WO2012142242A2 - Procédé d'essai pour plage dynamique étendue - Google Patents

Procédé d'essai pour plage dynamique étendue Download PDF

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Publication number
WO2012142242A2
WO2012142242A2 PCT/US2012/033252 US2012033252W WO2012142242A2 WO 2012142242 A2 WO2012142242 A2 WO 2012142242A2 US 2012033252 W US2012033252 W US 2012033252W WO 2012142242 A2 WO2012142242 A2 WO 2012142242A2
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Prior art keywords
analyte
detection
antibody
chamber
assay
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WO2012142242A3 (fr
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Junhai Kai
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SILOAM BIOSCIENCES Inc
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SILOAM BIOSCIENCES Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique

Definitions

  • This invention relates to an assay method that enables an extended detection range for an analyte of interest and is especially well suited for use in microfluidic or lateral flow assay devices that are especially suitable for use in point-of-care, e.g. physician's office or other health care provider facilities as well as in typical clinical laboratory facilities
  • Immunoassay techniques are widely used for a variety of applications, as described for example in "Quantitative Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting and Clinical Applications; James Wu; AACC Press; 2000".
  • the most common immunoassay techniques are the non-competitive assay, an example of which is the widely know sandwich immunoassay, wherein two binding agents are used to detect an analyte, and another example is the "one-step” or “direct” immunoassay wherein only one binding agent with or without reporter agent is used to interrogate an unknown analyte concentration; and the competitive assay, wherein only one binding agent is required to detect an analyte.
  • the sandwich immunoassay can be described as follows: a capture antibody, as a first binding agent, is coated (typically) on a solid-phase support.
  • the capture antibody is selected such that it offers a specific affinity to the analyte and ideally does not react with any other analytes.
  • a solution containing the target analyte is introduced over this area whereby the target analyte conjugates with the capture antibody.
  • a second detection antibody as a second binding agent, is added to this area.
  • the detection antibody also offers a specific affinity to the analyte and ideally does not react with any other analytes.
  • the detection antibody is typically "labeled" with a reporter agent.
  • the reporter agent is configured to be detectable by one of many detection techniques such as optical (fluorescence or chemiluminescence or large -area imaging), electrical, magnetic or other means.
  • the detection antibody further binds with the analyte-capture antibody complex. After removing the excess detection antibody; finally the reporter agent on the detection antibody is interrogated by means of a suitable technique.
  • the signal from the reporter agent is proportional to the concentration of the analyte within the sample. Note that the above description applies to most common forms of the assay technique - such as for detection of proteins.
  • Immunoassay techniques can also be used to detect other analytes of interest such as, but not limited to, enzymes, nucleic acids and more. Similar concepts have also been widely applied for other variations as well, including detection of an analyte antibody using a "capture" antigen and a detection analyte.
  • An advantage of the immunoassay technique is the specificity of detection towards the target analyte of interest that is provided by the use of binding agents. While a relatively narrow detection range is acceptable for a wide variety of clinical applications; a large detection range is very desirable for a select group of analytes. Examples of such analytes include HCG (human chorionic gonadotropin) and PSA (prostate specific antigen), which occur in concentration ranges spanning 5-6 orders of magnitude dependant on the clinical condition.
  • HCG human chorionic gonadotropin
  • PSA prote specific antigen
  • serial dilution methods wherein the sample is successively diluted and analyzed at different dilution levels. Since dilution effectively reduces the concentration of the target analyte in the sample; the operating range of the assay is shifted to a region where the test device offers a linear (or at least quantifiable) response.
  • sample processing steps such as serial dilution
  • POCT point-of-care test
  • LFA test devices have been developed for both qualitative and quantitative readouts. Of these the qualitative devices are typically easier to configure and operate. As described previously, a wide variety of techniques can be used to "read" the assay signal. For LFA's traditionally, the most common method has been the use of chromatographic techniques. A porous substrate with dried (or lyophilized) binding agent is deposited at an upstream location close to the sample introduction chamber.
  • This particular binding agent is typically mobile, insofar as when the sample is added to the test, the sample reconstitutes the binding agent which then links with a target analyte in the sample and the conjugate is transported downstream with the sample.
  • the porous substrate also consists of a fixed (or immobilized) binding agent downstream. As the (sample + mobile binding agent) conjugate is transported along the porous substrate by capillarity it eventually encounters the fixed binding agent. A portion of the conjugate then links with the fixed binding agent, via linkage of the analyte to the fixed binding agent.
  • the mobile binding agent is typically labeled with a reporter agent, which can then be interrogated to determine the concentration of the analyte in the sample. LFA techniques have been extensively researched and improved upon.
  • Multi-layer approaches to LFA wherein the flow substrates are arranged on different portions, on physically distinct layers, of the test device and combined during testing, have also been developed, can These techniques are applicable for a wide variety of detection mechanisms such as electrochemical, fluorescence, chemiluminescence or bioluminescent and by selectively controlling the label interrogation can also extend the dynamic range of the assay with high sensitivity detection.
