Method for measuring total protein content and detecting proteins via immunoassay in microfluidic devices

Abstract

Some embodiments described herein relate to systems and methods that can be operated to combine immunoassay and total protein techniques in a single-sample run. [Solution] Some embodiments described herein enable multiple sequential immunoassays to be performed in the same microfluidic device. Some embodiments described herein relate to stripping reagents that can be operated to remove primary antibodies associated with an immunoassay. Such stripping reagents enable additional immunoassays and / or total protein assays to be performed in the same sample.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and benefit of U.S. Provisional Patent Application No. 63 / 010,436, filed Apr. 15, 2020, and U.S. Provisional Patent Applications Nos. 16 / 932,441 and 16 / 932,445, filed Jul. 17, 2020, respectively. The entire disclosure of each of the foregoing patent applications is hereby incorporated by reference in its entirety herein.

[0002] Embodiments described herein generally relate to capillary electrophoresis methods for performing immunoassays and / or protein quantification assays on samples. Stripping reagents are disclosed that are operable to remove antibodies associated with an immunoassay, enabling additional assays to be performed on the same sample.

Background Art

[0003] An important part of protein research involves characterizing proteins within heterogeneous samples, such as cell lysates that can contain thousands of proteins. Western blotting is a commonly used immunoassay - based method that uses the specificity of antibodies to identify target proteins and analyze specific proteins within these complex samples. When performing an immunoassay in a Western blot format, it has become increasingly important to quantify the resulting immunoassay signal. One method for quantification is to normalize the immunoassay signal to the total protein content in the sample. This is increasingly required by journals to ensure the accuracy and precision of data when publishing Western blot results.

[0004] Existing systems can be operated to provide fully automated microfluidic-based (e.g., capillary-based) immunoassays, such as ProteinSimple's® Simple Western® instrument. Some such systems can combine immunoassays with size separation similar to conventional gel-based Western blots in a capillary. Samples, separation matrix, stacking matrix, antibodies, and reagents can be automatically loaded. The instrument may be operated to aspirate the separation matrix, and then the stacking matrix, into each capillary. Next, a sample, which may contain heterogeneous protein mixtures, can be loaded, and the capillaries can be brought into contact with electrophoresis buffer. Voltage can be applied to enable separation by molecular weight or other appropriate characteristics. Once separation is complete, proteins can be immobilized on the capillary wall by UV light. Immunoproving of the proteins can be performed, for example, to immobilize the proteins and remove the matrix from the capillaries. In addition, some existing systems can be operated to provide a "total protein" assay, which can be done through biotinylation of proteins immobilized on the inner surface of a capillary, followed by detection of a horseradish peroxidase (HRP) conjugated streptavidin and chemiluminescence reaction.

[0005] However, there is a need for methods that enable chemiluminescent detection for both immunoassays and total protein readout in the same capillary or other microfluidic devices. Furthermore, increasing multiplexing capability beyond the number of detection modalities / channels is desirable. [Overview of the project]

[0006] Some embodiments described herein relate to systems and methods that can be operated to combine immunoassay and total protein techniques in a single-sample run. Instruments having both chemiluminescent and fluorescence detection capabilities, such as Jess® by ProteinSimple®, can provide a “protein standardization” method in which a fluorescent dye is covalently attached to all separated and immobilized protein molecules, for example, via NHS esteramine coupling. In this method, a specific target can be measured using chemiluminescence associated with the immunoassay, for example, while the measured value of the packed total protein is determined from the fluorescence signal. Known techniques for protein standardization typically have a dynamic range similar to a typical Western blot-style immunoassay and cannot multiplex the immunoassay in the same capillary in which it was performed using chemiluminescent detection. In contrast, embodiments of protein standardization described herein can multiplex the chemiluminescent immunoassay in the same capillary and may have a reduced or different dynamic range compared to the immunoassay signal.

[0007] In addition to performing total protein and immunoassays in the same capillary or other microfluidic device, some embodiments described herein enable the performance of multiple sequential immunoassays in the same capillary. Instruments with both chemiluminescence and fluorescence detection capabilities (e.g., ProteinEasy's® Jess®) enable "multiplex" detection, i.e., detection of multiple targets in a capillary using a single mixture of antibodies conjugated with portions for either 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 cross-reactivity of antibodies used for immunoassays, or incompatible dynamic ranges for antibodies when used in the same capillary (e.g., different dynamic ranges for fluorescence versus chemiluminescence).

[0008] The embodiments described herein relate to the use of a total protein assay and / or a second immunoassay in the same capillary as a Western blot-style immunoassay, enabling highly sensitive total protein measurement and immunoassays combined with the low sample requirements and high throughput capabilities previously demonstrated with simple Western techniques. [Brief explanation of the drawing]

[0009] [Figure 1A] Figures 1A to 1H illustrate events that occur in a stripping and immunoassay reprobing method according to one embodiment. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 1H] Same as above.

[0010] [Figure 2A] Figures 2A - 2H show events that occur in a stripping and total protein reprobing method according to one embodiment. [Figure 2B] The same as above. [Figure 2C] The same as above. [Figure 2D] The same as above. [Figure 2E] The same as above. [Figure 2F] The same as above. [Figure 2G] The same as above. [Figure 2H] The same as above.

[0011] [Figure 3] Figure 3 illustrates stripping efficiency vs. pH for three different targets.

[0012] [Figure 4] Figure 4 illustrates stripping efficiency vs. a broader pH range and demonstrates a significant decrease in stripping efficiency at pH > 4.5.

[0013] [Figure 5A] Figures 5A and 5B show experimental data illustrating the reproducibility of probed analytes after introduction of the stripping reagent. [Figure 5B] The same as above.

