Method for measuring total protein content and detecting proteins by immunoassay in a microfluidic device

Abstract

Certain embodiments described herein relate to systems and methods operable to combine immunoassays and total protein techniques in a single sample run. Certain embodiments described herein allow for the performance of multiple sequential immunoassays in the same microfluidic device. Certain embodiments described herein relate to stripping reagents operable to remove primary antibodies associated with an immunoassay. Such stripping reagents can allow for additional immunoassays and / or total protein assays to be performed on the same sample.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 010,436, filed April 15, 2020, and U.S. Non-Provisional Patent Application Nos. 16 / 932,441 and 16 / 932,445, filed July 17, 2020. The entire disclosure of each of the aforementioned patent applications is hereby incorporated by reference. Technical Field

[0003] The embodiments described herein generally relate to capillary electrophoresis methods for performing immunoassays and / or protein assays on samples. Stripping reagents are disclosed that are operable to remove antibodies associated with the immunoassay, thereby enabling additional assays on the same sample. Background Technology

[0004] A significant part of protein research involves characterizing proteins in heterogeneous samples, such as cell lysates that can contain thousands of proteins. Western blotting is a commonly used immunoassay-based method for analyzing specific proteins in these complex samples, using the specificity of antibodies to identify proteins of interest. When performing immunoassays in the form of Western blotting, quantifying the resulting immunoassay signal becomes increasingly important. One method for quantification is to normalize the immunoassay signal to the total protein content in the sample. Journals are increasingly requiring this when publishing Western blotting results to ensure data accuracy and precision.

[0005] Existing systems are operable to provide fully automated microfluidic (e.g., capillary-based) immunoassays, such as ProteinSimple's ® Simple Western ® Instruments. Some such systems combine immunoassays with size separations similar to those of conventional gel-based protein blots in capillaries. Samples, separation matrices, stacking matrices, antibodies, and reagents can be loaded automatically. The instrument is operable to sequentially aspirate the separation matrices and stacking matrices into each capillary. Next, a sample, potentially containing a mixture of heterogeneous proteins, can be loaded, and the capillaries can be contacted with a running buffer. A voltage can be applied to enable separation by molecular weight or other suitable characteristics. Once separation is complete, ultraviolet light can immobilize the proteins to the capillary wall. For example, the immobilized proteins and the matrix removed from the capillary can be used for immunodetection of the proteins. Furthermore, some existing systems are operable to provide a “total protein” assay, which can be performed via biotinylation of proteins immobilized to the inner surface of the capillary, followed by detection of horseradish peroxidase (HRP)-conjugated streptavidin and a chemiluminescent reaction.

[0006] However, a method is needed that allows for chemiluminescent detection of immunoassay and total protein readout within the same capillary or other microfluidic device. Additionally, increased reusability beyond the number of detection modes / channels is desirable. Summary of the Invention

[0007] Some implementations described herein relate to systems and methods operable to combine immunoassay and total protein techniques in a single sample run. Instruments with chemiluminescence and fluorescence detection capabilities, such as ProteinSimple... ® Jess ® A “protein normalization” method can be provided in which a fluorescent dye is covalently linked to all isolated and immobilized protein molecules via, for example, NHS-esteramine coupling. In this way, a specific target can be measured, for example, using chemiluminescence associated with an immunoassay, while simultaneously determining the measurement of the total loaded protein from the fluorescence signal. Known techniques for protein normalization typically have a dynamic range similar to typical protein blot immunoassays and cannot be reused in the same capillary used for immunoassays with chemiluminescence detection. In contrast, the protein normalization implementation described herein can be reused in the same capillary as a chemiluminescent immunoassay and can have a reduced or different dynamic range compared to the immunoassay signal.

[0008] In addition to performing total protein and immunoassays in the same capillary or other microfluidic device, certain embodiments described herein allow for multiple sequential immunoassays to be performed in the same capillary. Instruments with chemiluminescence and fluorescence detection capabilities (e.g., ProteinSimple's) ® Jess ® This allows for “reusable” detection, meaning the detection of multiple targets within a capillary using a single mixture of antibodies partially conjugated to those used for chemiluminescence or fluorescence detection. However, combining antibodies in a single mixture limits which antibodies can be mixed, for example, due to nonspecific signals resulting from the cross-reactivity of antibodies used in immunoassays or the incompatible dynamic range of antibodies when used in the same capillary (e.g., the different dynamic range of chemiluminescence versus fluorescence).

[0009] The implementation scheme described herein relates to the application of total protein assays and / or a second immunoassay in the same capillary as a Western blot immunoassay, enabling highly sensitive total protein measurements and immunoassays, combined with the low sample requirements and high throughput capabilities previously demonstrated with simple Western blot techniques. Attached Figure Description

[0010] Figure 1A-1HThe events that occurred in the stripping and immunoassay reprobe method according to one implementation scheme are explained.

[0011] Figure 2A-2H The events that occurred in the stripping and total protein reproducibility method according to one implementation scheme are explained.

[0012] Figure 3 The relationship between stripping efficiency and pH for three different targets was explained.

[0013] Figure 4 The relationship between stripping efficiency and a wide pH range was explained, showing a significant decrease in stripping efficiency at pH > 4.5.

