Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks

Abstract

Devices and methods are provided for the detection of CSF in a biological sample. In certain embodiments the device is a lateral flow device comprising: a porous substrate; a sample addition zone disposed on or in said porous substrate; a detection zone disposed on or in said porous substrate where said detection zone comprises at least a first test line (T1) and a second test line (T2) each test line comprising binding moieties that bind a complex formed between beta-trace protein (betaTP) and an indicator attached to a betaTP binding molecule; wherein said porous substrate defines a flow path through which a sample applied to the sample addition zone flows under capillary action away from said sample addition zone into said detection zone.

Claims

1. A smartphone-based lateral flow assay (LFA) reader configured to read test lines on a lateral flow device for detecting a cerebrospinal fluid leak, where said lateral flow device comprises: a porous substrate; a sample addition zone disposed on or in said porous substrate; a detection zone disposed on or in said porous substrate where said detection zone comprises at least a first test line (T1) and a second test line (T2) each test line comprising binding moieties that bind a complex formed between beta-trace protein (βTP) and an indicator attached to a βTP binding molecule; wherein said porous substrate defines a flow path through which a sample applied to the sample addition zone flows under capillary action away from said sample addition zone into said detection zone; and wherein: said first test line (T1) and said second test line (T2) are configured so that either no test line signal or just a signal at the first test line (T1) is detectable when βTP concentration in a sample applied to said device is lower than the βTP level indicative of a cerebrospinal fluid (CSF) leak; and said second test line (T2) is configured so that a signal is detectable at said second test line when βTP concentration in a sample applied to said device is greater than the βTP level indicative of a CSF leak.

2. The smartphone-based LFA reader of claim 1, wherein said reader is configured to photograph the test lines on said lateral flow device in a controlled light setting to produce photographs of said test lines.

3. The smartphone-based LFA reader of claim 2, wherein said reader converts said photographs to grayscale, and locates the test lines on the LFA device by searching for peaks in pixel intensity.

4. The smartphone-based LFA reader of claim 3, wherein said reader determines the mean or median grayscale intensity of each line and, optionally, the sum of all test line intensities.

5. The smartphone-based LFA reader of claim 4, wherein said reader compares the measured line intensities with an internal standard curve and displays the concentration of βTP in the sample applied to the sample addition zone, and/or a clinical diagnosis.

6. The smartphone-based LFA reader of claim 1, wherein said detection zone comprises a third test line (T3) that comprises binding moieties that bind a complex formed between beta-trace protein (βTP) and an indicator attached to a βTP binding molecule, wherein: said first test line (T1) said second test line (T2) and said third test line (T3) are configured so that either no test line signal or just a signal at said first test line (T1) is detectable by said reader when βTP concentration in a sample applied to said device is lower than or equal to the βTP level indicative of the absence of a CSF leak; said first test line (T1) said second test line (T2) and said third test line (T3) are configured so that a signal is detectable by said reader at said first test line and said second test line, but not at said third test line when βTP concentration in a sample applied to said device is greater than the βTP level indicative of no CSF leak and less than a βTP level definitive of the presence of a CSF leak; and a signal is detectable by said reader at all three test lines T1, T2, and T3 when βTP concentration in a sample applied to said device is equal to or greater than a βTP level definitive of the presence of a CSF leak.

7. The smartphone-based LFA reader of claim 6, wherein said first test line (T1) said second test line (T2) and said third test line (T3) are configured so that no signal is detectable by said reader at test lines T1, T2, or T3, or a signal is detectable only at test line T1, when βTP concentration is less than about 0.7 mg/L in a nasal drip sample applied to said lateral flow device.

8. The smartphone-based LFA reader of claim 6, wherein said first test line (T1) said second test line (T2) and said third test line (T3) are configured so a signal is detectable by said reader at test lines T1, and T2, but not at test line T3, when βTP concentration ranges from about 0.7 mg/L up to about 1.3 mg/L in a nasal drip sample applied to said device.

9. The smartphone-based LFA reader of claim 6, wherein said first test line (T1) said second test line (T2) and said third test line (T3) are configured so a signal is detectable by said reader at test lines T1, T2, and T3, when βTP concentration is greater than about 1.3 mg/L in a nasal drip sample applied to said device.

