Patent Application
20190195865
Embodiments of the invention relate generally to assay systems and examples include methods, systems, and apparatus for conducting assays, including the detection and/or quantification of active toxin such as but not limited to Ricin toxin, Shiga-like toxins (SLT), and Staphylococcal Enterotoxin B (SEB).
Ricin, Shiga-like toxins (SLT) and Staphylococcal enterotoxin B (SEB) have either been used as bioterrorism agents or are considered a bioterrorism threat because of their extreme toxicity and ease of administration. These toxins can be easily administered by inhalation, injection or ingestion. In the event of a mass exposure to biological toxins, identification of the agent in question is important for accurate diagnostic assessment of affected patients. It may also, however, be important to determine the fraction of the toxin which is still active; for example, if a significant fraction of the toxin is inactive, a treatment may not be as aggressive as it would be when a large fraction of the toxin is active. Distinguishing between active and inactive toxin may be advantageous because of the possibility that genetically engineered toxins, including the enzymatic portion of the toxin and a binding domain of another protein, can be used as a bioweapon agent, and may not be captured by traditional qualitative toxin detection tests.
Ricin is a highly toxic protein produced by Ricinus communis or castor bean plant. It is a category B agent, under the Biological Select Agents or Toxins, as defined by the United States Department of Human and Health Services. The major symptoms of ricin poisoning depend on the route of exposure and the dose received, though many organs may be affected in severe cases. The likely symptoms of Ricin inhalation include respiratory distress (difficulty breathing), fever, cough, nausea, and tightness in the chest. Finally, low blood pressure and respiratory failure may occur, leading to death. Swallowing of Ricin would likely lead to vomiting and diarrhea. Severe dehydration may also result. Other signs or symptoms may include seizures, and blood in the urine. Within several days, the person's liver, spleen, and kidneys might stop working, and the person could die. Ricin is unlikely to be absorbed through normal skin. Death from ricin poisoning could take place within 36 to 72 hours of exposure, depending on the route of exposure (inhalation, ingestion, or injection) and the dose received.
Shiga-like toxins (SLTs) are a class of toxins produced by pathogenic Escherichia coli strains. They cause hemolytic uremic syndrome in humans, which may lead to death.
Currently, there are no portable quantitative activity assays available for determining activity of Ricin and Shiga-like toxins. The mechanism of action of these toxins generally does not cause a break in nucleic acid phosphodiester backbone, making it difficult to determine activity. Accordingly, available assays are qualitative in nature, only determining presence or absence of these toxins in a sample, without giving any information regarding their activity. Further, the few quantitative assays that are available include tedious processes and steps. For instance, cell-free translation assays for determining activity of Ricin toxin require cell-extracts that provide transcriptional and translational molecular machinery including RNA polymerases for mRNA transcription, ribosomes for polypeptide translation, tRNA and amino acids, enzymatic cofactors and energy source, and cellular components essential for protein folding, while cytotoxicity assays for determining biological activity require bacterial or tissue culture cell. Alternatively, mass-spectrometry may be used for detecting free adenine released in a sample after Ricin attack on ribosomes, or HPLC-ESI-MS is used for detecting Ricinine, a marker of Ricin. The processes involving mass-spectrometry suffer from background noise and reduced sensitivity due to presence of interfering components in a sample; not to mention cumbersome equipment that is not portable.
Not only are these processes labor-intensive, they are also time-intensive. For instance, the rapid detection tests used by Center for Disease Control and Prevention's Laboratory Response Network take 6-8 hours, while the toxin activity tests take about 48 hours. Although there have been reports of new detection assays that only take 1-2 hours, these assays are only qualitative in nature and do not give any information on activity of the toxin. Staphylococcal enterotoxin B (SEB) is an enterotoxin produced by the bacterium Staphylococcus aureus. It is a common cause of food poisoning, with severe diarrhea, nausea and intestinal cramping often starting within a few hours of ingestion. SEB is classified as an incapacitating agent because in most cases aerosol exposure does not result in death but in a temporary, though profoundly incapacitating, illness lasting as long as 2 weeks. SEB is a superantigen, which causes massive nonspecific activation of immune system causing release of large amounts of cytokines that lead to significant inflammation.
