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Student Number 93223054
Author Kai-Yi Wang(ͻ)
Author's Email Address No Public.
Statistics This thesis had been viewed 2023 times. Download 624 times.
Department Chemistry
Year 2005
Semester 2
Degree Master
Type of Document Master's Thesis
Language English
Title Nanoprobe-Based Affinity Mass Spectrometry for Multiplexed Immunoassay in Human Plasma
Date of Defense 2006-07-05
Page Count 94
Keyword
  • mass spectrometry
  • multiplexed immunoassay
  • nanoprobe
  • Abstract Human plasma is the most used specimen for diagnosis where the level and/or modification of some specific proteins are associated with the progression of disease. Because of the complex nature of disease, diagnosis using single protein assay often results in insufficient detection specificity. The development and application of a multiplexed assay provide advantages of reduced analytical time, lower cost of reagents and smaller sampling quantity. Unfortunately, the high protein content of plasma is present across an extraordinary dynamic range of concentration that is likely to span more than 1012, which result in analytical challenge.
    Recent success of mass spectrometry in clinic proteomics has shown the ability to screen and discover multiple biomarkers simultaneously that increases the sensitivity and specificity of disease diagnosis. We have developed a multiplexed immunoassay based on nanoprobe-based affinity mass spectrometry (NBAMS) to specifically isolate, preconcentrate, and characterize several disease-associated antigens in plasma simultaneously. To demonstrate the availability of the approach, three serum proteins, C-reactive protein (CRP), serum amyloid P component (SAP), and serum amyloid A (SAA) with different concentrations are chosen as model system. Based on the antibody-antigen interaction, antibody-conjugated magnetic nanoparticles (MNPs), a-CRP MNPs, a-SAP MNPs, and a-SAA MNPs, can be used as the affinity probe to specifically extract antigens from plasma and quantitativly analyze multiple proteins. When applying this multiplex assay to human plasma analysis, distinct pattern of the protein profile can be used to differentiate healthy controls, patients with surgical operation of cardiac catheter, and gastric cancer patients.
    Given the flexibility of manipulating functional groups on the nanoparticles, their low cost, and simplicity of the assay, this technique is capable for clinical proteomic diagnosis.
    Table of Content Table of Content
    Page
    Chinese Abstract KKKKKKKKKKKKKKKKKKKKK..
    Abstract KKKKKKKKKKKKKKKKKKKKKKKKK
    Acknowledgment KKKKKKKKKKKKKKKKKKKKK.
    List of Figures KKKKKKKKKKKKKKKKKKKKKK..
    Nonstandard Abbreviations KKKKKKKKKKKKKKKKK..
    Chapter 1: Introduction KKKKKKKKKKKKKKKKKKK1
     1-1. Clinic Proteomics KKKKKKKKKKKKKKKKKK..1
      1-1.1. Plasma in Clinic Diagnostic KKKKKKKKKKKK2
      1-1.2. Multiparameter Indicator KKKKKKKKKKKKK.4
     1-2. Affinity Detection KKKKKKKKKKKKKKKKKK.5
      1-2.1. Protein Array KKKKKKKKKKKKKKKKKK..6
      1-2.2. Mass Spectrometry KKKKKKKKKKKKKKKK7
       1-2.2.1. MALDI ..KKKKKKKKKKKKKKKKKK...7
