Patent Application
20080014657
Specific exemplary embodiments of the present invention will now be described by way of example only and with reference to the accompanying drawings.
a-c depict three schematics of the assay device according to exemplary embodiments of the invention, illustrating three different combinations of possible signals displayed by the detection and control zones.
a-b depict an illustration of a device constructed according to an exemplary embodiment of the invention.
The term “sample” generally refers to anything that may contain a bacterium. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, ocular lens fluid, sweat, milk, ascites fluid, synovial fluid, peritoneal fluid, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid, cervical mucus, vaginal or urethral secretions, mucus, amniotic fluid or the like. Fluid homogenates of cellular tissues such as, for example, hair, skin and nail scrapings, meat extracts and skins of fruits and nuts can also be considered biological fluids.
Biological tissues can be single cells or aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues include organs, tumors, lymph nodes, arteries and individual cell(s).
The term “sample” may also include water, food products, soil extracts, solids, surface swabs or wipes, and the like.
The sample can be used as obtained directly from the source or following a pretreatment so as to modify its character. The sample can be prepared in any convenient medium that does not interfere with the assay.
The sample can be treated prior to its application on the immunoassay device. Methods of treatment can involve, but are not be limited to, filtration, distillation, separation, concentration, inactivation of interfering components, and the like, as well as the addition of reagents. Methods utilized for the selection and pretreatment of biological, industrial, and environmental samples prior to testing are generally known by one of ordinary skill in the art.
The term “labeled receptor binding region,” as used herein, generally refers to a region on an analyte or analog wherein the receptor portion of a labeled receptor is capable of binding.
The term “antigenic determinant,” as used herein, generally refers to an epitope that is recognized by an antibody.
The term “analog,” as used herein, generally refers to a reagent comprising a domain or region that mimics the characteristics of a labeled receptor binding region of an analyte of interest, such labeled receptor binding region being recognized by a particular receptor portion of a labeled receptor utilized in the assay to detect the analyte of interest.
The term “similar,” as used herein, in the context of labeled receptor binding regions and antigenic determinants, generally refers to a region or binding motif on the analyte and analog that comprises sufficient similarity so that a receptor portion of a labeled receptor is capable of binding both the analog and analyte regions.
The term “Group A Streptococcus (GAS),” as used herein, refers to Streptococcus pyogenes, a gram-positive, nonmotile, nonsporeforming coccus that occurs in chains or in pairs of cells. GAS may cause serious infections associated with strep throat, tonsillitis, septicemias, and scarlet fever. The backbone structure of all GAS species comprises a group-specific carbohydrate antigen that contains β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone.
The term “comprehensive control,” as used herein, refers to a control signal that verifies to an operator that the conditions of the assay provide for the necessary chemical and immunological reactions to detect the analyte of interest, if present, in a sample. The control signal may utilize chemical and immunological reactions that mimic the interactions and reactions on which the device is relying on in detecting the presence or amount of the analyte of interest.
The present invention can provide immunoassay devices, kits and methods for displaying a comprehensive control signal when testing a sample for the presence of a bacterium in a sample. To test for the presence of a bacterium in the sample devices of embodiments of the invention may test for the presence of a bacterial carbohydrate analyte of interest known to be contained in the bacterium. The comprehensive control signals utilize analogs to the analyte of interest as control reagents, wherein the analogs may be an immobilized reagent comprising the same or similar labeled receptor binding regions as those of the analyte of interest. A principal advantage of using as a control reagent an analog with the same or similar labeled receptor binding regions to that of the analyte of interest is that the same immunological-type of reactions used in detecting the analyte of interest will be used in generating a control signal. Thus, a positive control signal generated verifies to the operator that the sample has passed through the immunoassay, and that the conditions of the immunoassay support the chemical and immunological reactions that generate accurate test results, such as the integrity of the immunological activity of the labeled receptors.
In particular embodiments, the invention can provide a device that may test a sample for the presence of a bacterium, the device comprising:
a) a receptor moiety attached to a label moiety thereby forming a labeled receptor, wherein the receptor moiety is capable of binding with a bacterial carbohydrate analyte of interest; and
b) a binder support medium comprising a detection zone and a control zone; wherein the detection zone provides for the detection of the analyte of interest, or derivative or homologue thereof, and the control zone is in fluid communication with the detection zone, the control zone comprising, in an immobilized form, an analog of the analyte of interest, wherein the analog comprises a labeled receptor binding region that is the same as or similar to that of the analyte of interest.
