Fluorescence Elman assay for free thiol detection

Abstract

The present disclosure relates to methods and kits for detecting free thiols in a substrate, and methods for quantifying the amount of free thiols in a substrate. In particular, the present disclosure provides a fluorescent Ellman's assay for increasing the sensitivity of the detection and quantification of free thiols.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 106,569, filed October 28, 2020, which is incorporated herein by reference in its entirety and which claims priority.

[0002] Technical field This disclosure relates to a method for detecting thiols. In particular, this disclosure provides an enhancement of the conventional Elman method to a fluorescent Elman method in order to increase the sensitivity of detecting free thiols. [Background technology]

[0003] Levels of low molecular weight thiols, such as cysteine, homocysteine, and glutathione, are often crucial for redox signaling and maintaining redox homeostasis. Thiol imbalances (particularly homocysteine) are associated with diseases such as cancer, Alzheimer's disease, and cardiovascular disease. Signs of oxidative stress and aging can also manifest as a distorted distribution between free thiols and other thiol forms (cysteine, glutathione, sulfinic acid, etc.) on low molecular weight thiol substrates and proteins such as human serum albumin. Quantitative analysis of free thiols or reactive sulfhydryls is an important analytical tool for biological and medical research. Quantitative analysis of free thiols is useful for high-throughput screening of novel inhibitors of acetyltransferases, a key class of enzymes involved in major metabolic pathways and epigenetic regulation. Quantitative methods for free thiols are also applied in the biopharmaceutical industry to monitor the quality of protein products, where free thiols may be incorporated into proteins to form bioconjugates for therapeutic or diagnostic purposes, or may be misrepresented as undesirable post-translational modifications, and are treated as a potentially important quality attribute.

[0004] Among the available quantitative methods, photodetection of free thiols is particularly used, and a large family of optical probes for thiol detection has been developed. However, a common drawback of fluorescent probes for thiol detection is that their sensitivity can be dependent on the thiol substrate. As a result, fluorescent probes for thiol detection usually require either calibration using the same standard as the test substrate, or determination of an appropriate response coefficient. Further drawbacks of fluorescent probes include their potential for sterically bulky structures and often limited solubility in water. On the other hand, the classic optical probe for free thiol detection, Elman's reagent or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), is, coincidentally, highly water-soluble, compact, and its sensitivity is independent of the thiol substrate. The main drawback of Elman's method is that its quantification limit is significantly higher (3-4 orders of magnitude) than most fluorescent methods for the quantification of free thiols. This can mean that large quantities of sample (e.g., milligrams of therapeutic antibody) are required to obtain quantitative measurements of free thiols, which is impractical and sometimes prohibitively expensive. [Overview of the Initiative]

[0005] In certain embodiments, the disclosure a) brings a thiol substrate into contact with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form TNB 2- b) liberating TNB stoichiometrically; 2- The free TNB 2- Contacting with a reagent that interacts with it to produce a fluorescent signal; and c) free TNB 2- The present invention relates to a method for detecting free thiols, comprising detecting a fluorescent signal released by interaction with a reagent, thereby detecting free thiols. In certain embodiments, the reagent used in connection with the method is a fluorescent probe. In certain embodiments, the reagent used in connection with the method is a fluorescent probe. In certain embodiments, TNB 2-By incubating the molecule with a fluorogenic probe or a fluorescent probe, formation of a fluorescent TNB probe adduct, or a deprotected fluorescent probe and a non-fluorescent TNB adduct is brought about. In certain embodiments, the fluorescent signal is emitted by the fluorescent TNB probe adduct. In certain embodiments, the fluorescent signal is emitted by the deprotected fluorescent probe. In certain embodiments, the fluorogenic probe or the fluorescent probe is a thiol-specific probe. In certain embodiments, the thiol-specific probe contains a maleimide functional group. In certain embodiments, the thiol-specific probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorogenic probe is methyl maleimide-benzocoumarin-carboxylic acid (MMBC). In certain embodiments, the fluorogenic probe is ThioFluor623.

[0006] In certain embodiments of the method for detecting free thiols described herein, the thiol is present on a low molecular weight thiol substrate. In certain embodiments, the thiol is present on a high molecular weight thiol substrate. In certain embodiments, the high molecular weight thiol substrate is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is IgG2. In certain embodiments, the antibody is half of a bispecific antibody. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug conjugate (ADC).

[0007] In certain embodiments, the present disclosure provides a method comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically liberate TNB; 2- b) incubating the liberated TNB with a reagent that interacts with the liberated TNB to generate a fluorescent signal; 2- c) the liberated TNB 2- and 2-Detecting a fluorescence signal emitted by interaction with a reagent, and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) with a known amount of a reference signal, a method for determining the free thiol content of a thiol substrate is targeted.

[0008] In certain embodiments of the method for detecting free thiols described herein, the reagent is a fluorogenic probe. In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, the thiol is present on a high molecular weight thiol substrate. In certain embodiments, the high molecular weight thiol substrate is a polypeptide. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug conjugate. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is IgG2. In certain embodiments, the antibody is half of a bispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

[0009] In certain embodiments of the method for detecting free thiols described herein, TNB 2- Incubation with a fluorogenic probe or a fluorescent probe results in the formation of a fluorescent TNB probe adduct, or a deprotected fluorescent probe and a non-fluorescent TNB adduct. In certain embodiments, the fluorescence signal is emitted by the fluorescent TNB probe adduct. In certain embodiments, the fluorescence signal is emitted by the deprotected fluorescent probe. In certain embodiments, the fluorogenic probe or the fluorescent probe is a thiol-specific probe. In certain embodiments, the thiol-specific probe contains a maleimide functional group. In certain embodiments, the thiol-specific probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorogenic probe is methyl maleimidobenzocoumarin carboxylic acid (MMBC). In certain embodiments, the fluorogenic probe is ThioFluor623.

[0010] In a particular embodiment of the free thiol detection method described herein, the free thiol content is calculated by determining the thiol concentration by comparing the fluorescence signal with a calibration curve and dividing the thiol concentration by the concentration of the thiol substrate.

