fluorescent ellman 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 assay for improving the sensitivity of free thiols detection and quantification.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 106,569, filed October 28, 2020, the entire contents of which are incorporated herein by reference, and claims priority thereto. Technical Field

[0003] This disclosure relates to methods for detecting thiols. Specifically, this disclosure provides enhancements to the conventional Ellman method into a fluorescent Ellman method to improve the sensitivity of free thiols detection. Background Technology

[0004] The levels of low molecular weight thiols (such as cysteine, homocysteine, and glutathione) are generally crucial for redox signaling and maintaining redox homeostasis. Imbalances in these thiols (especially 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 of free thiols on low molecular weight thiol substrates and proteins such as human serum albumin, between free thiols and other thiol forms (cysteine, glutathione, sulfenic acid, etc.). Quantitative methods for free thiols, or reactive sulfhydryl groups, are key analytical tools in biological and medical research. Quantitative methods for free thiols can be used for high-throughput screening of novel acetyltransferase inhibitors, acetyltransferases being important enzymes involved in major metabolic pathways and epigenetic regulation. The quantitative method for free thiols has also been applied in the biopharmaceutical industry to monitor the quality of protein products. In the biopharmaceutical industry, free thiols are sometimes engineered into proteins to produce bioconjugates for therapeutic or diagnostic purposes, or free thiols may inadvertently appear as undesirable post-translational modifications and be considered a potentially critical quality attribute.

[0005] Among the available quantitative methods, optical sensing methods for free thiols are used, and a large family of optical probes for thiol sensing has been developed. However, a common drawback of fluorescent probes for thiol sensing is that their sensitivity can depend on the thiol substrate. Therefore, fluorescent probes for thiol sensing often require calibration using the same standard as the substrate in the test, or require the determination of an appropriate response factor. Other drawbacks of fluorescent probes are that they can be spatially bulky, and often have limited solubility in water. On the other hand, Ellman’s reagent, or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the classic optical probe for free thiol sensing, happens to be highly water-soluble, not spatially bulky, and its sensitivity is independent of the thiol substrate. The main drawback of the Ellman’s method is that its limit of quantitation is significantly higher (3-4 orders of magnitude) than most fluorescent methods for free thiols. This can mean that large amounts of sample (e.g. milligrams of therapeutic antibodies) are required to obtain a quantitative free thiol measurement, which is impractical, and sometimes also very expensive. SUMMARY

[0006] In certain embodiments, the present disclosure relates to a method for detecting free thiols comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) contacting the released TNB 2- with a reagent to generate a fluorescent signal, the reagent interacting with the released TNB 2- ; and c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, thereby detecting free thiols. In certain embodiments, the reagent used in the context of the present method is a fluorescent probe. In certain embodiments, the reagent used in the context of the present method is a fluorescent probe. In certain embodiments, incubating the TNB 2- molecule with the fluorescent probe or the fluorescent probe allows 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 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 fluorescent probe is methylmaleimido-benzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the fluorescent probe is ThioFluor 623.

[0007] 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 one half of a bispecific antibody. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug-conjugate (ADC).

[0008] In certain embodiments, the present disclosure relates to a method for determining the free thiol content of a thiol substrate comprising: a) contacting the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) incubating the TNB 2- with a reagent to generate a fluorescent signal, the reagent interacting with the released TNB 2- ; c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) to a known amount of a reference signal.

[0009] 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 one half of a bispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

[0010] In certain embodiments of the free thiol detection methods described herein, the incubation of the TNB 2- with the fluorescent probe or the 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 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 fluorescent probe is maleimidobenzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the fluorescent probe is ThioFluor 623.

[0011] In certain embodiments of the free thiol detection methods described herein, the fluorescence signal is compared to a calibration curve to determine the thiol concentration and the free thiol content is calculated by dividing the thiol concentration by the concentration of the thiol substrate.

[0012] In certain embodiments, the present disclosure relates to a kit for the detection of a thiol compound comprising: a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorogenic probe or a fluorescent probe. 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 the group consisting of cysteine, glutathione, and N-acetylated cysteine. In certain embodiments, the fluorogenic probe or the fluorescent probe is a thiol-specific fluorogenic probe. In certain embodiments, the fluorogenic probe is maleimidobenzocycloheenecarboxylic acid methyl ester (MMBC). In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a maleimide functional group. In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorogenic probe is ThioFluor 623. In certain embodiments, the kit of the present disclosure comprises a denaturation buffer. In certain embodiments, the denaturation buffer is a 3-(N-morpholino)propanesulfonic acid hemisodium salt (MOPS) buffer containing guanidine hydrochloride. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 depicts A schematic of the fluorescent Ellman ("F. Ellman") assay is depicted. Incubation of the thiol substrate with DTNB (step I) stoichiometrically releases TNB 2- molecule, which then reacts with the thiol-specific fluorogenic probe (step II) to generate a fluorescent signal. These fluorogenic reactions proceed either by the formation of a fluorescent TNB-probe adduct (IIA) or by the generation of a deprotected fluorescent probe (IIB).

