Patent Application
20210364506
The present disclosure relates to methods for detecting an amount of an analyte in a solution, for example, methods for detecting an amount of an analyte in an immunoassay.
Suboptimal performance of protein assays is often due to sample complexity and non-specific interactions, especially for some immunogenicity assays and cytokine assays. For example, in an immunogenicity assay, drugs can bind to anti-drug antibodies (ADA), thereby forming drug-ADA complexes which can adversely affect an accurate quantification of the concentration of the ADA or the drug. In addition, cytokines and protein biomarkers often have native binding partners in blood and serum, and complexes can also be formed in an assay to affect an accurate detection of a target analyte. A pH treatment step can be performed to break up such complexes and other undesired interactions prior to assaying a sample. However, this step is often performed manually, which is not ideal for accurately measuring the concentration of a target analyte in the sample.
According to one embodiment, a method for detecting an amount of an analyte in a solution is disclosed. The method may include adding an electrochemically active agent to the solution containing molecules other than the electrochemically active agent. The electrochemically active agent can generate or consume hydrogen (H+) ions in the solution through an electrochemical redox reaction. The method may also include applying an electrical current to a working electrode in contact with the solution containing the electrochemically active agent to initiate the electrochemical redox reaction to change a pH of the solution from a first pH value to a second pH value, where the second pH value may be different from the first pH value. The method may further include incubating the solution at the second pH value to allow the analyte to dissociate from the other molecules in the solution, where the incubating step may further include incubating the solution at the second pH value to allow the analyte to bind to a capture molecule connected to the working electrode via a linker. The method may also include reacting a detecting probe with the analyte to allow the detecting probe to bind to the analyte. The detecting probe may have a signaling tag attached thereto and configured to produce a signal at the second pH value. The method may further include collecting the signal to calculate the amount of the analyte in the solution.
According to another embodiment, a method for detecting an amount of an analyte in a solution is disclosed. The method may include adding an electrochemically active agent to the solution containing molecules other than the electrochemically active agent. The electrochemically active agent can generate or consume hydrogen (H+) ions in the solution through an electrochemical redox reaction. The method may also include applying a first electrical current to a working electrode in contact with the solution containing the electrochemically active agent to initiate a first electrochemical redox reaction to change a pH of the solution from a first pH value to a second pH value, where the second pH value may be different from the first pH value. The method may further include incubating the solution at the second pH value to allow the analyte to dissociate from the other molecules in the solution. The method may also include applying a second electrical current to the working electrode to initiate a second electrochemical redox reaction to change the pH of the solution from the second pH value to a third pH value, where the third pH value may be different from the second pH value. The method may further include incubating the solution at the third pH value to allow the analyte to bind to a capture molecule connected to the working electrode via a linker. The method may also include reacting a detecting probe with the analyte to allow the detecting probe to bind to the analyte. The detecting probe may have a signaling tag attached thereto and configured to produce a signal at the third pH value. The method may further include collecting the signal to calculate the amount of the analyte in the solution.
According to yet another embodiment, a method for detecting an amount of an analyte in a solution with a first pH value is disclosed. The method may include adding a detecting probe to the solution. The detecting probe may have a signaling tag attached thereto and configured not to produce a signal at the first pH value. The method may also include adding an electrochemically active agent to the solution containing molecules other than the electrochemically active agent and the detecting probe. The electrochemically active agent can generate or consume hydrogen (H+) ions in the solution through an electrochemical redox reaction. The method may further include applying a first electrical current to a working electrode in contact with the solution to initiate a first redox reaction to change a pH of the solution from the first pH value to a second pH value, where the second pH value may be different from the first pH value. The method may also include incubating the solution at the second pH value to allow the analyte to dissociate from the other molecules in the solution and to bind to a capture molecule connected to the working electrode via a linker and to the detecting probe. The signaling tag attached to the detecting probe may be configured to produce a first signal at the second pH value. The method may further include collecting the first signal to calculate a first amount of the analyte in the solution. The method may also include applying a second electrical current to the working electrode in contact with the solution to initiate a second redox reaction to change the pH of the solution from the second pH value to a third pH value, where the third pH value may be different from the second pH value. The method may further include incubating the solution at the third pH value, where the signaling tag may be configured to produce a second signal at the third pH value. The second signal may be stronger than the first signal. The method may also include collecting the second signal to calculate a second amount of the analyte in the solution, where the second amount may be higher than the first amount.