[0011] In certain embodiments, the present disclosure relates to a kit for the detection of thiol compounds comprising a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorescent probe or fluorescent probe. In certain embodiments, the kit includes a thiol-containing standard for generating a calibration curve. In certain embodiments, the thiol-containing standard is selected from cysteine, glutathione, and N-acetylated cysteine. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific fluorescent probe. In certain embodiments, the fluorescent probe is maleimidebenzochromenecarboxylate methyl (MMBC). In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe contains a maleimide functional group. In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is ThioFluor623. In certain embodiments, the kit of the present disclosure includes a denatured buffer. In certain embodiments, the denatured buffer is a 3-(N-morpholino)propanesulfonate hemisodium (MOPS) buffer containing guanidine hydrochloride. [Brief explanation of the drawing]

[0012] [Figure 1] Schematic diagram of the fluorescent Elman ("F. Elman") assay. Incubation of a thiol substrate with DTNB (Step I) stoichiometrically releases a TNB2 molecule, which then reacts with a thiol-specific fluorescent probe (Step II) to generate a fluorescent signal. These fluorescence reactions proceed either by forming a fluorescent TNB probe adduct (IIA) or by generating a deprotected fluorescent probe (IIB). [Figure 2]Schematic diagram of the reaction between TNB2- and MMBC to produce a fluorescent TNB-MMBC adduct (exact mass: 578.0637 Da, expected structure at the far right) according to the pathway (IIA) shown in Figure 1. [Figure 3] Emission and excitation spectra of a fluorescent TNB-MMBC adduct. (A) Emission spectrum at an excitation wavelength fixed at 385 nm, and (B) Excitation spectrum at an emission wavelength fixed at 510 nm. [Figure 4] A) Reverse-phase chromatograms of the starting reagents (TNB, MMBC) and TNB-MMBC adducts, detected by UV detection at 214 nm and B) fluorescence detection at 375 nm / 510 nm. Peaks marked with an asterisk indicate impurities associated with the TNB or MMBC starting material. C) Mass spectra of the early-eluted and D) late-eluted TNB-MMBC adduct peaks. [Figure 5] A graph showing the time course of fluorescence of a cysteine ​​standard using the F. Elman assay (MMBC reagent was introduced at time 0). [Figure 6] Graphs showing F. Elman calibration curves for cysteine ​​(cys), N-acetylated cysteine ​​(NAC), and glutathione (gsh) under A) natural and B) denatured conditions. Fluorescence readings were taken 30 minutes after introduction of MMBC reagent. Substrate-independent linear regression (trend lines) for both plots were based on all plotted data points. [Figure 7] A graph showing bridging datasets between F. Elman (denatured) and NcHM-tagged RPLC assays, orthogonal methods for the quantification of total free thiols in antibodies. Intermediate precision for F. Elman was determined using the first seven molecules (IgG1-A / B / C / D / E, IgG4-A, bispecific IgG). Error bars represent one standard deviation. The coefficient of variation for F. Elman was <8% for each molecule used for intermediate precision. [Figure 8] A schematic diagram of the reaction between TNB2- and ThioFluor623 to produce deprotected fluorescent ThioFluor623 according to the pathway (IIB) shown in Figure 1. [Figure 9]This graph shows the sensitivity of MMBC (white circles, left y-axis) versus ThioFluor623 (black circles, right y-axis) after reacting with TNB2- for 30 minutes. Note that the magnitude of the obtained MMBC fluorescence is approximately 40 times greater than that of the obtained ThioFluor623 fluorescence. [Figure 10] This graph shows an acid / base titration comparing the buffering capacity of phosphate and MOPS buffer in the presence of 4M guanidine hydrochloride (Phosphate buffer: 100mM phosphate, 4M guanidine hydrochloride, 1mM EDTA, pH 7.4; MOPS buffer: 100mM MOPS, 4M guanidine hydrochloride, 1mM EDTA, pH 7.4). Acid titrations were performed using 1N HCl, and base titrations were performed using 1N NaOH. [Modes for carrying out the invention]

[0013] The following detailed description is given as an example, but is not intended to limit the subject matter of this disclosure to the specific embodiments described and can be understood in conjunction with the accompanying drawings.

[0014] The subject of this disclosure is a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form TNB 2- b) liberating TNB stoichiometrically; 2- The free TNB 2- Contacting with a reagent that interacts with it to produce a fluorescent signal, and c) releasing TNB 2- The present invention provides a method for detecting thiols, comprising detecting a fluorescent signal released by interaction with a reagent, thereby detecting the thiol. The subject of this disclosure also provides a method for quantifying the free thiol content of a thiol substrate, a) contacting the thiol substrate with 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and TNB 2- b) Stoichiometric liberation of TNB 2- TNB released 2- Incubate with a reagent that interacts with it to produce a fluorescent signal; c) Free TNB 2-The present invention also provides a method comprising detecting a fluorescent signal released by interaction with a reagent; and quantifying the free thiol content of a molecule by comparing the signal detected in d) and c) with a known reference signal. The subject matter of this disclosure also provides a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a kit for detecting thiol compounds including a fluorescent probe or a fluorescent probe.

[0015] The subject matter of this disclosure is, at least in part, based on the discovery that by adding an incubation step with the fluorescent probe maleimide benzochromenecarboxylate methyl (MMBC) to the end of the Elman method, the UV absorption signal can be effectively converted to a fluorescent signal, and even with a 2-fold dilution by MMBC addition, the quantification limit of the Elman method can be improved by approximately 4-fold.

[0016] Non-limiting embodiments of the subject matter of this disclosure are described herein and by examples.

[0017] 1.Definition Unless otherwise specified, all technical terms, notations, and other scientific terms used herein are intended to have meanings that are generally understood by those skilled in the art to which this disclosure relates. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those generally understood in the art.

[0018] As used herein, “equipment,” “includes,” “have,” “possess,” “can have,” “contain,” and their variations are intended to be unrestrictive transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of” the embodiments or elements presented herein, whether expressly described or not.

[0019] As used herein, the terms “about” or “approximately” mean within a range of tolerance for a particular value as determined by those skilled in the art, which in part depends on the method of measuring or determining the value, i.e., the limitations of the measuring system. For example, “about” may mean a standard deviation of 3 or more than 3, according to the practice of the art. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably up to five times, and even more preferably up to two times.