[0014] Figure 5A and 5B The experimental data explaining the reproducibility of the analyte after the introduction of the stripping reagent are explained. Detailed Implementation

[0015] Certain embodiments described herein relate to methods suitable for performing multiple immunoassays on samples separated by electrophoresis via a capillary or other suitable microfluidic device. Samples can be separated such that at least a first analyte and a second analyte are separated into different bands. The first and second analytes can be immobilized in a capillary. A first primary antibody configured to selectively bind the first analyte (and optionally, not the second analyte) can be introduced into the capillary. A first secondary antibody configured to selectively bind the first primary antibody can be introduced into the capillary. The first analyte can be detected based on optical characteristics associated with the first secondary antibody. For example, the first secondary antibody can be conjugated to horseradish peroxidase (HRP), and the first analyte can be detected based on a chemiluminescent reaction associated with HRP. A stripping reagent configured to remove the first primary antibody from the first analyte can be introduced into the capillary. After the stripping reagent is introduced and the first primary antibody (along with the first secondary antibody and / or HRP) is removed, the first and second analytes can remain immobilized in the capillary. A second primary antibody configured to bind the second analyte can then be introduced, for example, after the introduction of a stripping reagent. A secondary antibody configured to bind the second primary antibody can then be introduced, and the second analyte can be detected based on optical characteristics associated with the secondary antibody (e.g., chemiluminescent reaction and / or fluorescent tagging).

[0016] Some embodiments described herein relate to methods suitable for immunoassay and total protein determination of samples separated by electrophoresis via capillary or other suitable microfluidic devices. Analytes from the sample can be separated and immobilized in a capillary. Molecules having reactive portions configured to nonspecifically bind proteins (such as biotin) can be introduced into the capillary. Similarly, and for example, proteins can be biotinylated. Primary antibodies configured to bind at least one subset of analytes can be introduced into the capillary. Secondary antibodies configured to bind the primary antibodies can be introduced. A subset of analytes can be detected based on optical characteristics associated with the secondary antibodies. A stripping reagent configured to remove primary antibodies from the subset of analytes can be introduced. After the stripping reagent is introduced and the primary antibodies (along with the secondary antibodies) are removed, the immobilized analytes can be retained in the capillary. Optically detectable reagents configured to bind molecules can be introduced into the capillary. In embodiments where the molecule is biotin, streptavidin can be introduced. Antibiotic streptavidin can be conjugated to HRP or otherwise made optically detectable. Based on the optical signal associated with the optically detectable reagent, all biotinylated analytes (e.g., all proteins) in the capillary can be detected. Based on the optical signal associated with the optically detectable reagent (e.g., total protein signal), optical features associated with secondary antibodies (e.g., immunoassay signals) can be normalized. In some embodiments, it is important that the events in this section are performed in the order described.

[0017] By eliminating the influence of certain uncontrolled differences between samples from non-study subjects, normalizing the immunoassay signal (or other suitable signal) improves the instrument's and / or analyst's ability to accurately compare measurements from different samples. For immunoassays, normalizing to the total protein content in each sample can eliminate the effects of variability caused by sample composition (e.g., cell count, lysate dilution) or adsorption errors. Furthermore, since the expression levels of housekeeping proteins can be affected by experimental treatments or their immunoassay signals may not be within the same linear dynamic range as target proteins, normalizing to the total protein content is beneficial for normalizing to specific housekeeping proteins (e.g., β-actin or β-tubulin). In one embodiment, normalization is performed as follows: the amount of a specific protein determined by the immunoassay in the capillary is divided by the ratio of the total protein in the capillary to the total protein in a reference capillary.

[0018] Some embodiments described herein relate to the formulation of stripping reagents. Stripping reagents may include buffer solutions, tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP), and detergents. Stripping reagents may have a pH below 5.

[0019] Some embodiments described herein relate to methods for using a stripping reagent comprising TECP and having a pH between 3.0 and 4.5. The stripping reagent can be used to remove a first primary antibody associated with an immunoassay from an analyte. The analyte can be separated and immobilized by electrophoresis in a capillary. The first primary antibody and the first secondary antibody associated with the immunoassay can be introduced into the capillary. After removing the first primary antibody from the capillary using the stripping reagent, an antibiotic streptavidin or another suitable reagent configured to bind a biotinylated protein and / or a second primary antibody configured to bind the analyte in the sample can be introduced into the capillary.

[0020] Stripping and gravity detection methods

[0021] Stripping and gravity detection allow users to analyze the same immobilized protein in the same capillary (or other microfluidic device) and in the same run, saving time, money, and valuable samples.

[0022] Figure 1A-1H The events that occur in a stripping and gravity detection method according to one implementation scheme are explained. Stripping and gravity detection can be used to perform two or more immunoassays sequentially on a single sample (e.g., reuse of isolated and immobilized samples and / or eliminate the need for (re)loading and / or (re)separating additional samples for each immunoassay). Figure 1A This is a schematic diagram of a sample that has been separated and immobilized onto the surface of capillary 110. Analytes can be covalently bound to the surface of capillary 110, for example, using the apparatus and / or methods shown and described in U.S. Patent No. 7,846,676 and / or U.S. Patent Application Publication No. 2008 / 0017512, the entire disclosure of which is hereby incorporated by reference. As shown, the sample has been separated into two bands, 112 and 114. Each band represents a unique analyte species and is present in a unique portion of capillary 110. It should be understood that the sample may contain any number of analyte species and / or be separated into any number of bands.

[0023] As used herein, the term "analyte" means any molecule or compound to be separated by electrophoresis and / or detected using the methods, apparatus, and systems provided herein. Suitable analytes include, but are not limited to, small chemical molecules, such as, for example, environmental molecules, clinical molecules, chemical substances, pollutants, and / or biomolecules. More specifically, such chemical molecules may include, but are not limited to, pesticides, insecticides, toxins, therapeutic and / or abused drugs, antibiotics, organic materials, hormones, antibodies, antibody fragments, antibody-molecule conjugates (e.g., antibody-drug conjugates), antigens, cell membrane antigens, proteins (e.g., enzymes, immunoglobulins and / or glycoproteins), nucleic acids (e.g., DNA and / or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and / or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides (containing ribonucleic acid and / or deoxyribonucleic acid), transition state analogs, inhibitors, receptors, receptor ligands (e.g., neural receptors or ligands thereof, hormone receptors or ligands thereof, nutrient receptors or ligands thereof and / or cell surface receptors or ligands thereof), receptor-ligand complexes, nutrients, electrolytes, growth factors and other biomolecules and / or non-biomolecules, as well as fragments and combinations thereof. In some embodiments, the analyte is a protein or protein complex, and the sample is a cell lysate or purified protein. Other suitable analytes may include aggregates, agglomerates, flocs, and / or dispersed phase droplets or particles of colloids and / or emulsions. Once separated, the “band” of the analyte is referred to herein as an “analyte species.”