[0014] Figure 2 depicts A schematic of the reaction of TNB 2- and MMBC to generate a fluorescent TNB-MMBC adduct (exact mass: 578.0637 Da, expected structure on the far right) is depicted, following Figure 1 the pathway shown in IIA).

[0015] Figure 3 depicts The emission and excitation spectra of the fluorescent TNB-MMBC adduct are depicted. (A) Emission spectra with excitation wavelength fixed at 385 nm and (B) excitation spectra with emission wavelength fixed at 510 nm.

[0016] Figure 4 depicts reverse phase chromatograms of starting reagents (TNB, MMBC) and TNB-MMBC adduct with A) 214 nm UV detection and B) 375 nm / 510 nm fluorescence detection. Peaks with asterisks indicate impurities related to the TNB or MMBC starting materials. C) Mass spectra of the earlier eluting and D) later eluting TNB-MMBC adduct peaks.

[0017] Figure 5 depicts Plots of fluorescence spectra of cysteine standards over time (MMBC reagent introduced at 0) using the F. Ellman assay.

[0018] Figure 6 depicts F. Ellman calibration curves using cysteine (cys), N-acetylated cysteine (NAC), and glutathione (gsh) under A) native conditions and B) denaturing conditions. Fluorescence readings were taken 30 minutes after introduction of MMBC reagent. Linear regressions (trend lines) for the unknown substrates in both plots were based on all data points plotted.

[0019] Figure 7 depicts A bridging data set between F. Ellman (denaturing) and NcHM labeled RPLC assays (an orthogonal method for total free thiol quantitation of antibodies) is described. The intermediate precision for F. Ellman was determined with the first seven molecules (IgG1-A / B / C / D / E, IgG4-A, bispecific IgG) with error bars representing one standard deviation. The F. Ellman coefficient of variation was <8% for each molecule in the intermediate precision.

[0020] Figure 8 depicts Plots of TNB 2- and ThioFluor 623 to produce deprotected fluorescent ThioFluor 623 following the pathway shown in Figure 1 IIB) to produce deprotected fluorescent ThioFluor 623 following the pathway shown in

[0021] Figure 9 depicts Plots of TNB 2- Sensitivity of MMBC (open circles, left y-axis) and ThioFluor 623 (solid circles, right y-axis) after 30 minutes of reaction. Note that the resulting MMBC fluorescence intensity is approximately 40 times greater than the ThioFluor 623 fluorescence intensity.

[0022] Figure 10 depictsAn acid-base titration plot comparing the buffering capacity of phosphate and MOPS buffer in the presence of 4 M guanidine hydrochloride is described (phosphate buffer: 100 mM phosphate, 4 M guanidine hydrochloride, 1 mM EDTA, pH 7.4; MOPS buffer: 100 mM MOPS, 4 M guanidine hydrochloride, 1 mM EDTA, pH 7.4). Acid titration was performed with 1 N HC1, and base titration was performed with 1 N NaOH. DETAILED DESCRIPTION

[0023] The following detailed description can be understood when considered in connection with the accompanying drawings, which illustrate and serve to elucidate the presently disclosed subject matter.

[0024] The presently disclosed subject matter provides a method of detecting a thiol comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) contacting the released TNB 2- with a reagent that interacts with the released TNB 2- and c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, thereby detecting the free thiol. The presently disclosed subject matter also provides a method of quantifying the free thiol content of a thiol substrate, the method comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) incubating the TNB 2- with a reagent that interacts with the released TNB 2- ; c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent; and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) to a known amount of a reference signal. The presently disclosed subject matter also provides a kit for detecting a thiol compound, comprising: a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorescent probe or a fluorescent probe.

[0025] The presently disclosed subject matter is based, at least in part, on the discovery that by adding an incubation step with a fluorescent probe maleimidobenzocycloalkene-carboxylic acid methyl ester (MMBC) at the end of the Ellman method, the ultraviolet (UV) absorbance signal is effectively converted to a fluorescent signal and the limit of quantification of the Ellman method is improved by about 4-fold, even though there is a 2-fold dilution due to the addition of MMBC.

[0026] The specification and examples describe non-limiting embodiments of the presently disclosed subject matter.

[0027] 1. Definitions

[0028] Unless otherwise defined, all technical and scientific terms, acronyms, and other scientific or technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In certain instances, the terms defined herein have been intended to have meanings that are commonly understood by those of ordinary skill in the art in light of the teachings of the present disclosure. In some cases, a term can be defined herein in one or more places with the same or similar meaning.

[0029] The terms “comprise,” “comprising,” “include,” “including,” “contain,” “containing,” “have,” “having,” “may,” “may comprise,” “may including,” “may contain,” “may have,” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0030] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean 3 or more standard deviations away from the mean. Alternatively, “about” can 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. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a value.

[0031] As used herein, “polypeptide” generally refers to peptides and proteins having more than about ten amino acids. Polypeptides can be homologous to the host cell or preferably can be exogenous, meaning that the polypeptides are heterologous to the host cell utilized, i.e., foreign, 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 a mammalian organism) are used, more preferably those that are secreted directly into the culture medium.