  • detection mechanisms such as electrochemical, fluorescence, chemiluminescence or bioluminescent
  • By selectively controlling the label interrogation can also extend the dynamic range of the assay with high sensitivity detection.
  • POCT devices are the developments in control techniques that can be used to monitor the status of immunoassays.
  • the term "assays for POCT” can include all assay techniques wherein by a multitude of available techniques, the assay reagents are sequentially transported to a detection region by means of capillarity or by applied pressure on the fluid column.
  • the devices can include without limitation LFA, so called “through flow assays", and microfluidic devices (passive flow as well as active flow).
  • LFA so called “through flow assays”
  • microfluidic devices passive flow as well as active flow
  • the signal is proportional to the analyte concentration and increases with increasing analyte in sample. However, at very high analyte concentrations, the signal will actually start decreasing due to the hook-effect.
  • the analyte conjugates with the mobile binding agents and then the conjugate is transported to the fixed binding agent. In cases of very high analyte concentrations a significant portion of the mobile binding agent is bound with the analyte and still leaving a large portion of the analyte unbound. As the conjugate and sample travels to the fixed binding site, a larger fraction of unbound analyte links with the fixed binding agent than the conjugated analyte (with mobile binding agent).
  • the label on the mobile binding agent is responsible for generating the signal; this effect reduces the overall assay signal. Addressing the hook-effect problem is thus important to extend the dynamic range of the immunoassay technique being employed. Note that the hook effect only occurs in cases where the target analyte is first mixed with the detection (or mobile) binding agent and the (analyte + detection antibody) conjugate is then presented to the capture (or solid-phase supported) binding agent.
  • a device containing a series of "specific binding partners" with progressively higher affinity to the target analyte ⁇ actually to (target analyte + first specific binding partner i.e. capture antibody) ⁇ has been envisioned in the art.
  • the (target analyte + capture antibody) is transported to multiple capture (antibody) sites each of which has a different binding affinity to the analyte.
  • the analyte (+label) is bound to the first capture antibody it effectively depletes the analyte concentration reaching the second capture antibody and even lower concentration reaches the third capture antibody and so forth.
  • This technique relies on "reading" one or more of the detection zones (signal lines) and uses that to analyze the target analyte concentration with a manufacturer supplied interpretation chart. Thus, this technique relies on the "interfering" effect of the first capture zone on the second capture; the interfering effect of the first and second capture zones on the third capture zone; and so forth. Furthermore, this technique requires reading one or more detection zones (lines) depending on the concentration of the analyte.
  • the number of lines with detectable signal is proportional to the amount of analyte present in the sample.
  • the present invention seeks to address the shortcomings of the art described above, and seeks to provide an easy and reliable wide dynamic range assay technique that can be used with a variety of conventional assay techniques including, but not limited to, commonly used lateral flow assay and microfluidic assay devices.
  • microfluidic as used herein generally refers to the use of microchannels for transport of liquids or gases.
  • the microfluidic system consists of a multitude of microchannels forming a network and associated flow control components such as pumps, valves and filters. Microfluidic systems are ideally suited for controlling minute volume of liquids or gases. Typically, microfluidic systems can be configured to handle fluid volumes ranging from picoliter to milliliter range.
  • binding agents as used herein means chemical/biochemical molecules which can bind with high specificity to one or a very limited group of molecules. A wide variety of molecules are commonly used as binding agents including, but not limited to; protein, peptide, DNA, R A, or ligand to name only a few.
  • capture antibody as used herein means the binding agent typically coated on a solid- phase support in sandwich immunoassay procedures. This will then bind one or a specific group of molecules in the solution passed through.
  • detection antibody means the binding agent which binds to the specific analyte. It typically binds to the analyte at different binding sites from the capture antibody, so that it does not interact with with the capture antibody. Typically, the detection antibody will either bind with the analyte already bound to the capture antibody, or be mixed and bound with analyte before the capture antibody is introduced.
  • reporter agent means the molecule(s) or small particles labeled on the detection antibody, which is configured to be detectable by one of many detection techniques such as optical (fluorescence or chemiluminescence or large -area imaging), electrical, magnetic or other means. A wide variety of material are commonly used as report agents including, but not limited to; fluorescence molecule, enzyme, nano particle, to name only a few. The report agent can be attached on the detection antibody directly, or through another binding agent bound to the detection antibody.
  • microchannel refers to a groove or plurality of grooves created on a suitable substrate with at least one of the dimensions of the groove in the micrometer range. Microchannels can have widths, lengths, and/or depths usually ranging from 1 ⁇ to 1000 ⁇ . It should be noted that the terms “channel” and “microchannel” are used interchangeably in this description. Microchannels can be used as stand-alone units or in conjunction with other microchannels to form a network of channels with a plurality of flow paths and intersections.