Currently, there are no assays available for determining activity of SEB. The assays available, such as enzyme-linked immunosorbent assays (ELISA), chemiluminescence (ECL), and polymerase chain reaction (PCR), are quantitative in nature and only aid in detection of SEB.
Microfluidic systems, including “lab on a chip” or “lab on a disk” systems continue to be in development. See, Lee, B. S., et. al., “A fully automated immunoassay from whole blood on a disc,” Lab Chip 9, 1548-1555 (2009) and Madou, M. et. al., “Lab on a CD,” Annu. Rev. Biomed. Engr. 8, 601-628 (2006), which articles are hereby incorporated by reference in their entirety for any purpose.
Certain details are set forth below to provide a sufficient understanding of embodiments of the invention. However, it will be clear to one skilled in the art that embodiments of the invention may be practiced without various of these particular details. In some instances, well-known chemical structures, chemical components, molecules, materials, microfluidic components, electronic components, electronic circuits, control signals, timing protocols, and software operations have not been shown in detail in order to avoid unnecessarily obscuring the described embodiments of the invention.
Embodiments of the present invention include systems, apparatuses, and methods for detecting and/or quantifying Ricin, SLTs and SEB toxin in a sample. As mentioned above, existing methods for detecting these toxins are generally either limited to only qualitative detection, and not activity determination, or are cumbersome and time-intensive. Examples according to the current invention include analysis of target analytes including toxins such as but not limited to Ricin, SLTs or SEB. References will be made herein to and examples given of applications targeting Ricin, SLTs or SEB, but it should be understood that in other examples, other toxins may also be targeted for detection and/or quantification. In examples according to the present invention, the presence of active toxin may be detected and/or quantified. Active toxin generally refers to toxin that is able to act on its designated target or targets. Active toxin generally does not include toxin which may be present but, for whatever reason, is unable to act (e.g. cleave or bind) on its designated target or targets.
Assays described herein may be conducted using systems and devices that utilize sedimentation to perform assays. For example, co-pending U.S. application Ser. No. 12/891,977, filed Sep. 28, 2010, entitled “Devices, systems, and methods for conducting sandwich assays using sedimentation,” is hereby incorporated by reference in its entirety for any purpose. The aforementioned application includes examples of the formation of complexes including a capture agent, target analyte, and labeling agent on sedimentation particles. Thus, target analytes may be separated from sample by affinity with a capture agent, and the sedimentation particles passed through a density medium to pellet out at a detection region of a microfluidic disk. Examples of systems and devices described in the aforementioned application may be utilized to conduct assays described herein.
Examples of the present invention include methods, systems, and devices, for performing assays to detect ricin, shiga-like toxins, or both. Ricin generally acts by inhibiting protein synthesis. It is approximately 64 kDa in size and is composed of two chains joined by a single disulfide bond. The A chain is generally responsible for Ricin's catalytic enzymatic activity, while the B chain is generally responsible for binding to cell surface receptors on the cell membrane and facilitating entry of the toxin in to the cytosol. Ricin generally inhibits protein synthesis by inactivating ribosomes by attacking the sarcin-ricin loop. This loop is a highly conserved sequence of nucleotides generally found in the 28S RNA of the large subunit of ribosomes. It has a conformation of a loop and is generally cleaved by both α-sarcin and ricin. Ricin generally acts by specifically and irreversibly hydrolyzing the N-glycosidic bond of the single adenine residue at position 4324 (A4324) in the sarcin-ricin loop within the 28S rRNA, releasing the base, but leaving the phosphodiester backbone of the RNA intact. Inactive ricin may be unable to perform this action. The process of attacking and releasing A4324 is generally referred to as depurination. The sarcin-ricin loop may be important in binding elongation factors during protein synthesis; however, depurination by Ricin may prevent this binding causing inactivation of ribosome, resulting in toxicity by inhibition of protein synthesis.