[0032] The term "protein" means an amino acid sequence of chain length sufficient to give rise to higher levels of tertiary and / or quaternary structure. This is to be distinguished from "peptides" or other small molecular weight drugs that do not have such structure. Typically, a protein herein will have a molecular weight of at least about 15 to 20 kD, preferably at least about 20 kD. Examples of proteins encompassed by the definition herein include all mammalian proteins, particularly therapeutic proteins and diagnostic proteins, such as therapeutic antibodies and diagnostic antibodies, and generally proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and / or intra-chain disulfide bonds.

[0033] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies composed of a single heavy chain sequence and a single light chain sequence, including such paired multimers), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0034] 2. Methods

[0035] The presently disclosed subject matter provides methods for detecting thiols. In certain embodiments, the method comprises: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) contacting the released TNB 2- with a reagent that interacts with the released TNB 2- ; and c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, thereby detecting free thiols.

[0036] The presently disclosed subject matter also provides methods for quantifying the free thiol content of a thiol substrate, the method comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) incubating the TNB 2- with a reagent that interacts with the released TNB 2- ; c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent; and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) to a known amount of a reference signal.

[0037] In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, the reagent is a fluorescent probe. In certain embodiments, the TNB 2-Incubation of the molecule with the fluorescent probe or the 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 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 fluorescent probe is maleimido benzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the fluorescent probe is ThioFluor 623. In certain embodiments, 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 one 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 comparing the fluorescent signal to a calibration curve to determine the thiol concentration and dividing the thiol concentration by the concentration of the thiol substrate.

[0038] 3. Kit

[0039] The presently disclosed subject matter provides kits for detecting thiol compounds. In certain embodiments, the kits include 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and a fluorogenic probe or a fluorescent probe. In certain embodiments, the kits include a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, blisters, blister packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain embodiments, the kits include thiol-containing standards for generating a calibration curve. In certain embodiments, the thiol-containing standards are selected from the group consisting of cysteine, glutathione, and N-acetylcysteine. In certain embodiments, the fluorogenic probe or the fluorescent probe is a thiol-specific fluorogenic probe. In certain embodiments, the fluorogenic probe is maleimido benzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a maleimide functional group. In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorogenic probe is ThioFluor 623. In certain embodiments, the kits include a denaturation buffer. In certain embodiments, the denaturation buffer is 3-(N-morpholino)propanesulfonic acid hemisodium (MOPS) buffer containing guanidine hydrochloride.

[0040] 4. Exemplary embodiments of the present disclosure

[0041] In certain embodiments, the present disclosure relates to a method for detecting free thiols, comprising: a) contacting a thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) contacting the released TNB 2- with a reagent that interacts with the released TNB 2- ; and c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, thereby detecting free thiols. In certain embodiments, the reagent used in the context of the present method is a fluorogenic probe. In certain embodiments, the reagent used in the context of the present method is a fluorescent probe. In certain embodiments, the TNB 2-Incubation of the molecule with the fluorogenic probe or the 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 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 methylmaleimido-benzochromene-methylformate (MMBC). In certain embodiments, the fluorogenic probe is ThioFluor 623.

[0042] 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 one half of a bispecific antibody. In certain embodiments, the high molecular weight thiol substrate is an antibody-drug-conjugate (ADC).

[0043] In certain embodiments, the present disclosure relates to a method for determining the free thiol content of a thiol substrate, comprising: a) contacting the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB 2- ; b) incubating the TNB 2- with a reagent to generate a fluorescent signal, the reagent interacting with the released TNB 2- ; c) detecting the fluorescent signal emitted by the interaction of the released TNB 2- with the reagent, and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) to a known amount of a reference signal.

[0044] In certain embodiments of the free thiol detection methods 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 one half of a bispecific antibody. In certain embodiments, the antibody is a bispecific antibody.

[0045] In certain embodiments of the free thiol detection methods described herein, the TNB2- 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 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 maleimido benzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the fluorogenic probe is ThioFluor 623.

[0046] In certain embodiments of the free thiol detection methods described herein, the concentration of the thiol is determined by comparing the fluorescent signal to a calibration curve and the free thiol content is calculated by dividing the concentration of the thiol substrate by the concentration of the thiol.

[0047] In certain embodiments, the present disclosure relates to a kit for the detection of a thiol compound comprising: a) 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorogenic probe or a fluorescent probe. 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 the group consisting of cysteine, glutathione, and N-acetylated cysteine. In certain embodiments, the fluorogenic probe or the fluorescent probe is a thiol-specific fluorogenic probe. In certain embodiments, the fluorogenic probe is maleimido benzochromene-carboxylic acid methyl ester (MMBC). In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a maleimide functional group. In certain embodiments, the thiol-specific fluorogenic probe or the fluorescent probe contains a 2,4-dinitrobenzenesulfonamide (DNBS) functional group. In certain embodiments, the fluorogenic probe is ThioFluor 623. In certain embodiments, the kit of the present disclosure comprises a denaturation buffer. In certain embodiments, the denaturation buffer is a half-sodium 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0048] Examples