Mode for Carrying Out the Invention

[0014] Some embodiments described herein relate to appropriate methods for performing multiple immunoassays on samples separated by electrophoresis performed in a capillary or other suitable microfluidic device. The sample 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 to the first analyte (and optionally not to the second analyte) can be introduced into the capillary. A first secondary antibody configured to selectively bind to the first primary antibody can be introduced into the capillary. The first analyte can be detected based on optical properties associated with the first secondary antibody. For example, the first secondary antibody can be conjugated with 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. The first and second analytes can remain immobilized in the capillary after the stripping reagent has been introduced and the first primary antibody (along with the first secondary antibody and / or HRP) has been removed. A second primary antibody configured to bind to the second analyte can then be introduced, for example, after the introduction of the stripping reagent. A second secondary antibody configured to bind to the second primary antibody can then be introduced, and the second analyte can be detected based on optical properties associated with the second secondary antibody (e.g., chemiluminescence and / or fluorescent tag).

[0015] Some embodiments described herein relate to methods suitable for performing immunoassays and total protein assays on samples separated via electrophoresis performed in a capillary or other suitable microfluidic device. Analytes can be separated from a sample and immobilized in a capillary. Molecules having reactive moieties configured to non-specifically bind to proteins, such as biotin, can be introduced into the capillary. Similarly described, for example, proteins can be biotinylated. Primary antibodies configured to bind to at least a subset of the analytes can be introduced into the capillary. Secondary antibodies configured to bind to the primary antibodies can be introduced. A subset of the analytes can be detected based on optical properties associated with the secondary antibodies. Stripping reagents configured to remove the primary antibodies from the subset of the analytes can be introduced. The immobilized analytes can remain in the capillary after the stripping reagent is introduced and the primary antibodies (along with the secondary antibodies) are removed. Optically detectable agents configured to bind to the molecules can be introduced into the capillary. In an example where the molecule is biotin, streptavidin can be introduced. Streptavidin can be conjugated to HRP or otherwise made optically detectable. All biotinylated analytes (e.g., all proteins) in the capillary can be detected based on an optical signal associated with the optically detectable agent. The optical properties associated with the secondary antibodies (e.g., immunoassay signal) can be standardized based on an optical signal associated with the optically detectable agent (e.g., total protein signal). In some embodiments, the events of this paragraph may be important to perform in the order in which they are described.

[0016] Standardizing immunoassay signals (or other appropriate signals) can improve the instrument's and / or analyst's ability to accurately compare measurements from different samples by eliminating the influence of certain uncontrolled differences between samples not being tested. For immunoassays, standardization against the total protein content in each sample can eliminate the influence of variations due to sample composition (e.g., cell number, solubilizer dilution) or pipetting errors. In addition, standardization against total protein content is advantageous for standardization against specific housekeeping proteins (e.g., beta-actin or beta-tubulin) because the expression levels of housekeeping proteins may be affected by experimental procedures, or their immunoassay signals may not be in the same linear dynamic range as those of the target protein. In one embodiment, standardization is performed by dividing the amount of a specific protein determined by the immunoassay in the capillary by the ratio of the total protein in the capillary to the total protein in the reference capillary.

[0017] Some embodiments described herein relate to formulations of stripping reagents. A stripping reagent may comprise a buffer, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and a surfactant. The stripping reagent may have a pH below 5.

[0018] Some embodiments described herein relate to a method for using a stripping reagent comprising TECP and having a pH between 3.0 and 4.5. Using the stripping reagent, the primary antibody associated with the immunoassay can be removed from the analyte. The analyte can be separated electrophoretically and immobilized in a capillary. The primary antibody and secondary antibody associated with the immunoassay can be introduced into the capillary. After removing the primary antibody from the capillary using the stripping reagent, another suitable reagent configured to bind to streptavidin or biotinylated protein, and / or a secondary primary antibody configured to bind to the analyte in the sample, may be introduced into the capillary.

[0019] Stripping and reprobing methods Stripping and reprobing allow users to analyze the same immobilized protein in the same capillary (or other microfluidic device) in the same run, thereby saving time, money, and valuable samples.

[0020] Figures 1A–1H illustrate events occurring in a stripping and refroving method according to one embodiment. Stripping and refroving can be used to perform two or more immunoassays sequentially on a single sample (e.g., without requiring the reuse of separated and immobilized samples, and / or the (re)filling and / or (re)separation of additional samples for each immunoassay). Figure 1A is a schematic diagram of a separated sample immobilized on the surface of a capillary 110. For example, an analyte can be covalently bonded to the surface of a capillary 110 using devices and / or methods shown and described in, for example, U.S. Patent No. 7,846,676 and / or U.S. Patent Publication No. 2008 / 0017512, the entirety of each of those disclosures is incorporated herein by reference in whole. As shown, the sample is separated into two bands 112 and 114. Each band represents a distinct analyte and exists in a distinct part of the capillary 110. It should be understood that a sample can contain any number of analytes and / or be separated into any number of bands.

[0021] As used herein, the term “analyte” means any molecule or compound that is separated by electrophoretic techniques and / or detected by the methods, apparatus, and systems provided herein. Suitable analytes include, but are not limited to, small chemical molecules such as environmental molecules, clinical molecules, chemicals, contaminants, and / or biomolecules. More specifically, such chemical molecules include, but are not limited to, pesticides, insecticides, toxins, therapeutic drugs and / or drugs of abuse, antibiotics, organic materials, hormones, antibodies, antibody fragments, antibody-molecular 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, e.g., pathogenic bacteria and / or eukaryotic cells, e.g., mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides (including ribonucleic acid and / or deoxyribonucleic acid), transition state analogs, inhibitors, receptors, receptor ligands (e.g., nerve receptors or their ligands, hormone receptors or their ligands, nutrient receptors or their ligands, and / or cell surface receptors or their ligands), 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 solubilized or purified protein. Other suitable analytes may include aggregates, clusters, flocs, and / or dispersed phase droplets, or colloidal and / or emulsion particles. Once separated, the “bands” of the analyte are referred to herein as “analyte species.”