10. The smartphone-based LFA reader of claim 6, wherein said lateral flow device comprise a control line downstream from said first test line and said second test line, wherein said control line comprises binding moieties that can capture the said indicator, in the presence or absence of βTP.

11. The smartphone-based LFA reader of claim 1, wherein said lateral flow device comprises a conjugation zone comprising said indicator attached to a βTP binding molecule, where said conjugation zone is disposed in said sample addition zone and/or between said sample addition zone and said detection zone.

12. The smartphone-based LFA reader of claim 1, wherein said lateral flow device comprises an absorbent pad disposed downstream from said detection zone and when a control line is present said absorbent pad is disposed downstream from said control line.

13. The smartphone-based LFA reader of claim 1, wherein said indicator is selected from the group consisting of a colorimetric indicator, a fluorescent indicator, a radioactive indicator, and a magnetic indicator.

14. The smartphone-based LFA reader of claim 1, wherein said βTP binding molecule is an antibody or antibody fragment.

15. The smartphone-based LFA reader of claim 1, wherein said binding moieties that bind a complex formed between beta-trace protein (βTP) and an indicator comprise an antibody or antibody fragment.

16. The smartphone-based LFA reader of claim 15, wherein said binding moieties that bind a complex formed between beta-trace protein (βTP) and an indicator comprise an antibody that specifically binds βTP.

17. The smartphone-based LFA reader of claim 1, wherein said lateral flow device is configured to perform a sandwich assay.

18. The smartphone-based LFA reader of claim 1, wherein, said lateral flow device comprises a porous substrate comprising a material selected from the group consisting of sintered glass or sintered ceramic, a mineral, cellulose, a fiberglass, a nitrocellulose, polyvinylidene fluoride, a nylon, a charge modified nylon, a polyethersulfone, and combinations thereof.

19. A kit for semi-quantification of βTP in a biological sample, said kit comprising: a smartphone-based LFA reader of claim 1; and a container containing a lateral flow device for the semi-quantitative detection of a cerebrospinal fluid.

20. The kit of claim 19, wherein said kit contains two barcode-style LFA test devices, one for testing serum and the other for testing a nasal drip sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a schematic of a typical lateral-flow immunoassay test strip (top). The sandwich format of the lateral-flow immunoassay is illustrated in the bottom panel.

(2) FIG. 2 illustrates a schematic and illustrative βTP cutoff levels of a two-line LFA design.

(3) FIG. 3 illustrates dilutions of human CSF in human serum (HS). Negative control, HS, displays 1 visible test line. All tests containing CSF display 2 test lines. There were no control lines printed on these strips.

(4) FIG. 4 shows a schematic and illustrative βTP cutoff levels of a three-test line LFA design.

(5) FIG. 5 schematically illustrates a lateral-flow immunoassay incorporating a conjugation zone.

DETAILED DESCRIPTION

(6) In various embodiments devices and methods are provided for the rapid detection of cerebrospinal fluid (CSF) leaks in a subject. The devices and methods provide a rapid, robust, easy-to-use, and inexpensive point-of-care diagnostic device that can distinguish between samples containing CSF and those that do not.

(7) The current standards for diagnosing CSF leaks include invasive and expensive techniques such as CT and MM, as well as ELISA and beta-2 transferrin electrophoresis, which are rarely used due to their high cost and long time-to-result. More recently, researchers have looked into the detection of beta-trace protein (βTP) using a nephelometric assay. While rapid and highly sensitive, the βTP nephelometric assay still requires expensive equipment found in a centralized laboratory and extensive training.

(8) The devices described herein provide the ability to rapidly quantify the concentration of βTP in a sample in order to determine the presence or absence of a CSF with the use of a barcode-style lateral flow assay (LFA). This technology can allow for the rapid diagnosis of a CSF leak without the need for expensive equipment, invasive tests, or extensive training. Moreover the devices and methods facilitate rapid detection of a CSF leak before the subject decompensates as a consequence of the leakage.

(9) Accordingly the methods and devices described herein are directed to the detection of βTP in a subject (e.g., in a biological sample derived from a subject). In particular embodiments, lateral-flow assay devices (e.g., bar-code lateral flow devices) are provided for the detection and semi-quantitative determination of CSF in a sample thereby permitting the diagnosis of CSF leaks in a subject. It is noted that, to the extent that the following description is of a specific embodiment or a particular uses, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the claims.