[0020] As used herein, “polypeptide” generally refers to peptides and proteins having about 10 or more amino acids. Polypeptides may be homologous to host cells, or preferably exogenous, meaning they are heterogeneous, or foreign, to the host cell being utilized, such as human proteins produced by Chinese hamster ovary cells or yeast polypeptides produced by mammalian cells. In certain embodiments, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those secreted directly into the culture medium.

[0021] The term “protein” refers to a sequence of amino acids whose chain length is sufficient to produce a higher level of tertiary and / or quaternary structure. This is to distinguish it from “peptides” or other low molecular weight drugs that do not have such structures. Typically, proteins as used herein have a molecular weight of at least about 15–20 kD, preferably at least about 20 kD. Examples of proteins included within the definition herein include all mammalian proteins, particularly therapeutic and diagnostic proteins such as therapeutic and diagnostic antibodies, and proteins containing one or more disulfide bonds, including multi-chain polypeptides that generally contain one or more interchain and / or intrachain disulfide bonds.

[0022] The term "antibody" is used in its broadest sense herein and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence (including polymers of such pairings)), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0023] 2. Method The subject matter of this disclosure provides a method for detecting thiols. In a particular embodiment, the method involves a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to TNB 2- b) liberating TNB stoichiometrically; 2- The free TNB 2- Contacting with a reagent that interacts with it to produce a fluorescent signal; and c) free TNB 2- This method includes detecting a fluorescent signal released by the interaction of a reagent, thereby detecting a thiol.

[0024] The subject matter of this disclosure also provides a method for quantifying the free thiol content of a thiol substrate, the method comprising a) contacting the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and TNB 2-b) Stoichiometric liberation of TNB 2- TNB released 2- Incubate with a reagent that interacts with it to produce a fluorescent signal; c) Free TNB 2- This includes detecting a fluorescent signal released by interaction with a reagent, and quantifying the free thiol content of the molecule by comparing the signal detected in d) and c) with a known reference signal.

[0025] In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, TNB 2-Incubation of a molecule with a fluorescent probe or fluorescent probe results in the formation of a) a fluorescent TNB probe adduct; or b) a deprotected fluorescent probe and a non-fluorescent TNB adduct. In certain embodiments, the fluorescent signal is emitted by the fluorescent TNB probe adduct. In certain embodiments, the fluorescent signal is emitted by the deprotected fluorescent probe. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific probe. In certain embodiments, the thiol-specific probe contains a maleimide functional group. In certain embodiments, the thiol-specific probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is maleimide benzo-chromene carboxylate methyl (MMBC). In certain embodiments, the fluorescent probe is ThioFluor623. In certain embodiments, the thiol is located on a low molecular weight thiol substrate. In certain embodiments, the thiol is located on a high molecular weight thiol substrate. In certain embodiments, the high molecular weight thiol substrate is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is IgG2. In certain embodiments, the antibody is half of a bispecific antibody. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug conjugate (ADC). In certain embodiments, the free thiol content is calculated by determining the thiol concentration by comparing the fluorescence signal with a calibration curve and dividing the thiol concentration by the concentration of the thiol substrate.

[0026] 3. Kit The subject matter of this disclosure provides a kit for detecting thiol compounds. In certain embodiments, the kit comprises 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and a fluorescent probe or fluorescent probe. In certain embodiments, the kit comprises a sterile container; such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding pharmaceuticals. In certain embodiments, the kit comprises a thiol-containing standard for generating a calibration curve. In certain embodiments, the thiol-containing standard is selected from cysteine, glutathione, and N-acetylated cysteine. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific fluorescent probe. In certain embodiments, the fluorescent probe is maleimide benzo-chromene carboxylate methyl (MMBC). In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe comprises a maleimide functional group. In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is ThioFluor623. In certain embodiments, the kit contains a denatured buffer. In certain embodiments, the denatured buffer is a 3-(N-morpholino)propanesulfonic acid hemisodium (MOPS) buffer containing guanidine hydrochloride.

[0027] 4. Exemplary Embodiments of the Present Disclosure In certain embodiments, the disclosure a) brings a thiol substrate into contact with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form TNB 2- b) liberating TNB stoichiometrically; 2- The free TNB 2- Contacting with a reagent that interacts with it to produce a fluorescent signal; and c) free TNB 2-The present invention relates to a method for detecting free thiols, comprising detecting a fluorescent signal released by interaction with a reagent, thereby detecting free thiols. In certain embodiments, the reagent used in connection with the method is a fluorescent probe. In certain embodiments, the reagent used in connection with the method is a fluorescent probe. In certain embodiments, TNB 2- Incubating a molecule with a fluorescent probe or a fluorescent probe results in the formation of a fluorescent TNB probe adduct, or a deprotected fluorescent probe with a non-fluorescent TNB adduct. In certain embodiments, the fluorescent signal is emitted by the fluorescent TNB probe adduct. In certain embodiments, the fluorescent signal is emitted by the deprotected fluorescent probe. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific probe. In certain embodiments, the thiol-specific probe contains a maleimide functional group. In certain embodiments, the thiol-specific probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is maleimide-benzochromene-methyl carboxylate (MMBC). In certain embodiments, the fluorescent probe is ThioFluor623.

[0028] In certain embodiments of the free thiol detection methods described herein, the thiol is present on a low molecular weight thiol substrate. In certain embodiments, the thiol is present on a high molecular weight thiol substrate. In certain embodiments, the high molecular weight thiol substrate is a polypeptide. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is IgG2. In certain embodiments, the antibody is half of a bispecific antibody. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug conjugate (ADC).

[0029] In certain embodiments, the disclosure a) brings a thiol substrate into contact with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form TNB 2- b) Stoichiometric liberation of TNB 2-TNB released 2- Incubate with a reagent that interacts with it to produce a fluorescent signal; c) Free TNB 2- This invention relates to a method for determining the free thiol content of a thiol substrate, which includes detecting a fluorescent signal released by interaction with a reagent, and quantifying the free thiol content of the molecule by comparing the signal detected in d) and c) with a known reference signal.