[0022] As used herein, the term “sample” refers to the analyte to be detected or a composition containing the analyte. In some embodiments, a sample may be homogeneous and contain diverse components (e.g., different proteins), or heterogeneous and contain a single component (e.g., a population of a single protein). In some examples, a sample may be naturally occurring, biological material, and / or manufactured material. Furthermore, a sample may be in its natural form (e.g., a cell suspension) or in a modified form (e.g., a solubilized product). In some examples, a sample may be a single cell (or the contents of a single cell, e.g., as a cell lysate from a single cell, or as purified protein) or multiple cells (or the contents of multiple cells, e.g., as a cell lysate from multiple cells, or as purified protein from multiple cells), a blood sample, a tissue sample, a skin sample, a urine sample, a water sample, and / or a soil sample. In some examples, a sample may be from an organism, e.g., a eukaryote, a prokaryote, a mammal, a human, a yeast, and / or a bacterium, or a sample may be from a virus.

[0023] Samples can be separated based on 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 properties), isoelectric point, and / or similar. For example, in some embodiments, samples are subjected to electrophoretic separation in a capillary tube containing a separation matrix based on mobility parameters, such as molecular weight. The capillary tube may contain a separation matrix, which can be added in an automated manner. In some embodiments, the separation matrix is ​​an isoelectric focusing separation matrix and has similar or substantially the same properties as polymer gels used in conventional electrophoretic experiments, such as a pH gradient. Capillary electrophoresis in a separation matrix is ​​similar to separation in a polymer gel, such as a polyacrylamide gel or agarose gel, where molecules are separated based on the mobility parameters of molecules in the sample by providing porous channels through which molecules can move.

[0024] As shown in Figure 1B, the first primary antibody 120 can be introduced into the capillary 110, for example, after the separation and immobilization of the analytes. In some cases, after the analysis has started, the instrument may be operable 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 to one or more analytes within the capillary 120. That is, in some cases, the first primary antibody 120 can be configured to bind to a specific target analyte within the sample / capillary 120 without binding to other (e.g., non-target) analytes. In some cases, the unbound first primary antibody can be removed, for example, in a washing step after the incubation period.

[0025] The first secondary antibody 122 can be introduced into the capillary 110, as shown in Figure 1C. The first secondary antibody 122 can be configured to bind to the first primary antibody 120. In some examples, unbound first secondary antibody 122 can be removed, for example, in a washing step after the incubation period. As shown in Figures 1C and 1D, the secondary antibody 122 is conjugated with HRP before being introduced into the capillary 110. As shown in Figure 1D, a chemiluminescent substrate 124, such as 3,3',5,5'-tetramethylbenzidine (TAB), 3,3'-diaminobenzidine (DAB), luminol, peroxide, or any other suitable chromogenic substrate and / or (enhancing) chemiluminescent substrate, is introduced into the capillary. The HRP conjugated to the secondary antibody 122 can catalyze the chemiluminescent substrate 124 and generate an optical signal.

[0026] The first analyte 112 / analyte species labeled with the first primary antibody 120 can be detected based on the optical properties associated with the first secondary antibody 122. For example, the chemiluminescent reaction associated with HRP conjugated to the first secondary antibody 122 can be detected and / or recorded in images taken over time or in a series of images using 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 the capillary 110, as shown in Figure 1E. The stripping reagent may be operated to remove the first primary antibody 120, the first secondary antibody 122, and / or an optically detectable agent 124, while retaining the immobilized sample for another round of immunoassay.

[0027] Figure 1F-1H shows the following repetition of the above steps and subsequent second detection steps using a second primary antibody 130 and a second secondary antibody (labeled with HRP) 132. The second primary antibody 130 is configured to selectively bind to the second analyte 114, and the second 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 a different analyte than the first primary antibody 120; however, in some cases, the second primary antibody 130 can be identical to the first primary antibody 120, for example, to evaluate the reproducibility, stability, or characterization of the assay. The second secondary antibody 132 can be the same antibody as the first secondary antibody 122, or it can be a different secondary antibody. In some cases, the second primary antibody 130 and / or the second secondary antibody 132 can be introduced after the first primary antibody 110 has been removed by a stripping agent, thereby enabling sequential and separate immunoassays to be performed on a single sample. For example, the first and second secondary antibodies can be HRP-labeled and configured to produce an optically indistinguishable signal in the presence of a chemiluminescent substrate. Different analytes can be detected using the same optically detectable agent by stripping the first primary antibody 120 and the first secondary antibody 122 after detecting the first analyte 112 and before introducing the second secondary antibody 132.

[0028] Figures 1A-1H depict a first primary antibody 120 and a second primary antibody 130 that selectively bind to different (separated) analytes; however, in other embodiments, the first primary antibody 120 and the second primary antibody 130 can be configured to selectively bind to different epitopes of electrophoretically undistinguished analytes.

[0029] The chemiluminescent substrate 134 can be introduced into the capillary 110 so that the HRP-labeled secondary antibody 132 and, therefore, the second analyte 114 / analyte can be detected based on the optical signal associated with the interaction between the HRP and the chemiluminescent substrate.

[0030] As will be readily apparent to those skilled in the art, additional stripping and reprobing steps are possible, and alternative detection modalities (such as color detection and fluorescence detection) can be used in addition to or instead of chemiluminescence detection. While Figures 1A-1H describe two sequential immunoassays for detecting two distinct protein species via chemiluminescence, those skilled in the art will readily understand that, by alternative embodiments, two different antibodies may be used for two different epitopes of the same target or the same protein. Furthermore, fluorescence detection, absorbance, or any other suitable detection method may be used, including combinations of multiple detection modes.