(10) Lateral flow immunoassays are simple tests for rapid detection of the presence or absence of a target analyte in a sample for home testing, point of care testing, or laboratory applications. “Bar-code” or “ladder” lateral flow immunoassay provides a semi-quantitative measurement of the amount of analyte present in a sample typically by providing multiple detection bands (e.g., in a detection zone) zone each of which represents a different analyte concentration range.

(11) Lateral flow test strips typically utilize a solid support through which a mobile phase (e.g., a liquid sample) can flow through by capillary action to a reaction matrix where a detectable signal, such as color changes or color differences on the test strip, may be generated to indicate the presence or absence of the target analyte. As used herein, the term “capillary action” or “capillarity” means the process by which a molecule is drawn across the lateral test strip due to such properties as surface tension and attraction between molecules.

(12) In certain embodiments the concentration of βTP in a sample is quantified using a barcode-style lateral-flow immunoassay (LFA) in order to detect (e.g., determine the presence of or absence of, or severity of) a CSF leak. A typical LFA consists of at least 3 components: a sample pad where the sample is applied to the test strip, a detection zone where there is binding and where results can be observed, and an absorbent pad which acts as a sink for excess sample (FIG. 1, top). In certain embodiments of a sandwich assay format, the LFA indicator (which can be colorimetric, fluorescent, radioactive, etc.) decorated with binding molecules (e.g., antibodies, aptamers, etc.) can be first added to the sample which is then applied to the LFA device. Alternatively, in certain embodiments the LFA comprise a conjugation zone containing the indicator attached to binding molecules and combination of the sample with the indicator can occur in the conjugation zone. If the target, in this case βTP, is present, it will bind to the indicator decorated with the binding molecule. These complexes are flow through the strip into the detection zone towards the absorbent pad when present. If βTP is present, they associate with the binding molecules immobilized on the test line(s) and become sandwiched between the indicator and the membrane providing a signal at the particular test line(s). For example, if the indicator is colorimetric, the colorimetric indicator will exhibit a strong color, and a visual band forms as the βTP-indicator complex accumulates at the test line, indicating a positive result. Alternatively, if no βTP is present, the indicator does not attach to the test line, and the absence of the test line indicates a negative result. Regardless of the presence of βTP, the binding molecule decorated on the indicator can associate with and accumulate on the control line when a control line is present. A band at the control line signifies that the sample has flowed through the strip, indicating a valid test. In certain embodiments then, a positive result is therefore indicated by two bands, one at the test line and one at the control line, while a negative result is indicated by a single band at the control line (FIG. 1, bottom).

(13) While the traditional LFA gives a qualitative “yes” or “no” readout, a more quantitative device would be instrumental in the accurate diagnosis of a CSF leak, where lower concentrations of βTP are indicative of a negative result, and higher concentrations of βTP are indicative of a positive result for a CSF leak. An alternative format of the LFA that provides a more quantitative output is called the barcode-style or ladder LFA, in which multiple test lines are printed on the detection zone. Each test line will have a cut-off, which is the minimum concentration of βTP in a sample necessary for that test line to become visible. These cut-offs can be adjusted by varying the density of the binding molecule that is immobilized, as well as altering the affinity that binding molecule has for βTP. After running the assay, the number of test lines that are visible on the LFA strip can be correlated with an approximate βTP concentration in the sample and thus a clinical diagnosis.

(14) Accordingly, in certain embodiments, a semi-quantitative LFA assay is provided with two test lines to quantify βTP levels. To develop the two test line assay, an antibody to βTP, the capture antibody, is immobilized on the detection zone of the LFA membrane in two locations: test line 1 (T1), and test line 2 (T2). Previous studies have shown that a βTP concentration of >1.3 mg/L, in a nasal drip sample, is a good indicator of a CSF leak.

(15) Accordingly, in certain embodiments, capture moieties (e.g., capture antibodies) are immobilized on the detection zone such that 0 or 1 test line will be detectable (e.g., visible) when the concentration of βTP is below 1.3 mg/L and 2 test lines will be detectable (e.g., visible) when the concentration of βTP is above 1.3 mg/L (FIG. 2). A secondary antibody that can capture the colorimetric indicator, in the presence or absence of βTP, can be immobilized as the control line, downstream from the test lines. In the following example of the two test line barcode-style LFA, gold nanoparticles decorated with anti-βTP antibodies (GNPs) were used as the colorimetric indicator.