[0024] As used herein, the term "sample" refers to a composition containing one or more analytes to be detected. In some embodiments, the sample is heterogeneous, i.e., containing multiple components (e.g., different proteins), or homogeneous, i.e., containing a single component (e.g., a group of proteins). In some cases, the sample can be naturally occurring, biological, and / or artificial materials. Furthermore, the sample can be in a natural (e.g., cell suspension) or denatured form (e.g., lysate). In some cases, the sample can be a single cell (or the contents of a single cell, e.g., as cell lysate from a single cell, or purified protein) or multiple cells (or the contents of multiple cells, e.g., as cell lysate from multiple cells, or purified protein from multiple cells), blood samples, tissue samples, skin samples, urine samples, water samples, and / or soil samples. In some cases, the sample can be derived from a living organism, such as a eukaryote, prokaryote, mammal, human, yeast, and / or bacteria, or the sample can be derived from a virus.

[0025] Samples can be separated using any suitable mobility parameter, such as charge, molecular weight, electrophoretic mobility (e.g., influenced by molecular weight, characteristic length, area or volume, oligonucleotide length, or other suitable characteristics), isoelectric point, etc. For example, in some embodiments, samples are electrophoretically separated in a capillary containing a separation matrix based on mobility parameters such as molecular weight. The capillary may include the separation matrix, which can be added automatically. In some embodiments, the separation matrix is ​​an isoelectric separation matrix and has properties similar to or substantially the same as those of polymer gels used in conventional electrophoresis experiments, such as pH gradients. Capillary electrophoresis in the separation matrix is ​​analogous to separation in polymer gels (such as polyacrylamide gels or agarose gels), where molecules are separated based on mobility parameters of molecules in the sample by providing porous channels in which molecules can move.

[0026] As in Figure 1B As shown, a first primary antibody 120 can be introduced into capillary 110, for example, after the separation and immobilization of analytes. In some cases, after the start of analysis, the instrument can be operated to automatically separate, immobilize, and / or introduce the first primary antibody 120 without any further user intervention. The first primary antibody 120 can be configured to selectively bind one or more analyte species within capillary 120. That is, in some cases, the first primary antibody 120 can be configured to bind certain target analytes within the sample / capillary 120 without binding other (e.g., non-target) analytes. In some cases, unbound first primary antibody can be removed in a washing step, for example, after an incubation period.

[0027] The primary antibody 122 can be introduced into the capillary 110, such as in... Figure 1C As shown in the diagram. Primary antibody 122 can be configured to bind primary antibody 120. In some cases, unbound primary antibody 122 can be removed during a washing step, for example, after the incubation period. (As shown in...) Figure 1C and 1D As shown, HRP is conjugated to secondary antibody 122 before being introduced into capillary 110. (As in...) Figure 1D As shown, a chemiluminescent substrate 124, such as 3,3′,5,5′-tetramethylbenzidine (TAB), 3,3′-diaminobenzidine (DAB), luminol, peroxide, or any other suitable chromogenic and / or (enhancing) chemiluminescent substrate, is introduced into the capillary. HRP conjugated to secondary antibody 122 can catalyze the chemiluminescent substrate 124, thereby generating an optical signal.

[0028] Based on the optical characteristics associated with the primary antibody 122, the first analyte 112 / analyte species labeled with the first primary antibody 120 can be detected. For example, the chemiluminescent reaction associated with the HRP conjugated to the primary antibody 122 can be detected and / or recorded in a single image or a series of images taken over time by a CCD camera or another suitable detector. After detecting the analyte / analyte species labeled with the first primary antibody 120, a stripping reagent can be introduced into capillary 110, as in... Figure 1E As shown in the diagram. The stripping reagent is operable to remove the first primary antibody 120, the first secondary antibody 122, and / or the optically detectable reagent 124, while retaining the immobilized sample for another round of immunoassay.

[0029] Figure 1F-1H The above steps are then repeated using a second primary antibody 130, a secondary antibody (labeled with HRP) 132, and a subsequent second detection step. The second primary antibody 130 is configured to selectively bind to the second analyte 114, and the secondary antibody 132 is configured to bind to the second primary antibody 130. Typically, the second primary antibody 130 is configured to selectively bind to an analyte different from the first primary antibody 120, but in some cases, the second primary antibody 130 may be the same as the first primary antibody 120, for example, to assess the reproducibility, stability, or characterization of the assay. The secondary antibody 132 may be the same antibody as the first primary antibody 122, or it may be a different secondary antibody. Optionally, the second primary antibody 130 and / or the secondary antibody 132 may be introduced after the stripping reagent has removed the first primary antibody 110, which allows for sequential and different immunoassays of a single sample. For example, both the primary and secondary antibodies may be HRP-labeled and configured to produce optically indistinguishable signals in the presence of a chemiluminescent substrate. Different analytes can be detected using the same optically detectable reagent by stripping the first primary antibody 120 and the first secondary antibody 122 after detecting the first analyte 112 and before introducing the secondary antibody 132.

[0030] although Figure 1A-1H A first primary antibody 120 and a second primary antibody 130 are described as selectively binding to different (separated) analyte species. In other embodiments, the first primary antibody 120 and the second primary antibody 130 may be configured to selectively bind to different epitopes of analyte species that are not distinguished by electrophoresis.