[0049] Example 1: Materials and Methods

[0050] Materials

[0051] All antibodies and antibody-drug-conjugates (ADCs) tested were produced by Genentech (South San Francisco, CA). Two ADCs tested included one conjugated to a drug with a reduced interchain disulfide (ADC-A) and another conjugated to a drug with an engineered unpaired cysteine (ADC-B). DTNB, cysteine, glutathione, N-acetyl cysteine, dimethyl sulfoxide (DMSO), sodium phosphate, 3-(N-morpholino)propanesulfonic acid hemisodium (MOPS), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO). Guanidine hydrochloride was purchased from EMD Millipore (Burlington, MA). TNB 2- were purchased from Biovision (Milpitas, CA). MMBC (maleimidobenzocycloalkene-carboxylic acid methyl ester) was purchased from Combi-Blocks (San Diego, CA); a 1 mM stock of MMBC was prepared in DMSO and stored frozen at -20 °C until use. Working solutions of MMBC (20 mM) were prepared by diluting the 1 mM stock with 100 mM sodium phosphate, pH 7.4. Other probes tested were purchased from the vendors listed in Table 1. Other consumables and equipment used included NBS 96-well half-volume black plates and 96-well half-volume clear bottom plates from Corning (Corning, NY), Poroshell 120-C18 reverse phase columns (2.7 pm particle size, 3.0 x 100 mm) and 1290 UHPLC from Agilent (Santa Clara, CA), Nanodrop 2000 and Fusion Tribrid mass spectrometer from Thermo Scientific (Waltham, MA), SpectraMax i3 microplate reader (San Jose, CA), and Mettler-Toledo InLab Ultra-micro pH meter (Columbus, OH).

[0052] Table 1. List of fluorescent probes / fluorogenic probes tested

[0053]

[0054]

[0055] Screening of fluorogenic probes and fluorescent probes

[0056] Seven fluorescent probes or fluorogenic probes including MMBC (Table 1) were evaluated for their ability to convert TNB 2- into a fluorescent signal Figure 1,Step II). Briefly, the probe was reacted with various concentrations of TNB 2- (0 - 2 mM range) which was dissolved in water 2- directly prepared) according to the manufacturer's recommendations. Fluorescence was monitored over 45 minutes using the manufacturer's recommended excitation / emission wavelength pair (Table 1).

[0057] Characterisation of the TNB-MMBC adduct

[0058] To verify the properties of the resulting TNB-MMBC adduct, the emission and excitation spectra of the TNB-MMBC adduct were determined. 10 mM TNB 2- The standard was diluted 1 : 1 with 20 mM MMBC solution and incubated at room temperature in the dark for 30 minutes before reading using a microplate reader. A range of emission wavelengths from 425 nm to 600 nm were tested while the excitation was fixed at the value recommended by the manufacturer of the fluorescent probe, 385 nm Figure 3) The maximum fluorescence intensity was achieved by monitoring the emission at 510 nm, so a range of excitation wavelengths from 325 nm to 475 nm were subsequently tested with the emission fixed at 510 nm. The maximum fluorescence intensity was achieved by excitation at 375 nm.

[0059] The structure of the TNB-MMBC adduct was further interrogated using LC-MS. An equal volume mixture of 10 mM TNB 2- An equal volume mixture of the standard and 20 mM MMBC solution (both prepared in 20 mM sodium phosphate, pH 7.4) was incubated at room temperature in the dark for 30 minutes. The reaction was quenched by acidifying 10% formic acid to a final concentration of 1% formic acid prior to injection onto a Poroshell 120 EC-C18 reverse phase chromatography column pre-equilibrated with 10% solvent B (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile). After 2 minutes of initial conditions, the starting reagents and reaction products were separated using a gradient from 10% to 60% solvent B over 5 minutes. The LC eluent was coupled to a fluorescence detector and a Thermo mass spectrometer equipped with an electrospray source (Orbitrap Fusion). Notably, the 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 1e5 counts.

[0060] Traditional Ellman method

[0061] Cysteine standards (range from 0 to 40 mM) and protein samples (targeting a free thiol concentration of 20 mM) were prepared in native conditions (1 1 1 mM MOPS, 1 mM EDTA, pH 7.25) or denaturing conditions (native conditions + 4 M guanidine hydrochloride) respectively. Protein concentration of samples was measured using Nanodrop (spectrophotometer blanked with matching matrix) and extinction coefficient of the respective protein. Samples and standards were then incubated with 0.58 mM DTNB for 60 minutes at room temperature in the dark. One hundred microliter of each sample and standard was transferred into Corning 96 well half volume clear bottom plates in triplicates. Absorbance readings at 412 nm were acquired on a Spectramax i3 microplate reader. Thiol concentration in each protein sample was determined by referencing its absorbance readings to a calibration curve (linear) derived using cysteine standards. Free thiol content on a molar per mole basis can be calculated by dividing the sample thiol concentration by the sample protein concentration determined by Nanodrop. Whenever possible, multi-channel pipetting was used in this process.