(16) In certain embodiments prior to running the assay, the sample is diluted, e.g. 300-fold in phosphate-buffered saline (PBS). The diluted sample is mixed with GNPs and is then applied to the sample pad, where it will begin to flow through the LFA strip. Any βTP in the sample will bind to the GNPs to form βTP-GNP complexes. When these βTP-GNP complexes cross the detection zone, they will first bind to T1. βTP-GNP complexes that do not bind to T1 will be able to bind to T2. Finally, unbound GNPs and any remaining βTP-GNP complexes will bind to the control line to indicate a valid test. After, e.g., 20 minutes, the number of detectable (e.g., visible) test lines is counted. If 0 or 1 test line is present, the sample is considered negative for a CSF leak. If 2 test lines are present, the sample is considered positive for a CSF leak.

(17) To demonstrate the two-test line LFA device, a CSF sample obtained from a lumbar drain was diluted in human serum to simulate a clinical nasal drip sample containing trace amounts of CSF. When run on the 2 test line LFA device, all samples containing CSF displayed 2 visible test lines, indicating positive results for the presence of CSF. When the negative control, human serum without CSF, was run, only one test line appeared, indicating a negative result for the presence of CSF (FIG. 3). Control lines were not printed in the results shown.

(18) In another illustrative, but non-limiting embodiments, a semi-quantitative LFA device with three test lines to quantify βTP levels is provided. A βTP concentration of >1.3 mg/L has been shown to have relatively high sensitivity when detecting for CSF leaks using the nephelometric assay. However, in the region from 0.7-1.3 mg/L, which we will refer to as a “grey zone”, it has been reported that approximately 1 in 3 samples will contain CSF. As a result, if all βTP concentrations of <1.3 mg/L are classified as negative for CSF, potentially life-threatening CSF leaks will be misdiagnosed. Therefore, it is beneficial to develop a device that can identify samples in this grey zone, so that further testing can be performed before ruling out the presence of a CSF leak. To develop this assay, an antibody to βTP, the capture antibody, is immobilized on the detection zone of the LFA membrane in three locations: test line 1 (T1), test line 2 (T2), and test line 3 (T3). The capture moieties (e.g., antibodies) will be immobilized such that 0 or 1 test line will be visible when the concentration of βTP in a sample is below 0.7 mg/L, 2 test lines will be visible when the concentration is 0.7-1.3 mg/L, and 3 test lines will be visible when the concentration is greater than 1.3 mg/L (FIG. 4). A secondary antibody, that can capture the indicator bound with detection antibodies, in the presence or absence of βTP, is immobilized as the control line, downstream from the test lines. In the following example of the three test line barcode-style LFA, gold nanoparticles decorated with anti-βTP antibodies (GNPs) are used as the colorimetric indicator.

(19) In certain embodiments prior to running the assay, the sample to be run is diluted, e.g., 300-fold in phosphate-buffered saline (PBS). The diluted sample is then applied to the sample pad, where it begins to flow through the LFA strip. Any βTP in the sample binds to the GNPs to form βTP-GNP complexes. When these βTP-GNP complexes cross the detection zone, they will first bind to T1. βTP-GNP complexes that do not bind to T1 will be able to bind to T2. βTP-GNP complexes that do not bind to T1 or T2 will then be able to bind to T3. Finally, the unbound GNPs and any remaining βTP-GNP complexes will bind to the control line to indicate a valid test. After, e.g., 20 minutes, the number of colored test lines is counted. If 0 or 1 test line is present, the sample is considered negative for a CSF leak. If 2 test lines are present, the sample is considered in the grey zone, where further testing is required and it is recommended to rerun the assay with a newly collected sample. If 3 test lines are present, the sample is considered positive for a CSF leak.