Shiga-like toxins have a similar mechanism of action as Ricin; they also generally act by depurinating A4324 and thereby inhibiting protein synthesis. Since both ricin and shiga-like toxins act by similar mechanisms, generally the same activity assays may be utilized for both Ricin and SLTs. Further, there may be other toxins that also act by attacking highly conserved sequences of different conformations. The same activity assays may also be utilized for such toxins.
In an example of an activity assay suitable for detection of active ricin, SLTs or combinations thereof, depurination caused by the toxins may be used to determine their activity. Generally, a labeled enzyme may be used to bind to a site created by an active toxin attack. For example, a fluorescently-labeled human Apurinic/apyrimidinic (AP) endonuclease (APE1) enzyme may be used to bind to the abasic site (i.e., a site without a base, for example, without A4324) left after an attack by the toxins to detect depurination, hence the activity of the toxin. The enzyme may generally be labeled with quantum dots (e.g. QDot 585 from Molecular Probes), alexa fluor 647 or 10 nm latex particles loaded with dye. Typically, APEI participates in the DNA base excision repair (BER) pathway by nicking the phosphodiester backbone at an AP site (a site without a base, for example, without A4324) through acyl substitution after a DNA glycosylase removes a damaged or inappropriate base. This process is mediated by Mg2+ ions, which stabilize the AP site and cause release of APE1 from the site to allow for other enzymes to continue with the BER pathway. In ricin, SLT, or both, activity assays, binding of fluorescently labeled APE1 is desired while preventing enzyme turnover. This is achieved, for example, by depriving APE1 of Mg2+ ions. Thus, APE1 binds to the damaged DNA substrate without acting to complete the repair, leading to detection of depurination caused by Ricin/SLTs activity. Another advantage of using APE1 in detection assays is its higher affinity for an abasic site. Unlike other enzymes that may recognize an abasic site, APE1 is involved in the BER pathway, which makes it a very strong candidate for detection of depurination.
In block 103, complexes including the target analyte (e.g., a toxin), and labeling agent may be formed on beads in a fluid sample. Any sedimentation particles with appropriate surface properties, including beads, may be used, including but not limited to, polystyrene beads, silica beads or poly(methyl methacrylate) (PMMA) beads. Substantially any bead radii may be used. Examples of beads may include beads having a radius from 150 nanometers to 10 microns.
In Ricin/SLTs activity assays described herein, the beads used to implement the method described with reference to
In
In some examples, it may be of interest to directly measure active toxin levels in whole blood samples.
Generally, blood cells may have a density less than or equal to 1.095 g/cc, and the silica beads may have a density of about 2.05 g/cc. Accordingly, the density media 510 may have a density of between about 1.095 g/cc and 2.05 g/cc. In one example, the density media 510 has a density of 1.11 g/cc.
Sedimentation described herein may occur under the influence of a natural gravitational force, such as by allowing the assay to sit, unpowered, under the influence of a gravitational force. Sedimentation may also occur using centrifugal force, such as by spinning a microfluidic disk. For examples, silica beads on the order of 10-30 microns in diameter may sediment in minutes under a normal gravitational force. Following sedimentation, as shown in
Ricin and/or SLT activity assays conducted in accordance with embodiments of the present invention may accordingly separate interfering matrix components from the active Ricin and/or SLT in complexes. For example, inactive Ricin and/or SLT or other components in a sample may not cause depurination of the DNA (or RNA) substrate covalently linked to surface of silica beads. The lack of depurination may prevent attachment of fluorescently labeled APE1 to the substrate, and, thus, formation of substrate-label complexes. Other components and inactive Ricin and/or SLT may not cause formation of complexes, and may not travel through the density media due to the density of the particle being less than the media. Accordingly, sensitivity of determination of active Ricin and/or SLT may be improved through use of sedimentation assays described herein. The sensitivity of detection and/or quantification may also be improved relative to standard techniques by determination and/or quantification of the active Ricin and/or SLT in the sample, concentration in a pellet, and/or enhancement of fluorescent signal during transport through the density medium. Still further Ricin and/or SLT assays in accordance with the present invention may be performed in a relatively short period of time.