Embodiments of the present disclosure are described herein. It is to be understood, however, that the disclosed embodiments are merely examples and other embodiments can take various and alternative forms. The figures are not necessarily to scale; some features could be exaggerated or minimized to show details of components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the embodiments. As those of ordinary skill in the art will understand, various features illustrated and described with reference to any one of the figures can be combined with features illustrated in one or more other figures to produce embodiments that are not explicitly illustrated or described. The combinations of features illustrated provide representative embodiments for typical applications. Various combinations and modifications of the features consistent with the teachings of this disclosure, however, could be desired for applications or implementations.
This present disclosure is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing embodiments of the present disclosure and is not intended to be limiting in any way.
As used in the specification and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.
The description of a group or class of materials as suitable for a given purpose in connection with one or more embodiments implies that mixtures of any two or more of the members of the group or class are suitable. Description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description and does not necessarily preclude chemical interactions among constituents of the mixture once mixed.
Except where expressly indicated, all numerical quantities in this description indicating dimensions or material properties are to be understood as modified by the word “about” in describing the broadest scope of the present disclosure.
The first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation. Unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.
Reference is being made in detail to compositions, embodiments, and methods of embodiments known to the inventors. The disclosed embodiments are merely exemplary of the present disclosure which may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, rather merely as representative bases for teaching one skilled in the art to variously employ the present disclosure.
Biotherapeutics, such as proteins and monoclonal antibodies (mAbs), are growing fast on the pharmaceutical market. Currently, over 250 approved biotherapeutics are on the market, and more than 500 are under development. Biotherapeutics, compared to relatively small molecule drugs, are prone to the risk of immune responses. For example, an unwanted immunogenicity may lead to a production of anti-drug antibodies (ADAs), which can negatively impact drug levels in circulation and drug efficacy. In some cases, immune responses can be severe and life threatening. Therefore, to assess the risk of immune responses against biotherapeutics, pharmacokinetics (PK) and pharmacodynamics (PD) characterizations are necessary during pre-clinical development stages and clinical trials.
To characterize PK and/or PD profiles for a biotherapeutic, it is important to accurately measure the concentration of ADAs, drugs, or both in an aqueous environment. However, high drug levels in the aqueous environment can adversely interfere with the detection of ADAs, especially in bridging assay formats. For example, in an immunogenicity assay, ADAs can form complexes with drugs in a sample solution, thereby preventing an accurate quantification of the concentration of the ADAs, the drugs, or both. Such an issue may be more common in multiple-dose studies, where drug concentrations remain high for a significant period of the studies.
To overcome the issue, drug-tolerant assays have been developed. The drug-tolerant assays can measure the concentration of ADAs in the presence of drugs by adopting an acid treatment step. The acid treatment step can break up ADA-drug complexes and release ADAs and drugs to a sample solution. Some examples of the drug-tolerant assays include enzyme-linked immunosorbent assay (ELISA), affinity capture elution ELISA (ACE), biotin-drug extraction with acid dissociation (BEAD) assay, sample pretreatment bridging ELISA, acid dissociation radioimmunoassay, homogeneous mobility shift assay (HMSA), and precipitation and acid dissociation method.
Besides ADAs, soluble drug targets may also present a challenge for immunoassays. When the ratio of drugs to drug targets is low, very few binding sites of the drugs are available to engage the drug targets that are immobilized in an immunoassay. However, when the ratio of drugs to drug targets are high, a reverse effect may be observed (i.e. very few binding sites of the drug targets are available to bind the drugs). Therefore, different ratios of drugs to drug targets in an immunoassay may also have an impact on the accuracy of measuring the PK and/or PD profiles for a biotherapeutic.
To resolve the problem, an acid treatment step has been employed in performing an immunoassay. The acid treatment step may be conducted by modulating a pH of a sample solution, thereby dissociating drugs from drug targets and denaturing drug-binding epitopes on drug targets. The sample solution may then be neutralized and ready for analysis in an ELISA format. This method has been used for the detection of immunoglobulin G1 (IgG1) mAb in the presence of angiopoietin-2 (Ang2).