[0030] In certain embodiments of the free thiol detection methods described herein, the reagent is a fluorescent probe. In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, the thiol is present on a high molecular weight thiol substrate. In certain embodiments, the high molecular weight thiol substrate is a polypeptide. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug conjugate. In certain embodiments, the polypeptide is an antibody. In certain embodiments, the antibody is IgG2. In certain embodiments, the antibody is half of a bispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

[0031] In certain embodiments of the free thiol detection method described herein, TNB 2- Incubation with a fluorescent probe or fluorescent probe results in the formation of a fluorescent TNB probe adduct, or a deprotected fluorescent probe and a non-fluorescent TNB adduct. In certain embodiments, the fluorescent signal is emitted by the fluorescent TNB probe adduct. In certain embodiments, the fluorescent signal is emitted by the deprotected fluorescent probe. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific probe. In certain embodiments, the thiol-specific probe contains a maleimide functional group. In certain embodiments, the thiol-specific probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is maleimidebenzochromenecarboxylate methyl (MMBC). In certain embodiments, the fluorescent probe is ThioFluor623.

[0032] In a particular embodiment of the free thiol detection method described herein, the free thiol content is calculated by determining the thiol concentration by comparing the fluorescence signal with a calibration curve and dividing the thiol concentration by the concentration of the thiol substrate.

[0033] In certain embodiments, the present disclosure relates to a kit for the detection of thiol compounds comprising a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorescent probe or fluorescent probe. In certain embodiments, the kit includes a thiol-containing standard for generating a calibration curve. In certain embodiments, the thiol-containing standard is selected from cysteine, glutathione, and N-acetylated cysteine. In certain embodiments, the fluorescent probe or fluorescent probe is a thiol-specific fluorescent probe. In certain embodiments, the fluorescent probe is maleimidebenzochromenecarboxylate methyl (MMBC). In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe contains a maleimide functional group. In certain embodiments, the thiol-specific fluorescent probe or fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorescent probe is ThioFluor623. In certain embodiments, the kit of the present disclosure includes a denatured buffer. In certain embodiments, the denaturation buffer is 3-(N-morpholino)propanesulfonic acid hemisodium salt (MOPS) buffer. [Examples]

[0034] Example 1: Materials and Method material All tested antibodies and antibody-drug conjugates (ADCs) were manufactured by Genentech, Inc. (South San Francisco, California, USA). The two ADCs tested contained one drug conjugated to a reduced interchain disulfide (ADC-A) and the other conjugated to a modified unpaired cysteine ​​(ADC-B). DTNB, cysteine, glutathione, N-acetylcysteine, dimethyl sulfoxide (DMSO), sodium phosphate, 3-(N-morpholino)propanesulfonate hemisodium salt (MOPS), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich, Inc. (St. Louis, Missouri, USA). Guanidine hydrochloride was purchased from EMD Millipore, Inc. (Burlington, Massachusetts, USA). TNB 2- The probes were purchased from Biovision, Inc. (Millipitas, California, USA). MMBC (Methyl Maleimide Benzochromenecarboxylate) was purchased from Combi-Blocks, Inc. (San Diego, California, USA); a 1 mM stock of MMBC was prepared with DMSO and stored frozen at -20°C until use. A diluted standard solution (20 μM) of MMBC was prepared by diluting the 1 mM stock with 100 mM sodium phosphate, pH 7.4. Other probes tested were purchased from the suppliers listed in Table 1. Other consumables and equipment used included Corning's NBS 96-well half-area black plates and 96-well half-area clear bottom plates; Agilent's Poroshell 120-C18 reverse-phase column (2.7 μm particle size, 3.0 × 100 mm) and 1290 UHPLC; Thermo Scientific's Nanodrop 2000 and Fusion hybrid mass spectrometers; SpectraMax i3 plate reader (San Jose, California); and Mettler-Toledo InLab ultramicro pH meter (Columbus, Ohio). JPEG0007840325000001.jpg150170

[0035] Screening of fluorescent probes and fluorescence-emitting probes Seven fluorescent or fluorescent probes, including MMBC (Table 1), were used in TNB. 2- The ability to convert the signal into a fluorescent signal was evaluated (Figure 1, Step II). In short, the probe was converted into a TNB solution in water. 2- TNB of various concentrations, prepared directly from powder. 2- The reaction was carried out using a range of 0–2 μM, as recommended by the manufacturer. Fluorescence was monitored for 45 minutes using the manufacturer's recommended excitation / emission wavelength pair (Table 1).

[0036] Characterization of TNB-MMBC adducts To verify the properties of the obtained TNB-MMBC adduct, the emission and excitation spectra of the TNB-MMBC adduct were determined. 10 μM TNB 2- The standard was diluted 1:1 with 20 μM MMBC solution and incubated in the dark at room temperature for 30 minutes, after which readings were performed using a plate reader. A range of emission wavelengths from 425 to 600 nm were tested while the excitation was fixed at 385 nm, the manufacturer's recommended value for the fluorescent probe (Figure 3). Since the maximum fluorescence intensity was achieved by monitoring the emission at 510 nm, a range of excitation wavelengths from 325 to 475 nm was then tested, this time with the emission fixed at 510 nm. The maximum fluorescence intensity was achieved by excitation at 375 nm.

[0037] The structure of the TNB-MMBC adduct was further investigated using LC-MS. 10 μM TNB 2-Equivolent mixtures of the standard and 20 μM MMBC solution (both prepared in 20 mM sodium phosphate, pH 7.4) were incubated in the dark at room temperature for 30 minutes. The reaction was quenched by acidifying with 10% formic acid until the final concentration was 1% formic acid, and then injected into a Poroshell 120 EC-C18 reversed-phase column pre-equilibrated with 10% solvent B (mobile phase A: water, 0.1% formic acid; mobile phase B: acetonitrile, 0.1% formic acid). After holding at the initial conditions for 2 minutes, the starting reagent and reaction product were separated over 5 minutes using a 10% to 60% gradient of solvent B. The LC eluent was coupled to a fluorescence detector and a Thermo mass spectrometer equipped with an electrospray source (Orbitrap Fusion). Notable mass spectrometer parameters included a spray voltage of 3700 V, an ion transfer temperature of 325 °C, an Orbitrap resolution of 60 K, and an AGC target of 1 e5 counts.