[0031] Chemiluminescent substrate 134 can be introduced into capillary 110, enabling the detection of HRP-labeled secondary antibody 132 based on optical signals associated with the HRP / chemiluminescent substrate interaction, and thus the detection of second analyte 114 / analyte species.

[0032] As will be readily understood by those skilled in the art, additional stripping and gravity detection steps are possible, and alternative detection modes (color detection, fluorescence detection, etc.) can be used in addition to or in place of chemiluminescence detection. Although Figure 1A-1H Two successive immunoassays for the detection of two different protein species by chemiluminescence are described. Those skilled in the art will readily understand that alternative embodiments could employ two different antibodies against the same target or two different epitopes of the same protein. Furthermore, fluorescence detection, absorbance, or any other suitable detection method, including combinations of multiple detection modalities, can be used.

[0033] In addition, although Figure 1A-1H The application of primary antibodies configured to selectively bind certain target analytes, HRP-labeled secondary antibodies configured to bind primary antibodies, and chemiluminescent substrates is described. Skilled technicians will understand that other detection techniques are possible. For example, see the references below. Figure 2D The secondary antibody discussed may be fluorescently labeled instead of HRP-labeled. In such an embodiment, a chemiluminescent substrate alone may not be necessary. In such an embodiment, the fluorescently labeled secondary antibody can be excited by an instrument and its emission detected. In other embodiments, the primary antibody may be labeled with HRP, a fluorescent tag, or otherwise optically detectable (e.g., by natural fluorescence or absorbance techniques). In such an embodiment, a secondary antibody alone may not be necessary. In other embodiments, third, fourth, or similar reagents may be used. For example, the secondary antibody may be biotinylated, and a third antibiotic conjugated to an optically detectable reagent (e.g., HRP or a fluorescent tag) may increase the detectable signal associated with the analyte. Those skilled in the art will further understand that different detection techniques can be used to evaluate different analyte species. For example, as referenced... Figure 1B-1D The first analyte species 112 is detected as shown and described (e.g., by using a primary antibody, an HRP-conjugated secondary antibody, and a chemiluminescent substrate), while the second analyte species 114 can be detected by introducing a fluorescently labeled primary antibody without using HRP or a secondary antibody.

[0034] In some implementations, in addition to the indication to initiate an immunoassay, reference may be performed automatically and / or without additional user intervention. Figure 1A-1H Some or all of the events shown and described. Furthermore, while other orders are possible and within the scope of this disclosure, references... Figure 1A-1H The events described can be executed in the order described.

[0035] Figure 2A-2HThe events that occur in the stripping and gravity detection methods according to one implementation scheme are explained. Figure 2A-2H The embodiments explained herein can be used to combine one or more immunoassays with a total protein assay. As shown in the figure, the total protein assay can be performed in the same capillary and / or on the same sample as the Western blot immunoassay. While other sequences are possible and within the scope of this disclosure, in some cases it may be preferred to perform the assay on... Figure 2A-2H The events shown and described below in the order indicated reduce reagent degradation.

[0036] Figure 2A This is a schematic diagram of a sample that has been separated and immobilized onto the surface of capillary 210. For example, the analyte may be covalently bound to the surface of capillary 210. As shown, the sample has been separated into three bands 212, 214, and 216. Each band represents a different analyte species. It should be understood that the sample may contain any number of analyte species and / or be separated into any number of bands. For example, in some cases, the sample may be a homogeneous mixture of a single analyte species.

[0037] After the sample has been separated and / or immobilized, biotinylated reagent 220 can be introduced into capillary 210, such as in Figure 2BAs shown in the diagram. Biotinylated reagent 220 can be configured to bind all proteins, enabling total protein assays (discussed in further detail below), that is, allowing determination of the total protein content in the sample / capillary 210. It is preferable to add biotinylated reagent 220 to capillary 210 prior to any immunoassay, as the biotinylated reagent may not be stable enough to be introduced into the capillary after the immunoassay. For example, in some cases, the user may load the biotinylated reagent (e.g., onto the sample plate) just before starting the run (e.g., less than 20 minutes before introducing the sample into the capillary), because in some cases, the biotinylated reagent may begin to degrade immediately after loading. In some cases, the biotinylated reagent is introduced into the capillary immediately after the sample has been isolated and / or immobilized (e.g., within 5 minutes of immobilization and / or within 90 minutes of introducing the sample into the capillary). Optionally, excess (e.g., unbound) biotin can be washed from the capillary after introduction. As discussed in further detail herein, the instrument is operable to run a reference sample in parallel lanes. Similarly, the instrument is operable to load the same or different samples into multiple capillaries, including capillary 210 and at least one reference capillary (not shown). Biotinylated reagent 220 can be introduced into the reference capillary simultaneously with or, for example, within 5 minutes of the introduction of biotinylated reagent into capillary 210. Typically, given the stability profile of the biotinylated reagent, it is introduced into both the reference capillary and capillary 210 before any immunoassay is performed on the sample in capillary 210.

[0038] By introducing one or more primary antibodies, immunoassays can be performed on samples immobilized in capillary 210. For example, in Figure 2C As shown, a first primary antibody 222 configured to selectively bind to a first analyte species 212 and a second primary antibody 224 configured to selectively bind to a second analyte species 214 are introduced. However, it should be understood that any number of primary antibodies having any suitable selective binding characteristics can be introduced. Furthermore, the first primary antibody 222, the second primary antibody 224, and / or any other primary antibodies can be introduced sequentially or substantially simultaneously (e.g., mixed together and / or drawn from a common reagent reservoir). Optionally, excess (e.g., unbound) primary antibodies can be washed from capillary 210.