Similarly, a sample may be subject to a mild acid treatment, followed by a direct analysis on the sample using a sandwich ELISA. This method does not denature drug-binding epitopes on drug targets, but it utilizes the ability of capture antibodies to bind to drugs at a specific pH value where the drugs and the drug targets remain dissociated (i.e. drugs are unbound to drug targets). This method has been applied to the detection of immunoglobulin G4 (IgG4) in the presence of a soluble circulating target.
Further, an improved assay conducted in an acidic pH value has been utilized for the detection of interleukin 13 (IL-13) in human plasma and serum. Specifically, incubation of a sample at the acidic pH value can reduce non-specific binding and interference from IL-13 binding proteins. Examples of biomarkers known to have soluble binding partners in blood and serum are, but not limited to, interleukin 1 (IL-1), interleukin 18 (IL-18), insulin-like growth factor 1 (IGF-1), tumor necrosis factor-beta (TNF-β), and testosterone.
To perform a bridging ELISA protocol, a sample solution may be diluted in acetic acid and then incubated for 1 hour. Thereafter, the solution may be mixed with a basic buffer (e.g. Tris pH 9.5) and then added to an assay plate which contains a surface-bound capture molecule (e.g. antigen). The solution may then be incubated for another 1 to 5 hours, followed by washing and signal amplification steps (e.g. using horseradish peroxidase, or alkaline phosphatase).
In view of the foregoing, the acid treatment step introduced in each method is performed manually. Manual operations can unavoidably introduce human errors, causing a low reproducibility of an assay. Therefore, there is a need to detect an amount of an analyte, such as an ADA, in an immunoassay in a more efficient and accurate manner.
In contrast to manually adjusting a pH of a sample solution, the pH of the sample solution may be modulated automatically. Methods of electrochemically modulating a pH of a sample solution, especially varying the pH of the sample solution near an electrode surface, have been disclosed in U.S. patent application Ser. Nos. 14/792,569 and 13/543,300, which are hereby incorporated by reference in their entirety. Generally, modulating pH electrochemically can give advantages toward performing an assay, including reducing the number of manual steps and reagents, shortening assay times, allowing multiplexing, and improving measurement accuracy. A system for electrochemically modulating a pH of a sample solution may include a working electrode and a counter electrode. The system may also include an electrochemically active agent, such as quinone, which may be added to the sample solution. The electrochemically active agent may undergo an electrochemical redox reaction (i.e. oxidation or reduction) under the influence of an electrical current. The electrochemical redox reaction of the electrochemically active agent may generate or consume hydrogen (H+) ions, causing a pH change in the sample solution. By controlling the electrical current applied to the working electrode, only the sample solution near a surface of the working electrode may undergo the pH change while the pH of other areas of the sample solution that are not near the surface of the working electrode may not be affected. In addition, the system may also include a reference electrode and a sensing electrode. The sensing electrode may actively measure the pH of the sample solution in real time. Signals generated from the sensing electrode may be transmitted to electronics and fed into a closed loop algorithm, which may be used to control the electrical current applied to the working electrode and configured to either change or maintain the pH in the sample solution. Such an automatic method of pH modulation may be applied to analyzing immunoassays and other bioassays which may involve biomarkers that have soluble binding partners.
Aspects of the present disclosure are directed to methods for detecting an amount of an analyte in a solution using an electrochemical pH modulation technology. The solution may include soluble binding partners of the analyte, which may bind to analytes in the solution to form complexes. The soluble binding partners may be drugs in the solution. The solution may also include detecting probes configured to bind to the analytes in the solution for detecting the amount of the analyte in the solution. Each detecting probe may include a signaling tag attached thereto. In one embodiment, aspects of the present disclosure include an acid pre-treatment step where analytes, such as anti-drug antibodies (ADAs), may be dissociated from their binding partners, followed by binding to capture molecules and to detecting probes for detecting an amount of an analyte in a solution. In another embodiment, aspects of the present disclosure include an acid pre-treatment step where analytes, such as ADAs, may be dissociated from their binding partners, followed by electrochemically modulating (e.g. neutralizing) a pH of a solution to allow the analytes to bind to capture molecules and to detecting probes for detecting an amount of an analyte in the solution. In yet another embodiment, aspects of the present disclosure utilize detecting probes having pH-dependent signaling tags attached thereto for detecting an amount of an analyte, such as an ADA, in a solution, where a pH-dependent signaling tag may produce a first signal when the solution is at a first pH value, and may produce a second signal when the solution is at a second pH value different from the first pH value.