  • microfiuidic as used herein generally refers to the use of microchannels for transport of liquids or gases.
  • microfiuidic system consists of a multitude of microchannels forming a network and associated flow control components such as pumps, valves and filters.
  • Microfiuidic systems are ideally suited for controlling minute volume of liquids or gases.
  • microfiuidic systems can be configured to handle fluid volumes ranging from picoliter to milliliter range.
  • This invention provides novel methods for extending the detection range (dynamic range) of immunoassays and other assays, and is particularly suitable for such assays that are performed on point-of-care test devices.
  • the invention also provides methods for performing such
  • the present invention provides novel assay methods that in particular can be used in conjunction with quantitative sandwich immunoassay based test devices to extend the dynamic detection range for a target analyte.
  • two or more detection chambers are arranged in a serial configuration such that each downstream detection chamber has capture antibodies with progressively higher affinity to the target analyte.
  • a target analyte or (target analyte + detection antibody conjugate) sequentially encounters the multiple serially configured detection chambers.
  • the assay method relies on difference in binding affinity of the capture antibodies such that the first detection chamber with the lowest affinity capture antibody will only capture the analyte or (target analyte + detection antibody conjugate); such that the assay signal is above a pre-defined threshold; if the analyte is present in high concentrations. In this case, i.e. with very high analyte
  • the second downstream detection chamber with a higher (than first) affinity capture antibody will capture the analyte or (target analyte + detection antibody conjugate); such that the assay signal is above a pre-defined threshold; also in cases where the analyte is present in concentrations lower than can be detected by the first capture antibody. In this case, the second and all further downstream detection chambers will produce a detectable signal.
  • the third downstream detection chamber with a higher (than first and second) affinity capture antibody will capture the analyte or (target analyte + detection antibody conjugate); such that the assay signal is above a pre-defined threshold; also in cases where the analyte is present in
  • the third and all further downstream detection chambers will produce a detectable signal and so forth.
  • the amount of analyte or (target analyte + detection antibody conjugate) captured by the various capture antibodies; and effectively the signal from the various detection chambers will be proportional to the concentration of the analyte in solution and also be proportional to the binding affinity of the capture antibodies.
  • the quantitative signal from all the detection chambers will be monitored simultaneously after the assay sequence is completed.
  • the target analyte is present at low concentrations it can only be detected by the most downstream detection chamber with the highest affinity capture antibody such that the assay signal is above a specific threshold.
  • the signal from the most downstream chamber will be used to quantify the analyte concentration using a stored calibration table.
  • the analyte concentration is higher than the first instance and such that the assay signal is above a specific threshold in the last and second to last downstream detection chambers; only the signal from the second to last downstream detection chamber will be used to quantify the analyte concentration.
  • the signal from the last downstream detection chamber will be ignored and not used in the analysis.
  • the analyte concentration when the analyte concentration is higher than the second instance and such that the assay signal is above a specific threshold in the last and second to last and third to last downstream detection chambers; only the signal from the third to last downstream detection chamber will be used to quantify the analyte concentration. In this third instance, the signal from the last and second to last downstream detection chamber will be ignored and not used in the analysis.
  • This concept can be extended to the last case wherein the analyte concentration is high enough that even the first detection chamber produces an assay signal above a specific threshold. In this case, only the signal from the first detection chamber will be used for quantifying the analyte concentration and signals from all other downstream chambers will be ignored.
  • the assay device By ensuring that the assay signal from only the "most upstream" detection chamber is the signal that is above a set threshold, the cross-talk effect can effectively be eliminated and the assay device provided by the invention thus yields reproducible signals. Furthermore, the use of a single flow path simplifies the device configuration and removes uncertainties associated with variations of different flow paths.
  • the above described method of the present invention can be seen to be a novel and effective approach to extending the dynamic detection range of assays, particularly in point-of- care test devices, while maintaining an overall relatively non-complex assay device
  • FIG. 1 shows a schematic sketch of the preferred embodiment of an assay device in accordance with the present invention
  • FIG. 2 shows schematic sketches of a prior art assay device which can also benefit from being improved by the assay methods provided by the present invention
  • FIG. 3 shows the theoretical calculation results for the "cross-talk" effect when two capture antibodies of similar affinities are arranged in a serial configuration
  • FIG. 4 shows concept validation results on a microplate format and effect of setting thresholds on assay sensitivity and dynamic range.
  • FIG. 5 shows test results for the present invention, using a microfluidic lab-on-a-chip for a wide dynamic range assay
  • this invention provides assay methods and devices which are particularly advantageous for improving sandwich immunoassay methods for a target analyte (for example, HCG).
  • a target analyte for example, HCG
  • this invention is by no means limited to this particular assay methodology or analyte, or even to a particular class of analytes.