In certain embodiments, the device may test a sample for the presence of a GAS bacterium, wherein the analyte of interest is a bacterial carbohydrate found in the GAS bacterium, and the control reagent may be an analog of the analyte of interest. In a particular embodiment, the analyte of interest may be a GAS group-specific carbohydrate antigen that contains β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone, and the control reagent may be Albumin, Bovine, p-Aminophenyl N-Acetyl β-D Glucosaminide (NAGBSA), an analog of β-N-Acetyl-D-Glucosamine.
The detection zone of the invention may be capable of displaying a detectable signal, indicating the presence or amount of a particular analyte of interest in the sample. The detection zone may comprise an immobilized binder, capable of binding with the analyte of interest. The immobilized binder may be a ligand capable of binding an analyte of interest. Alternatively, it may be a ligand capable of binding an analyte-labeled receptor complex.
The control zone of the invention may be capable of displaying a detectable signal, verifying whether or not the sample has passed through the detection zone and reached the control zone, and whether or not the conditions of the immunoassay support the chemical and immunological reactions that generate accurate test results.
In exemplary embodiments, the immunoassay device may further comprise a sample pad, a sump, a neutralizing pad, a support housing, and/or a tracer pad. If the device comprises a tracer pad, the tracer pad may comprise a labeled receptor. If present, the support housing of the invention may comprise apertures that can allow access to the sample pad or tracer pad, where applicable, for the application of the sample. In addition, the housing may comprise apertures to allow visual access to the detection zone and control zone, wherein an operator can analyze the signals displayed in the detector and control zones to determine both the presence or amount of the bacterial carbohydrate in the sample, and whether or not the sample reached the control zone and whether or not the conditions of the immunoassay provided for the chemical and immunological reactions that would generate accurate test results.
The invention is not limited to any particular arrangement of the component parts. For example, a sample pad, neutralizing pad, tracer pad, binder support medium, and sump may be placed in lateral juxtaposition with one another. In such an embodiment, the sample may flow across the device by capillary action. In another example, a sample pad, neutralizing pad, tracer pad, binder support medium, and sump may be placed in vertical juxtaposition with one another, wherein the sample may flow through the device using gravitational forces, capillary forces, or both. These and other arrangements are generally known by one of ordinary skill in the art.
The invention is not limited to the type of analysis. For example, the analysis of the bacterial carbohydrate may be qualitative, semi-quantitative, or quantitative. Qualitative, semi-quantitative, and quantitative assays are known to one of ordinary skill in the art. Semi-quantitative or quantitative analysis of the concentration or amount of a bacterial carbohydrate in a sample requires the use of a calibration mechanism. For example, the calibration mechanism may be a known amount of the bacterial carbohydrate. The calibrated analog may be placed on the binder support medium in a separate zone (calibration zone) from the detection zone, wherein the tracer pad if present, comprises an amount of labeled receptor that is capable of binding to the bacterial carbohydrate in the calibration zone. To determine semi-quantitative or quantitative amounts, the intensity of label in the detection zone is compared to the intensity of label in the calibration zone. Such calibration mechanisms are generally known by one of ordinary skill in the art.
Utilization of the invention can allow for a sample containing or lacking a bacterial carbohydrate analyte of interest to be prepared and applied to the device, interacting with a mobile labeled receptor comprising i) a label moiety capable of detection attached to ii) a receptor, wherein the receptor is capable of binding to a labeled receptor binding region contained in the analyte. In certain embodiments, the receptor utilized in the invention may be an antibody to the analyte of interest, or a derivative or homologue thereof.
When the sample comes into contact with the labeled receptor, the labeled receptor may be bound to the analyte of interest, if present in the sample, and may form a mobile analyte-labeled receptor complex. The sample may then flow to a detection zone and a control zone located on a binder support medium. The detection zone may comprise an immobilized binder that is capable of binding the analyte-labeled receptor complex. In certain embodiments, the immobilized binder utilized in the invention may be an antibody to the analyte of interest, capable of binding the analyte on the same or different antigenic determinant from where the analyte is bound with the labeled receptor. The detection zone may allow for the concentration of the analyte-labeled receptor complex, wherein the label provides an indication of the presence or amount of the analyte.