(20) Another aspect of this invention is the development of a kit that contains two separate LFA tests to quantify the level of βTP in both a patient's serum and nasal drip sample. Previous studies have demonstrated that the ratio of βTP concentrations in nasal drip sample to serum is also a sensitive and specific indicator of the presence or absence of a CSF leak. This invention looks to use the barcode-style LFA to quantify the βTP concentration in both serum and nasal drip samples. In certain embodiments the kit will contain two barcode-style LFA test devices, one for testing serum and the other for testing the nasal drip sample. In certain embodiments it can also contain a reference card that correlates the number and intensity of visible test lines with the concentrations of βTP in the sample. After running a patient's serum and nasal drip samples on their respective LFA test strips and obtaining the βTP concentrations in each samples, the nasal drip to serum βTP concentration ratio will be taken. In certain embodiments a ratio of less than 2 (<2) is classified as negative for the presence of CSF and a ratio of greater than 2 (>2) or greater than or equal to 2 is positive.

(21) Other embodiments contemplated herein involve the integration of a smartphone-based LFA reader with the LFA test strips described above. An optional smartphone-based LFA reader can further improve the ease-of-interpretation and accuracy of the tests for the detection of CSF leaks. Photographs of the LFA test strips are taken (in certain embodiments in a controlled light setting, such as an external photo light box or an integrated phone attachment that blocks outside light and uses the smartphone flash as a lighting source). A smartphone application then converts the photos to grayscale (e.g., 8-bit, 16-bit, 32-bit grayscale). Each test line on the LFA strip can be located by searching for peaks in pixel intensity. Once the location of each test line is determined, the mean grayscale intensity of each line can be calculated and/or as the sum of all test line intensities. The measured intensities can be compared with an internal standard curve and the concentration of βTP in the sample and/or the clinical diagnosis can be displayed on the smartphone screen.”

(22) Lateral Flow Assay Components.

(23) In various embodiments the lateral flow assay comprises a porous substrate, a sample addition zone disposed on or in the porous substrate; and a detection zone disposed on or in said porous substrate where said detection zone comprises at least a first test line (T1) and a second test line (T2) and in certain embodiments a third test line (T3) as described above. In certain embodiments the lateral flow assay additionally comprises a conjugation zone containing the indicator attached to a moiety that binds βTP. The lateral flow device can additionally comprise a control line and/or an absorbent pad (e.g., sink).

(24) Sample Receiving Zone

(25) The LFA devices described herein typically comprise a sample receiving zone for application of the biological sample to the device. In certain embodiments the sample receiving zone comprise a sample pad disposed on or in the porous substrate. In certain embodiments the sample pad can act as a filter that can remove debris, contaminants, and mucus from the collected fluid. It can also store dried reagents, and when rehydrated, these reagents can (i) adjust the solution for optimal detection conditions (pH, ionic strength, etc); and (ii) break down mucus, glycoproteins, and other viscous materials in the collected specimen that may affect detection. Illustrative materials for the sample pad include, but are not limited to, cellulose, nitrocellulose, fiberglass, cotton, woven or nonwoven paper, etc. Reagents on the pad may include, but are not limited to, surfactants such as Triton X-100, Tween 20, or sodium dodecyl sulfate, etc.; polymers such as polyethylene glycol, poloxamer, polyvinylpyrrolidone (PVP), etc.; buffers such as phosphate-buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris(hydroxymethyl)aminomethane (Tris), sodium borate, TRICINE, etc.; proteins such as albumin, etc.; enzymes such as protease, etc.; salts such as sodium chloride, sodium phosphate, sodium cholate, potassium phosphate, etc. In various embodiments these reagents can be applied to the sample pad by (i) soaking the paper material in the reagent solution, or (ii) through wicking the membrane via capillary flow. The treated sample pad can be dried by (i) air dry (let sit in room temperature); (ii) baking (place in high temperature using an oven or heating device); (iii) vacuum; or (iv) lyophilization.

(26) Conjugation Zone

(27) In certain embodiments the LFA devices described herein can comprise a conjugation zone for mixing the sample with an indicator attached to a βTP binding molecule (see, e.g., FIG. 5). In certain embodiments the conjugation zone comprises a conjugate pad. In certain embodiments the conjugation zone, when present can contain dehydrated indicators (e.g., colorimetric indicators, a fluorescent indicators, a radioactive indicators, magnetic indicators, etc.) decorated with binding moieties that bind the βTP target analyte. In certain embodiments the binding moieties are specific binding moieties that have high affinity towards βTP. When the sample solution reaches the conjugate pad, the indicators (e.g., colorimetric indicators) are rehydrated. The binding moieties on the indicators can then bind to the βTP the resulting complexes can flow to the detection zone. In certain embodiments the indicators can comprise colorimetric indicators that can comprise metallic particles such as gold, silver particles, polymeric particles such as latex beads, and polystyrene particles encapsulating visible or fluorescent dyes. Illustrative materials material for the conjugation zone (e.g., conjugate pad) include, but are not limited to, cellulose, nitrocellulose, fiberglass, cotton, woven or nonwoven paper etc. In certain embodiments the colorimetric indicators can be applied and dehydrated onto the pad as described above.