  • FIG. 1 shows a schematic sketch of the preferred embodiment of a microfluidic lab-on-a-chip (“biochip”) that can be used to implement the assay methods of the invention disclosed herein.
  • the biochip consists of multiple liquid reagent reservoirs connected on one end to a series of detection chambers and on the other end to a solid propellant actuator (SPA) module. The other end of the series of detection chambers is connected to a waste chamber.
  • the detection chambers are loaded with beads coated with the capture antibody.
  • the capture antibody can be directly coated on the surface of the microfluidic channel forming the detection chamber via covalent, hydrophobic binding or other suitable methods.
  • the beads can be magnetic beads that injected and then entrapped in position by an array of permanent magnets or electromagnets.
  • a wide variety of techniques are well-known and practiced in the art to deposit and/or link antibodies to a solid-phase support. All these techniques are considered within the scope of this invention insofar as the technique is amenable to deposition of different capture antibodies, or mixtures thereof, in the various detection chambers.
  • the techniques for depositing and/or linking the capture antibody can include any and all techniques that can deposit and/or link the capture antibody within a enclosed chamber; microfluidic or otherwise; as well as all techniques that can deposit and/or link the capture antibody on an open surface; for instance as in the case of spray coating capture antibody lines for LFA devices.
  • the beads can be the Ultralink Biosupport beads.
  • the essential requirement for the beads is reasonably uniform size distribution and the ability to bind the capture antibody; preferably in a non -reversible format although methods such as non-specific adsorption can also be acceptable.
  • beads or other polymeric/non-polymeric supports can be used for this device.
  • the beads are suspended in a solution; linked to the capture antibody, and then the functional capture sites of the beads are de-activated using standard protocols. Finally, the beads are rinsed and re-suspended in a buffer medium suitable for the linked capture antibody stability.
  • the bead solution (with capture antibody linked to the beads) is then injected into the detection chamber of the biochip.
  • the detection chamber fluidic structure is configured such that when the beads are injected from one end, they are trapped by a narrow constriction section at the terminal end. An excess of bead solution is injected into each detection chamber to ensure that the detection chamber is completely packed with beads. Importantly, this configuration feature ensures that different types of beads and/or beads with different molecules on the surface can be independently and separately loaded into the multiple detection chambers.
  • the beads After loading the beads in the detection chamber, they are still surrounded by the solution used to suspend them in the final stage. Based on the inventor's experience, this ensures better stability for the linked capture antibody; however, if applicable the solution surrounding the beads can be expelled after the beads are loaded into the detection chamber.
  • Each chamber can have beads with a different capture antibody coated on them.
  • each detection chamber can have a different capture antibody for HCG.
  • the capture antibodies are selected such that they bind preferably to the beta chain of the HCG (beta-HCG) molecule.
  • the capture antibodies can also be targeted to the alpha chain of the HCG molecule although this configuration is less preferred. This is to ensure that the capture antibodies do not capture other molecules such as the Luteinizing Hormone (LH) which has an identical alpha chain as the HCG molecule.
  • the capture antibodies can be selected such that a few of the capture antibodies target the whole (or intact) form of HCG whereas other antibodies target the hyperglycosylated form of HCG.
  • Detecting the normal and hyperglycosylated forms of HCG can yield a more accurate diagnosis of early pregnancy.
  • the test targets other molecules, for example such as Thyroid Stimulating Hormone (TSH)
  • Thyroid Stimulating Hormone (TSH) antibodies with selectivity to that target analyte can be used.
  • the various detection chambers can contain beads coated with capture agent for different molecules; for example when the capture antibody in one detection chamber is targeted towards beta-HCG and the capture antibody in another chamber is targeted towards progesterone.
  • the capture antibodies targeted towards the beta chain of the HCG molecule can further be differentiated by their affinity towards beta-HCG.
  • the antibodies can target different epitopes of beta-HCG to ensure that the there is a distinct difference in the affinity of the antibodies towards beta-HCG.
  • the assay reagents loaded and stored on-chip include the detection antibody solution, washing buffer, and chemiluminescence substrate.
  • the detection antibody is labeled with preferably an enzyme label.
  • the detection antibody can have an Alkaline Phosphates (AP) label although other enzymes; such as Horse Radish Peroxidase (HRP) or even other non-enzymatic labels can be used; depending on choice of chemiluminescence substrate.
  • AP Alkaline Phosphates
  • the reagents loaded on the chip can vary per the detecting scheme; for instance in the case of fluorescence detection only the detection antibody and washing buffer can be present.
  • the reservoir configured to hold the blood sample can remain empty during manufacture of the test device.
  • the biochip can be further enclosed in a cartridge (not shown in FIG. 1).
  • the cartridge can hold the SPA and the SPA can be in fluidic connection to the biochip using an appropriate interface.
  • the chip and cartridge assembly can then be packed in an appropriate pouch and stored at a pre-defined temperature until the test is to be used.
  • the user can add a controlled volume of blood sample into a cavity on the cartridge.