[0039] Figure 2DThe introduction of primary antibody 226 and secondary antibody 228 into capillary 210 is explained. Primary antibody 226 is configured to selectively bind to a first primary antibody 222, and secondary antibody 228 is configured to selectively bind to a second primary antibody 224. Primary antibody 226 and / or secondary antibody 228 may have or be modified to have optically detectable features. For example, the secondary antibodies may be labeled with an optically detectable reagent before or after introduction into the capillary. In some cases, it may be desirable for different secondary antibodies to have different optical features, said secondary antibodies being configured to be associated with a particular primary antibody and therefore with a particular analyte species. For example, Figure 2D The primary antibody 226 is labeled with an optically detectable marker (e.g., a fluorescent dye) before being introduced into capillary 210. Optionally, unbound secondary antibody and / or optically detectable reagent can be washed from capillary 210. The secondary antibody 228 may be labeled, for example, with HRP before being introduced into capillary 210. Figure 2E The introduction of a chemiluminescent substrate is explained, which is configured to interact with HRP-labeled secondary antibody 228 to generate an optically detectable signal. Optical features associated with the secondary antibody, such as chemiluminescence and fluorescence signals, can be collected transiently and / or over time by a CCD camera or another suitable detector. Such optical signals can be used to identify some or all analyte species present in the sample. For example, as in... Figure 2E As shown, analyte species 212 and 214 will be detectable during the immunoassay. This is especially true in cases where one analyte species is associated with HRP and the other with a fluorescent dye (e.g., as in...). Figure 2C-2E As shown in the diagram, chemiluminescent and fluorescent signals can be detected simultaneously or sequentially. For example, fluorescently labeled primary antibody 226 can be excited before, during, or after the introduction of a chemiluminescent substrate.

[0040] As in Figure 2F As shown, once the optical characteristics of the secondary antibody are detected, a stripping reagent configured to remove primary and / or secondary antibodies from the analyte can be introduced into capillary 210. The stripping reagent is configured to bind biotin 220 to the analyte. Figure 2G The introduction of streptavidin 232, avidin, and / or other suitable reagents configured to specifically bind biotin into the capillary is explained. The streptavidin may be HRP-conjugated (before or after introduction into capillary 210) and / or otherwise labeled or optically detectable. For example, total protein detection (e.g., determining the amount of each biotin-labeled protein) can be performed by loading a chemiluminescent substrate and detecting the chemiluminescent signal, as in... Figure 2HAs shown in the diagram. In some cases, the amount of protein in each band can be determined individually.

[0041] Determining the total protein amount allows the immunoassay signal to be normalized to the total protein content. Similarly, based on optical signals indicating protein quantity (e.g., optical signals associated with streptomycin bound to proteins via biotin), optical signals associated with the immunoassay (e.g., signals associated with secondary antibodies bound to analyte species via primary antibodies) can be corrected or normalized. In some cases, a reference sample suitable for correcting the immunoassay signal can be loaded onto a reference capillary (not shown). For example, samples can be loaded sequentially or in parallel onto a tube containing multiple capillaries (e.g., capillary 210 and a reference capillary). Proteins in each capillary can be biotinylated (sequentially or in parallel). HRP-conjugated streptomycin or other suitable reagents can be introduced into the capillaries (sequentially or in parallel). Chemiluminescent substrates can also be introduced into the capillaries (sequentially or in parallel) so that the total protein quantity in each capillary can be determined. In some embodiments, the analyte detected by immunoassay in capillary 210 can be normalized based on the total protein content in the reference capillary. For example, the ratio of total protein in the reference capillary to total protein in capillary 210 can be determined. This ratio can be used to correct for signals associated with immunoassays of individual protein species. Such techniques can be used to correct immunoassay signals to compensate for loading heterogeneity. Similarly, a strong immunoassay signal may be the result of a “true” signal associated with a high concentration of the protein of interest relative to other proteins in the sample, or it may be associated with a large total amount of protein, for example, if more cellular contents than expected are loaded into the capillary.

[0042] Typically, before initiating the immunoassay and / or total protein measurement, the analyst prepares the sample and / or suitable reagents and loads the reagent / sample plate. In some embodiments, after initiating the immunoassay and / or total protein measurement, some or all subsequent events (e.g., sample loading, separation, immunoassay and / or total protein, detection) can be performed automatically and / or without further analyst interaction. As will be readily apparent to those skilled in the art, additional stripping and reprobe steps are possible, additional intermediate washing steps can be performed to rinse unbound reagents from the capillary, different combinations of detection modes can be used, and / or alternative detection modes (color detection, fluorescence detection, etc.) can be used instead of the precise combinations described in the above embodiments. Although Figure 2A-2HThe method described involves detecting two different protein species using chemiluminescence and fluorescence, followed by a total protein measurement. However, those skilled in the art will readily understand that alternative implementations may employ two different antibodies targeting the same protein or two different epitopes of the same protein. Furthermore, fluorescence detection, absorbance, or any suitable, well-known detection method, including combinations of various detection modalities, may be used.

[0043] Figure 2C and 2D This explains the essentially simultaneous (e.g., as a mixture) introduction of multiple primary antibodies 222 and 224, and also explains the essentially simultaneous introduction of multiple secondary antibodies 226 and 228. Conversely, Figure 1B-1G This explains the sequential introduction of different primary and secondary antibodies, with a stripping event occurring between the introduction of the first primary antibody 122 and the introduction of the second primary antibody 130. However, it should be understood that regarding... Figure 1A The methods shown and described in -H may include the introduction of a mixture of primary and / or secondary antibodies. Similarly, regarding Figure 2A-2H The methods shown and described may include successive immunoassays, for example, with additional stripping events between immunoassays.