[0062] Fluorescent Ellman method

[0063] Cysteine standards (range from 0 to 10 mM) and protein samples (targeting a free thiol concentration of 5 mM) were prepared in native conditions (1 1 1 mM MOPS, 1 mM EDTA, pH 7.25) or denaturing conditions (native conditions + 4 M guanidine hydrochloride) respectively. Protein concentration was measured using Nanodrop (spectrophotometer blanked with matching matrix) and extinction coefficient of the respective protein. Samples and standards were then incubated with 0.58 mM DTNB for 60 minutes at room temperature in the dark. Fifty microliter of each sample and standard was transferred into Corning NBS 96 well half volume black plates in triplicates, where the target wells contained 50 pL of a 20 mM MMBC solution. Mixing was achieved by insertion of the pipette. Fluorescence readings (emission wavelength: 375 nm, excitation wavelength: 510 nm) were acquired on a Spectramax i3 microplate reader. Thiol concentration in each protein sample was determined by referencing its fluorescence readings to a calibration curve (linear) derived using cysteine standards. Free thiol content on a molar per mole basis can be calculated by dividing the sample thiol concentration by the sample protein concentration determined by Nanodrop 2000. Whenever possible, multi-channel pipetting was used in this process.

[0064] Evaluation of method attributes

[0065] The limit of detection (LOD) and limit of quantitation (LOQ) for the F. Ellman or traditional Ellman method were calculated using the following mathematical relationships:

[0066]

[0067]

[0068] where σ h is the standard deviation of 8 blank measurements, and m is the slope of the cysteine calibration curve.

[0069] The sensitivity of the F. Ellman method to different thiol substrates was next evaluated. Using cysteine, N-acetyl cysteine, or glutathione as the thiol substrate, a calibration curve ranging from 0-10 mM was generated using the F. Ellman method. This experiment was performed under native and denaturing conditions.

[0070] To evaluate method accuracy, the free thiol content on 11 molecules (including 7 IgGl antibodies, 2 IgG4 antibodies, 1 bispecific antibody, and 1 antibody Fab fragment) was determined using the F. Ellman method under denaturing conditions. The F. Ellman results were compared to the free thiol values determined for the same 11 molecules using a previously described NcHM labeled 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 chosen as a suitable control because it relies on a free thiol detection mechanism that is orthogonal to the F. Ellman method (i.e., derivatization of free thiols using NcHM followed by reversed-phase separation with enhanced selectivity due to NcHM hydrophobicity and UV absorbance peak detection). The intermediate precision of the F. Ellman on a reduced sample set (7 of the 11 molecules) was determined by two different analysts on different days using different reagents.

[0071] Comparison of the buffering capacity of phosphate buffer in the presence of 4M guanidine hydrochloride to MOPS buffer

[0072] Figure 1 shows

[0073] Acid titrations were performed on 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) using 1 N hydrochloric acid (10 μL each time, maximum 40 μL). Separate one mL aliquots of the same MOPS buffer and phosphate buffer were titrated using 1 N sodium hydroxide (10 μL each time, maximum 40 μL). pH measurements were taken using a miniature pH meter after each addition of acid / base titrant.

[0074] Example 2: Fluorescence Ellman assay

[0075] result

[0076] This disclosure describes an enhancement to the Ellman method, referred to as the F. Ellman assay. An incubation step at the end of the Ellman method, incorporating a fluorescent probe (e.g., methyl maleimide benzo[a]methyl benzo[a]methyl benzo[a]methyl maleimide (MMBC) or a fluorescent probe, effectively converts the UV absorption signal to a fluorescence signal and improves the limit of quantitation of the Ellman method by approximately 4-fold, even with a 2-fold dilution due to the addition of MMBC. The accuracy and general effectiveness of the F. Ellman assay are demonstrated in its application to a wide range 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).

[0077] A schematic diagram of the exemplary two-step reaction for this determination is shown in Figure 1, Out. In the first step ( Figure 1, In step I), free thiols in the sample are exchanged with DTNB to stoichiometrically generate TNB. 2- (As in the traditional Ellman method). In the second step ( Figure 1, In step II), a thiol-specific fluorescent probe was introduced, which interacts with TNB. 2- The reaction proceeds by forming a fluorescent TNB-probe adduct ( Figure 1, Step IIA) or by releasing the deprotected fluorescent probe ( Figure 9) Step IIB) generates a fluorescence signal. To develop a feasible F. Ellman assay, commercially available thiol-specific fluorescent probes were screened. The feasibility of the F. Ellman method was demonstrated, the increased sensitivity gained from converting UV absorption signals to fluorescence signals was assessed, and the effectiveness of the method was evaluated by determining the free thiol content in complex therapeutic antibody samples.

[0078] with TNB 2- Seven commercially available thiol-specific fluorescent / profluorescent probes were tested to demonstrate the feasibility of the F. Ellman assay. Five of these probes failed to produce any dose-dependent fluorescence. Two of the fluorescent probes (ThioFluor 623 and MMBC) successfully produced dose-dependent fluorescence, demonstrating proof of concept 2- Figure 3) Of these two probes, MMBC (also known as ThioGlo-1) produced approximately 40-fold more signal and was selected as the probe for further development.