Examples of the present invention include assays for detection and/or quantification of active Staphylococcal enterotoxin B (SEB). SEB is a protein toxin and generally functions as a superantigen. SEB may be responsible for a number of extensive pathophysiological changes in humans and mammals and may trigger an excessive cellular immune response leading to toxic shock. SEB may be called a superantigen because it interacts with the immune system to produce an excessive response, activate a very high percentage of T-cells, which may lead to toxic shock. It is approximately 24-29 kDa in size. It causes non-specific cross linking of major histocompatibility complex (MHC) II and T cell receptors (TCR). This may cause rapid proliferation of T-cells and production of cytokines, which then lead to significant inflammation. Exposure to SEB may cause severe diarrhea, vomiting, cramping, inflammation of skin, eye, fever, headache, and even toxic shock.
In an example of SEB activity assay described herein, crosslinking of MHC II and T-cell receptors by SEB is used to determine its activity. For example, an immortalized T-cell line, such as Jurkat cells, which express T-cell receptors on the cell surface may be used to cross-link with MHC II via SEB. In one embodiment, fixed Jurkat cells may be used, making them readily available and precluding maintenance of live cell cultures prior to the assay. In one embodiment, the Jurkat cells used may be stained by acrinidine orange. The MHC II may be conjugated to 1 μm silica microparticles. In presence of SEB a complex may form between the Jurkat cells and MHC II conjugated to silica microparticles, through SEB. These complexes may be separated from unbound Jurkat cells and MHC II-silica microparticles conjugates by using a density media denser than the Jurkat cells.
In an example of a SEB activity sedimentation assay, when Jurkat cells and silica microparticles conjugated to MHC II are placed together in the presence of SEB, the dense microparticles may act as a sink, and drive the Jurkat cells down through the density media by sedimentation force (e.g. gravitational or centrifugal force). In the absence of active SEB, the Jurkat cells may stay above the density media.
SEB activity assays conducted in accordance with embodiments of the present invention may accordingly separate interfering matrix components from the active SEB the in complexes. For example, inactive SEB or other components in a sample may not cause cross-linking of cells expressing TCR and MHC II conjugated to silica beads. The lack of cross-linking will prevent attachment of stained cells to the substrate, and, thus, formation of substrate-label complexes. Other components and inactive SEB may not cause formation of complexes, and may not travel through the density media due to the density of the interfering components being less than the media. Accordingly, sensitivity of determination of active SEB may be improved. The sensitivity of detection and/or quantification may also be improved relative to standard techniques by determination of only the active SEB in the sample, concentration in a pellet, and/or enhancement of fluorescent signal during transport through the density medium. Still further SEB assays in accordance with the present invention may be performed in a relatively short period of time.
In some embodiments, methods described herein may be implemented using a microfluidic disk.
The substrate 610 may be implemented using any of a variety of suitable substrate materials. In some embodiments, the substrate may be a solid transparent material. Transparent plastics, quartz, glass, fused-silica, PDMS, PMMA and other transparent substrates may be desired in some embodiments to allow optical observation of sample within the channels and chambers of the disk 600. In some embodiments, however, opaque plastic, metal or semiconductor substrates may be used. In some embodiments, multiple materials may be used to implement the substrate 610. The substrate 610 may include surface treatments or other coatings, which may in some embodiments enhance compatibility with fluids placed on the substrate 610. In some embodiments surface treatments or other coatings may be provided to control fluid interaction with the substrate 610. While shown as a round disk in
In some embodiments, as will be described further below, the substrate 610 may itself be coupled to a motor for rotation. In some embodiments, the substrate may be mounted on another substrate or base for rotation. For example, a microfluidic chip fabricated at least partially in a substrate may be mounted on another substrate for spinning. In some examples, the microfluidic chip may be disposable while the substrate or base it is mounted on may be reusable. In some examples, the entire disk may be disposable. In some examples, a disposable cartridge including one or more microfluidic channels may be inserted into disk or other mechanical rotor that forms part of a detection system.