Labeled receptor that remains unbound in the assay may flow with the sample to the control zone portion of the binder support medium. The control zone may comprise immobilized analog to the analyte. In certain exemplary embodiments, the sample may then flow to a sump pad in fluid communication with the binder support medium, wherein the sump prevents the sample from flowing over the control zone again. The immobilized analog in the control zone may bind with the previously unbound labeled receptors, according to the same labeled receptor binding region as in the formation of the analyte-labeled receptor complexes. The concentration of labeled receptor bound to the analog in the control zone may verify to an operator of the device that the sample has reached the control zone, and the conditions present in the assay allow for the analyte to bind with the labeled receptors. This verification informs the operator that the presence or amount of a signal in the detection zone will correspond with a presence or amount of the analyte of interest in the sample.
One aspect of the invention provides a method of testing a sample for the presence or amount of a bacterium, the method comprising:
a) preparing a sample for application to an assay device;
b) applying the sample to the assay device, wherein the assay device comprises an analyte-specific labeled receptor;
c) binding a bacterial carbohydrate analyte of interest, if present in the sample, to an analyte-specific labeled receptor, forming an analyte-labeled receptor complex, wherein the amount of labeled receptor and or binding kinetics of the assay provide for an amount of unbound labeled receptor remaining in the device;
d) binding the analyte-labeled receptor complex to an immobilized binder capable of binding the analyte-labeled receptor complex in the sample, and incapable of binding the unbound labeled receptor, wherein the immobilized binder is located in a detection zone on a binder support medium;
e) binding the unbound labeled receptor to an immobilized analyte analog, wherein the analyte analog is located in a control zone on a binder support medium, the analog comprising a labeled receptor binding region that is the same as or similar to the labeled receptor binding region of the analyte of interest;
f) determining whether or not conditions in the immunoassay provide for accurate determination of the presence or amount of the analyte in the sample and whether or not the sample passed through the detection zone and control zone by analyzing the control zone for a signal, wherein the presence of a signal verifies proper conditions that will support the binding of the labeled receptors and analyte of interest, if present; and
g) determining the presence or amount of the analyte of interest in the sample by analyzing the detection zone for a signal, wherein a positive signal in the detection zone indicates the presence of the analyte of interest in the sample.
In certain embodiments, the method may determine the presence or amount of GAS bacterium in a sample, the analyte of interest may be a bacterial carbohydrate contained in a GAS bacterium, and the control zone may comprise an immobilized analog of the bacterial carbohydrate. In more particular embodiments, the bacterial carbohydrate may comprise β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone, which is found in GAS bacterium, and the control zone may comprise NAGBSA, an analog of β-N-Acetyl-D-Glucosamine.
In certain embodiments, the method may determine the presence or amount of a GAS bacterium in the sample, and the method may further comprise obtaining a sample, possibly by swabbing the back of a throat that may contain a bacterial carbohydrate found in GAS bacterium. In certain embodiments, the step of preparing the sample may comprise mixing the sample with an acidic or enzymatic reagent, wherein the reaction between the reagent and the sample may release any analyte contained in the sample and enable it to bind with antibodies in the device. Acidic and enzymatic reagents capable of reacting with the sample as described are well-known to those skilled in the art.
In an exemplary embodiment, a sample obtained by means such as swabbing the back of a throat may be prepared by steps comprising admixing 5.0M sodium nitrite with 0.03M citric acid in a container, placing the sample in the container, squeezing the sample to expel as much liquid as possible from the sample, and adding the contents of the container to an assay device according to an exemplary embodiment of the invention.
Referring now to the drawings, in which like numerals represent like elements throughout the several Figures, aspects of the invention and the illustrative operating environment will be described. The Figures, while representative of certain exemplary embodiments of the invention, are not intended to limit the invention in any way.
The receptor 48 portion of the labeled receptor 12 may be capable of specifically binding or complexing with the analyte of interest 6. In certain embodiments, the analyte of interest 6 may be a bacterial carbohydrate that is found in the GAS bacterium. In more particular embodiments, the analyte of interest 6 may be β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone, wherein an antibody specific for that antigen may be used as the receptor 48, or immunologically reactive fragments of the antibody, such as F(ab′)2, Fab or Fab′ may be used as the receptor 48. These receptors 48 coupled to the label 50 may then bind to the analyte 6 if present in the sample 4 as the sample 4 passes through the labeled receptors 12, and form an analyte-labeled receptor complex 22. These analyte-labeled receptor complexes 22 may then be carried into the detection zone 14 on the binder support medium 8 by fluid flow 30 through the device 2. When the analyte-labeled receptor complex 22 reaches the detection zone 14 it may be captured by an immobilized binder 16.