(28) Detection Zone

(29) In certain embodiments the reaction pad, when present, can comprise immobilized reagents, and when the immobilized reagents react with the sample solution, they may produce signals (e.g., visual signals) to indicate the presence or absence or quantity of the target analyte(s) at particular test lines. Illustrative materials for the detection zone include, but are not limited to cellulose, nitrocellulose, fiberglass, cotton, woven or nonwoven paper etc.

(30) In certain embodiments for a lateral-flow test strip, the reagents in the detection zone are immobilized in the form of lines perpendicular to the direction of flow to ensure all samples can interact with the immobilized reagents. The concentrations of the reagents can be optimized to control the signal intensities, and thus, control the sensitivity of the assay. For example, a semi-quantitative assay can be designed by immobilizing multiple lines of the same reagent with various concentrations. Each line therefore will yield signals only when a specific concentration of target biomolecules is reached. The concentration of the target biomolecules can then be interpreted by counting the number of lines that are visible, e.g., as described above.

(31) Absorbent Pad/Sink

(32) In certain embodiments the lateral flow device comprises an absorbent pad disposed downstream from the detection zone and when said control line is present the absorbent pad is disposed downstream from the control line. In certain embodiments the sink, when present, can comprise an absorbent pad that collect excess fluid and prevents back-flow which can affect the test performance. Illustrative materials for the sink include, but are not limited to cellulose, nitrocellulose, fiberglass, cotton, woven and nonwoven paper etc.

(33) Lateral Flow Assay (LFA)or Flow-Through (Spot) Assay

(34) As explained above, in certain embodiments, the devices and systems described herein are configured to provide a lateral flow assay (LFA) for detection of the target analyte (βTP) in a sample. The LFA typically comprises a porous matrix into which is disposed sample and assay components, e.g., as described above. The porous matrix is configured to and has porosity sufficient to allow the assay reagents to flow through the porous matrix when the components are in a fluid phase. Such porous LFA devices are can be referred to as paper or paper fluidic devices and these terms are used interchangeably.

(35) The term “paper”, as used herein, is not limited to thin sheets from the pulp of wood or other fibrous plant substances although, in certain embodiments the use of such papers in the devices described herein is contemplated. Papers more generally refer to porous materials often in sheet form, but not limited thereto that allow a fluid to flow through.

(36) In some embodiments, the porous matrix is sufficiently porous to allow the assay components and target analyte(s) to flow through the LFA. In some embodiments the porous matrix comprises inter alia a material such as a scintered glass ceramic, a mineral, cellulose, a fiberglass, a nitrocellulose, polyvinylidene fluoride, a nylon, a charge modified nylon, a polyethersulfone, combinations thereof, and the like.

(37) Sandwich Assay

(38) In some embodiments, the LFA is configured to provide or run a sandwich assay (see e.g., FIG. 1, bottom left, in copending PCT Application No: PCT/US2015/019297, filed on Mar. 6, 2015, which is hereby incorporated by reference for the LFA configurations described therein). In some embodiments, the sandwich assay comprises a capture moiety that binds the target analyte. In some embodiments, the device comprises an indicator attached to a moiety that binds to the analyte of interest βTP). In some embodiments, the indicator comprises a detectable property (colorimetric, fluorescent, radioactive, etc.). In some embodiments, the probe comprises a binding moiety that interacts with the target analyte (e.g. an antibody). In some embodiments, the indicator is added to the sample before application to the device and binds the target analyte to form a probe-analyte complex. In some embodiments, the indicator is combined with the sample in a conjugation zone in the LFA device after the sample is added to the device and binds the target analyte to form a probe-analyte complex.