The substrate 610 may generally at least partially define a variety of fluidic features. The fluidic features may be microfluidic features. Generally, microfluidic, as used herein, refers to a system, device, or feature having a dimension of around 1 mm or less and suitable for at least partially containing a fluid. In some embodiments, 500 μm or less. In some embodiments, the microfluidic features may have a dimension of around 100 μm or less. Other dimensions may be used. The substrate 410 may define one or more fluidic features, including any number of channels, chambers, inlet/outlet ports, or other features.
Microscale fabrication techniques, generally known in the art, may be utilized to fabricate the microfluidic disk 600. The microscale fabrication techniques employed to fabricate the disk 600 may include, for example, embossing, etching, injection molding, surface treatments, photolithography, bonding and other techniques.
A fluid inlet port 625 may be provided to receive a fluid (e.g. sample) that may be analyzed using the microfluidic disk 600. The fluid inlet port 625 may have generally any configuration, and a fluid sample may enter the fluid inlet port 625 utilizing substantially any fluid transport mechanism, including pipetting, pumping, or capillary action. The fluid inlet port 425 may take substantially any shape. Generally, the fluid inlet port 625 is in fluid communication with at least one assay area, and may be in fluid communication with multiple assay areas 620-623 in
The assay area 620 generally may include one or more channels in fluid communication with the fluid inlet port 625. Although four assay areas 620-623 are shown in
As the microfluidic disk 600 is rotated in the direction indicated by the arrow 635 (or in the opposite direction), a centrifugal force may be generated. The centrifugal force may generally transport fluid from the inlet port 625 into one or more of the assay areas 620-623.
Accordingly, the microfluidic disk 600 may be used to perform assays described herein. Centrifugal forces may be used to generate sedimentation forces described herein. In other examples, however, gravity may be used to generate sedimentation forces, and assays described herein may be conducted in a vial or other container.
The detection region 730 and reservoir 735 may generally be implemented using any size and shape, and may contain one or more reagents including solids and/or fluids which may interact with fluid entering and/or exiting the features.
The detection region 730 may be configured to contain a density media. Constituents of an appropriate density media are explained previously. In some embodiments, the density media may include a detergent, such as Tween 20. The detergent may enhance a wash function of transport through the density media.
Sample, sedimentation particles, and labeling agent may first be mixed and/or incubated in the reservoir 735, as shown in
On opening the valve 740, as shown in
The detection region 730 may be a channel or chamber and may vary in configuration in accordance with the detection technique employed, as will be described further below. The detection region 730 may generally be configured to allow for detection of a signal emitted by labeling agents in a complex. The complex may include active toxin and labeling agent in embodiments pertaining to toxin activity assays. In some examples, the complex may not itself include active toxin, but the complex may have formed due in part to the presence of active toxin in a sample. The complexes include sedimentation particles (e.g. microparticles, beads).
Centrifugal forces may generally be used to transport the mixture from the inlet port 725 toward the detection region 730. Additionally, centrifugal forces may be used to transport density media from the reservoir 735 to the detection region 730. In other examples, pressure-driven or other flow drivers may be used to load fluids into the device and transport the mixture to the detection region 730.
Incubation of sedimentation particles and labeling agents with the sample may take place within a microfluidic disk. Referring again to
The detection region 730 of
Once the mixture of sample, bead, and labeling agent is in the detection region 730 above the density media (e.g. closer to the center of the disk), sedimentation forces may be used to transport the beads through the density media to form a bead pellet, as shown in
Accordingly, a sample fluid including: 1) beads; 2) target analytes; and 3) labeling agents may be transported to an interface with a density media and sedimentation forces used to transport the beads, along with any bound complexes, through the density media to form a pellet at the end of the detection region.