[0031] Furthermore, while Figures 1A-1H depict the use of a primary antibody configured to selectively bind to a specific target analyte, an HRP-labeled secondary antibody configured to bind to the primary antibody, and a chemiluminescent substrate, those skilled in the art will understand that other detection techniques are possible. For example, as discussed below with reference to Figure 2D, the secondary antibody may be fluorescently labeled rather than HRP-labeled. In such embodiments, a separate chemiluminescent substrate may not be necessary. In such embodiments, the fluorescently labeled secondary antibody can be excited by an instrument and its luminescence can be detected. In yet another embodiment, the primary antibody may be labeled with HRP, a fluorescent tag, or otherwise optically detectable (e.g., via natural fluorescence techniques or absorbance techniques). In such embodiments, a separate secondary antibody may not be necessary. In yet another embodiment, third, fourth, and so on-label agents may be used. For example, the secondary antibody may be biotinylated, and a third streptavidin conjugated with an optically detectable agent (e.g., HRP or a fluorescent tag) can 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 analytes. For example, the first analyte 112 can be detected as shown and described with reference to Figure 1B-1D (e.g., through the use of a primary antibody, an HRP-conjugated secondary antibody, and a chemiluminescent substrate), while the second analyte 114 can be detected through the introduction of a fluorescently labeled primary antibody without the use of HRP or a secondary antibody.

[0032] In some embodiments, some or all of the events shown and described with reference to Figures 1A-1H can be performed automatically and / or without additional user intervention other than the instructions to initiate the immunoassay. In addition, other sequences are possible and within the scope of this disclosure, although the events shown with reference to Figure 1A-1H can be performed in the order described.

[0033] Figures 2A-2H illustrate events that occur in a stripping and reprobing method according to one embodiment. The embodiment illustrated in Figure 2A-2H can be used to combine one or more immunoassays with a total protein assay. As shown, the total protein assay can be performed in the same capillary and / or the same sample as the Western blot-style immunoassay. Other orders are possible and within the scope of this disclosure, but in some examples, it may be preferable to perform the events illustrated in Figure 2A-2H and described below in the order shown, thereby reducing reagent degradation.

[0034] Figure 2A is a schematic diagram of a sample that has been separated and immobilized on the surface of capillary 210. For example, an analyte can be covalently bonded to the surface of capillary 210. As shown, the sample is separated into three bands 212, 214, and 216. Each band represents a distinct analyte. It should be understood that a sample can contain any number of analytes and / or be separated into any number of bands. For example, in some cases, the sample can be a homogeneous mixture of a single analyte.

[0035] After the sample has been separated and / or immobilized, the biotinylation reagent 220 can be introduced into the capillary 210, as shown in Figure 2B. The biotinylation reagent 220 can be configured to bind to all proteins so that a total protein assay can be performed (as will be discussed in more detail below), i.e., so that the total amount of protein in the sample / capillary 210 can be determined. It may be preferable to add the biotinylation reagent 220 to the capillary 210 before performing any immunoassay, because the biotinylation reagent is not stable enough to be introduced into the capillary after the immunoassay. For example, in some cases, the user may pack the biotinylation reagent (e.g., onto the sample plate) immediately before starting electrophoresis (e.g., less than 20 minutes before introducing the sample into the capillary), because in some situations, the biotinylation reagent may begin to degrade once packed. In some cases, the biotinylating reagent is introduced into the capillary immediately after the sample is separated and / or immobilized (e.g., within 5 minutes of immobilization and / or within 90 minutes of introducing the sample into the capillary). If necessary, excess (e.g., unbound) biotin can be washed out of the capillary after it has been introduced. As will be discussed in more detail herein, the instrument may be operable to run a reference sample in parallel lanes. As will also be described, the instrument may be operable to fill the same or different samples into multiple capillaries, including capillary 210 and at least one reference capillary (not shown). The biotinylating reagent 220 can be introduced into the reference capillary at the same time as the introduction of the biotinylating reagent into capillary 210, or, for example, within 5 minutes of the introduction of the biotinylating reagent into capillary 210. Typically, considering the stability profile of the biotinylation reagent, it is introduced into the reference capillary and capillary 210 before performing any immunoassay with the sample in capillary 210.

[0036] The immunoassay can be performed on a sample immobilized in a capillary 210 by introducing one or more primary antibodies. As shown in Figure 2C, a first primary antibody 222 configured to selectively bind to a first analyte 212 and a second primary antibody 224 configured to selectively bind to a second analyte 214 are introduced. However, it should be understood that any number of primary antibodies with any suitable selective binding properties can be introduced. In addition, 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 taken from a common reagent reservoir). If necessary, excess (e.g., unbound) primary antibodies can be washed from the capillary 210.

[0037] Figure 2D illustrates the introduction of a first secondary antibody 226 and a second secondary antibody 228 into a capillary 210. The first secondary antibody 226 is configured to selectively bind to the first primary antibody 222, and the second secondary antibody 228 is configured to selectively bind to the second primary antibody 224. The first secondary antibody 226 and / or the second secondary antibody 228 may have, or can be modified to have, optically detectable properties. For example, the secondary antibodies may be labeled with an optically detectable agent before or after introduction into the capillary. In some cases, it may be desirable that different secondary antibodies, configured to be associated with a particular primary antibody and therefore a particular analyte, have different optical properties. For example, Figure 2D illustrates the first secondary antibody 226 labeled with an optically detectable marker (e.g., a fluorescent dye) before introduction into the capillary 210. Optionally, the unbound secondary antibody and / or the optically detectable agent may be washed out of the capillary 210. The second secondary antibody 228 can be labeled with HRP, for example, before being introduced into the capillary 210. Figure 2E illustrates the introduction of a chemiluminescent substrate configured to interact with the HRP-labeled second secondary antibody 228 and produce an optically detectable signal. The optical properties of the secondary antibody, e.g., associated with the chemiluminescent and fluorescent signals, can be collected immediately and / or over time using a CCD camera or another suitable detector. Using such optical signals, some or all of the analytes present in the sample can be identified. For example, as shown in Figure 2E, analytes 212 and 214 would be detectable during the immunoassay. In cases where one analyte is associated with HRP and another with a fluorescent dye (e.g., as shown in Figures 2C-2E), the chemiluminescent and fluorescent signals can be detected simultaneously or sequentially. For example, the fluorescently labeled first secondary antibody 226 can be excited before, during, or after the introduction of the chemiluminescent substrate.