(39) Indicator-analyte complex flows through the LFA or through the flow-through device towards the absorbent pad. In some embodiments, the target analyte of the indicator-analyte complex binds to the capture moiety. In some embodiments, the capture moiety is immobilized on a test line or a test region (e.g., a test layer in a flow-through device) and the indicator-analyte complex becomes immobilized on the test line or in the test region. In some embodiments, the indicator is colorimetric, and the test line or test region will exhibit a strong color (e.g. detectable signal) as the indicator-analyte complex accumulates at the test line or in the test region, indicating a positive result. In some embodiments, there is no target analyte present in the sample, and the indicator of the indicator-analyte complex does not interact with the capture moiety, and the absence of the test line or signal in the test region indicates a negative result at that test line. In some embodiments, the LFA comprises an indicator capture moiety on a control line (or in a control region, e.g., of a flow-through assay device) that interacts directly with the indicator and/or the binding moiety, and thus, regardless of the presence of the target analyte in the sample, the indicator/binding moiety binds to the probe capture moiety and accumulates on the control line or in the control region. In some embodiments, the indicator capture moiety is a secondary antibody that binds the binding moiety, wherein the binding moiety is a primary antibody that binds that target analyte. In some embodiments, the indicator becomes immobilized and detected on the control line or in the control region, indicating a valid test. In some embodiments, a positive result (e.g. target analyte is present in sample) is indicated by a detectable signal at the test line(s) as described above. In some embodiments, a negative result is indicated by a detectable signal at the control line or in the control region in the absence of test line signal(s) as described above.

(40) Indicators

(41) In certain embodiments the systems and/or devices described herein and/or the methods described herein utilize an indicator, where the indicator comprises a binding moiety that binds the target analyte to form an indicator-analyte complex.

(42) In some embodiments, the indicator comprises a material selected from the group consisting of a synthetic polymer, a metal, a mineral, a glass, a quartz, a ceramic, a biological polymer, a plastic, and combinations thereof. In some embodiments, the indicator comprises a polymer selected from the group consisting of polyethylene, polypropylene, nylon (DELRIN®), polytetrafluoroethylene (TEFLON®), dextran and polyvinyl chloride. In some embodiments, the polyethylene is polyethylene glycol. In some embodiments, the polypropylene is polypropylene glycol. In some embodiments, the indicator comprises a biological polymer selected from the group consisting of collagen, cellulose, and chitin. In some embodiments, the indicator comprises a metal selected from the group consisting of gold, silver, platinum titanium, stainless steel, aluminum, and alloys thereof. In some embodiments, the indicator comprises a nanoparticle (e.g., a gold nanoparticle, a silver nanoparticle, etc.).

(43) In some embodiments, the indicator comprises a detectable label. Detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Illustrative useful labels include, but are not limited to, fluorescent nanoparticles (e.g., quantum dots (Qdots)), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,.sup.14C, .sup.32P, .sup.99Tc, .sup.203Pb, .sup.67Ga, .sup.68Ga, .sup.72As, .sup.111In, .sup.113mIn, .sup.97Ru, .sup.62Cu, 641Cu, .sup.52Fe, .sup.52mMn, .sup.51Cr, .sup.186Re, .sup.188Re, .sup.77As, .sup.90Y, .sup.67Cu, .sup.169Er, .sup.121Sn, .sup.127Te, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.161Tb, .sup.109Pd, .sup.165Dy, .sup.149Pm, .sup.151Pm, .sup.153Sm, .sup.157Gd, .sup.159Gd, .sup.166Ho, .sup.172Tm, .sup.169Yb, .sup.175Yb, .sup.177Lu, .sup.105Rb, .sup.111Ag, and the like), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), various colorimetric labels, magnetic or paramagnetic labels (e.g., magnetic and/or paramagnetic nanoparticles), spin labels, radio-opaque labels, and the like.

(44) Alternatively or additionally, the indicator can bind to another particle that comprises a detectable label. In some embodiments, the indicator provide a detectable signal at the detection zone (e.g. test line, control line, test region, control region). In some embodiments, the detectable label/property is selected from the group consisting of a colorimetric label/property, a fluorescent label/property, an enzymatic label/property, a colorigenic label/property and a radioactive label/property. In some embodiments, the indicator comprises a gold nanoparticle and the detectable property is a color. In some embodiments, the color is selected from orange, red and purple.