The sample fluid may then be incubated.
The beads may then be transported through the density media. The beads are transported through the density media using centrifugal force, such as that which may be applied by motor, described further below. Following a period of centrifugal force, the beads may be concentrated in a detection location.
The motor 1101 may be implemented using a centrifugation and/or stepper motor. The motor 1101 may be positioned relative to the detection module 1102 such that, when the disk 600 is situated on the motor 1101, the disk is positioned such that a detection region of the assay area 620 is exposed to the detection module 1102.
The detection module 1102 may include a detector suitable for detecting signal from labeling agents in complexes that may include labeling agent. In toxin activity assays (e.g., Ricin, SLT, and/or SEB) assays, the complexes may include active toxin and labeling agent or may form in response to the presence of an active toxin and/or label agent designed to be incorporated into complexes responsive to the presence of active toxin. The complexes may be formed on the surface of one or more sedimentation particles (e.g., beads), as described further below. The detector may include, for example, a laser and optics suitable for optical detection of fluorescence from fluorescent labeling agents. The detection module may include one or more photomultiplier tubes. In other examples, other detectors, such as photodiodes or CCD cameras, may be used. The actuator 1101 may move the detector in some examples where signal may be detected from a variety of locations of the microfluidic disk 600.
The processing device 1104 may include one or more processing units, such as one or more processors. In some examples, the processing device 1104 may include a controller, logic circuitry, and/or software for performing functionalities described herein. The processing device 1104 may be coupled to one or more memories, input devices, and/or output devices including, but not limited to, disk drives, keyboards, mice, and displays. The processing device may provide control signals to the motor 1101 to rotate the disk 600 at selected speeds for selected times. The processing device may provide control signals to the detection module 1102, including one or more detectors and/or actuators, to detect signals from the labeling agents and/or move the detector to particular locations. The processing device may develop these control signals in accordance with input from an operator and/or in accordance with software including instructions encoded in one or more memories, where the instructions, when executed by one or more processing units, may cause the processing device to output a predetermined sequence of control signals. The processing device 1104 may receive electronic signals from the detection module 910 indicative of the detected signal from labeling agents. The processing device 1104 may detect a target analyte and/or calculate a quantity of a target analyte in a fluid sample based on the signals received from the detection module 1102. Accordingly, the processing device 1104 may perform calculations in accordance with software including one or more executable instructions stored on a memory causing the processing device to perform the calculations. Results may be stored in memory, communicated over a network, and/or displayed. It is to be understood that the configuration of the processing device 1104 and related components is quite flexible, and any of a variety of computing systems may be used including server systems, desktops, laptops, controllers, and the like.
Having described examples of micro fluidic disks and systems in accordance with embodiments of the present invention, methods for conducting assays will now be described. Some discussion will also be provided regarding mechanisms for sedimentation and centrifugation. The discussion regarding mechanisms is provided as an aid to understanding examples of the present invention, but is in no way intended to limit embodiments of the present invention. That is, embodiments of the present invention may not employ the described mechanism.
Sedimentation of spheres may occur within a viscous fluid under the influence of a gravitational field (which may be natural or induced by centrifugation). The settling velocity of approximately spherical particles may be described by Stoke's flow equations:
where Vs is the sedimentation velocity, μ is the fluid viscosity, ρρ is the density of the particle, ρf is the density of the fluid, g is acceleration due to effective gravity, and R is the particle radius. Note that sedimentation rate scales with the square of particle radius and therefore a small difference in radius may form a basis for separation of particles in some examples, as they may sediment at a different rate. There is also a linear dependence of sedimentation rate with the difference in density between the particle and the surrounding fluid, which may also be an effective mechanism for separation. Accordingly, beads or other particles may be separated according to their density and/or radius based on different sedimentation velocities. Separation of particles using these principles may be referred to as “rate zonal centrifugation.”
