Patent Application
20080025871
The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate specific embodiments of the invention and, together with the general description of the invention given above, and the detailed description of the specific embodiments, serve to explain the principles of the invention.
Referring to the drawings, to the following detailed description, and to incorporated materials, detailed information about the invention is provided including the description of specific embodiments. The detailed description serves to explain the principles of the invention. The invention is susceptible to modifications and alternative forms. The invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
Referring to the drawings and in particular to
The system 10 maintains the liquid slurry of small particles 14, i.e., microspheres or “beads” for use in biological or chemical analysis ready for use. In the system 10, the slurry is kept in the collapsible bag reservoir 11 instead of a rigid container reservoir. The collapsible bag 11 minimizes the interface between the walls of the bag 11, the liquid 12, and air space 15, which then minimizes the loss of particles 14 from sticking to the walls and other agglomeration effects. Agitation of the bag 11 can be used to make the slurry ready for use over an extended period of time without refilling or changing reservoirs.
The existing systems for maintaining liquid slurries of small particles for use in biological or chemical analysis use a rigid reservoir. With the rigid reservoir the beads tend to be lost over time as a film on the walls as the liquid level drops and also lost at the water/air/wall interface even if the liquid level is kept constant. The rigid reservoir requires charging the system with excess beads (which are expensive) and currently requires weekly refilling of the reservoir.
In the system of the present invention, the bag 11 is filled with the liquid 12 and the microspheres or beads 14. The system 10 maintains the liquid slurry of small particles by keeping 10 to 100 mL of bead slurries suspended for periods of one to several weeks while frequently (hourly) withdrawing aliquots for use in biological assays. The bag 11 is similar to an intravenous (IV) bag used to administer fluids in medical applications. The bag 11 is watertight and sealed except for ports (usually at the bottom) where tubing can be attached for filling and withdrawing fluids. The bag 11 is filled with the liquid slurry from a port at the bottom of the bag. A small fraction (e.g., about 5%) of the fill volume can be air, which allows the particles to be kept from settling by shaking or tipping the bag. Other methods of keeping the particles ready for use include squeezing the bag 11, pumping liquid in and out of the bag 11, or agitating the particles with sonic energy.
The collapsible bag 11 minimizes the interface between the container, liquid, and air space (if any), which then minimizes the loss of particles from sticking to the walls and other agglomeration effects. As aliquots of the liquid slurry are withdrawn from the bag 11 for use, there is little change in the air/water/wall contact line and the wetted wall area, and no change in the air volume over the liquid. Particles that might be stuck to the wall are readily recovered by shaking or tipping the bag. Continuous or frequent agitation of the bag keeps the slurry ready for use over an extended period of time. Another advantage of this approach is that no ambient air needs be introduced to the collapsible bag, so the risk of contamination from the environment is eliminated.
The system 10 of the present invention has many uses. For example the system 10 has use in bioterrorism analysis, medical clinical diagnostics, and environmental analysis. The system 10 has use with the Luminex flow cytometer. Since the Luminex platform is a bench-top laboratory instrument the system 10 has wide laboratory, commercial, and industrial use. Ion exchange resin beads and glass beads have similar characteristics and the system 10 has use with ion exchange resin beads and glass beads.
The system 10 has use with Scintillation Proximity Assays (SPA). Scintillation Proximity Assays utilize microscopic beads which contain a scintillant that can be stimulated to emit light. This stimulation event only occurs when radiolabeled molecules of interest are bound to the surface of the bead then blue light is emitted that can be detected on standard scintillation counters. SPA Scintillation beads are microspheres containing scintillant which emit light in the blue region of the visible spectrum. As a result, these beads are ideally suited to use with photomultiplier tube (PMT) counters such as the TopCount or MicroBeta. Two types of core SPA Scintillation bead are available—yttrium silicate (YSi) and Polyvinyltoluene (PVT). A number of biological coatings also exist for each core bead type to enable receptor binding, kinase, molecular interaction and radioimmunoassays to be investigated. The system 10 incorporating Scintillation Proximity Assays has use in Enzme SPA applications that can be used to identify potential new enzyme inhibitors, measure enzyme activity, and perform kinetic analysis. Kinases play pivotal roles in many signal transduction cascades and consequently kinase activities remain a key focus of academic and pharmaceutical research. The SPA beads can be used in the study of tyrosine and serine/threonine kinases using both peptide and protein substrates. Receptor-binding SPAs can be configured to determine receptor kinetics, saturation binding, or to detect inhibitors of radioligand binding. SPA has been successfully applied to receptor-binding assays by immobilizing receptors directly to SPA beads via a number of coupling methods. SPA technology has been successfully applied to the study of a variety of molecular interactions. Protein: protein interactions. Protein: peptide interactions. Protein: DNA interactions Cell adhesion molecule interactions. SPA radioimmunoassay is based on the reaction of antibody-bound ligand with SPA beads coated with either a secondary antibody or Protein A.