The linker 14 is configured to immobilize the capture molecule 10 and to preserve the functionality thereof. Specifically, the linker 14 may be a polymeric material configured to immobilize the capture molecule through adsorption. The polymeric material may be, but not limited to, polystyrene, polymethyl methacrylate (PMMA), polypropylene, cyclic olefin copolymer, agarose, dextran, or nitrocellulose. The linker may also be a peptide sequence or a protein configured to immobilize the capture molecule through affinity. The peptide sequence or the protein may be, but not limited to, streptavidin, protein A, or protein G, an aptamer, or a polyhistidine tag (His-Tag). In addition, the linker may be a polynucleic acid configured to immobilize the capture molecule through hybridization. Further, the linker may be an organic molecule for immobilizing the capture molecule through covalent bonding. The organic molecule may be, but not limited to, maleimide, succinimide, (trifluoromethyl)phenyldiaziridine, amine, hydrazide, boronic acid, iodoacetyl, epoxide, thiol, azide, or alkyne.
The signaling tag 20 may be pH-dependent or pH-independent. Basically, a pH-dependent signaling tag may change its behavior as a function of a pH of a solution. For example, a pH-dependent signaling tag may produce a first signal at a first pH value and may produce a second signal at a second pH value, where the first and the second pH value are different. On the contrary, a pH-independent signaling tag may not change its behavior as a function of a pH of a solution, but rather produce a signal when it presents in a solution.
As to compositions, the signaling tag 20 may be a fluorescent tag, a fluorescent dye, a fluorescent protein, an electroluminescent dye, a chemiluminescent dye, or an enzyme.
Specifically, fluorescent tags can be pH-dependent or pH-independent, which may be, but not limited to, organic dyes, quantum dots, lanthanide ions, or proteins. Fluorescent dyes may be, but not limited to, fluorescein, coumarin, rhodamin, cyanine, or derivatives thereof. Examples of commonly used fluorescent dyes include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Alexa Fluor, Cy-3, Cy-5, and ATTO dyes. Examples of pH-dependent fluorescent dyes may include, but not limited to, pHrodo, Protonex, Oregon Green, LysoSensor Green, pHAb, fluorescein, FAM, rhodamine B derivatives, and SNARF. In addition, fluorescent proteins can be pH-dependent or pH-independent, which may be, but not limited to, green fluorescent proteins, yellow fluorescent proteins, or cyan fluorescent proteins. Further, electroluminescent dyes may be fluolid dyes, such as Fluolid-Green, Fluolid-Orange, and Fluolid-Red. Chemiluminescent dyes may be, but not limited to, luminol, luciferin, or xanthene dyes. Examples of pH-dependent enzymes include horseradish peroxidase (HRP), glucose oxidase, and alkaline phosphatase.
Referring to
Referring to
HQ→BQ+2H++2e− (1)
The oxidization reaction of HQ may thus modify the pH of the solution, especially an area in the solution that is close to the electrode 56, from pH 7 to pH 4. Incubating the solution at pH 4 may allow the analyte 50 to be dissociated from the binding partners 52.
Other electrochemically active agents may also be utilized for electrochemically modulating a pH of a solution, such as quinone, catechol, aminophenol, hydrazine, naphthoquinone, derivatives thereof, or combinations thereof. Each of the electrochemically active agents may undergo an electrochemical redox reaction (i.e. oxidation or reduction) under the influence of an electrical current, which may either generate or consume H+ ions in the solution for modulating the pH of the solution.
To accurately measure the amount of the analyte 74 in the assay, an excess amount of detecting probes 82 may be added to the solution at pH 4. Each detecting probe 82 may have a signaling tag 84 attached thereto and configured to produce a signal at pH 4. As shown in
To afford optimal bindings between the analyte 104 and a capture molecule 108 in the assay, and between the analyte 104 and a detecting probe 110, the pH of the solution may be further adjusted. As shown in
BQ+2H++2e−→HQ (2)
Thereafter, the solution may be transferred to an assay chamber, where the analyte 104 may bind to the capture molecule 108. The capture molecule 108 may be attached to a solid substrate 110 in the assay chamber via a linker 112. The linker 112 is configured to immobilize the capture molecule 108 through adsorption, affinity, hybridization, or covalent bonding.