[0038] Conventional Elman method Cysteine ​​standards (ranging from 0 to 40 μM) and protein samples (targeting a free thiol concentration of 20 μM) were prepared under either natural conditions (111 mM MOPS, 1 mM EDTA, pH 7.25) or denatured conditions (natural conditions + 4 M guanidine hydrochloride). Protein concentrations in the samples were measured using Nanodrop (by blanking the spectrophotometer with a matched matrix), and the extinction coefficient of the corresponding protein was determined. The samples and standards were then incubated with 0.58 mM DTNB in ​​the dark at room temperature for 60 minutes. 100 microliters of each sample and standard were transferred in triples to Corning 96-well half-area transparent bottom plates. Absorbance at 412 nm was read using a Spectramax i3 plate reader. The thiol concentration in each protein sample was determined by referring to the absorbance readings relative to a calibration curve (linear) derived using the cysteine ​​standard. The free thiol content can be calculated in moles / moles by dividing the sample thiol concentration by the sample protein concentration determined by Nanodrop. Multichannel pipetting was used whenever possible in this procedure.

[0039] Fluorescence Elman method Cysteine ​​standards (ranging from 0 to 10 μM) and protein samples (targeting a free thiol concentration of 5 μM) were prepared under either natural conditions (111 mM MOPS, 1 mM EDTA, pH 7.25) or denatured conditions (natural conditions + 4 M guanidine hydrochloride). Protein concentrations, along with the extinction coefficient of the corresponding protein, were measured using Nanodrop (by blanking the spectrophotometer with a matched matrix). The samples and standards were then incubated with 0.58 mM DTNB in ​​the dark at room temperature for 60 minutes. 50 microliters of each sample and standard were transferred in triples to a Corning NBS 96-well half-area black plate. Each well of interest contained 50 μL of 20 μM MMBC solution. Mixing was achieved by pipetting. Fluorescence readings (excitation: 375 nm, emission: 510 nm) were acquired using a Spectramax i3 plate reader. The thiol concentration in each protein sample was determined by referring to the fluorescence readings against a calibration curve (linear) derived using a cysteine ​​standard. The free thiol content could be calculated in moles / moles by dividing the sample thiol concentration by the sample protein concentration determined using Nanodrop2000. Multichannel pipetting was used whenever possible in this procedure.

[0040] Evaluation of method characteristics The limits of quantification and detection were determined by creating a calibration curve (0–10 μM cysteine) using the F. Elman method and measuring eight replicates (individual sample preparations) with a 0 μM cysteine ​​standard, i.e., a blank. For comparison, the conventional Elman method was used to create a calibration curve (0–40 μM cysteine) and measure eight replicates with a 0 μM cysteine ​​standard. The limit of detection (LOD) and limit of quantification (LOQ) were calculated using either the F. Elman method or the conventional Elman method using the following mathematical relationship: TIFF0007840325000002.tif23170 In the formula, σ bis the standard deviation of 8 blank measurements, and m is the slope of the cysteine ​​calibration curve.

[0041] Next, the sensitivity of the F. Elman method to different thiol substrates was evaluated. Calibration curves in the range of 0–10 μM were created using the F. Elman method with cysteine, N-acetylcysteine, or glutathione as the thiol substrate. This experiment was performed under both natural and denatured conditions.

[0042] To evaluate the accuracy of the method, the free thiol content of 11 molecules (including 7 IgG1 antibodies, 2 IgG4 antibodies, 1 bispecific antibody, and 1 antibody Fab fragment) was determined using the F. Elman method under denaturing conditions. The F. Elman results were compared with the free thiol values ​​for the same 11 molecules determined using the aforementioned NcHM-tagged reversed-phase liquid chromatography assay (Wei, B et al. Development of a Rapid Reversed-Phase Liquid Chromatographic Method for Total Free Thiol Quantitation in Protein Therapeutics. J. Pharm. Biomed. Anal. 2020, No. June, 113434). The latter assay was selected as a suitable comparator because it relies on a free thiol detection mechanism orthogonal to the F. Elman method (i.e., derivatization of free thiols using NcHM, followed by reversed-phase separation with enhanced selectivity due to NcHM hydrophobicity, and detection of UV absorption peaks). F. Elman's intermediate precision for a simplified sample panel (7 out of 11 molecules) was determined by two different analysts on different days using different reagents.

[0043] Buffering capacity of phosphate buffer compared to MOPS buffer in the presence of 4M guanidine hydrochloride One mL aliquots of denatured MOPS buffer (100 mM MOPS, 4 M guanidine hydrochloride, 1 mM EDTA, pH 7.4) and denatured phosphate buffer (100 mM sodium phosphate, 4 M guanidine hydrochloride, 1 mM EDTA, pH 7.4) were each titrated with 1 N hydrochloric acid (10 μL at a time, up to a maximum of 40 μL). One mL aliquots of the same MOPS buffer and phosphate buffer were each titrated with 1 N sodium hydroxide (10 μL at a time, up to a maximum of 40 μL). After adding each acid / base titrant, pH measurements were obtained using a micro pH meter.

[0044] Example 2: Fluorescence Elman assay result This disclosure describes an enhancement of the Elman assay, known as the F. Elman assay. By adding an incubation step with a fluorescent probe, such as maleimidebenzochromenecarboxylate methyl (MMBC), or a fluorescent probe, at the end of the Elman assay, the UV absorption signal is effectively converted to a fluorescent signal, improving the quantification limit of the Elman assay by approximately four times, even with a twofold dilution by MMBC addition. The accuracy and general utility of the F. Elman assay have been demonstrated in its application to a variety of low molecular weight thiol substrates (i.e., cysteine, N-acetylated cysteine, glutathione) as well as several complex high molecular weight thiol substrates such as monoclonal antibodies.

[0045] A schematic diagram of an exemplary two-step reaction of the assay is shown in Figure 1. In the first step (Figure 1, Step I), free thiols in the sample are exchanged for DTNB, and TNB is stoichiometrically, as in the conventional Elman method. 2- This is obtained. In the second step (Figure 1, Step II), TNB 2-A thiol-specific fluorescent probe that reacts with the thiol to generate a fluorescent signal is introduced (either by forming a fluorescent TNB probe adduct (Figure 1, step IIA) or by releasing a deprotected fluorescent probe (Figure 1, step IIB)). Commercially available thiol-specific fluorescent probes were screened to develop a viable F. Elman assay. The feasibility of F. Elman's method was demonstrated, the improved sensitivity obtained by converting the UV absorption signal to a fluorescent signal was evaluated, and the usefulness of the method in determining the free thiol content in complex therapeutic antibody samples was assessed.