(45) In some embodiments, the indicator further comprises a coating. In some embodiments, the coating comprises polyethylene glycol or polypropylene glycol. In some embodiments, the coating comprises polypropylene. In some embodiments, the coating comprises polypropylene glycol. In some embodiments, the coating comprises dextran. In some embodiments, the coating comprises a hydrophilic protein. In some embodiments, the coating comprises serum albumin. In some embodiments, the coating has an affinity for the first phase solution or the second phase solution.

(46) Binding Moiety

(47) In some embodiments, the binding moiety is a molecule that binds the target analyte (βTP). In some embodiments, the binding moiety is a molecule that specifically binds βTP. In some embodiments, “specifically binds” indicates that the molecule binds preferentially to βTP or binds with greater affinity to the target analyte than to other molecules. By way of non-limiting example, an antibody will selectively bind to an antigen against which it was raised. Also, by way of non-limiting example, a DNA molecule will bind to a substantially complementary sequence and not to unrelated sequences under stringent conditions. In some embodiments, “specific binding” can refer to a binding reaction that is determinative of the presence of a target analyte in a heterogeneous population of molecules (e.g., proteins and other biologics). In some embodiments, the binding moiety binds to its particular target analyte and does not bind in a significant amount to other molecules present in the sample.

(48) In some embodiments, the binding moiety is selected from the group consisting of an antibody, a lectin, a protein, a glycoprotein, a nucleic acid, monomeric nucleic acid, a polymeric nucleic acid, an aptamer, an aptazyme, a small molecule, a polymer, a lectin, a carbohydrate, a polysaccharide, a sugar, a lipid, and any combination thereof. In some embodiments, the binding moiety is a molecule capable binding pair the target analyte.

(49) In some embodiments, the binding moiety is an antibody or antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′).sub.2, Fv, Fv′, Fd, Fd′, scFv, hsFv fragments, single-chain antibodies, cameloid antibodies, diabodies, and other fragments described herein.

(50) In certain embodiments, the binding moiety comprises an aptamer. In some embodiments, the aptamer comprises an antibody-analogue formed from nucleic acids. In some embodiments, the aptamer does not require binding of a label to be detected in some assays, such as nano-CHEM-FET, where the reconfiguration would be detected directly. In some embodiments, the binding moiety comprises an aptazyme. In some embodiments, the aptazyme comprises an enzyme analogue, formed from nucleic acids. In some embodiments, the aptazyme functions to change configuration to capture a specific molecule, only in the presence of a second, specific, analyte.

(51) Sample Collection

(52) In various embodiments the sample to be assayed using the devices and methods described herein comprises a biological sample. Illustrative biological samples include, but are not limited to biofluids such as blood or blood fractions, lymph, nasal or oral fluids, and the like.

(53) Where the biological sample comprises a tissue, in certain embodiments, the tissue may be lysed, homogenized, and/or ground and, optionally suspended in a sample solution. Where the biological sample comprise a biological fluid the fluid may be assayed directly or suspended in a sample solution prior to assay. In certain embodiments the sample solution may act to preserve or stabilize the biological sample or components thereof, and/or may act to extract or concentrate the biological sample or components thereof. In certain embodiments the sample solution may comprise a buffer, optionally containing preservatives, and/or enzymes (protease, nuclease, etc.), and/or surfactants, and/or ATPS components.

(54) In certain embodiments, particular in point-of-care embodiments, the sample may be applied to the assay device immediately or after a modest time interval. In certain embodiments the sample may be delivered to a remote testing facility where the assay is run.

(55) Methods and devices for collecting biological samples are well known to those of skill in the art.

(56) Kits.

(57) In certain embodiments a kit for semi-quantification of βTP in a biological sample is provided. In certain embodiments the kits comprise a container containing a lateral flow device for the semi-quantitative detection of a cerebrospinal fluid leak as described herein. In certain embodiments the kit contains two barcode-style LFA test devices as described herein, one for testing serum and the other for testing a nasal drip sample. In certain embodiments the kit comprises a reference card that correlates the number and intensity of visible test lines with the concentrations of βTP in the sample. In certain embodiments the kit further comprises a smartphone LSA reader for the LSA device(s). In certain embodiments the kit comprises one or more sample collection devices (e.g., devices for collecting blood or plasma, or nasal or oral material).

(58) In certain embodiments the kit comprises instructions (instructional materials) for using the kit for semi-quantification of βTP in a biological sample.

(59) While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

(60) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.