To accurately measure the amount of the analyte 104 in the assay, an excess amount of detecting probes 114 may be added to the solution at pH 7. Each detecting probe 114 may have a signaling tag 116 attached thereto and configured to produce a signal at pH 7. As shown in
To achieve optimal bindings between the analyte 130 and a capture molecule 140 in the assay, and between the analyte 130 and a detecting probe 134, the pH of the solution may be further adjusted. As shown in
In one embodiment, the signaling tag 136 may be pH-independent, where the signaling tag 136 may produce the same signal when the pH of the solution is at either 4 or 7. In this case, upon the completion of binding at pH 7, a wash step may be performed to remove any unbound detecting probes 134 in the solution. The method may then measure the signals produced by the signaling tags 136 remained in the solution and calculate the amount of the analyte 130 in the assay based on the signals.
In another embodiment, the signaling tag 136 may be pH-dependent, where the signaling tag 136 may produce a first signal at pH 4 and may produce a second signal at pH 7, the second signal being different from the first signal. In this embodiment, an optimal pH for the signaling tag 136 to produce a signal is 7. In other words, the second signal produced at pH 7 may be stronger than the first signal produced at pH 4. In this case, upon the completion of binding at pH 7, a wash step may be performed to remove any unbound detecting probes 134 in the solution. The method may then measure the second signal produced by the signaling tags 136 remained in the solution and calculate the amount of the analyte 130 in the assay based on the second signal.
As discussed above, under the influence of an electrical current applied to an electrode in contact with a solution, electrochemically active agents can subsequently modulate a pH of the solution in an automatic manner. The solution may include both HQ and BQ. Therefore, referring to
In this method embodiment, an optimal pH for the analyte 150 to bind to the capture molecule 160 and to the detecting probe 154 may be 7. However, an optimal pH for the signaling tag 156 to produce a signal may be, for example, 8. Therefore, as shown in
Similar to
Thereafter, applying a second electrical current (I2) to the electrode 178 may trigger a second electrochemically active agent (not shown) in the solution to undergo a reduction reaction, thereby changing the pH of the solution at Area III back to pH 7. At this stage, both areas of the solution, Area III and Area IV, are at pH 7, and an analyte 170 may bind to a capture molecule 180 in the assay chamber. The capture molecule 180 may be attached to the electrode 178 via a linker 182. The linker 182 is configured to immobilize the capture molecule 160 through adsorption, affinity, hybridization, or covalent bonding. However, as shown in
To ensure as much as analytes 170 in the solution to be dissociated from their binding partners 172, a third electrical current (I3) may then be applied to the electrode 178 to initiate an oxidation reaction of a third electrochemically active agent (not shown) in the solution. The oxidation reaction may reduce the pH of the solution back to pH 4, especially the solution in Area III that is near to the surface of the electrode 178. Incubating the solution at pH 4 can afford more analytes 170 to be dissociated from the binding partners 172. Thereafter, applying a fourth electrical current (I4) may once again bring the pH of the solution back to pH 7. Such a process of oxidation and reduction reactions of electrochemically active agents may repeat several times to maximize the number of analytes 170 that are not bound by binding partners 172 in the solution such that the analytes 170 are available to be detected by the detecting probes 174.
Referring to
While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms encompassed by the claims. The words used in the specification are words of description rather than limitation, and it is understood that various changes can be made without departing from the spirit and scope of the disclosure. As previously described, the features of various embodiments can be combined to form further embodiments of the present disclosure that may not be explicitly described or illustrated. While various embodiments could have been described as providing advantages or being preferred over other embodiments or prior art implementations with respect to one or more desired characteristics, those of ordinary skill in the art recognize that one or more features or characteristics can be compromised to achieve desired overall system attributes, which depend on the specific application and implementation. These attributes can include, but are not limited to cost, strength, durability, life cycle cost, marketability, appearance, packaging, size, serviceability, weight, manufacturability, ease of assembly, etc. As such, to the extent any embodiments are described as less desirable than other embodiments or prior art implementations with respect to one or more characteristics, these embodiments are not outside the scope of the disclosure and can be desirable for particular applications.
