bind to the antibodies of infected patients and not to the antibodies of uninfected patients. From this list of twenty-four candidate antigens, we selected four and from these we selected the one that worked the best in conventional immunodiagnostic tests. This antigen, ISG65, was used to make lateral flow devices, where a small sample of patient serum is added to a pad and thirty minutes later infection can be inferred by simple optical read out. This simple prototype device works as well as the CATT test and may be developed and optimized for clinical use in the field.
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DOI：
10.1186/1475-9292-5-1
发表时间：
2006-04-25
期刊：
Kinetoplastid biology and disease
影响因子：
--
作者：
Steverding D
通讯作者：
Steverding D
影响因子：
3.7
作者：
Manful T;Mulindwa J;Frank FM;Clayton CE;Matovu E
通讯作者：
Matovu E
影响因子：
3.8
作者：
Buescher, Philippe;Ngoyi, Dieudonne Mumba;Bieler, Sylvain
通讯作者：
Bieler, Sylvain
DOI：
10.1007/978-1-4419-7273-6_9
发表时间：
2011-01-01
期刊：
FOLDING OF DISULFIDE PROTEINS
影响因子：
--
作者：
Arredondo, Silvia A.;Georgiou, George
通讯作者：
Georgiou, George
影响因子：
2.9
作者：
Burnouf, Thierry;Goubran, Hadi Alphonse;El-Ekiaby, Magdy
通讯作者：
El-Ekiaby, Magdy