[0044] According to certain embodiments, the methods described herein can be performed on instruments suitable for protein content measurement and / or immunoassay in the same capillary and / or in an automated manner, such as the Simple Western® platform of ProteinSimple®. Unlike other known instruments and techniques, the embodiments described herein are generally simpler than conventional methods used for total protein measurement in conventional Western blotting. Immunoassay and total protein measurement can be performed using chemiluminescent or fluorescent methods or other methods known in the art. Furthermore, the stripping reagent used to remove antibodies from the immunoassay improves the accuracy of detecting the total protein content immobilized on the capillary.

[0045] As referenced above Figure 1A-1H The discussion is for experienced technicians to understand and refer to. Figure 2A-2H The embodiments shown and described are examples and not limitations. Specifically, those skilled in the art will understand that analyte species can be detected by any combination of chemiluminescence, fluorescence, and / or absorbance techniques. Those skilled in the art will understand that more or fewer antibodies than primary and secondary antibodies can be used. Those skilled in the art will understand that antibodies can be pre-labeled with optically detectable reagents, that optically detectable reagents can be introduced into capillaries to selectively bind antibodies and / or analyte species, and / or analyte species and / or antibodies can be inherently detectable (e.g., unlabeled antibodies can be detected, for example, based on their absorbance characteristics).

[0046] Operation sequence

[0047] As previously mentioned, when measuring total protein and immunoassay signals in a capillary, a specific order of reagent addition is preferred to improve performance. Experimental evidence suggests that adding biotinylated reagent after the immunoassay results in a signal that is 70% lower than when biotinylated reagent is added before the immunoassay. It is important to maintain a higher signal and corresponding sensitivity in this assay to obtain the preferred detection level for the assay. One could attempt to perform a complete total protein assay before the immunoassay (e.g., biotinylation and detection using HRP-conjugated streptavidin with luminol / peroxide), however, this is a less than ideal assay configuration, likely due to the difficulty in removing HRP-conjugated streptavidin, which has a very high binding affinity to biotin. Incomplete removal of HRP-conjugated streptavidin can negatively impact immunoassay performance, for example, by preventing antibody binding to the target protein through residual HRP-conjugated streptavidin bound to biotinylated proteins.

[0048] Stripping reagents

[0049] Various formulations for removing antibodies from protein blot membranes are known in the art. These formulations typically contain buffer components, detergents, denaturants, acidic or alkaline pH, and / or reducing agents. Most stripping buffers known in the art use β-mercaptoethanol as a reducing agent; however, β-mercaptoethanol is toxic, unstable in solution, has an unpleasant odor, and its use is currently restricted or prohibited in some countries. Therefore, there is a need for stripping reagents for removing antibodies bound to analytes from capillaries.

[0050] Stripping reagent formulations using tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) as a phosphonic reducing agent in place of β-mercaptoethanol have been developed and are shown in Table 1. While most of the formulations in Table 1 serve to remove antibodies to some extent, it is desirable to always remove at least 95% of the residual signal in the immunoassay, for example, as performed using a Simple Western® instrument. Preferably, the stripping reagent should also be stable in solution, i.e., it should not precipitate or decompose during storage over a period of time (e.g., 1 day, 1 week, 1 month, 6 months, 1 year, or any other suitable time range). As shown in Table 1, using only TCEP instead of β-mercaptoethanol cannot consistently achieve a stripping efficiency >95% without precipitation. Achieving high stripping efficiency is important for many antibodies because retained antibodies will generate noise and reduce the detection limit of subsequent immunoassay steps.

[0051] Further optimization was carried out through extensive titration and novel combinations of the components in Table 1 with additional components, such as different detergents and reducing agents. Key factors influencing stripping efficiency were identified as Trizma (Tris base; CAS: 77-86-1) concentration, TCEP concentration, and pH, while stripping efficiency was relatively insensitive to SDS concentration. As shown in Table 2, antibody removal efficiency of the formulation was analyzed on Simple Western® in combination with various immunoassays (different antibodies, different target proteins). Further testing was conducted on the formulation's precipitation, degradation, and ≥95% removal efficiency against various antibodies. Several candidates in Table 2 met these criteria. Formulations with neutral or alkaline pH performed significantly worse than those with acidic pH. This observation is surprising, as TCEP is the preferred reducing agent in the formulation, and by conventional wisdom, TCEP is considered effective over a wide pH range of 1.5 to 8.5, with optimal reducing performance at near-neutral pH (i.e., close to 7) (see Han, JC and Han, YH). A Procedure for Quantitative Determination of tris(2-carboxyethyl)phosphine, an Odorless Reducing Agent More Stable and Effective Than Dithiothreitol Anal Biochem. July 1994; 220(1):5-10, which is hereby incorporated in its entirety by reference. Additionally, such as Figure 3 As shown, the preferred formulation was determined to operate with the highest antibody removal efficiency over a narrow pH range, indicating that the preferred formulation capable of producing a stripping efficiency greater than 97% has a pH of 4.05 ± 0.3. Figure 3 The stripping efficiency against three different targets, park7, β-actin, and HSP60, was explained.

[0052] The stripping reagents in Tables 1 and 2 are prepared by mixing the specified components with water at the specified molar concentration or percentage (by weight). If the pH of the buffer species or TCEP stock is reported, these pH values ​​indicate the pH of the component before combination to form the stripping reagent. The stripping reagent is then adjusted to the indicated final pH (e.g., by adding hydrochloric acid, sodium hydroxide, or other suitable acid or base) after all components are mixed. In some cases, no pH adjustment is performed, and the final pH is simply the pH measured after combining the components. The final pH value indicated by an asterisk is an estimated final pH (i.e., not measured). It should be understood that the concentration of the components may vary by 1%, 5%, or 10%, and remains within the range of stripping buffer reagent compositions considered by the inventors. The indicated pH or final pH of the buffer species, TCEP stock may vary by 0.1, 0.3, 0.5, or 1, and remains within the range of stripping buffer reagent compositions considered by the inventors. The reported stripping efficiency information is observed experimentally. It should be understood that buffers prepared as shown may not perfectly replicate the observed stripping efficiency.

