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The Antigens, Volume VI is a comprehensive treatise covering all aspects of antigens, including their chemistry and biology as well as their immunologic role .
Table of contents
- Antigen Binding Molecules: Antibodies and T-Cell Receptors, Volume 49
- The Antigens - 1st Edition
- 1st Edition
The chip array aluminium carrier contains immobilized GAD 65 antibodies, enabling it to focus specially on the GAD 65 antigen. The sample molecules are mixed with small matrix molecules, facilitating desorption and ionization of the proteins in the mixture.
The laser pulse excites the matrix molecules; the serum molecules are liberated from the array and protonized. Inside the vacuum chamber, an electric field along the direction r is generated by an electrostatic accelerating potential U :. The electric field is switched on synchronously with the end of the laser pulse. The protonated molecules are then accelerated in the electrical field by a force:. Where z is the net charge of the ionized molecule and e is the elementary electron charge 1. The molecules are accelerated into a field-free region, the TOF tube Figure Inside the TOF tube, the ionized molecules move toward the ion detector.
The physical principle of the TOF analyser is that proteins, which have a different mass over charge ratio m z are accelerated differently and enter the TOF tube with different velocities. The ratio of each protein molecule is recorded on the basis of the time required to pass through the tube. A protein with a net charge z , which passes through a potential difference of U , acquires an energy of:. The net charge is always a multiple whole number of the elementary electron charge e. The potential energy is converted, during the acceleration of the charged protein, into kinetic energy:.
Antigen Binding Molecules: Antibodies and T-Cell Receptors, Volume 49
Where m is the protein mass and v is its velocity. Then the velocity of the protein inside the TOF tube equals:. Because the molecules gain the same kinetic energy in the electric field, the velocity of the molecules with a small mass is bigger than the velocity of molecules with greater masses. If the TOF tube has a length of d , the time of flight to the detector is given by:. Then, the mass over charge ratio can be expressed as a function of the time of flight of the molecule:.
The detector measures the time interval between the switch on of the electric field and the moment a charged molecule hits the detector. When the molecules strike the detector plates, the plates release a certain multiple of electrons. The release of electrons is generally amplified by a cascade of successive releases avalanche effect. The detector signal is then defined by the ratio of released electrons and the number of molecules striking the detector plate.
The counted totals per time interval are displayed in the spectrum Figure The detection of molecules with the same molecular weight and the same electric charge produce a signal which is called a singleton peak. A peak in the spectrum is then the signal induced by neighbouring singleton peaks. The peak area is proportional to the number of detected molecules. The baseline of the spectrum is formed by the dark current inside the detector and the detected air molecules. In practice, linear deviations from the expected theoretical relation are observed.
Therefore a calibration must be performed before the measurement. The calibration equation is given by:. The factor a is a temporary dummy variable. PS20 Protein chip arrays are 8 spot chips with 2 mm diameter spots, spatially compatible with one column of a standard well microplate. The serum was applied on the array and covered with the matrix from the laboratory of the Pasteur Institute in Paris. In a first experiment, sera without dilution were used and in a second experiment, the sera were diluted. To realize a diagnostic procedure, every serum was tested in duplicate with different successive dilutions , , The concentration of albumin in sera is of course tremendously higher than the concentration of the expected GAD By use of the protein chip PS20 with fixation by covalent bonds of the GAD 65 antibodies, the contamination of the exuberant albumin is less disturbing.
The sera from 2 paediatric patients also contained in the sample set of the FCS measurements with early onset of diabetes mellitus type 1 were kindly provided from the serum bank of the Department of Paediatrics of the Medical University of Graz. Clean and spiking experiments allowed a minimal precisely detectable amount of the GAD 65 at the concentration of 2.
The Antigens - 1st Edition
The measured diffusion times are shown in figure 9. This enabled a sufficient serum dilution and avoids interference by auto fluorescence and other proteins in the FCS system. With diffusion times of resp. GAD 65 was found in different concentrations, where the values range from 0.
This leads to an average concentration of The correlation between the GAD 65 concentrations from samples stored at room temperature and the same samples stored in a refrigerator cooled at C are shown in Figure Correlation between the GAD 65 concentrations from samples stored at room temperature and cooled at C.
The high value of the correlation coefficient 0. Additionally, the correlation between the GAD 65 concentrations taken from samples stored at room temperature and taken from the same samples after a time delay of one week was calculated. During this week, the samples were stored at room temperature Figure The value of the correlation coefficient 0. Correlation analysis between the GAD 65 concentrations taken from samples stored at room temperature and taken from the same samples after a time delay of one week.
Evidence for GAD 65 in one serum sample in the mass spectrum was found, giving a peak very close to albumin with a molecular weight of 65 kDa. This serum was from a juvenile patient with an early onset of diabetes mellitus type 1. Peak evaluation after spiking experiments was done to have comparable results as with FCS.
The spectra always show the peaks of the antibodies at 50, 75 and kDa. The spectra of the diluted sera Figure 16 show a much diminished peak of the reference albumin. For the interpretation of the results, the peak intensity at 65 kDa was calibrated in proportion to the intensity of the albumin peak in the reference. The intensity of the appropriate peak in the spectrum is greater than the peak obtained for albumin by a factor of 8.
The sera are diluted at The spectrum of the diluted serum shows a peak at 65kDa which may results from GAD The intensity of the peak is greater, by a factor of 8. This serum is from a paediatric patient with diabetes mellitus type 1 at an early stage.
Functional specificity and biological function of a protein are linked to its structure. Due to the 3-D folding structure, the residues, which are responsible for the protein function, are brought into a precise geometric arrangement. The rest of the protein structure is mainly necessary to enable and maintain the correct spatial position between the amino acids on the active site.
Therefore, to understand a protein function, the 3-D structure of the protein reveals far more information than its sequence. For an analysis and planning of neutralization of GAD 65 with antibodies in the sera, the locations and shapes of the epitopes on the GAD 65 molecule are important.
The structural visualization of the GAD 65 molecule and its epitope locations is illustrative and may help the reader to speculate on the antigenic sites of the molecule, enabling an understanding of the interaction with the immune-receptor, which is the basis for the development of active or passive immunotherapy in the near future. The epitopes are well known in literature and were identified by homolog-scanning mutagenesis, where segments of sequences from a homologous molecule GAD 67 known not to bind to the specific antibodies are systematically substituted throughout the GAD 65 gene [ 57 , 58 ].
A complete or partial loss of antibody reactivity by the resulting mutated molecules chimeras suggests that GAD65 specific sequences required for contact with the antibody has been exchanged by the point mutation. The quaternary structure of GAD 65 was determined experimentally by X-ray diffraction [ 59 ].
Bioinformatics is an interdisciplinary discipline between computer science and molecular biology. On one side, bioinformatics is a science with interconnected data banks, connecting sequence information to structural, biomedical and clinical data. On the other side, bioinformatics is a science which provides mathematical algorithms and computational tools for: detecting sequence similarities and finding homologous sequences; prediction, analysis and visualization of protein 3-D structure etc.
Structural information about proteins is available in the PDB structure database, an international repository for 3-D structure files [ 61 ]. Once a protein structure has been determined experimentally, either with crystallographic methods or by nuclear magnetic resonance spectroscopy, the structural information is deposited into the PDB.
The structural information is stored in the PDB as data files, which contain mainly the Cartesian coordinates of all atoms involved in the protein Figure The database is accessed via Internet and the selected PDB data files, containing the atomic coordinates, are downloaded. The data files are used as input for protein visualization and the viewer software transforms the information in the PDB data file into an appropriate representation of the protein 3-D structure.