The label 50 of the analyte-specific labeled receptor 12 may be any one of a wide variety of detectable labels known to one of ordinary skill in the art. The label 50 attached to the receptor 48 may be any substance which is capable of detection by visual or instrumental means. Various labels 50 suitable for use in the invention include labels that are detectable through either chemical or physical means. Such labels 50 may include, but are not limited to, enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, and radioactive labels. Other suitable labels 50 include particulate labels such as colloidal metallic particles such as gold, colloidal non-metallic particles such as selenium or tellurium, dyed or colored particles such as a dyed plastic or a stained microorganism, colored organic polymer latex particles and liposomes, colored beads, polymer microcapsules, sacs, erythrocytes, erythrocyte ghosts, or other vesicles containing directly visible substances, and the like.
As shown in
In exemplary embodiments, the detection zone 14 on the binder support medium 8 may comprise an immobilized capture reagent 16, known as a binder, capable of binding the analyte-labeled receptor complex 22, rendering the complex 22 immobile on the medium 8. By “immobilized,” it is generally meant that the binder 16, once on the binder support medium 8, may not be capable of substantial movement to positions elsewhere within the binder support medium 8. Thus, the analyte-labeled receptor complex 22 may be trapped at the detection zone 14 through the binding of the binder 16 to the complex 22, thereby forming a bound analyte-labeled receptor complex 24.
The immobilized binder 16 of certain embodiments of the invention may include any moiety or compound capable of binding the analyte-labeled receptor complex 22, analyte of interest 6, or similar detectable complex contemplated in the type of assay employed. Because one determinant of the analyte 6 may be occupied by the labeled receptor 12 in forming the analyte-labeled receptor complex 22, the binder 16 may consist of any ligand capable of binding to another determinant of the analyte 6. Alternatively, the binder 16 may consist of any ligand capable of binding to the same determinant of the analyte 6 as the labeled receptor 12. Specific binding reagents useful with the invention are known by one of ordinary skill in the art and are generally readily identifiable. In certain embodiments, the device 2 may test for the presence of the GAS bacterium, the analyte of interest 6 may be a bacterial carbohydrate that is found in the GAS bacterium, and the immobilized binder 16 may be an antibody directed at the bacterial carbohydrate. In more particular embodiments, the bacterial carbohydrate may comprise β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone, which is found in GAS bacterium, and the immobilized binder 16 may be an antibody specifically directed at the β-N-Acetyl-D-Glucosamine.
Because the binder support medium 8 of the device 2 is preferably chemically inert, it can be activated at the detection zone 14 where it is desired to immobilize a specific binding reagent 16. Various methods can render the binder reagent 16 immobilized according to the particular chemical nature of the binder support medium 8 material. The immobilized binder 16 may be supported on the binder support medium 8 in a manner which immobilizes the binder 16. The binder 16 can be immobilized to the binder support medium 8 directly or indirectly. Direct attachment methods may include adsorption, absorption and covalent binding such as by use of (i) a cyanogen halide, e.g., cyanogen bromide or (ii) by use of glutaraldehyde. Other methods may include treatment with Schiff bases and borohydride for reduction of aldehydic, carbonyl and amino groups. DNA, RNA and certain antigens may be immobilized against solvent transport by baking onto the binder support medium 8.
Alternatively, it may be preferred to retain or immobilize the binder reagent 16 on the binder support medium 8 material indirectly through the use of insoluble microparticles to which the binder reagent 16 has been attached. The methods of attaching an reagent to the microparticles encompass both covalent and non-covalent mechanisms including adherence, absorption, or adsorption. Microparticles are generally known by one of ordinary skill in the art. For example, microparticles may be selected from any suitable type of particulate material composed or polystyrene, polymethylacrylate, polyacrylamide, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, glass or similar material.
In certain embodiments, the binder 16 may be deposited singly or in various combinations on or in the detection zone 14 of the binder support medium 8 in a variety of configurations to produce different detection or measurement formats. Such configurations and measurement formats are generally known by one of ordinary skill in the art.