[0038] As shown in Figure 2F, once the optical properties of the secondary antibody are detected, a stripping reagent configured to remove the primary and / or secondary antibody from the analyte can be introduced into the capillary 210. The stripping reagent is configured to leave biotin 220 bound to the analyte. Figure 2G illustrates the introduction of streptavidin 232, avidin, and / or other suitable reagents configured to specifically bind to biotin into the capillary. Streptavidin can be HRP conjugated (before or after introduction into the capillary 210) and / or labeled in other ways, or optionally optically detectable. Detection of total protein can occur, for example, by packing a chemiluminescent substrate and detecting the chemiluminescent signal, as shown in Figure 2H (e.g., the amount of each biotin-labeled protein can be determined). In some examples, the amount of protein in each band can be determined separately.

[0039] Determining the total amount of protein allows for the standardization of the immunoassay signal relative to the total protein content. Similarly, as described, optical signals associated with the immunoassay (e.g., signals associated with a secondary antibody conjugated to an analyte via a primary antibody) can be corrected or standardized based on optical signals indicating the amount of protein (e.g., optical signals associated with streptavidin conjugated to a protein via biotin). In some cases, a reference capillary (not shown) can be filled with a reference sample suitable for correcting the immunoassay signal. For example, a cartridge containing multiple capillaries (e.g., capillary 210 and a reference capillary) can be filled with samples sequentially or in parallel. Proteins in each capillary can be biotinylated (sequentially or in parallel). HRP-conjugated streptavidin or another suitable reagent can be introduced into the capillaries (sequentially or in parallel). A chemiluminescent substrate can also be introduced into the capillaries (sequentially or in parallel) so that the total amount of protein in each capillary can be determined. In some embodiments, the analytes detected via immunoassay in capillary 210 can be standardized 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 the signals associated with the immunoassay of individual protein species. Using such techniques, the immunoassay signals can be corrected to account for packing heterogeneity. As similarly described, a strong immunoassay signal may be the result of a “true” signal associated with a high concentration of the target protein compared to other proteins in the sample, or it may be associated with a large total amount of protein, for example, if more cell contents than expected are packed into the capillary.

[0040] Typically, the analyst prepares the sample and / or appropriate reagents and fills the reagent / sample plate before initiating the immunoassay and / or total protein measurement. In some embodiments, after initiating the immunoassay and / or total protein measurement, some or all events (e.g., sample filling, separation, immunoassay and / or total protein, detection) can be carried out automatically and / or without further interaction by the analyst. As will be readily apparent to those skilled in the art, additional stripping and reprobing steps are possible, additional intermediate washing steps may be performed to flush unbound reagents from the capillary, different combinations of detection modes may be used, and / or alternative detection modalities (e.g., color detection, fluorescence detection) may be used instead of the exact combinations described in the embodiments above. While Figures 2A-2H describe the detection of two distinct protein species via chemiluminescence and fluorescence, followed by a total protein measurement, those skilled in the art will readily understand that alternative embodiments may use two different antibodies for two different epitopes of the same target or the same protein. Furthermore, fluorescence detection, absorbance, or any suitable and well-known detection method may be used, and this may include combinations of multiple detection modes.

[0041] Figures 2C and 2D illustrate the substantially simultaneous introduction of multiple primary antibodies 222, 224 (e.g., as a mixture), and the substantially simultaneous introduction of multiple secondary antibodies 226, 228. In contrast, Figures 1B–1G illustrate the sequential introduction of different primary and secondary antibodies, with a stripping event occurring between the introduction of the first secondary antibody 122 and the introduction of the second primary antibody 130. However, it should be understood that the methods shown and described in Figures 1A–H may include the introduction of a mixture of primary and / or secondary antibodies. Similarly, the methods shown and described in Figures 2A–2H may include sequential immunoassays, for example, with additional striping events between immunoassays.

[0042] According to some embodiments, the methods described herein can be performed on appropriate instruments for measuring protein content and / or performing immunoassays in the same capillary and / or in an automated manner, such as the Simple Western® platform by ProteinEasy®. Unlike other known instruments and techniques, the embodiments described herein are generally simpler than conventional methods used for total protein measurement for conventional Western blots. Immunoassays and total protein measurements can be performed using chemiluminescent or fluorescent methods, or other methods known in the art. Furthermore, the accuracy of detecting total protein content immobilized on the capillary can be improved by using stripping reagents used to remove antibodies from the immunoassay.

[0043] As discussed above with reference to Figures 1A-1H, those skilled in the art will understand that the embodiments shown and described with reference to Figures 2A-2H are illustrative and not limiting. Specifically, those skilled in the art will understand that analytes can be detected through any combination of chemiluminescence, fluorescence, and / or absorbance techniques. Those skilled in the art will understand that additional or fewer primary and secondary antibodies can be used. Those skilled in the art will understand that antibodies can be pre-labeled with optically detectable agents, that optically detectable agents can be introduced into capillaries to selectively bind to antibodies and / or analytes, and that / or analytes and / or antibodies are inherently detectable (e.g., unlabeled antibodies can be detected, for example, based on their absorbance properties).

[0044] Order of operations As previously described, a specific order of reagent addition is preferred for improved performance when measuring total protein and immunoassay signals in the capillary. Experimental evidence demonstrates that adding the biotinylation reagent after the immunoassay is complete results in a signal 70% lower than that obtained when the biotinylation reagent is added before the immunoassay. To obtain a preferred detection level for the assay, it is important to maintain a higher signal and thus sensitivity in this assay. While it may be possible to attempt to perform complete total protein detection before the immunoassay (e.g., biotinylation and detection using HRP-conjugated streptavidin and luminol / peroxide), this is an undesirable assay configuration, likely due to the difficulty in removing HRP-conjugated streptavidin, which has an extremely high affinity for binding to biotin. Incomplete removal of HRP-conjugated streptavidin can negatively impact immunoassay performance, for example, through residual HRP-conjugated streptavidin bound to biotinylated proteins that prevent antibody binding to target proteins.

[0045] Stripping reagent preparation There are a wide variety of preparations known in the art for removing antibodies from Western blot membranes. These preparations typically contain buffering components, surfactants, denaturants, acidic or basic 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 to remove antibodies bound to analytes from capillaries.