For nanometer scale particles, such as active toxins, gravitational forces may act in conjunction with Brownian diffusion, but neither will generally cause motion of these nanometer scale particles over significant distances during typical centrifugal conditions (<100,000 g). Accordingly, beads with affinity for active toxins, due to surface modifications, may be used to separate active toxins from a fluid sample containing mixture of other small molecules. By capturing active toxin on the bead surface, and separating the beads from the remaining sample using sedimentation (e.g. centrifugal or gravitational) forces, the need for wash steps may be reduced or eliminated, because unbound labeling agents and/or other molecules may be dissociated from the beads by fluid flow.
For ricin and/or shiga-like toxin activity assay, a sample may be incubated with microparticle-conjugated stem-loop substrates for 20 or 240 min in 10 mM potassium citrate at pH 4.0. To this suspension, fluorescently labeled APE1 may be added to a final concentration of 100 nM, as well as PBS (pH 7.4) with between 50 mM EDTA and 250 mM EDTA. The fluorescent label may be an organic dye, such as Alexa Fluor 647 from Molecular Probes, a quantum dot, such as QDot 585 from Molecular Probes, dye-loaded latex particles, such as 10 nm FluoSpheres from Life Technologies. The suspension may be allowed to react for 20 min. After incubation, 4 μL of the suspension may be added to a channel pre-loaded with density medium. Density medium may be 95% Percoll and 5% (v/v) concentrate of additives in distilled water containing 50 mM sodium phosphate, 125 mM EDTA, 0.01% Tween 20 (w/v) and 100 mM sodium chloride. The channel may be spun at 8000 rpm for 45 s, transporting the microparticles through the density medium to form a pellet in a detection area. The pellet may be imaged using a fluorescent microscope.
For SEB activity assay, MHC II may be immobilized to 1 μm carboxylic acid functionalized microparticles. Jurkat cells may be fixed in ice-cold methanol for 5 min, washed in PBS, and stained with 100 μM acrinidine orange for 10 min. Following which the cells may be washed in PBS and resuspended to a concentration of 3×107 cells/mL. To 7 μL of a 5% solid suspension of MHC II-conjugated microparticles, 7 μL of SEB toxin and 1 μL of stained Jurkat cells may be added.
Hela cells may be used as a negative control. HeLa cells may be treated with trypsin and EDTA for 5 min at 37° C., washed with PBS, fixed in ice-cold methanol for 5 min, stained with 100 100 μM acrinidine orange for 10 min, and resuspended at a concentration of 3.8×107 cell/mL. To 7 μL of a 5% solids suspension of MHC II-conjugated microparticles 7 μL of SEB toxin and 1 μL of HeLa cells may be added.
The channel may be spun at 8000 rpm for 45 s, transporting the microparticles through the density medium to form a pellet in a detection area. Resultant bead pellets (for both Jurkat cells and, negative control, HeLA cells) may be imaged on a fluorescent microscope using 488 nm excitation and 525 nm emission.
This application is a divisional application of, and discloses subject matter that is related to subject matters disclosed in, co-pending parent application U.S. Ser. No. 14/157,278, filed Jan. 16, 2014 and entitled “TOXIN ACTIVITY ASSAYS, DEVICES, METHODS AND SYSTEMS THEREFOR” which claimed benefit under 35 U.S.C. 119(e) to U.S. provisional patent application Ser. No. 61/759,486, entitled “METHOD FOR DETECTION OF TOXIN ACTIVITY IN MICROFLIUDIC DISKS” filed Feb. 1, 2013. The present application claims the priority of its parent application, which is incorporated herein by reference in its entirety for any purpose.
This invention was developed under Contract DE-AC04-94AL85000 between Sandia Corporation and the U.S. Department of Energy. The U.S. Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 20160178619 A1 | Jun 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61759486 | Feb 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14157278 | Jan 2014 | US |
| Child | 15056870 | US |