  • This cavity can be in fluidic connection to the biochip and the blood can be transported to the biochip and fill the sample reservoir.
  • whole blood sample can be added to the test device to minimize sample prep steps required by the user.
  • the device configuration is equally amenable to other samples such as serum, plasma, urine, saliva, sweat or other bodily fluids.
  • the test device can also be used for analyzing non-human samples such as those from veterinary sources. Following the sample loading step, the sample loading port can be sealed and the test device can be inserted into an appropriate reader.
  • the reader can verify the test device and initiate the assay operational sequence.
  • the assay sequence is initiated by activating a heater within the reader.
  • the reader can be in close proximity and in thermal contact with the area of the biochip that encapsulates the SPA.
  • Applying heat to the SPA causes dissociation of the SPA chemical.
  • the gas released by the thermal decomposition of the SPA can result in an increase in the pressure within the SPA chamber and after the pressure crosses a certain threshold; as defined by the microfluidic biochip configuration; the serially connected assay reagents can be transported towards the detection chambers.
  • the waste chamber configuration can include an air-vent to ensure that as the liquids are transported to the waste chamber there is no build up of pressure within the device.
  • the entire liquid column and intervening air gaps can start moving towards the detection chambers and subsequently to the waste chamber.
  • the liquid surrounding the beads in the detection chambers can be sequentially expelled and transported to the waste chamber.
  • the detection antibody solution can be transported to the detection chambers.
  • detection antibody solution As the detection antibody solution is transported through the detection chambers a certain amount of detection antibodies can link with the analyte captured by the capture antibodies.
  • the amount of detection antibody captured in the different detection chambers can be proportional to the amount of the analyte captured in the detection chambers.
  • the detection antibody solution can consist of one detection antibody with high affinity towards a particular binding site of the analyte or it can consist of a mixture of detection antibodies each of which binds to a different site on the target analyte.
  • washing buffer can remove any excess and/or unbound detection antibody and/or analyte from the detection chambers.
  • test device and more specifically the SPA is configured such that the flow can stop when the
  • the flow sequence can be configured such that even the
  • the chemiluminescence substrate is expelled from the detection chamber to the waste chamber. In the latter embodiment, it has been observed that it is impossible to flush out all the chemiluminescence substrate (or for that matter any liquid) completely from the packed bead column.
  • the residual chemiluminescence substrate solution can also generate an optical signal.
  • the chemiluminescence substrate can interact with the enzyme label on the detection antibody and generate a light signal.
  • the signal can be proportional to the concentration of the analyte captured in the various detection chambers and in the preferred embodiment the signal can be of varying intensity from each of the detection chambers.
  • the device in another embodiment, can be configured such that a start -stop flow sequence is possible. Specifically in Step 3, when the sample is transported through the detection chambers, and Step 4, when the detection antibody solution is transported through the detection chambers, the flow can be stopped to allow additional incubation time for the analyte and detection antibody respectively.
  • the illustrative device configuration shows a test device in accordance with the present invention with five serially arranged detection chambers. The number of detection chambers can be varied depending on the need for detection range and the device can include more or less detection chambers.
  • the device can contain multiple detection chambers and only a portion of these detection chambers are used for the analyte test whereas other detection chambers are empty (not filled with packed bead column). In yet another embodiment, the device can contain multiple detection chambers and only a portion of these detection chambers are used for the analyte test whereas other detection chambers are packed with dummy bead columns that do not contain any capture antibodies linked to the surface. In yet another embodiment, the device can contain multiple detection chambers and only a portion of these detection chambers are used for the analyte test whereas other detection chambers are used to verify the assay flow sequence as control chambers.
  • control chamber can be wherein the detection chamber is packed with beads that are linked to a capture antibody not suitable to capture the target analyte.
  • the control chamber can contain beads linked with an anti-(detection antibody) antibody.
  • the control chamber can be the last chamber (most downstream) to ensure that the assay performance can be detected at all of the preceding chambers. Techniques for use of control zones are well known in the art, and any combination thereof can be used herein.
  • this assay sequence is very similar to the assay sequence performed by a Lateral Flow Assay (LFA) or a Through Flow Assay (TFA) (or membrane assay) device.
  • LFA Lateral Flow Assay
  • TFA Through Flow Assay
  • the device shown in FIG. 2 has a very similar flow sequence. Briefly, in this device - the sample is added at the sample pad whereupon it is transported by capillary action to the conjugate pad. If the sample is a whole blood sample, the sample pad additionally filters out the cellular components and only transports the serum/plasma further.
  • the conjugate pad contains the detection antibody (linked to an appropriate reporter molecule). As the sample passes through the conjugation pad, the detection antibody is reconstituted and links to the target analyte within the sample.
  • the conjugate is then further transported by the flow substrate and passes the detection zones.
  • the detection zones contain appropriate capture antibodies which entrap the (analyte + detection antibody) conjugate.