[0079] TNB 2- reacts with MMBC to produce a product that fluoresces at 375 nm / 510 nm Figure 2 shows with excitation and emission. Figure 4B Possible reaction products based on the nucleophilic attack of thiol (TNB 2- ) on maleimide (MMBC) are shown. Reverse phase liquid chromatography coupled with fluorescence detection and mass spectrometry were used to monitor the reaction and confirm the reaction product. TNB 2- and MMBC starting materials do not have significant fluorescence, but the combination of TNB 2- and MMBC produces two strongly fluorescent chromatographic peaks Figure 4C ) via reverse phase separation. The masses corresponding to the two fluorescent peaks are identical, the two peaks are linked as isomers, and the mass (579.0706 m / z, MH + , Figure 5 shows ) is within 1 ppm of the theoretical mass (579.0710 m / z, MH + ) of the proposed TNB-MMBC adduct. The later retention time of the fluorescent peaks is also consistent with the TNB-MMBC adduct, which can be more hydrophobic than either TNB 2- or MMBC starting materials.

[0080] Figure 1 in shows the change in fluorescence emission over time when the F. Ellman assay is applied to a set of cysteine standards (at 1 hour Fluorescent Ellman ​(In step II). The F. Ellman assay yielded a dose-dependent fluorescence signal in response to the thiol substrate, and the fluorescence readings began to stabilize 30 minutes after the introduction of MMBC, as the reaction was complete at this point. By selecting the 30-minute time point as the endpoint reading, a linear calibration curve was constructed that correlated cysteine ​​concentration with fluorescence (Figure 6). The slope of this calibration curve, along with the variation in blank measurements (i.e., noise), yielded a limit of quantitation (LOQ) of 0.4 μM cysteine, a 4-fold improvement compared to the LOQ (1.6 μM cysteine) of our current conventional Ellman method (Table 2).

[0081] Table 2. Limits of detection and limits of quantitation of F. Ellman and conventional Ellman.

[0082] Traditional Ellman LOD (μΜ SH) LOQ (μΜ SH) 0.14 0.55 Figure 1 in 0.42 1.67

[0083] As shown in Figure 6, comparable calibration curves can be generated using F. Ellman assays with other thiol substrates (i.e., glutathione and N-acetylated cysteine). Fluorescence readings stabilized 30 minutes after the introduction of these other thiol substrates into MMBC, indicating that the thiol exchange reaction with DTNB (… Figure 7. Step II) has been completed, and MMBC is compatible with TNB in ​​all cases. 2- Reaction. Given the similar sensitivity of the F. Ellman assay to different thiol substrates and the proposed mechanism of the F. Ellman assay, assuming the assay is performed as described, the 0.4 μM F. Ellman assay LOQ can be universally applied to all thiols regardless of substrate properties. Furthermore, the F. Ellman assay performs similarly under native and denaturing conditions (Figure 6). In samples exhibiting tertiary structures (such as protein samples), the F. Ellman assay can be used to selectively query solvent-accessible free thiols in native folded substrates, or to query total free thiols (embedded + solvent-accessible).

[0084] The accuracy and precision of the F. Ellman assay were evaluated using a panel of antibody-based proteins (under denaturing conditions), and the total (embedded + solvent-accessible) free thiols per mole were presented as shown in [data missing]. Figure 7) The fluorescence readings of these protein samples remained stable 30 minutes after the introduction of the MMBC probe. The intermediate precision of the F. Ellman assay was demonstrated using a simplified set (7 out of 11 proteins), with coefficients of variation all <8%. Figure 7) The entire protein profile was evaluated using orthogonal NcHM-labeled reversed-phase chromatography, and the total free thiol values ​​were consistent with those determined by F. Ellman. Molecule This indicates that the measurement accuracy of the two methods is comparable, despite their significant differences in detection modes.

[0085] Similar to the traditional Ellman, the F.Ellman assay was able to determine the free thiol content of chromatographically challenging molecules (Table 3) which can include ADC and IgG2 molecules. ADCs have conjugated hydrophobic drugs which often prevent reversed phase separation due to significant non-specific interactions, and IgG2 molecules contain disulfide bond isoforms which result in multiple peaks on reversed phase separation and complicate analysis. Both types of samples were amenable to analysis by the F.Ellman assay but not by the NcHM labeled reversed phase chromatography assay. Table 3 shows the free thiol values of chromatographically challenging molecules using the denaturing F.Ellman assay. Chromatographically challenging molecules can include: antibody-drug conjugates (ADCs) which contain conjugated hydrophobic drugs that often prevent reversed phase separation due to non-specific interactions; and IgG2 molecules which contain disulfide bond isoforms that result in multiple peaks on reversed phase separation and complicate analysis.

[0086] Table 3.

[0087] SH: Protein ADC-A ADC-B 0.39 IgG2-A 0.29 Discussion 0.61

[0088] Figure 9)

[0089] While the F.Ellman assay is a simple conceptual enhancement of the traditional Ellman method, it is completely unpredictable whether it can be practically implemented given the properties of TNB 2- . The electron withdrawing functionality and electron delocalization - which make DTNB have an exceptionally low bond dissociation energy (Oae, S. Organic Sulfur Chemistry: Structure and Mechanism, 1st Ed.; Doi, J., Ed.; CRC press: Boca Raton, FL, 1992) and make it well suited as a thiol exchange reagent - also make TNB 2- a weak nucleophile. Furthermore, the electron withdrawing functionality and electron delocalization of TNB 2- may potentially quench the fluorescence of conjugated TNB-fluorescent probes through various mechanisms including photo-induced electron transfer, internal charge transfer, etc. (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). Thus, five of the seven probes tested failed to work with TNB 2-It is not surprising that a suitable dose-dependent response was generated. However, the results outlined herein suggest that TNB 2- can be converted into a fluorescent signal with the help of the fluorescent probe ThioFluor 623 or MMBC Figure 8) .