[0053] Table 1

[0054]

[0055] (THPP is tris(hydroxypropyl)phosphine; TCEP is tris(2-carboxyethyl)phosphine hydrochloride); Tween is polysorbate 20 (CAS: 9005-64-5); Triton X-100 is... Uncle Octylphenoxy polyethoxyethanol (CAS: 9002-93-1)

[0056] Table 2

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] (BRIJ 35 (also known as BRIJ L23) is a 30% solution of polyoxyethylene (23) lauryl ether (CAS: 9002-92-0))

[0064] Appendix Figure 4 The performance of the formulations shown in Table 3 is explained, and the even greater decrease in stripping efficiency is shown when pH is greater than 4.5. When pH is less than 3, the decrease in stripping efficiency is less severe, but still noticeable (data not shown). Figure 4 (As is evident from the rows in Table 2). Surprisingly, a narrow and specific pH range was found to be required to achieve optimal antibody removal (≥95%) in Simple Western® capillaries. This is likely due to the unique properties and / or internal environment of Simple Western capillaries compared to protein blot membranes. Furthermore, it was determined that multiple incubations of the stripping reagent in the capillary improved antibody removal efficiency.

[0065] Table 3

[0066]

[0067] Figure 5A and 5B These are graphs displaying experimental data, explaining the reproducibility of the analytes (ATK1 and ATK2) after the introduction of the stripping reagent. Similarly, based on the reference... Figure 1A-1H The method shown and described is similar to the method that produces Figure 5A and 5B The results are shown. Figure 5B The peak area changes before and after reprobe detection are explained (e.g., including the introduction of stripping reagent). ATK1 and ATK2 are detected and reprobeted in multiple capillaries, and the order in which the analytes are detected is different. In this case, ATK1 is detected first in six capillaries, and ATK2 is detected first in six capillaries. Stripping reagent is introduced into all twelve capillaries after the first probe to remove primary and secondary antibodies from the first target analyte, and then the other target analyte is detected. (See also...) Figure 5B As shown, the peak area of ​​the target analyte is not significantly affected by the stripping reagent or the order in which the analyte is detected. This indicates that the stripping reagent does not remove a significant amount of the analyte.

[0068] Although different implementations have been described above, it should be understood that they are presented merely as examples and not as limiting. For example, while the implementations described herein generally describe capillary-based techniques, it should be understood that any suitable microfluidic device or other electrophoresis technique can be used. For example, Yao, S. Anex, DS, Valdwell, WB, Arnold DW, Smith KB, and Schultz, PG, SDS Capillary Gel Electrophoresis of Proteins in Microfabricated Channels, 96(10) Proc NatlAcad Sci USA 5372-77 (May 11, 1999) (the entire contents of which are hereby incorporated by reference) describes a chip fabricated to include microchannels and other microfluidic structures suitable for performing protein size separation. Such a chip can be adapted to perform the methods and / or assays described herein. As another example, the embodiments described herein related to total protein labeling generally describe biotinylation. However, it should be understood that other suitable techniques for nonspecific protein labeling and detection are also possible, such as his-tag / anti-his, glutathione / glutathione S-transferase, maltose / maltose-binding protein, chitin / chitin-binding protein, etc. Those skilled in the art will understand that any suitable molecule having an NHS-ester or other moiety to chemically react with proteins (e.g., amino acids such as lysine, protein backbone such as nitrogen, post-translational modifications such as glycans, etc.) can be used for total protein labeling. Furthermore, while techniques for identifying total protein content and protein-based normalization have been described, it should be understood that similar techniques exist for many other analytes. For example, the total amount of nucleic acids, lipids, and / or glycoproteins may be measured and then used for capillary-based normalization measurements. For instance, molecules with amino-oxygen reactive groups can chemically react with sugars such as polysaccharide or glycan groups. Similarly, molecules with psoralen groups can bind to DNA or RNA via UV-activated insertion of thymine and other pyrimidine bases into the psoralen group. Nucleic acid binding chemistry includes the carbodiimide crosslinking agent EDC / imidazole and other chemical and enzymatic ligation methods known in the art. Likewise, methods for biotinylated lipids are known in the art, for example, see Henry, Stephen, et al. 'Rapid one-step biotinylation of biological and non-biological surfaces.' Scientific reports 8.1 (2018): 1-6”, the entire disclosure of which is hereby incorporated by reference.

[0069] Where certain components are arranged in certain orientations or locations as indicated in the above diagrams and / or embodiments, the arrangement of components may be modified. Although embodiments have been specifically shown and described, it should be understood that various variations in form and detail may be made. Although various embodiments have been described as combinations of specific features and / or components, other embodiments may have any combination of features and / or components from any of the embodiments discussed above.

[0070] Where the methods and / or events indicate that certain events and / or procedures occur in a certain order, the order of certain events and / or procedures may be modified. Furthermore, certain events and / or procedures may, where possible, be executed simultaneously in parallel processes, as well as sequentially as described above.

Claims

1. An immunoassay method, comprising: Samples containing the first and second analytes are separated by capillary electrophoresis. After electrophoretic separation, the first analyte and the second analyte are immobilized in the capillary; A first primary antibody configured to bind the first analyte is introduced into the capillary. A primary antibody configured to bind the first primary antibody is introduced into the capillary. The first analyte is detected based on optical characteristics associated with the primary antibody. A stripping reagent configured to remove the first primary antibody from the first analyte is introduced into the capillary while the first and second analytes remain immobilized in the capillary, wherein the stripping reagent comprises tris(2-carboxyethyl)phosphonic acid hydrochloride and has a pH between 3 and 4.