Following interaction with the detection zone 14, the sample 4 may flow 30 away from the detection zone 14 to the control zone 18. The control zone 18 may be juxtaposed and in fluid communication with the detection zone 14. The control zone 18 may be capable of verifying to the operator that the sample 4 has flowed 30 to the control zone 18, and thus through the detection zone 14 because the detection zone 14 precedes the control zone 18.
Significantly, the control zone 18 of preferred embodiments of the present invention also may verify that the performance of the assay was within acceptable conditions when used, because the control zone 18 may contain, in immobilized form, an analog 20 to the analyte of interest 6 as a control reagent. The analog 20 may be immobilized using any of the techniques for immobilizing binders 16, as described above. The immobilized analog 20 may react with labeled receptor 12 that did not bind the analyte of interest 6 in the tracer pad 10, thereby forming an analog-labeled receptor complex 26. The label portion of the analog-labeled receptor complex 26 may display a signal capable of being detected visually or with a machine. A signal in the control zone 18 is indicative that the conditions of the assay provide for the binding of the labeled receptors 12 to the analyte of interest 6.
In exemplary embodiments, the analyte of interest 6 may be a GAS group-specific carbohydrate antigen that contains β-N-Acetyl-D-Glucosamine attached to a poly-rhamnose backbone. In these embodiments, the control zone 18 may comprise an analog 20 of β-N-Acetyl-D-Glucosamine, such as NAGBSA, in an immobilized form. The signal generated in the control zone 18 may verify to the operator that the conditions of the assay provide for the binding of the labeled receptors 12 to the β-N-Acetyl-D-Glucosamine. NAGBSA used in the control zone 18 may be obtained from vendors known to those skilled in the art. When supplied from such vendors, the NAGBSA may be lyophilized, which makes it stable when stored at temperatures under 0° C. The NAGBSA may then be hydrated and aliquoted, and then stored at a temperature under 0° C.
In preparation for application of the analog 20 to the control zone 18 of an assay device 2, a suitable stable spotting solution may then be prepared, comprising the analog 20 in a buffer. The solution may then be applied to the control zone 18 of the binder support medium 8 by a suitable spray applicator and dried for an appropriate amount of time.
The type of binder support medium 8 that may be utilized in the invention includes, but is not limited to, substrate materials having capillarity and the capacity for chromatographic solvent transport of non-immobilized reagents and reactive sample components by means of a selected chromatographic solvent. The binder support medium 8 of the assay device 2 of the invention may be any suitably absorbent, porous or capillary possessing material through which a sample 4 containing the analyte of interest 6 may be transported by a wicking action.
In certain embodiments, as shown in
In one exemplary embodiment, the size of the upper housing 44 and lower housing 46 may allow for convenient handling and packaging of the device 2. The upper housing 44 and lower housing 46 of the invention may be made of plastic, glass or other suitably rigid material. The upper housing 44 and lower housing 46 may serve other functions as well, including providing a handle or displaying information such as bar codes, fluorescent marks, or colored marks that may aid in the calibration of the assay. The upper housing 44 and lower housing 46 material may be in sheet or roll form, and may be manufactured from an opaque plastic sheet material of appropriate color, thickness, and rigidity.
a-c illustrate three exemplary views of the invention in an exemplary embodiment. Each figure depicts a view of the upper housing 44 of the device 2, after sufficient time has elapsed following the application 28 of the sample 4 to the device 2 through the aperture 38 with access to the tracer pad 10.