Cytostar-T™ Scintillating Microplates have been developed specifically for the study of cell-based assays using SPA technology. Cytostar-T Scintillating Microplates can be utilized in non-invasive and real time analysis of cellular events. DMFK assays are a range of kits for use in drug metabolism and pharmacokinetic studies. DMPK assays provide an economical and higher throughput alternative to conventional assay methodologies such as equilibrium dialysis, HFLC, and ultrafiltration.
Referring to
The embodiment of a system for maintaining liquid slurries of small particles for use in biological or chemical analysis is a system used for the bead reagents in the Autonomous Pathogen Detection System 20, or APDS developed by the Lawrence Livermore National Laboratory. The Autonomous Pathogen Detection System 20 is described in the article, “Reducing the Threat of Biological Weapons” in the June 1998 issue of Science and Technology Review. The disclosure provided in the article, “Reducing the Threat of Biological Weapons” in the June 1998 issue of Science and Technology Review is incorporated herein by this reference. The Autonomous Pathogen Detection System 20 is also described in the article “Detecting Bioaersols When Time is of the Essence” in the October 2004 issue of Science and Technology Review. The disclosure in the article “Detecting Bioaersols When Time is of the Essence” in the October 2004 issue of Science and Technology Review is incorporated herein by reference.
The Lawrence Livermore National Laboratory researchers received seed funding from the Laboratory Directed Research and Development Program to develop an instrument that counters bioterrorism by providing a rapid early warning system for pathogens, such as anthrax. That instrument, the Autonomous Pathogen Detection System (APDS) 20, is now ready for deployment to better protect the public from a bioaerosol attack, and the development team has been honored with a 2004 R&D 100 Award.
As illustrated in
As the Autonomous Pathogen Detection System 20 collects air samples, it first runs them through an immunoassay detector. If that detector returns a positive result, the Autonomous Pathogen Detection System 20 performs a second assay based on nucleic-acid amplification and detection. Having two different assay systems increases system reliability and minimizes the possibility of false positives. In another configuration, the Autonomous Pathogen Detection System 20 can perform the nucleic-acid amplification and detection as the first assay.
The immunoassay detector incorporates liquid arrays, a multiplexed assay that uses small-diameter polystyrene beads (microbeads) coated with thousands of antibodies. Each microbead is colored with a unique combination of red- and orange-emitting dyes. The number of agents that can be detected in a sample is limited only by the number of colored bead sets. When the sample is exposed to the beads, a bioagent, if present, binds to the bead with the appropriate antibody. A second fluorescently labeled antibody is then added to the sample, resulting in a highly fluorescent target for flow analysis. Preparing the sample and performing this first analysis can take less than 30 minutes.
Another way the Autonomous Pathogen Detection System 20 makes use of microbeads is in multiplexed nucleic-acid detection. After the nucleic acid sequences are amplified, the products of this amplification are bound to microbeads and identified by the same flow detector used for the immunoassay.
The Autonomous Pathogen Detection System 20 monitors the air for the three types of biological threat agents: bacteria, viruses, and toxins. Because it operates continuously, the system can detect low concentrations of bioagents that might go undetected by a system that is triggered only when the overall number of particles in the air is high. The Autonomous Pathogen Detection System 20 collects aerosol samples, prepares them for analysis, and tests for multiple biological agents simultaneously. This automation reduces the cost and staffing that would be required to manually analyze samples. The lectern-size Autonomous Pathogen Detection System 20 can be placed in airports, office buildings, performing arts centers, mass transit systems, sporting arenas—anywhere an attack might be launched.
The article, “Autonomous Detection of Aerosolized Bacillus anthracis and Yersinia pestis” by Mary T. McBride, Don Masquelier, Benjamin J. Hindson, Anthony J. Makarewicz, Steve Brown, Keith Burris, Thomas Metz, Richard G. Langlois, Kar Wing Tsang, Ruth Bryan, Doug A. Anderson, Kodumudi S. Venkateswaran, Fred P. Milanovich, and Bill W. Colston, Jr. in Analytical Chemistry, vol. 75, pages 5293-5299 (2003) describes a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The disclosure in the article, “Autonomous Detection of Aerosolized Bacillus anthracis and Yersinia pestis” by Mary T. McBride, Don Masquelier, Benjamin J. Hindson, Anthony J. Makarewicz, Steve Brown, Keith Burris, Thomas Metz, Richard G. Langlois, Kar Wing Tsang, Ruth Bryan, Doug A. Anderson, Kodumudi S. Venkateswaran, Fred P. Milanovich, and Bill W. Colston, Jr. in Analytical Chemistry, vol. 75, pages 5293-5299 (2003) is incorporated herein by reference.