5. A second primary antibody configured to bind the second analyte is introduced into the capillary. A secondary antibody configured to bind the second primary antibody is introduced into the capillary. and The second analyte is detected based on optical characteristics associated with the secondary antibody. in: The first primary antibody is introduced before the stripping reagent is introduced; and The second primary antibody is introduced after the stripping reagent is introduced.

2. The method according to claim 1, wherein the primary antibody and the secondary antibody are the same secondary antibody.

3. The method according to claim 1, wherein: The primary antibody and the secondary antibody are configured to produce indistinguishable optical features; and The stripping reagent is introduced after the detection of the first analyte and before the introduction of the secondary antibody.

4. The method of claim 1, wherein the primary antibody is conjugated to horseradish peroxidase such that the optical feature associated with the primary antibody is a chemiluminescent reaction associated with the horseradish peroxidase.

5. The method of claim 1, wherein the primary antibody is conjugated to horseradish peroxidase, the method further comprising: When a chemiluminescent substrate is introduced into a capillary, the optical characteristics associated with the primary antibody are chemiluminescent signals related to the interaction between the chemiluminescent substrate and horseradish peroxidase conjugated to the primary antibody.

6. The method according to claim 1, wherein: The primary antibody is conjugated to horseradish peroxidase such that the optical characteristic associated with the primary antibody is a chemiluminescent signal; and The secondary antibody is conjugated to a fluorescent dye such that the optical characteristic associated with the secondary antibody is a fluorescent signal.

7. The method of claim 1, further comprising washing unbound primary antibody prior to detection of the first analyte.

8. The method of claim 1, further comprising washing the first primary antibody from the capillary after introducing the stripping reagent and before introducing the second primary antibody, while simultaneously immobilizing the first analyte and the second analyte in the capillary.

9. The method of claim 1, wherein the stripping reagent is configured to remove the first primary antibody and the first-level antibody.

10. The method of claim 1, wherein electrophoretic separation of the sample causes the first analyte to migrate to a first portion of the capillary, and the second analyte to migrate to a second portion of the capillary, the first analyte and the second analyte being immobilized in the first portion and the second portion of the capillary, respectively.

11. The method of claim 1, wherein the introduction of the first primary antibody, the introduction of the first secondary antibody, the detection of the first analyte, the introduction of the stripping reagent, the introduction of the second primary antibody, and the introduction of the second secondary antibody occur in the above order and without user intervention.

12. The method of claim 1, wherein the stripping agent has a stripping efficiency of greater than 95% and has a composition selected from: 200 mM Tris base, 100 mM TCEP, 2% SDS, 2% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 2% tert-octylphenoxypolyethoxyethanol; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 2% polysorbate 20; 60 mM 2-(N-morpholino)ethanesulfonic acid, 100 mM TCEP, 2% SDS; 60 mM 3-(N-morpholino)propanesulfonic acid, 100 mM TCEP, 2% SDS; 60 mM Tris hydrochloride, 50 mM TCEP, 2% SDS; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 2% polyoxyethylene 23 lauryl ether; 60 mM tris(hydroxymethyl)methylglycine, 100 mM TCEP, 2% SDS; 50 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 100 mM TCEP, 2% SDS; 200 mM Tris base, 100 mM TCEP, 2% SDS, 2% polysorbate 20; 200 mM Tris base, 100 mM TCEP, 2% SDS, 2% polyoxyethylene 23 lauryl ether; 200 mM Tris base, 100 mM TCEP, 2% tert-octylphenoxy polyethylene glycol; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 0.5% polysorbate 20; 75 mM Tris base, 100 mM TCEP, 2% SDS; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 0.5% tert-octylphenoxypolyethoxyethanol; 50 mM Tris base, 100 mM TCEP, 2% SDS; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 0.5% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; 200 mM Tris base, 100 mM TCEP, 2% SDS, 0.5% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; 200 mM glycine HCl, 100 mM TCEP, 2% SDS, 0.5% polyoxyethylene 23 lauryl ether; 200 mM Tris base, 100 mM TCEP, 2% SDS, 0.5% tert-octylphenoxypolyethoxyethanol; 200 mM glycine HCl, 100 mM TCEP, 1% lithium dodecyl sulfate; 50 mM N-di(hydroxyethyl)glycine, 100 mM TCEP, 2% SDS; 200 mM glycine HCl, 100 mM TCEP, 1% SDS; 200 mM glycine HCl, 100 mM TCEP, 2% lithium dodecyl sulfate; 200 mM glycine HCl, 100 mM TCEP, 2% SDS; 200 mM Tris base, 100 mM TCEP, 1% SDS; 200 mM Tris base, 100 mM TCEP, 5% lithium dodecyl sulfate; 200 mM Tris base, 100 mM TCEP, 2% SDS, 0.5% polysorbate 20; 200 mM Tris base, 100 mM TCEP, 2% lithium dodecyl sulfate; 100 mM Tris base, 100 mM TCEP, 2% SDS; 150 mM N-di(hydroxyethyl)glycine, 100 mM TCEP, 2% SDS; 200 mM Tris base, 100 mM TCEP, 1% lithium dodecyl sulfate; 100 mM N-di(hydroxyethyl)glycine, 100 mM TCEP, 2% SDS; 200 mM Tris base, 100 mM TCEP, 2% SDS, 0.5% polyoxyethylene 23 lauryl ether; 150 mM Tris base, 100 mM TCEP, 2% SDS; 200 mM glycine HCl, 100 mM TCEP, 5% SDS; 200 mM Tris base, 100 mM TCEP, 2% SDS; and 200 mM glycine HCl, 100 mM TCEP, 5% lithium dodecyl sulfate.

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