[0046] To establish the feasibility of the F. Elman assay, seven commercially available thiol-specific fluorescent / fluorescent probes were used in TNB. 2- The tests were conducted at TNB. Five of the probes failed to produce dose-dependent fluorescence. Two fluorescent probes (ThioFluor623 and MMBC) did not produce fluorescence. 2- We successfully demonstrated proof of concept by generating dose-dependent fluorescence against (Figure 9). Of the two, MMBC (also known as ThioGlo-1) generated a signal ~40 times stronger and was selected as the probe for further development.

[0047] TZ 2- The reaction between and MMBC yields a product that fluoresces with excitation and emission pairs at 375 nm / 510 nm (Figure 3). Figure 2 shows the reaction between maleimide (MMBC) and thiol (TNB). 2- This shows a likely reaction product based on nucleophilic attack by ). The reaction was monitored and confirmed using reversed-phase liquid chromatography combined with fluorescence detection and mass spectrometry. TNB 2- Neither MMBC nor the starting material has fluorescence strong enough to be detected, but TNB 2- When combined with MMBC, two strong fluorescence chromatography peaks are obtained by reverse-phase separation (Figure 4B). The masses corresponding to both fluorescence peaks are the same, linking the two peaks as isomers, with a mass of (579.0706 m / z, MH). +Figure 4C) shows the proposed TNB-MMBC adduct and its theoretical mass (579.0710 m / z, MH). + The concentration is consistent within 1 ppm. The retention time after the fluorescence peak is also consistent with that of the TNB-MMBC adduct, and TNB 2- Alternatively, it may have higher hydrophobicity than the MMBC starting material.

[0048] Figure 5 shows the time-dependent fluorescence emission (in step II of Figure 1) when the F. Elman assay is applied to a series of cysteine ​​standards. The F. Elman assay gives a dose-dependent fluorescence signal in response to a thiol substrate, and at 30 minutes after introduction of MMBC, the reaction is complete, and the fluorescence readings begin to plateau. By selecting the 30-minute point as the endpoint reading, a linear calibration curve relating cysteine ​​concentration to fluorescence was created (Figure 6). From the slope of this calibration curve and the variation in blank measurements (i.e., noise), the limit of quantification (LOQ) for 0.4 μM cysteine ​​can be obtained. This is a four-fold improvement compared to the LOQ of our conventional Elman method (1.6 μM cysteine) (Table 2). TIFF0007840325000003.tif36170

[0049] As shown in Figure 6, equivalent calibration curves can be created using the F. Elman assay with other thiol substrates, namely glutathione and N-acetylated cysteine. Similarly, for these other thiol substrates, the fluorescence readings plateau 30 minutes after introduction of MMBC. This completes the thiol exchange reaction with DTNB (Step II in Figure 1), and in all cases MMBC is converted to TNB 2-This suggests that a reaction is occurring. Given the similar sensitivity of the F. Elman assay for different thiol substrates and the proposed mechanism of the F. Elman assay, assuming the assay is performed as described, the LOQ of the 0.4 μM F. Elman assay can be generalized to all thiols, regardless of the properties of the substrate. Furthermore, the F. Elman assay is performed similarly under both native and denatured conditions (Figure 6). In samples exhibiting tertiary structures, such as protein samples, the F. Elman assay can be used to selectively examine solvent-accessible free thiols in naturally folded substrates, or to examine total free thiols (embedded + solvent-accessible).

[0050] The accuracy and precision of the F. Elman assay were evaluated using a panel of antibody-based proteins (under denaturation conditions), and their total free thiols (buried + solvent-accessible) in molar / molar basis are shown in Figure 7. Fluorescence readings of these protein samples also plateaued 30 minutes after introduction of the MMBC probe. The intermediate precision of the F. Elman assay was demonstrated using a simplified panel (7 out of 11 proteins), with all coefficients of variation <8% (Figure 7). A complete panel of proteins was investigated using orthogonal NcHM-tagged reversed-phase chromatography assay, and the total free thiol values ​​were consistent with the results of the F. Elman assay (Figure 7), suggesting that the measurement precision of these two methods is comparable despite significant differences in detection modes.

[0051] Similar to the conventional Elman assay, the F. Elman assay can determine the free thiol content of chromatographically challenging molecules, including ADCs and IgG2 molecules (Table 3). ADCs contain conjugated hydrophobic drugs that often interfere with reversed-phase separation due to significant nonspecific interactions, while IgG2 contains disulfide-bonded isoforms that produce multiple peaks in reversed-phase separation, complicating the analysis. Both types of samples are suitable for analysis by the F. Elman assay but not by NcHM-tagged reversed-phase chromatography assays. Table 3 shows the free thiol values ​​of chromatographically challenging molecules using the denatured F. Elman assay. Chromatographically challenging molecules may include antibody-drug conjugates (ADCs) (containing conjugated hydrophobic drugs that often interfere with reversed-phase separation due to nonspecific interactions) and IgG2 molecules (containing disulfide-bonded isoforms that produce multiple peaks in reversed-phase separation, complicating the analysis). TIFF0007840325000004.tif33170

[0052] Consideration The F. Elman assay is a conceptually simple extension of the conventional Elman method, but TNB 2- Given its properties, it was impossible to predict whether it could actually be implemented. The electron-withdrawing functional group and electron delocalization (which gives DTNB a uniquely low bond dissociation energy (Oae, S. Organic Sulfur Chemistry: Structure and Mechanism, 1st Ed.; Doi, J., Ed.; CRC press: Boca Raton, FL, 1992), making it successful as a thiol exchanger), TNB 2- The nucleophilicity of the electron-withdrawing functional group and TNB decreases. 2-Electron delocalization can quench the fluorescence of conjugated TNB-emitting fluorescent probes through various mechanisms, including photo-induced electron transfer and internal charge transfer (Chen, X et al., Chem. Soc. Rev. 2010, 39 (6), 2120-2135; Escudero, D. Acc. Chem. Res. 2016, 49 (9), 1816-1824; Daly, B. et al., Chem. Soc. Rev. 2015, 44 (13), 4203-4211). Therefore, five of the seven probes tested were TNB-emitting. 2- It was not surprising that a suitable dose-dependent response to TNB could not be generated. However, the results outlined herein, with the assistance of the fluorescent probe ThioFluor623 or MMBC, 2- We demonstrated that this can be converted into a fluorescent signal (Figure 9).