The APDS is completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The APDS uses polystyrene microbeads. The beads are embedded with precise ratios of red and infrared fluorescent dyes yielding an array of 100 different bead classes, where each class has a unique spectral address. The immunoassays employ a sandwich immunoassay format, where antigen-specific capture antibodies are immobilized on the beads, antigen is introduced and allowed to bind the beads, and the bound analyte is subsequently detected using secondary antibodies labeled with the fluorescent reporter, phycoerythrin (PE). Each optically encoded and fluorescently labeled microbead is then interrogated by the flow cytometer. A classification laser (635 nm) excites the dye molecules inside the beads, and a reporter laser (532 nm) excites the fluorescent molecules bound to the bead surfaces. The flow cytometer is capable of reading hundreds of beads per second; analysis is completed in 60 s. Upon completion of the automated immunoassay, the fluidics module dispenses the sample to the flow cytometer for analysis. After analysis, the sample is pumped to waste and the system is flushed in preparation for the next sample.
Referring now to
The bag 30 is filled with a liquid. The microbeads are contained within the bag 30 in the liquid. An air space is located above the surface of the liquid. A system 31 for shaking the bag 30 provides agitation of the contents of the bag 30 to keep the slurry ready for use over an extended period of time without refilling or changing reservoirs. The bag 30 is held by a moveable arm 32. The moveable arm 32 is connected to a moveable frame 33. The moveable frame 33 and movable arm 32 are driven by a motor located in the main housing 34.
The reservoir 25 maintains the liquid slurry of small microspheres or beads ready for use. The beads are small-diameter polystyrene beads (microbeads) coated with thousands of antibodies. Each microbead is colored with a unique combination of red- and orange-emitting dyes. The number of agents that can be detected in a sample is limited only by the number of colored bead sets. When the sample is exposed to the beads, a bioagent, if present, binds to the bead with the appropriate antibody. A second fluorescently labeled antibody is then added to the sample, resulting in a highly fluorescent target for flow analysis. Preparing the sample and performing this first analysis can take less than 30 minutes.
Referring to
As shown, the portable pathogen detection system 40 is a handheld device and comprises a casing or housing which can be held in a human hand, the housing includes the mixing chamber 43 within which bead packs 41 and 42 are located. The mixing chamber 43 is carried by the vibration or mixing unit 44. The analyzer is generally indicated at 45.
Samples are added to the bead packs 41 and 42 containing optically encoded microbeads. Each microbead contains a capture ligand and bioagent-specific antibodies. Each microbead, in addition to the standard sample capture assay, contains special attachment sites. The bead packs 41 and 42 are then placed in the mixing chamber 43.
The portable pathogen detection system 40 and the bead packs 41 and 42 can be the type of systems described and illustrated in U.S. Pat. No. 6,905,885 issued Jan. 14, 2005 to Billy W. Colston, Matthew Everett, Fred P. Milanovich, Steve B. Brown, Kodumudi Vendateswaran, and Jonathan N. Simon for a portable pathogen detection system which describes and illustrates the detection of pathogens and toxins, particularly a highly flexible liquid array that utilizes optically encoded microbeads as the templates for biological assays, and more particularly to a micro-immunoassay (handheld) system wherein target biological samples are optically labeled and captured on microbeads, which are in turn captured on an ordered array or disposable capture substrate and optically read. U.S. Pat. No. 6,905,885 issued Jan. 14, 2005 to Billy W. Colston, Matthew Everett, Fred P. Milanovich, Steve B. Brown, Kodumudi Vendateswaran, and Jonathan N. Simon for a portable pathogen detection system is incorporated herein by reference.
Referring now to
Referring now to
The bag 60 is filled with a liquid 64. The microbeads 63 are contained within the bag 60 in the liquid 64. An air space is located above the surface of the liquid 64. An impeller 65 provides agitation of the contents of the bag 60 to keep the slurry ready for use over an extended period of time without refilling or changing reservoirs. The impeller 65 is driven by a motor 66 through the shaft 67.
While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.
The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.