[0046] Stripping reagent formulations using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a phosphine reducing agent instead of β-mercaptoethanol have been developed and are shown in Table 1. While most of the formulations in Table 1 tested functioned to some extent in removing antibodies, it is desirable to consistently 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., precipitation or degradation should not occur during storage over a certain period (e.g., 1 day, 1 week, 1 month, 6 months, 1 year, or any other suitable time frame). As can be seen in Table 1, simply replacing β-mercaptoethanol with TCEP did not consistently achieve >95% stripping efficiency without precipitation. It is important to achieve high stripping efficiency across many antibodies because retained antibodies can introduce noise and lower the detection limit for subsequent immunoassay steps.

[0047] Further optimization was carried out through broader titrations and novel combinations of components from Table 1 with additional components (e.g., different surfactants and reducing agents). It was determined that stripping efficiency is relatively insensitive to SDS concentration, while important effects on stripping efficiency include Trizma (Tris base; CAS: 77-86-1), TCEP concentration, and pH. The formulations were analyzed for antibody removal efficiency in Simple Western® in combination with various immunoassays (different antibodies, different target proteins), as shown in Table 2. The formulations were further tested for precipitation, degradation, and antibody removal efficiency of ≥95% for multiple antibodies. Several candidates in Table 2 meet these criteria. Formulations with neutral or basic pH performed significantly worse than those with acidic pH. This observation was surprising. This is because TCEP is the reducing agent in the preferred formulation, and conventional wisdom suggests that TCEP is effective over a wide pH range of 1.5 to 8.5, exhibiting best reducing performance near neutral pH (i.e., near pH 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. 1994 Jul;220(1):5-10, which is incorporated herein by reference in its entirety). Furthermore, the preferred formulation is determined to operate with the highest antibody removal efficiency in a narrow pH range, as shown in Figure 3, thereby illustrating that a preferred formulation capable of producing a >97% stripping efficiency has a pH of 4.05 ± 0.3. Figure 3 illustrates the stripping efficiency against three different targets: park7, beta-actin, and HSP60.

[0048] The stripping reagents in Tables 1 and 2 were prepared by mixing the specified components with water at the specified molar concentrations or percentages (by weight). Where pH is reported for a buffer species or TCEP stock, these pH values ​​represent the pH of the component before it is combined to form the stripping reagent. The stripping reagent is then adjusted to the indicated final pH after all components have been mixed (e.g., by adding hydrochloric acid, sodium hydroxide, or other suitable acid or base). In some examples, no pH adjustment is performed, and the final pH is simply the measured pH after the components have been combined. Final pH values ​​indicated with an asterisk are estimated final pH values ​​(i.e., no measurement was performed). It should be understood that component concentrations may vary by 1%, 5%, or 10%, but still remain within the range of the stripping buffer reagent compositions intended by the inventors. The buffer type, TCEP stock, or the indicated pH of the final pH may vary by 0.1, 0.3, 0.5, or 1, and remain within the range of the stripping buffer composition intended by the inventors. The reported stripping efficiency information was observed experimentally. It should be understood that the formulated buffer, as shown, may not accurately reproduce the observed stripping efficiency. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0049] Figure 4 illustrates the performance of the formulations shown in Table 3, demonstrating a greater decrease in stripping efficiency when the pH is greater than 4.5. A less severe, but still significant decrease in stripping efficiency is observed when the pH is less than 3 (data is not shown in Figure 4, but is evident from the individual rows in Table 2). It was surprising to discover that a narrow and specific pH range is required for optimal antibody removal (≥95%) in Simple Western® capillaries. This may be due to the inherent properties and / or internal environment of the Simple Western capillary versus the Western blot membrane. It was also determined that multiple incubations of the stripping reagent in the capillary improved antibody removal efficiency. [Table 3]

[0050] Figures 5A and 5B are charts showing experimental data illustrating the reproducibility of the probing analytes (ATK1 and ATK2) after the introduction of the stripping reagent. As similarly described, the results shown in Figures 5A and 5B were generated according to the same methods as shown and described with reference to Figures 1A-1H. Figure 5B illustrates the change in peak area before and after reprobing (e.g., including the introduction of the stripping reagent). ATK1 and ATK2 were probed and reprobed in multiple capillaries, but the order in which the analytes were probed was varied. In this example, ATK1 was first probed in six capillaries, and ATK2 was first probed in six capillaries. The stripping reagent was introduced into all 12 capillaries after the initial probing to remove the primary and secondary antibodies from the first target analytes, and then the other target analytes were probed. As shown in Figure 5B, the peak area of ​​the target analyte is not significantly affected by the stripping reagent or the order in which the analytes are probed. This illustrates that the stripping reagent does not remove a significant amount of the analyte.

[0051] While various embodiments have been described above, it should be understood that they are presented only as examples and are not limiting. For example, while the embodiments described herein generally describe capillary-based techniques, it should be understood that any suitable microfluidic device or other electrophoretic technique can be used. For example, Yao, S. Anex, DS, Valdwell, WB, Arnold DW, Smith KB, & Schultz, PG, SDS, Capillary Gel Electrophoresis of Proteins in Microfabricated Channels, 96(10) Proc Natl Acad Sci USA 5372-77 (May 11, 1999) (the entire disclosure is incorporated herein by reference) describes a chip fabricated to include suitable microchannels and other microfluidic structures for performing protein sizing separation. Such a chip can be adapted to perform the methods and / or assays described herein. As another example, biotinylation is generally described by the embodiments described herein relating to total protein labeling. However, it should be understood that other suitable techniques for non-specific 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 part for chemically reacting with a protein (e.g., amino acids, e.g., lysine, etc., protein backbone, e.g., nitrogen, etc., post-translational modifications such as glycans, etc.) may be suitable for total protein labeling. It should also be understood that while techniques for determining the total amount of protein and standardizing based on protein amount have been described, similar techniques exist for many other analytes. For example, it is possible to measure the total amount of nucleic acids, lipids, and / or glycoproteins, and then use this to standardize the measurement among capillaries.For example, molecules having an aminooxy reactive group can chemically react with sugars, such as polysaccharides or glycan groups. Similarly, molecules having a psoralen group can bind to DNA or RNA via UV photoactivated intercalation of the psoralen group with thymine and other pyrimidine-containing bases. Nucleic acid binding chemistry includes the carbodiimide crosslinking agent EDC / imidazole and other chemical and enzymatic attachment methods known in the art. Similarly, methods for biotinylating lipids are known in the art. See, for example, 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 incorporated herein by reference.