[0053] ThioFluor623 is a fluorescent compound containing a 2,4-dinitrobenzenesulfonamide (DNBS) functional group, and its fluorescence is conditional on the release of this DNBS group by a thiol substrate (Figure 8). Deprotected ThioFluor623 fluoresces moderately in aqueous media with a quantum yield of 0.01 (Bouffard, J. et al., Org. Lett. 2008, 10 (1), 37-40). Alternative DNBS-containing fluorescent probes that selectively react with thiophenol are known and can be used in immediate strategies with significantly better quantum yields than ThioFluor623 (Wang, Z. et al., Anal. Chem. 2012, 84 (11), 4915-4920; Jiang, W. et al., Angew. Chemie - Int. Ed. 2007, 46 (44), 8445-8448; Lin, W. et al., ; Long, L.; Tan, W. A Highly Sensitive Fluorescent Probe for Detection of Benzenethiols in Environmental Samples and Living Cells W. 2010, 1503-1505). MMBC is a fluorescent compound with a maleimide functional group, and its fluorescence is conditional on nucleophilic attack of this maleimide by a thiol substrate (Figure 1A). When reacted with cysteine, MMBC emits strong fluorescence with a quantum yield of 0.65 (Yang, J. -R et al., Journal of Heterocyclic Chemistry. 1991, pp 1177-1180).

[0054] The LOQs for the F. Elman assay and the conventional Elman assay were determined to be 0.4 μM and 1.6 μM SH, respectively, suggesting that the enhancement with MMBC improved the limit of quantification by approximately four times (Table 2). While this observed performance improvement can be explained by the low background signal of the fluorescence and UV absorbance measurements, the magnitude of the enhancement appears modest, considering that the fluorescence assay typically achieves an LOQ three to four orders of magnitude lower than the UV absorbance assay. Further improvement in the LOQ of the F. Elman assay may have been limited by background DTNB hydrolysis, which introduces more noise into the fluorescence measurements. Nevertheless, the improved LOQ, coupled with the fact that the F. Elman assay requires half the amount of sample, means that approximately eight times less material is needed when determining thiols in the F. Elman assay compared to the conventional Elman assay.

[0055] It is important to note that the determination of the LOQ using the conventional Elman method at 1.6 μM is higher than the previously reported LOQs using the conventional Elman method, which ranged from 0.6 to 0.9 μM (Wright, SK et al., Anal. Biochem. 1998, 265 (1), 8-14; Riener, CK et al., Anal. Bioanal. Chem. 2002, 373 (4-5), 266-276). However, a key methodological difference is that the sample was incubated with DTNB for 60 minutes (in contrast to 5 minutes in previous studies). The longer incubation time was intentionally chosen to allow free thiols on the protein to react completely with DTNB (Wright, SK et al., Anal. Biochem. 1998, 265 (1), 8-14). However, because longer incubation times increase the likelihood of DTNB hydrolysis and background noise, the conventional Elman method yields a higher LOQ in our study. To ensure a fair comparison of the performance of these methods within the scope of this study, we kept the methodology (i.e., reagents, consumables, equipment, etc.) as identical as possible between the F. Elman assay and the conventional Elman method. To our knowledge, the augmentation of the F. Elman assay represents the greatest improvement in sensitivity of the conventional Elman method since its inception more than 60 years ago.

[0056] In particular, the F. Elman assay successfully maintains one of the key advantages of the conventional Elman method more effectively than other fluorescent free thiol assays: the sensitivity of the F. Elman assay is independent of the properties of the thiol substrate, assuming sufficient time is given for the DTNB and thiol substrate to react (Figure 6). As a result, regardless of the properties of the (one or more) substrates or the (one or more) microenvironments of the thiols, a single universal calibration curve can be used for the F. Elman assay (e.g., using cysteine) to quantify thiols in a diverse set of samples. For example, this method is not possible when directly assaying thiol substrates using MMBC (without DTNB) because MMBC exhibits different fluorescence efficiencies when conjugated to different substrates (Hoff, S. et al., Analyst 2013, 138, 2096-2103). In this study, we successfully determined the thiol content in seven different IgG1, bispecific IgG, IgG2, two IgG4, two antibody-drug conjugates, Fab fragments, and various small molecules using a single cysteine ​​calibration curve in the F. Elman assay.

[0057] Several limitations exist in the F. Elman assay using MMBC. First, the solubility of MMBC is 20 μM in aqueous solution, which limits the upper limit of the substrate range for the F. Elman assay. Second, the F. Elman assay must be performed using a calibration curve to control background DTNB hydrolysis and calibrate the fluorescence response, whereas TNB... 2- If we rely on the extinction coefficient, a calibration curve is not necessary in the conventional Elman method (however, this is not advisable because the matrix effect on the extinction coefficient of TNB at 412 nm is known (Riddles, PW et al., Methods Enzymol. 1983, 91 (1979), 49-60)).