  • the sample solution continues to flow in the flow substrate till it also reaches the control zone which performs similarly as described above.
  • the sample continues to flow and is collected in the absorbent pad and the continued flow removes excess analyte and excess unbound detection antibody.
  • the test strip can then be interrogated using a wide variety of techniques well known in the art.
  • the capture antibodies are chosen such that in instances when the analyte concentration is very low only the highest affinity capture antibody; located in the most downstream chamber; can capture sufficient number of analyte molecules to generate a detectable response.
  • the target analyte concentration in the sample solution increases capture antibodies with lower affinities; in successively upstream detection chambers; also capture sufficient target analyte to generate a detectable response.
  • the most upstream detection chamber and capture antibody therein is configured such that a detectable signal is only observed in this chamber at the high end of the target analyte concentration.
  • the most downstream detection chamber and capture antibody therein is configured such that only this detection chamber produces a detectable signal at the low end of the target analyte concentration.
  • the first element is the relative positions or configuration of the serially configured detection chambers with varying affinity antibodies; specifically from lowest to highest affinity.
  • the second element is the method used to interpret the immunoassay data.
  • Detection chamber 1 is located upstream such that all assay reagents first pass through detection chamber 1 and then flow into detection chamber 2 which is located downstream of chamber 1.
  • the detection chambers are loaded with a packed bead column as described earlier wherein the bead packing density is equivalent in both chambers.
  • beads in both chambers are coated with an identical capture antibody such that both detection chambers are essentially identical in all respects. In this case:
  • Detection antibody concentration 1000 ng/ml
  • the concentration of captured detection antibody in detection chamber 1 can be calculated, which in turn will produce the light signal.
  • equations, constant, and capture antibody concentration are same as those for chamber 1..
  • the effective antigen concentration is the original target antigen concentration minus the antigen captured by chamber 1.
  • the effective detection antibody concentration is the original detection antibody concentration minus the detection antibody captured by chamber 1.
  • the concentration of captured detection antibody in detection chamber 2 can be calculated, which in turn will produce the light signal.
  • the capture and detection antibodies are both typically at much higher concentrations than the antigen, the difference is signal is not very pronounced at low antigen concentrations. However, especially at high antigen concentrations a significant number of antigen molecules are captured in chamber 1 , and these in turn bind a significant number of detection antibody molecules. As a result there is a significant decrease in the detection antibody concentration as it passes through chamber 1 and a lower concentration reaches chamber 2. The combined effects of depletion of the antigen and the detection antibody lead to a reduction in signal from detection chamber 2. As shown in FIG. 3B, the effect is also present at low antigen concentration but at a much reduced scale. This effect would be even further exacerbated if the capture antibody in detection chamber 1 has a higher affinity to the antigen than the antibody is the second detection chamber.
  • One method of accomplishing this objective is by positioning the lowest affinity capture antibody in the first (most upstream detection chamber) and capture antibodies with increasing affinities in the second and subsequent downstream detection chambers.
  • a signal from the downstream chamber should only be used if the signal from the first (or other upstream chambers) is approximately equal to the "background" signal.
  • the background signal is generated primarily by non-specific adsorption of the detection antibody and is relatively constant for a given assay device configuration under controlled operating conditions. If the signal from a detection chamber is close to the background signal it indicates that there is very little to none capture of the target analyte. Hence, almost all the target analyte is being delivered to the downstream chambers ensuring that the response of the assays in the downstream chambers is indeed in response to the "true" target analyte concentration.
  • the second element of this invention namely the method to interpret the assay data can be understood better by the following example.
  • an extended range assay for Myoglobin was conducted on a 96 well microplate format. As explained earlier, in a 96 well format, all the assays are essentially independent of each another and there is no
  • Example 1 96-well sandwich immunoassay for Myoglobin
  • the highest affinity antibody (G-125-C) is added in wells collectively called Well 3. The signal from all the wells is averaged and interpreted as a single value.
  • the medium affinity antibody (M236) is added in wells collectively called Well 2. The signal from all the wells is averaged and interpreted as a single value.
  • the lowest affinity antibody (7001) is added in wells collectively called Well 1.
  • the signal from all the wells is averaged and interpreted as a single value.
  • Step 11 will only include one (unknown) sample concentration.
  • Step 11 will only include one (unknown) sample concentration.
  • the results (calibration curves) from the assay test are plotted in FIGS. 4 A, 4B and 4C. Note that the data is identical in all three figures, and the only difference is the manner in which the data is interpreted. As expected, the assay response curves for all three antibodies are similar and are offset on the X-axis indicating different sensitivity towards the analyte. In conventional immunoassay analysis techniques, the detection range for any one of the antibodies would be interpreted as the lowest detectable signal to the highest responsive signal. The lowest detectable signal can be the limit of detection (LoD) or the limit of quantitation (LoQ) or can be another value commonly accepted in practice.