b shows no signal generated in the detection zone 14 that can be seen through the corresponding aperture 40, but shows a signal generated in the control zone 18. Where, as in
c shows no signal generated in the detection zone 14 nor in the control zone 18 that can be seen through the corresponding apertures 40, 42. Where, as in
In step 100, initiate the process by preparing a sample and applying it to the optional sample pad of the assay device. In step 200, the sample pad receives the sample applied thereto, and the sample moves through lateral flow to an optional tracer pad. In step 300, the tracer pad receives the sample from the sample pad. In step 400, attach the labeled receptor capable of binding to an analyte of interest to the analyte, if present in the sample, to form an analyte-labeled receptor complex. In step 500, move the analyte-labeled receptor complex and unbound labeled receptor to the detection zone of the binder support medium through lateral flow from the tracer pad, wherein the detection zone is juxtaposed and in fluid communication with the tracer pad. In step 600, capture the analyte-labeled receptor complex with the immobilized binder of the detection zone. In certain embodiments, a portion of the analyte-labeled receptor may not bind with the immobilized binder of the detection zone. In such embodiments, these unbound complexes may move towards the control zone, along with the sample and unbound labeled receptor. In step 700, move the sample and unbound labeled receptor to the control zone of the binder support medium through flow from the detection zone, wherein the control zone is juxtaposed and in fluid communication with the detection zone. In step 800, capture the unbound labeled receptor with immobilized analog in the control zone. In step 900, display visual indicators with the labeled receptors now immobilized in the detection zone and control zone. In step 1000, analyze visual indicator in the control zone to determine if the sample has reached the detection and control zones, and whether or not the conditions of the immunoassay support the chemical and immunological reactions needed to generate accurate results. If the appropriate signal is displayed, analyze visual indicator in the detection zone to determine the presence or amount of analyte of interest in the sample.
The process allows the operator to determine if the sample has reached the detection and control zones of the device, whether or not the conditions are appropriate for generating accurate test results, and whether or not the sample contains an analyte of interest. Analyte-labeled receptor complexes that are captured by the immobilized binder may be capable of detection, either visually by the eye of the operator or via a measuring device. After an appropriate time, the operator may analyze the control zone visually or with the aid of a detection device to ascertain whether or not the assay has been completed, and then may ascertain the result of the assay by observing the detection zone. Certain steps in the method described above must naturally precede others for the invention to function as described. However, the invention is not limited to the order of the steps described if such order or sequence does not alter the functionality of the invention. That is, it is recognized that some steps may be performed before or after or in parallel with other steps without departing from the scope and spirit of the invention.
Kits
The invention further provides kits for carrying out immunoassays utilizing the device as described herein. In one embodiment, a kit according to the invention may comprise the assay device with its incorporated reagents as well as a wicking solution and/or test sample pretreatment reagents. Other assay components known to one of ordinary skill in the art, such as buffers, stabilizers, detergents, bacteria inhibiting agents and the like may also be present in the kit. In addition, the kit may contain packaging materials and directions on how to use the device.
It should be understood that the foregoing, as well as the example below, relate only to illustrate the embodiments of the invention, and that numerous changes may be made therein without departing from the scope and spirit of the invention as defined by the following claims.
The following example illustrates, but is not intended to limit the invention.
a. Construction of Immunoassay Device
a-b illustrate representations of a lateral flow GAS immunoassay device constructed according to the invention. As shown in
The detection zone 14 comprised a line of antibody directed against GAS antigen in a suitable buffer. The control zone 18 comprised a line of NAGBSA (Sigma A1034 or equivalent) in a suitable buffer. These lines were sprayed down by an applicator so that they were immobilized onto the nitrocellulose binder support medium 8.
b. Detection of GAS Antigen Under Varying Conditions
Three drops of extracted sample containing GAS antigen were applied to the sample pad via a dispense tube tip to the sample opening of the top of a device constructed as described above in Part (a). The sample was neutralized in the sample pad of the device. After neutralization the conditions were favorable to allow for binding of the GAS antigen and an antibody to the GAS antigen. The assays were then allowed to proceed. Signals in the control and detection zones were then visualized in the device through an aperture in the top of the device.
Three drops of an extracted sample not containing GAS antigen were applied to the sample pad via a dispense tube tip to the sample opening of the top of a second device constructed as described above in Part (a). The sample was neutralized in the sample pad of the device. After neutralization the conditions were favorable to allow for binding of the GAS antigen and an antibody to the GAS antigen. The assays were then allowed to proceed. Only a signal in the control zone was visualized in the device through an aperture in the top of the device.
Three drops of extracted sample containing GAS antigen were applied to the sample pad via a dispense tube tip to the sample opening of the top of a third device constructed as described above in Part (a). The sample was neutralized in the sample pad of the device. The assays were then allowed to proceed. Signals were not visualized in either the control or detection zone, indicating that either the sample did not reach the detection and control zone or that the conditions did not allow binding of the antigen or antibody to the detection or control zones. Because no signal was observed in the control zone, the lack of signal in the detection zone was deemed unreliable.
| Number | Date | Country | |
|---|---|---|---|
| 60807089 | Jul 2006 | US |