[0052] Where the schematic diagrams and / or embodiments described above show specific components arranged in a particular orientation or position, the arrangement of the components may be changed. While embodiments are specifically shown and described, it will be understood that various variations in form and detail are possible. Various embodiments have been described as having particular features and / or combinations of components, but as discussed above, other embodiments are possible having any combination of features and / or components from any of the embodiments.

[0053] If the methods and / or events described above indicate specific events and / or procedures occurring in a particular order, the order of those events and / or procedures may be modified. In addition, the specific events and / or procedures may be performed simultaneously in parallel processes where possible, as well as sequentially as described above.

Claims

1. Electrophoretic separation of a sample containing a first analyte and a second analyte in a microfluidic device; Immobilizing the first analyte and the second analyte in the microfluidic device; Introducing a first primary antibody configured to bind to the first analyte; Introducing a first secondary antibody configured to bind to the first primary antibody; To detect the first analyte based on the optical properties associated with the first secondary antibody; Introducing a stripping reagent configured to remove the first primary antibody from the first analyte while the first and second analytes remain immobilized in the microfluidic device; Introducing a second primary antibody configured to bind to the second analyte; Introducing a second secondary antibody configured to bind to the second primary antibody; and A method comprising detecting the second analyte based on optical properties associated with the second secondary antibody.

2. The first primary antibody is introduced before the stripping agent is introduced; and The method according to claim 1, wherein the second primary antibody is introduced after the stripping agent has been introduced.

3. The method according to claim 1, wherein the first secondary antibody and the second secondary antibody are the same secondary antibody.

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

5. The method according to claim 1, wherein the first secondary antibody is conjugated to horseradish peroxidase (HRP) such that the optical property associated with the first secondary antibody is a chemiluminescent reaction associated with horseradish peroxidase.

6. The first secondary antibody is conjugated with horseradish peroxidase (HRP), and the method is as follows: The method according to claim 1, further comprising introducing a chemiluminescent substrate into a capillary, wherein the optical property associated with the first secondary antibody is a chemiluminescent signal associated with the interaction between the chemiluminescent substrate and HRP conjugated to the first secondary antibody.

7. The first secondary antibody is conjugated to horseradish peroxidase (HRP) such that the optical property associated with the first secondary antibody is a chemiluminescent signal; and The method according to claim 1, wherein the second secondary antibody is conjugated with a fluorescent dye such that the optical property associated with the second secondary antibody is a fluorescent signal.

8. The method according to claim 7, wherein the first analyte and the second analyte are detected before the introduction of the stripping reagent.

9. The method according to claim 1, further comprising washing the unbound first secondary antibody before detecting the first analyte.

10. The method according to claim 1, further comprising washing the first primary antibody and the second primary antibody from the microfluidic device after introducing the stripping reagent and before introducing the second primary antibody, and while the first analyte and the second analyte remain immobilized in the microfluidic device.

11. The method according to claim 1, wherein the stripping agent is configured to remove the first secondary antibody.

12. The method according to claim 1, wherein the sample is separated electrophoretically, so that the first analyte moves to the first part of the microfluidic device and the second analyte moves to the second part of the microfluidic device, and the first analyte and the second analyte are immobilized in the first part of the microfluidic device and the second part of the microfluidic device, respectively.

13. The method according to claim 1, wherein the introduction of the first primary antibody, the introduction of the first secondary antibody, the detection of the first analyte, and the introduction of the stripping reagent occur before the introduction of the second primary antibody and the introduction of the second secondary antibody.

14. The method according to 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 that order and without user intervention.

15. The method according to claim 1, wherein the stripping reagent comprises tris(2-carboxyethyl)phosphine hydrochloride and has a pH of 3 to 4.

5.

16. The method according to claim 1, wherein the stripping reagent has a composition selected from Table 2, has a stripping efficiency of more than 95%, and does not exhibit precipitation.

17. Electrophoretic separation of samples containing multiple proteins in microfluidic devices; Immobilizing the multiple proteins in the microfluidic device after electrophoretic separation; Introducing a molecule containing a reactive moiety configured to bind nonspecifically to a protein; Introducing a primary antibody configured to bind to at least a subset of the aforementioned multiple proteins; Introducing a secondary antibody configured to bind to the primary antibody; Detecting the subset of proteins based on the optical properties associated with the secondary antibody; Introducing a stripping reagent configured to remove the primary antibody from the aforementioned subset of proteins; Introducing an optically detectable agent configured to bind to the aforementioned molecule; Detection of optical signals associated with the optically detectable drug; and A method comprising standardizing the optical properties associated with the secondary antibody based on the optical signal associated with the optically detectable drug.

18. The molecule is biotin such that introducing the molecule includes biotinylating the plurality of proteins; and The optically detectable agent is horseradish peroxidase (HRP) conjugate streptavidin, and the method is The method according to claim 17, further comprising introducing a chemiluminescent substrate configured to interact with the HRP and generate the optical signal associated with the optically detectable agent.

19. The optically detectable agent is horseradish peroxidase (HRP), and the method is The method according to claim 17, further comprising introducing a chemiluminescent substrate configured to interact with the HRP and generate the optical signal associated with the optically detectable agent.

20. The total amount of the plurality of proteins based on the optical signal associated with the optically detectable drug, The method of claim 17, further comprising determining the optical properties associated with the secondary antibody, which are standardized based on the total amount of the plurality of proteins.