  • LiD limit of detection
  • LoQ limit of quantitation
  • the highest responsive signal would be the value of the analyte concentration wherein further increases in analyte concentration would yield no further increase in assay signal; owing to saturation effects.
  • FIG. 4A there can also be an arbitrarily defined threshold for defining the lower limit of the data. Although not shown and not discussed further, it is also evident that a similar rationale can apply for the higher limit.
  • the detection threshold is set at 300,000 units. Using this detection threshold, the detection ranges for the three antibodies can be interpreted as:
  • ⁇ Detection range of lowest affinity antibody approximately 250 ng/ml to 28,000 ng/ml
  • Detection range of highest affinity antibody approximately 9.5 ng/ml to 50 ng/ml
  • the data interpretation method for an unknown sample can further be defined such that: "Signal from a particular well will only be used for analysis if (a) the signal from the well in question is at least above the pre-defined threshold AND (b) the signal from all wells of lower value is lower than the threshold". Hence in this illustrative example when testing for an unknown sample; signal from well 3 (highest affinity antibody) would only be used for analysis if it crosses the
  • 300,000 unit threshold (analyte concentration > 9.5 ng/ml) AND signal from well 2 is lower than 300,000 units (analyte concentration ⁇ 50 ng/ml). Beyond analyte concentrations of 50 ng/ml; only signal from Well 2 (mid affinity antibody) would be used until Well 1 (lowest affinity antibody) also generates a signal higher than 300,000 units (analyte concentration > 250 ng/ml). Beyond analyte concentrations of 250 ng/ml only signal from Well 1 would be used for data analysis. Hence, in this method signal from unknown concentration of analyte is ONLY acquired from one well and signals from other two wells are neglected. By using one of the three wells, the TOTAL detection range is higher than that of any one well.
  • the detection range is thus also governed by the threshold value.
  • reducing the threshold from 300,000 to 100,000 units changes the TOTAL detection range to 5 ng/ml to 28,000 ng/ml.
  • it can seem advantageous to use the lowest possible threshold it might not always be the optimal choice.
  • reducing the threshold to 100,000 reduces the dynamic operating range of Well 3 (highest affinity) and Well 2 (mid level affinity) and increase the dynamic operating range required from Well 1 (lowest affinity).
  • this is possible by comparing unknown sample data with calibration data generated for each experiment - however in a point-of-care test extending the dynamic range presents challenges for repeatability.
  • setting the detection threshold is a compromise between detection sensitivity and detection range and will need to be optimized for each individual assay.
  • Another method of using the threshold value is to set a different threshold for each well. As shown in FIG. 4C, three different threshold levels are set for each of the wells. In this case, the data interpretation method would be slightly modified so that signal from a particular well will only be used for analysis if (a) the signal from the well in question is at least above the predefined threshold for the given well ; and (b) the signal from all wells of lower value is lower than the threshold of those wells. By employing this variation, the total detection range is now approximately 1 ng/ml to 28,000 ng/ml. Comparison with total ranges of FIG. 4A and FIG.
  • the device shown in FIG. 1 is used for this example.
  • the device assembly and loading sequence is followed as described previously.
  • the reagent preparation is described below:
  • Coating incubation weigh 20 mg dry beads (Ultralink Bio-support, Pierce, 53110) in 2 mL tube, add 200 uL antibody coating solution directly to the dry beads. Briefly vortex sample at medium speed to suspend beads. Incubate for 2 hours at 25°C.
  • 2nd washing resuspend beads in 1.5 mL TBS buffer. Vortex sample at medium speed to resuspend beads in the wash solution and gently rock or rotate sample for 15 minutes. Centrifuge sample at 1,200 x g for 8 minutes to pellet the beads. Remove and discard supernatant.
  • Removing nonspecific bonded protein Resuspend bead pellet in 1.5 mL 1.0 M NaCl, to remove nonspecifically attached protein. Vortex and gently rock or rotate sample for 15 minutes. Centrifuge sample at 1,200 x g for 8 minutes to pellet the beads.
  • Biochip bead and reagent loading
  • Detection chamber These chambers are loaded with micro-beads coated with capture antibody. Note that in this example, for illustration purposes, only 2 of the 5 detection chambers are used. The remaining chambers are left empty. Micro -beads in chamber 1 are coated with low affinity anti-beta HCG antibody (Medix, 5014). Microbeads in chamber 2 are coated with high affinity anti-beta HCG antibody (Calbioreagents, 41-3-9). Chamber 1 is defined as the first chamber following the storage reservoir for sample as shown in FIG. 1. Chamber 2 is defined as the next adjoining detection chamber which is downstream of Chamber 1.
  • Detection antibody solution Reservoir filled with 5 ⁇ ⁇ AP conjugated anti-beta HCG antibody (antibody: Medix, 5008; AP conjugation service: Columbia Biosciences), diluted to 3 ⁇ g/ml in buffer. Washing buffer: Reservoir filled with 70 ⁇ _, TBS buffer solution, PH 8.0 (Sigma, T6664).