[0090] ThioFluor 623 is a fluorescent compound with a 2,4-dinitrobenzenesulfonamide (DNBS) functional group and its fluorescence depends on the release of this DNBS group by a thiol substrate Figure 1A . Deprotected ThioFluor 623 emits modest fluorescence 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 react selectively with benzene thiols are known and can be used in a one-shot strategy with significantly better quantum yield than ThioFluor 623 (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 depends on the nucleophilic attack of a thiol substrate to the maleimide Figure 10 in . After reaction 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).

[0091] We determined the LOQs for the F. Ellman and traditional Ellman assays to be 0.4 μΜ and 1.6 μΜ SH, respectively, indicating that the MMBC enhancement increased the limit of quantification by approximately 4-fold (Table 2). This observed enhancement in performance can be explained by the lower background signal in the fluorescence measurement compared to the UV absorbance measurement, although the magnitude of the enhancement seems small when considering that fluorescence assays typically achieve LOQs that are 3-4 orders of magnitude lower than UV absorbance assays. Further improvement in the LOQ of the F. Ellman assay can be limited by background DTNB hydrolysis, which introduces more noise in the fluorescence measurement. Nonetheless, the improved LOQ combined with the fact that the F. Ellman assay requires half the sample volume compared to the traditional Ellman assay, only ~1 / 8 of the material is required for thiol determination using the F. Ellman assay compared to the traditional Ellman assay.

[0092] It is worth noting that our LOQ determination for the traditional Ellman method at 1.6 μΜ is higher than previously reported LOQs for the traditional Ellman method, which were in the range of 0.6-0.9 μΜ (Wright, S. K. et al. Anal. Biochem. 1998, 265(1), 8-14; Riener, C. K. et al. Anal. Bioanal. Chem. 2002, 373(4-5), 266-276). However, one important difference in the methods is that we incubated our samples with DTNB for 60 minutes (as opposed to 5 minutes in those previous studies). We deliberately chose the longer incubation time to allow free thiols on the protein to fully react with DTNB (Wright, S. K. et al. Anal. Biochem. 1998, 265(1), 8-14). But because of the longer incubation time, there is more opportunity for DTNB hydrolysis and background noise, so the LOQ for the traditional Ellman method is higher in our study. To fairly compare the performance of their methods within the scope of this study, we maintained the same methods (i.e., reagents, consumables, instrumentation, etc.) between the F. Ellman assay and the traditional Ellman method where possible. To our knowledge, the enhancement of the F. Ellman assay represents the largest improvement in sensitivity for the traditional Ellman method since its inception over 60 years ago.

[0093] Notably, the F. Ellman assay successfully retains one of the main advantages of the traditional Ellman assay over other fluorescent free thiol assays: the sensitivity of the F. Ellman assay is independent of the nature of the thiol substrate, provided that DTNB and the thiol substrate have sufficient time to react (Figure 6). Thus, a single universal calibration curve (e.g. with cysteine) can be used to quantify thiols in a diverse set of samples in the F. Ellman assay, regardless of the nature of the substrate or thiol microenvironment. For example, this approach would not be possible if we used MMBC (without DTNB) to directly assay the thiol substrates, as MMBC exhibits different fluorescence efficiencies when conjugated to different substrates (Hoff, S. et al., Analyst 2013, 138, 2096-2103). In this study, we successfully used a single cysteine calibration curve and the F. Ellman assay to accurately determine the thiol content in 7 different IgGl, one bispecific IgG, one IgG2, 2 IgG4, 2 antibody-drug conjugates, one Fab fragment, and various small molecules.

[0094] There are some limitations to the F. Ellman assay using MMBC. First, the solubility of MMBC in aqueous solution is 20 mM, and this limits the upper substrate range of the F. Ellman assay. Second, the F. Ellman assay should be performed with a calibration curve to control for background DTNB hydrolysis and to calibrate the fluorescence response, whereas the traditional Ellman assay does not require a calibration curve if it relies on the extinction coefficient of TNB 2- , although this can not be advisable due to the known matrix effects on the extinction coefficient of TNB at 412 nm (Riddles, P. W. et al., Methods Enzymol. 1983, 91 (1979), 49-60).