21. The subset of the protein is a first protein, the primary antibody is a first primary antibody, and the secondary antibody is a first secondary antibody, and the method is Introducing a second primary antibody configured to bind to a second protein from the aforementioned multiple proteins; Introducing a second secondary antibody configured to bind to the second primary antibody; Detecting the second protein based on the optical properties associated with the second secondary antibody; and The method according to claim 17, comprising standardizing the optical properties associated with the second secondary antibody based on the optical signal associated with the optically detectable drug.

22. The method according to claim 21, wherein the first secondary antibody is labeled with horseradish peroxidase, and the second secondary antibody is labeled with a fluorescent dye.

23. The method according to claim 21, wherein introducing the molecule, introducing the first primary antibody, introducing the first secondary antibody, detecting the first protein, introducing the second primary antibody, introducing the second secondary antibody, detecting the second protein, introducing the stripping reagent, introducing the optically detectable agent, and detecting the optical signal associated with the protein all occur while the plurality of proteins are immobilized in the microfluidic device.

24. The method according to claim 21, wherein the following steps are taken in that order: introducing the molecule, introducing the first primary antibody, detecting the first protein, introducing the stripping reagent, introducing the optically detectable agent, and detecting the optical signal associated with the protein.

25. The second primary antibody is introduced substantially simultaneously with the introduction of the first primary antibody; and The method according to claim 24, wherein the second secondary antibody is introduced substantially simultaneously with the introduction of the second secondary antibody.

26. The method of claim 17, wherein the introduction of the molecule, the introduction of the primary antibody, the introduction of the secondary antibody, the detection of the subset of proteins, the introduction of the stripping reagent, the introduction of the optically detectable agent, and the detection of the optical signal associated with the proteins all occur while the plurality of proteins are immobilized in the microfluidic device.

27. The method according to claim 17, wherein the introduction of the molecule, the introduction of the primary antibody, the detection of the subset of the protein, the introduction of the stripping reagent, the introduction of the optically detectable agent, and the detection of the optical signal associated with the protein occur in that order without user intervention.

28. The method according to claim 17, wherein the stripping reagent comprises tris(2-carboxyethyl)phosphine hydrochloride and has a pH of 3 to 4.

5.

29. The method according to claim 17, wherein the stripping reagent has a composition selected from Table 2, has a stripping efficiency of more than 95%, and does not exhibit precipitation.

30. The method according to claim 17, wherein each of the proteins from the plurality of proteins is a molecule of a common protein species.

31. The method according to claim 17, wherein the plurality of proteins comprises a plurality of protein species.

32. The method according to claim 17, further comprising washing the primary antibody and the secondary antibody from the microfluidic device after introducing the stripping reagent and before introducing the optically detectable agent, and while the plurality of proteins remain immobilized in the microfluidic device.

33. The microfluidic device is the first capillary; The aforementioned sample is the first sample; and The optical signal associated with the optically detectable drug is the first optical signal, and the method is Introducing the second sample into the second capillary; Introducing the molecule, which includes the reactive portion configured to bind nonspecifically to a protein, into the second capillary; Introducing the optically detectable agent configured to bind to the molecule into the second capillary; and Detecting a second optical signal, wherein the second optical signal is associated with the optically detectable drug and the second capillary. The method according to claim 17, further comprising standardizing the optical properties associated with the secondary antibody, which includes correcting the optical properties associated with the secondary antibody based on the ratio of the first optical signal to the second optical signal.

34. A stripping reagent, Buffer; Tris(2-carboxyethyl)phosphine hydrochloride (TCEP); and Contains surfactants, A stripping reagent with a pH below 5.

35. The stripping reagent according to claim 34, wherein the pH is 3 to 4.

5.

36. The aforementioned buffer solution Trizma with a pH greater than 8; Glycine HCl with a pH less than 5; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); and A stripping reagent according to claim 34, selected from the group consisting of bicin.

37. The stripping agent according to claim 35, wherein the concentration of the buffer species is 100 to 200 mmol.

38. The stripping agent according to claim 34, wherein the stripping reagent has a composition selected from Table 2 and has a stripping efficiency of more than 95%.

39. The stripping agent according to claim 34, wherein the stripping reagent has a composition selected from Table 2, has a stripping efficiency of more than 95%, and does not exhibit precipitation.

40. Electrophoretic separation of a sample containing multiple analytes in a microfluidic device; Immobilizing the plurality of analytes in the microfluidic device; Introducing a first primary antibody configured to bind to a first analyte from the aforementioned plurality of analytes; Introducing a secondary antibody configured to bind to the first primary antibody; Introducing a stripping reagent configured to remove the first primary antibody from the first analyte, wherein the stripping reagent has a pH of 3 to 4.5 and contains tris(2-carboxyethyl)phosphine hydrochloride (TCEP); and A method comprising introducing, after introducing the stripping reagent, at least one of a second primary antibody configured to bind to a second analyte from the plurality of analytes, or streptavidin configured to bind to biotin complexed with the plurality of analytes.

41. The aforementioned stripping reagent Trizma with a pH greater than 8; Glycine HCl with a pH less than 5; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); and The method according to claim 40, comprising a buffer selected from the group consisting of bicin.

42. The method according to claim 40, wherein the stripping reagent has a composition selected from Table 2 and has a stripping efficiency of more than 95%.

43. Biotinylation of the plurality of analytes after immobilization and before introduction of the first primary antibody; and The method according to claim 40, further comprising introducing streptavidin after introducing the stripping reagent.

44. Biotinylation of the plurality of analytes after immobilization and before introduction of the first primary antibody; Detecting the first analyte based on the optical signal associated with the secondary antibody; The process involves introducing streptavidin after introducing the stripping reagent, wherein the streptavidin is conjugated with a luminescent agent configured to bind to biotin; To detect an optical signal associated with the luminescent agent; Determining the amount of the plurality of analytes based on the optical signal associated with the luminescent agent; and The method according to claim 40, further comprising standardizing the optical signal associated with the secondary antibody based on the amounts of the plurality of analytes.

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