  • AP substrate 30 ⁇ ⁇ AP chemiluminescence substrate (Ultrasensitive 450nm AP
  • Sample 15 ⁇ of sample (or calibration) solution is added in reservoir prior to test.
  • the optical signal was measured by a custom-configured photodetection unit designed for this experiment, although commercially available instruments such as the GlorRunner luminometer can also be used by suitable modifications that one skilled in the art can understand and readily accomplish.
  • the test device was first calibrated for HCG concentrations ranging from 1.7 ng/ml to 300 ⁇ g/ml.
  • a calibration curve was developed for each of the two antibodies as shown in FIG. 5.
  • the variable threshold method was used to analyze unknown sample concentrations. As shown in FIG. 5, the minimum threshold for chambers 1 and 2 is considerably lower than the set threshold.
  • the minimum threshold was defined as:
  • the detection range for HCG was defined as 15 ng/ml to 100,000 ng/ml.
  • the range is selected to simulate the clinically relevant range for HCG from about 10 mlU/ml to about 100,000 mlU/ml; although it can be varied without affecting the principle of this disclosure.
  • the detection threshold for Chamber 1 (low affinity antibody) and Chamber 2 (high affinity antibody) were set to 0.5 and 0.44 units respectively. Based on these thresholds, the detection range for Chamber 2 (high affinity, downstream) is about 15 ng/ml to about 3,700 ng/ml and the detection range for Chamber 1 (low affinity, upstream) is about 3,700 ng/ml to about 100,000 ng/ml.
  • the data interpretation rule is defined as: "Signal from a chamber 2 will only be used for analysis if (a) the signal from chamber 2 is at least above the pre-defined threshold for chamber 2 and (b) the signal from chamber 1 is lower than the threshold of chamber 1". Hence, for unknown analyte concentration analysis, the following cases are possible:
  • the data interpretation rule defines that signal from Chamber 2 (low concentration range of analyte) is only used when the signal from Chamber 1 is below 0.44 units. Furthermore, at the lower limit of analyte concentrations that can be detected in Chamber 2 (about 15 ng/ml); the signal from chamber 1 is approximately the same as the background signal. This indicates that little or none of the analyte is being depleted in Chamber 1 and essentially almost all the analyte is delivered to Chamber 2. At the high end of the analyte concentrations that can be detected in Chamber 2 (about 3,700 ng/ml); the signal from chamber 1 is higher than the background signal but still considerably lower than the signal from Chamber 2.
  • this method of the instant invention allows for detection of a target analyte across a wide detection range while ensuring that the results are accurate and repeatable.
  • the device and the assay method disclosed herein thus offer an improved method for point-of-care test devices using immunoassay based detection approach to detect a target analyte across a wide dynamic range while maintaining an easy and reliable device configuration and accurate and repeatable assay performance.
  • the assay device of the invention is inherently well suited to wide dynamic range detection, owing to its ability to avoid the hook effect by avoiding mixture of the analyte solution with detection antibody solution.
  • the device and more specifically the assay method are explicitly developed for quantitative detection application and point-of-care test devices using qualitative detection approach are not envisioned to benefit from this invention.
  • this assay method and device of this invention are well suited for immunoassay based diagnostics of molecules such as HCG and Myoglobin.
  • the examples are by no means intended to limit the application of this invention or the assay methods and devices contemplated thereby.
  • a wide variety of protocols based on the sandwich immunoassay principle can be practiced using this invention.
  • the sandwich assay protocol can be used to analyze other molecules such as drugs, haptens, nucleic acids to name a few.
  • the device configuration can be modified to include multiple "lanes" wherein each lane is similar to the structure described above and the modified device can be used to even further extend the detection range for one analyte, or to achieve narrow discrimination within a limited range by increasing the number of detection ranges.
  • a similar concept can be used to simultaneously detect two or even more target analytes all across a wide dynamic range, more than would be possible by detecting in a single chamber.
  • a similar concept can be used to simultaneously detect two or even more target analytes wherein only analyte needs to be detected across a wide dynamic range, more than would be possible by detecting in a single chamber; whereas other analytes can be detected across a narrow range by monitoring the signal from only one chamber.

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Abstract

L'invention concerne des procédés d'immunoessai pour étendre la plage de détection d'un analyte en utilisant deux chambres de détection ou plus agencées en série ; chaque chambre de détection offrant une réponse différente à l'analyte cible. La réponse d'une des multiples chambres de détection est utilisée pour déterminer la concentration d'un analyte dans un échantillon, en utilisant des données d'étalonnage stockées.
PCT/US2012/033252 2011-04-12 2012-04-12 Procédé d'essai pour plage dynamique étendue Ceased WO2012142242A2 (fr)

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