Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-08-28
2003-05-06
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C250S281000, C250S287000, C250S288000, C210S198200
Reexamination Certificate
active
06558946
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to biomarkers, more particularly to methods and apparatus for rapidly identifying biological agents through the automated detection of biomarkers such as proteins released from extracts or whole intact biological agents that may be present in an environmental or biological sample.
BACKGROUND OF THE INVENTION
Rapid and accurate identification of biological agents is essential in diagnosing diseases, anticipating epidemic outbreaks, monitoring food supplies for contamination, regulating bioprocessing operations, and detecting threats of warfare. It is highly desirable not only to rapidly distinguish between related biological agents especially pathogenic agents, but also to unmistakably identify species and strains in complex matrices especially for the purpose of risk assessment in field situations.
The classification of biological agents such as bacteria and viruses has traditionally been based on biochemical and morphological tests. Recently, several analytical techniques have been developed which enhance the speed and accuracy of the identification of such biological agents. In these techniques, the biochemical components of biological agents are examined to determine chemotaxonomic markers, which are specific for each species. The chemotaxonomic markers, or biomarkers, may include any one or a combination of the classes of molecules present in the cells such as lipids, phospholipids, lipopolysaccharides, oligosaccharides, proteins, DNA, and the like.
Recently, mass spectrometric techniques have been developed for generating specific protein profiles for various biological agents. These techniques generally employ electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) of protein extracts followed by mass spectrometric (MS) or tandem mass spectrometric (MS/MS) analysis. ESI and MALDI are ionization techniques that have enabled dramatic progress to be made in performing mass spectrometry on large biomolecules including proteins. For example, the MALDI technique, combined with time-of-flight mass spectrometry (TOF-MS), has been used to differentiate biological agents using a crude protein extract.
The MALDI-MS technique is based on the discovery in the late 1980's that desorption/ionization of large, nonvolatile molecules such as proteins and the like can be made when a sample of such molecules is irradiated after being co-deposited with a large molar excess of an energy-absorbing “matrix” material, even though the molecule may not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the molecule of the sample. The ionized molecules are accelerated toward a detector through a flight tube. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass. Thus low-mass ions have a shorter time of flight (TOF) than heavier molecules. Detailed descriptions of the MALDI-TOF-MS technique and its applications may be found in review articles written by E. J. Zaluzec et al. (
Protein Expression and Purification,
Vol. 6, pp. 109-23 (1995)) and D. J. Harvey (
Journal of Chromatography A,
Vol. 720, pp. 429-4446 (1996)), and in U.S. Pat. No. 6,177,266, each of which is incorporated herein by reference.
Aside from the means for desorption/ionization, the ESI-MS technique is similar to the MALDI-MS technique in principle. In ESI, a dilute solution of an analyte containing large, nonvolatile molecules such as proteins and the like is slowly supplied through a short length of capillary tubing. The capillary tubing is held at a few kilovolts with respect to the counter electrode, positioned about a centimeter away. The strong electric field at the end of the capillary tubing draws the solution into a cone-shaped form, and at the tip of the cone the solution is nebulized into small charged droplets. As the charged droplets travel towards the counter electrode, the solvent evaporates, thus ultimately yielding molecular ions. The ions are drawn into the vacuum chamber through a small aperture or another piece of capillary tubing, which is usually heated to ensure that the ions are completely desolvated. The molecular ions are then extracted into a mass spectrometer for analysis.
In both techniques, ionization is a critical event in mass spectrometry where the masses of the ionized particles can be accurately measured by the mass spectrometer. The mass spectrometer is a highly sensitive analysis instrument which provides the user with information on the molecular weight and structure of organic compounds and the like. Once the mass of the ion is known, the chemical composition and structure can further be determined through the use of tandem mass spectrometric techniques as known in the art. The utilization of ESI and MALDI when combined with mass spectrometry provides accurate analysis of large biological molecules such as proteins and DNA. The detection limits with mass spectrometry, especially MALDI, depend largely on concentrating the sample and reducing the volume thereof. Sensitivity will increase as ultramicro methods for concentrating and transferring ever smaller-volume samples are developed.
Typically, samples containing biomarkers released from intact biological agents can be directly subjected to mass spectrometric analysis using the above techniques. However, the presence of contaminants, ionizable impurities and non-polar detergents that may be present, undesirably suppresses ionization efficiency under the ESI and MALDI conditions. The removal of such contaminants substantially improves the sensitivity and thus improves the reliability of the identification of the biological agents while usefully extending the operating life of the MALDI and ESI equipment. Furthermore, during field use, the identification and detection of the biological agents, should be carried out in a continuous automated manner with little intervention by human personnel for efficient implementation and operation such as routine monitoring of suspected sites over extended periods of time.
Accordingly, there is a need to develop methods for rapidly identifying biological agents through the automated detection of biomarkers such as proteins released from extracts or whole intact biological agents that may be present in an environmental or biological sample. Such methods would permit protective measures or countermeasures to be quickly implemented in the event of, for example, an attack by weapons employing the same. There is also a need to provide an apparatus that performs the above-mentioned methods as a single operational system capable of routine field, hospital, bioprocessing and manufacturing, and clinical laboratory use.
It would also be desirable to optimize and improve the sensitivity and resolution of the detected ions using ESI-MS and MALDI-MS techniques, thus providing improved reliability in detection and identification. Still further, it would be desirable to provide detection and identification through protein sequencing for greater discrimination and sensitivity. Moreover, the demand for methods and apparatus capable of rapidly identifying biological agents has applications beyond those of the military such as in the pharmaceutical, medical, food and public safety industries, and the like.
SUMMARY OF THE INVENTION
The present invention is generally directed to methods for rapidly identifying biological agents through the automated detection of biomarkers preferably proteinaceous biomarkers released from extracts or whole intact biological agents that may be present in a sample suspected of containing the same, and to an apparatus for conducting the same. The apparatus of the present invention provides means for rapid and automated processing and identification of biological agents. Upon proper identification, a specific therapy or preventative measure may be implemented in respons
Biffoni Ulysses John
Redding David A.
The United States of America as represented by the Secretary of
LandOfFree
Automated sample processing for identification of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Automated sample processing for identification of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Automated sample processing for identification of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3081447