[0058] Finally, it is worth discussing important buffer considerations when performing the F. Elman assay (or the conventional Elman assay) under denaturing conditions. Previous reports have described the use of high concentrations of guanidine hydrochloride in neutral pH phosphate buffer to create a denaturing environment (Robotham, AC et al., MAbs 2019, 0 (0), 1-10; Riddles, PW; et al., Methods Enzymol. 1983, 91 (1979), 49-60; Aitken, A. et al., In Protein Protocols Handbook, The; Walker, JM, Ed.; Humana Press: New Jersey, 2002; pp 595-596). However, given that the pKa of phosphates is dramatically dependent on ionic strength, phosphates are not a suitable choice as a buffer system under these conditions (Scatchard, G. Chem. Rev. 1936, 19 (3), 309-327; Pitzer, KS; Pitzer, KS Thermodynamics of Electrolytes. 1972, 3965 (1969), 268-277). Figure 10 compares the buffering capacity of phosphate buffer and MOPS buffer in the presence of 4M guanidine hydrochloride. MOPS buffer, a good buffer, was able to reasonably buffer both acidic and baseic perturbations, while phosphate buffer had virtually no buffering capacity at neutral pH in such a strong electrolyte environment. In contrast to guanidine hydrochloride, high concentrations of urea do not contribute significantly to the ionic strength of the solution and therefore should be compatible with phosphate buffer at neutral pH. Buffer considerations are often overlooked, but incompatible buffer systems will significantly impact the accuracy and precision of F. Elman assays and conventional Elman assays.

Claims

1. A method for detecting free thiols in a thiol substrate, a) Contact the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) ２－ To stoichiometrically liberate, b) Free TNB ２－ the freed TNB ２－ Contacting a reagent that interacts with it to produce a fluorescent signal, and c) Free TNB ２－ The fluorescent signal released by the interaction between the reagent and the reagent is detected, thereby enabling the detection of free thiols. A method that includes this.

2. The method according to claim 1, wherein the reagent is a fluorescent probe.

3. The method according to claim 1, wherein the reagent is a fluorescent probe.

4. TNB ２－ By incubating the molecule with a fluorescent probe or fluorescent probe, a) Fluorescent TNB probe adduct, or b) Deprotected fluorescent probe and non-fluorescent TNB adduct The method according to claim 2 or 3, which brings about the formation of

5. The method according to claim 2, wherein the fluorescent signal is emitted by a fluorescent TNB probe adduct.

6. The method according to claim 2, wherein a fluorescent signal is emitted by a deprotected fluorescent probe.

7. The method according to claim 2 or 3, wherein the fluorescent probe or fluorescent probe is a thiol-specific probe.

8. The method according to claim 7, wherein the thiol-specific probe comprises a maleimide functional group.

9. The method according to claim 7, wherein the thiol-specific probe comprises a 2,4-dinitrobenzenesulfonamide (DNBS) functional group.

10. The method according to claim 7, wherein the fluorescent probe is maleimidebenzochromenecarboxylate methyl (MMBC).

11. The method according to claim 7, wherein the fluorescent probe is ThioFluor 623.

12. The method according to claim 1, wherein the thiol is present on a low molecular weight thiol substrate.

13. The method according to claim 1, wherein the thiol is present on a high molecular weight thiol substrate.

14. The method according to claim 9, wherein the high molecular weight thiol substrate is a polypeptide.

15. The method according to claim 10, wherein the polypeptide is an antibody.

16. The method according to claim 15, wherein the antibody is IgG2.

17. The method according to claim 15, wherein the antibody is half the number of bispecific antibodies.

18. The method according to claim 14, wherein the high molecular weight thiol substrate is an antibody-drug conjugate (ADC).

19. A method for quantifying the free thiol content of a thiol substrate, a) Contact the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) ２－ To stoichiometrically liberate, b) TNB ２－ free TNB ２－ Incubate with a reagent that interacts with it to produce a fluorescent signal. c) Detecting the fluorescence signal released by the interaction between the released TNB ２－ and the reagent, and d) Quantify the free thiol content of the molecule by comparing the signal detected in c) with a known reference signal. A method that includes this.

20. The method according to claim 19, wherein the reagent is a fluorescent probe.

21. The method according to claim 19, wherein the reagent is a fluorescent probe.

22. The method according to claim 19, wherein the thiol is present on a high molecular weight thiol substrate.

23. The method according to claim 22, wherein the high molecular weight thiol substrate is a polypeptide.

24. The method according to claim 23, wherein the high molecular weight thiol substrate is an antibody-drug conjugate.

25. The method according to claim 23, wherein the polypeptide is an antibody.

26. The method according to claim 25, wherein the antibody is IgG2.

27. The method according to claim 25, wherein the antibody is half of a bispecific antibody.

28. The method according to claim 25, wherein the antibody is a bispecific antibody.

29. TNB ２－ Through incubation with a fluorescent probe or fluorescent probe, a) Fluorescent TNB probe adduct, or b) Deprotected fluorescent probe and non-fluorescent TNB adduct The method according to claim 20 or 21, which brings about the formation of

30. The method according to claim 29, wherein the fluorescent signal is emitted by a fluorescent TNB probe adduct.

31. The method according to claim 29, wherein the fluorescent signal is emitted by a deprotected fluorescent probe.

32. The method according to claim 20 or 21, wherein the fluorescent probe or fluorescent probe is a thiol-specific probe.

33. The method according to claim 32, wherein the thiol-specific probe comprises a maleimide functional group.

34. The method according to claim 32, wherein the thiol-specific probe comprises a 2,4-dinitrobenzenesulfonamide (DNBS) functional group.

35. The method according to claim 32, wherein the fluorescent probe is maleimidebenzochromenecarboxylate methyl (MMBC).

36. The method according to claim 32, wherein the fluorescent probe is ThioFluor 623.

37. The method according to claim 19, wherein the free thiol content is calculated by determining the thiol concentration by comparing the fluorescence signal with a calibration curve and dividing the thiol concentration by the concentration of the thiol substrate.

38. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe Includes, The fluorescent probe or the fluorescent probe is a thiol-specific fluorescent probe. kit.

39. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe Includes, A kit in which the fluorescent probe is maleimide benzochromenecarboxylate methyl (MMBC).

40. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe Includes, A thiol-specific fluorescent probe or a fluorescent probe containing a maleimide functional group, kit.

41. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe Includes, A thiol-specific fluorescent probe or a fluorescent probe containing a 2,4-dinitrobenzenesulfonamide (DNBS) functional group, kit.

42. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe Includes, The fluorescent probe is ThioFluor 623. kit.

43. A kit for detecting thiol compounds, a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and b) Fluorescent probe or fluorescent probe including and Includes a denaturation buffer, kit.

44. The kit according to claim 43, wherein the denatured buffer is a 3-(N-morpholino)propanesulfonic acid hemisodium salt (MOPS) buffer containing guanidine hydrochloride.

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