Chin, Kai Ling
(2016)
Identification Of Antigenic Proteins Of Salmonella Enterica Subspecies Enterica Serovar Typhi In Sera Of Patients With Acute Typhoid Fever.
PhD thesis, Universiti Sains Malaysia.
Abstract
Demam kepialu disebabkan oleh sejenis bakteria Gram-negatif, Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). Penyakit ini berleluasa di seluruh dunia terutamanya di negara-negara yang mundur atau sedang membangun. Oleh sebab kekurangan biopenanda yang sensitif dan spesifik di pasaran, diagnosis demam kepialu masih kekal sebagai satu masalah kepada pengamal kesihatan. Ini mungkin kerana biopenanda tersebut dikenalpasti dalam keadaan in vitro menggunakan kultur mikrobiologi. Infeksi adalah satu proses dinamik dan penghasilan antigen bergantung kepada cabaran persekitaran yang dihadapi oleh bakteria. Dalam kajian ini, serum pesakit demam kepialu digunakan sebagai sumber antigen in vivo kerana ia mengandungi produk fisiologi daripada perumah dan bakteria yang dihasilkan ketika infeksi. Namun demikian, kewujudan protein manusia dalam kuantiti yang banyak (HAPs) di dalam serum pesakit akan menghalang pengesanan protein antigenik yang wujud dalam kuantiti yang sedikit (LAPs) dan berat molekul rendah, yang mungkin mengandungi biopenanda yang berkaitan dengan penyakit tertentu. Serum daripada pesakit demam kepialu (PTFS) dan serum daripada manusia normal (PNHS) diproses menggunakan 2 kaedah, iaitu 1) afiniti kromatografi untuk memisahkan albumin dan immunoglobulin G (IgG) daripada serum, dan 2) ultraturasan membran untuk memisahkan protein berat molekul lebih daripada 100 kDa daripada serum.
Typhoid fever is caused by a Gram-negative bacteria, Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). It is a worldwide disease infecting mostly the under-developed and developing countries. Due to a lack of sensitive and specific biomarkers available in the market, diagnosis of typhoid fever remains a problem for the clinician. This may be due to the fact that these biomarkers were identified using pure microbiological cultures grown in in vitro conditions. Infection is a dynamic process and the expression of antigens depend on the environmental challenges faced by the bacteria. This study used serum as a source of in vivo antigens as it contains physiological signatures from both host and bacterial origin that are produced in vivo during infection. However, the presence of high abundance proteins (HAPs) in human serum masks the detection of low abundance proteins (LAPs) and low molecular weight proteins, which contain candidate protein biomarkers relevant to a particular disease state. Pooled Typhoid Fever Sera (PTFS) and Pooled Normal Human Sera (PNHS) were subjected to 2 different serum separation methods, i.e. 1) affinity chromatography to remove albumin and immunoglobulin G (IgG), and 2) membrane ultrafiltration to remove proteins with molecular weights greater than 100 kDa. These protein preparations were then subjected to 2 protein analysis methods, i.e. 1) SDS-PAGE-Western blot, and 2) 2D-PAGE-Western blot.
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