       1-2.2.2. TOF KK..KKKKKKKKKKKKKKKKKK.9
       1-2.2.3. MALDI-TOF Mass Spectrometer K...KKKKKKK..10
       1-2.2.4. Affinity Mass Spectrometry ..KKKKKKKKKK 11
     1-3. Biomolecular Nanotechnology KKKKKKKKKKKKK. 13
      1-3.1. Nanoscale Affinity Probe KK.KKKKKKKKKKK.13
     1-4. Purpose KKKKKKKKKKKKKKKKKKKKKK.15
     1-5. Disease-Related Proteins KKKKKKKKKKKKKKK..15
      1-5.1. Acute Phase Response KKKKKKKKKKKKKK...15
      1-5.2. Overview of Targeted Proteins KKKKKKKKKKK.16
       1-5.2.1. Sereum amyloid A protein KKK..KKKKKKKK.16
       1-5.2.2. C-reactive protein KK..KKKKKKKKKKKK.16
       1-5.2.3. Serum amyloid P component K..KKKKKKKKK..17
    Chapter 2: Experiment K...KKKKKKKKKKKKKKKKK..18
     2-1. Materials ..KKKKKKKKKKKKKKKKKKKKK.18
      2-1.1. Chemicals and Materials KKKKKKKKKKKKKK18
      2-1.2. Synthesis of Antibody-Conjugated Magnetic Nanoparticles 18
      2-1.3. Human Plasma Preparation KKKKKKKKKKKKK19
     2-2. Instrument KKKKKKKKKKKKKKKKKKKKK.20
     2-3. Method KKKKKKKKKKKKKKKKKKKKKK..20
      2-3.1. Matrix Selection KKKKKKKKKKKKKKK.KK20
      2-3.2. Determination of Spectra Acquisition Method .KKK.21
      2-3.3. Immunoaffinity Extraction K...KKKKKKKKKK.21
      2-3.4. Optimization of NBAMS for Single Targeted Protein Assay.22
       2-3.4.1. Test of Specificity and MEG blocking K.KKKKK22
       2-3.4.2. Sensitivity KK..KKKKKKKKKKKKKKK..22
      2-3.5. Optimization of NBAMS for Multiplex Analysis KKKK22
       2-3.5.1. Specificity ..KKKKKKKKKKKKKKKKK..22
       2-3.5.2. Sensitivity KK..KKKKKKKKKKKKKKK..23
       2-3.5.3. Dynamic Range of Quantitative Analysis KKKK...23
       2-3.5.4. Concentration Effect in Multiplex Detection K... 23
       2-3.5.5. Response Curve for Multiplex Quantification K..K24
       2-3.5.6. Human Plasma Analysis KKKK..KKKKKKKK.24
    Chapter 3: Results and Discussion KKKKKKKKKKKKKK..26
     3-1. Optimization of MALDI-TOF MS Detection KKKKKKK..26
      3-1.1. Matrix Selection KKKKKKKKKKKKKKKKK26
      3-1.2. Spectra Acquisition KKKKKKKKKKKKKKKK28
     3-2. Optimization of Nanoprobe-Based Affinity Mass 
       Spectrometry Immunoassay K...KKKKKKKKKKKK.. 29
      3-2.1. Specificity KKKKKKKKKKKKKKKKKKK..30
      3-2.2. Sensitivity KKKKKKKKKKKKKKKKKKK..32
     3-3. Multiplexed Immunoassay for Detection of SAA, CRP, and SAP 34
      3-3.1. Specificity KKKKKKKKKKKKKKKKKKK..35
      3-3.2. Sensitivity K...KKKKKKKKKKKKKKKKK..36
      3-3.3. Dynamic Range of Quantitative Analysis KK...KKK37
      3-3.4. Concentration Effect in Multiplex Detection K...K.40
      3-3.5. Response Curve for Multiplex Quantification K...KK42
      3-3.6. Human Plasma Analysis K...KKKKKKKKKKKK.45
    Chapter 4: Conclusion KKKKKKKKKKKKKKKKKKK..47
    Figures KKKKKKKKKKKKKKKKKKKKKKKKK..49
    ReferencesK...KKKKKKKKKKKKKKKKKKKKK... 72
    Appendix A K...KKKKKKKKKKKKKKKKKKKKK... 81
    Appendix B .....KKKKKKKKKKKKKKKKKKKKKK. 82
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    Advisor
  • Duen-Ren Hou(J)
  • Yu-Ju Chen(ɦp)
  • Files
  • 93223054.pdf
  • approve in 2 years
    Date of Submission 2006-07-21

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