[0095] Finally, when performing the F. Ellman assay (or traditional Ellman) under denaturing conditions, it is worth discussing important considerations regarding the buffer. Previous reports describe the use of high concentrations of guanidine hydrochloride in a neutral pH phosphate buffer to create a denaturing environment (Robotham, A. C. et al. MAbs 2019, 0(0), 1-10; Riddles, P. W. et al. Methods Enzymol. 1983, 91 (1979), 49-60; Aitken, A. et al. In Protein Protocols Handbook, The; Walker, J. M., Ed.; Humana Press: New Jersey, 2002; pp 595-596). However, given the significant dependence of the pKa of phosphate on ionic strength, phosphate is a poor choice as a buffer system under these conditions (Scatchard, G. Chem. Rev. 1936, 19(3), 309-327; Pitzer, K. S.; Thermodynamics of Electrolytes. 1972, 3965 (1969), 268-277). In ​ we compared the buffering capacity of phosphate and MOPS buffers in the presence of 4 M guanidine hydrochloride. While MOPS buffer (Good’s buffer) is able to reasonably buffer the acid and base perturbations, phosphate buffer has little to no buffering capacity at neutral pH in such a strong electrolyte environment. High concentrations of urea do not significantly increase the ionic strength of the solution compared to guanidine hydrochloride and should therefore be compatible with neutral pH phosphate buffers. Buffer considerations can easily be overlooked, however, incompatible buffer systems will have a significant impact on the accuracy and precision of the F. Ellman and traditional Ellman assays.

Claims

1. A method for detecting free thiols, the method comprising: a) Contacting the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB. 2- ; b) To release TNB 2- Contact with a reagent to generate a fluorescent signal, the reagent reacting with the released TNB 2- Interaction; and c) Detect the TNB released 2- The fluorescence signal emitted by the interaction with the reagent is used to detect the free thiol. The reagent described therein is a thiol-specific probe containing maleimide or 2,4-dinitrobenzenesulfonamide (DNBS) functional groups.

2. The method of claim 1, wherein TNB is... 2- Incubation of the molecule with the thiol-specific probe results in the formation of: a) Fluorescent TNB-probe adduct; or b) Deprotected fluorescent probe and non-fluorescent TNB adduct.

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

4. The method of claim 2, wherein the fluorescent signal is emitted by the deprotected fluorescent probe.

5. The method according to claim 1, wherein the thiol-specific probe is methyl maleimide benzoxene-formate (MMBC).

6. The method according to claim 1, wherein the thiol-specific probe is ThioFluor 623.

7. The method of claim 1, wherein the thiol is present on a low molecular weight thiol substrate.

8. The method of claim 1, wherein the thiol is present on a high molecular weight thiol substrate.

9. The method according to claim 8, wherein the high molecular weight thiol substrate is a polypeptide.

10. The method of claim 9, wherein the polypeptide is an antibody.

11. The method of claim 10, wherein the antibody is IgG2.

12. The method of claim 10, wherein the antibody is half of a bispecific antibody.

13. The method of claim 8, wherein the high molecular weight thiol substrate is an antibody-drug conjugate (ADC).

14. A method for quantifying the free thiol content of a thiol substrate, the method comprising: a) Contact the thiol substrate with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically release TNB. 2- ; b) The TNB 2- Incubated with a reagent to generate a fluorescent signal, the reagent reacts with the released TNB 2- interaction; c) Detect the TNB released 2- Fluorescent signals emitted by the interaction with the reagent; as well as d) The free thiol content of the molecule is quantified by comparing the signal detected in c) with a known amount of a reference signal. The reagent described therein is a thiol-specific probe containing maleimide or 2,4-dinitrobenzenesulfonamide (DNBS) functional groups.

15. The method of claim 14, wherein the thiol is present on a high molecular weight thiol substrate.

16. The method of claim 15, wherein the high molecular weight thiol substrate is a polypeptide.

17. The method of claim 16, wherein the high molecular weight thiol substrate is an antibody-drug conjugate.

18. The method of claim 16, wherein the polypeptide is an antibody.

19. The method of claim 18, wherein the antibody is IgG2.

20. The method of claim 18, wherein the antibody is half of a bispecific antibody.

21. The method of claim 18, wherein the antibody is a bispecific antibody.

22. The method of claim 14, wherein TNB is... 2- Incubation with the thiol-specific probe results in the formation of: a) Fluorescent TNB-probe adduct; or b) Deprotected fluorescent probe and non-fluorescent TNB adduct.

23. The method of claim 22, wherein the fluorescent signal is emitted by the fluorescent TNB-probe adduct.

24. The method of claim 22, wherein the fluorescent signal is emitted by the deprotected fluorescent probe.

25. The method of claim 14, wherein the thiol-specific probe is methyl maleimide benzoxeno-formate (MMBC).

26. The method of claim 14, wherein the thiol-specific probe is ThioFluor 623.

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

28. A kit for detecting thiol compounds, comprising: a) 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB); and b) Thiol-specific probes, The thiol-specific probe contains a maleimide functional group or a 2,4-dinitrobenzenesulfonamide (DNBS) functional group.

29. The kit according to claim 28, further comprising a thiol-containing standard for generating a calibration curve.

30. The kit according to claim 29, wherein the thiol-containing standard is selected from cysteine, glutathione, and N-acetylated cysteine.

31. The kit according to claim 28, wherein the thiol-specific probe is methyl maleimide benzoxene-formate (MMBC).

32. The kit according to claim 28, wherein the thiol-specific probe is ThioFluor 623.

33. The kit according to claim 28, comprising a denaturing buffer.

34. The kit according to claim 33, wherein the denaturing buffer is a hemisodium 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

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