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US-12618779-B2 - Fluorescent ellman assay for free thiol detection

US12618779B2US 12618779 B2US12618779 B2US 12618779B2US-12618779-B2

Abstract

The present disclosure relates to methods and kits for detecting a free thiol in a substrate as well as methods for quantifying the amount of free thiol in a substrate. In particular, the present disclosure provides a fluorescent Ellman assay for enhanced sensitivity of free thiol detection and quantification.

Inventors

  • Aron Lee
  • Rashmi Sharma
  • Justin Joo-Ho Jeong
  • Michael Tae-Jong Kim

Assignees

  • GENENTECH, INC.

Dates

Publication Date
20260505
Application Date
20230428

Claims (20)

  1. 1 . A method for detecting a free thiol, the method comprising: a) contacting a thiol substrate with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically liberate TNB 2− b) contacting the liberated TNB 2− with a reagent that interacts with the liberated TNB 2 − to produce a fluorescent signal; c) detecting the fluorescent signal emitted by the interaction of the liberated TNB 2 − with the reagent to thereby detect the free thiol; and wherein the reagent is a fluorogenic probe or a fluorescent probe.
  2. 2 . The method of 1 , wherein incubating the TNB 2− molecules with the fluorogenic probe or the fluorescent probe results in formation of: a) a fluorescent TNB-probe adduct; or b) a deprotected fluorescent probe and a non-fluorescent TNB adduct.
  3. 3 . The method of claim 2 , wherein the fluorescent signal is emitted by: a) the fluorescent TNB-probe adduct; or b) the deprotected fluorescent probe.
  4. 4 . The method of claim 2 , wherein the fluorogenic probe or the fluorescent probe is a thiol-specific probe.
  5. 5 . The method of claim 4 , wherein the thiol-specific probe contains: a) a maleimide functional group; or b) a 2,4-dinitrobenzene sulfonamide (DNBS) function group.
  6. 6 . The method of claim 4 , wherein the fluorogenic probe: a) is methyl maleimidobenzochromene-carboxylate (MMBC); or b) is ThioFluor 623.
  7. 7 . The method of claim 1 , wherein the thiol; a) is present on a low molecular weight thiol substrate; or b) is present on a high molecular weight thiol substrate.
  8. 8 . The method of claim 7 , wherein the high molecular weight thiol substrate is a polypeptide, an antibody, or an antibody-drug-conjugate (ADC).
  9. 9 . The method of claim 8 , wherein the antibody is: a) an IgG2; b) one half of a bispecific antibody; or c) a bispecific antibody.
  10. 10 . A method of 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) to stoichiometrically liberate TNB2−; b) incubating the TNB2− with a reagent that interacts with the liberated TNB2− to produce a fluorescent signal; c) detecting a fluorescent signal emitted by the interaction of the liberated TNB2− with the reagent; and d) quantifying the free thiol content of the molecule by comparing the signal detected in c) with a reference signal of known quantity; and wherein the reagent is a fluorogenic probe or a fluorescent probe.
  11. 11 . The method of 10 , wherein incubation of TNB 2− molecules with the fluorogenic probe or the fluorescent probe results in formation of: a) a fluorescent TNB-probe adduct; or b) a deprotected fluorescent probe and a non-fluorescent TNB adduct.
  12. 12 . The method of claim 10 , wherein the thiol; a) is present on a low molecular weight thiol substrate; or b) is present on a high molecular weight thiol substrate.
  13. 13 . The method of claim 12 , wherein the high molecular weight thiol substrate is a polypeptide, an antibody, or an antibody-drug-conjugate (ADC).
  14. 14 . The method of claim 13 , wherein the antibody is: a) an IgG2; b) one half of a bispecific antibody; or c) a bispecific antibody.
  15. 15 . The method of claim 11 , wherein the fluorescent signal is emitted by: a) the fluorescent TNB-probe adduct; or b) the deprotected fluorescent probe.
  16. 16 . The method of claim 11 , wherein the fluorogenic probe or the fluorescent probe is a thiol-specific probe.
  17. 17 . The method of claim 16 , wherein the thiol-specific probe contains: a) a maleimide functional group; or b) a 2,4-dinitrobenzene sulfonamide (DNBS) function group.
  18. 18 . The method of claim 16 , wherein the fluorogenic probe is: a) methyl maleimidobenzochromene-carboxylate (MMBC); or b) ThioFluor 623.
  19. 19 . The method of claim 10 , wherein the free thiol content is calculated by comparing the fluorescence signal against a calibration curve to determine the thiol concentration and dividing the thiol concentration by the concentration of the thiol substrate.
  20. 20 . A kit for detecting a thiol compound, comprising: a) 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB); and b) a fluorogenic probe or a fluorescent probe.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Patent Application No. PCT/US2021/056978, filed Oct. 28, 2021, which claims priority to U.S. Provisional Application No. 63/106,569 filed Oct. 28, 2020, the contents of each of which are incorporated by reference in their entireties, and to each of which priority is claimed. FIELD OF INVENTION The present disclosure relates to method for detecting a thiol. In particular, the present disclosure provides augmenting traditional Ellman's method to fluorescent Ellman's for enhanced sensitivity of free thiol detection. BACKGROUND Levels of low molecular weight thiols such as cysteine, homocysteine, and glutathione, are often critical for redox signaling and maintaining redox homeostasis. Imbalances of these thiols—homocysteine in particular—have been implicated in diseases such as cancer Alzheimer's, and cardiovascular disease. Signs of oxidative stress and aging can also manifest in distorted distributions between free thiols and other thiol forms (cysteinylation, glutathionylation, sulfeinic acid, etc.) on low molecular weight thiol substrates as well as on proteins such as human serum albumin. Quantitation methods for free thiols, or reactive sulfhydryls, are key analytical tools to study biology and medicine. Free thiol quantitation methods are useful in high throughput screening for new inhibitors of acetyltransferases, which are an important class of enzymes involved in major metabolic pathways and epigenetic regulation. Free thiol quantitation methods have also been applied in monitoring protein product quality in biopharmaceutical industry, where free thiols are sometimes engineered into proteins to produce bioconjugates for therapeutic or diagnostic purposes, or where they inadvertently appear as undesired post-translational modifications and are treated as potential critical quality attributes. Among the available quantitation methods, optical sensing methods for free thiol are used and a large family of optical probes for thiol sensing have been developed. However, a common drawback to fluorogenic probes for thiol sensing is that their sensitivities can depend on the thiol substrate. As a result, fluorogenic probes for thiol sensing usually require either calibration using standards that are identical to the substrates in the test articles or determination of appropriate response factors. Additional drawbacks to fluorogenic probes are that they can be sterically 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, is not bulky, and its sensitivity is agnostic to the thiol substrate. The main disadvantage of the Ellman's method is that the quantitation limits are significantly higher (3-4 orders of magnitude) than most fluorogenic methods for free thiol quantitation. This can mean that large quantities of sample are required (e.g. milligrams of a therapeutic antibody) in order to obtain a quantitative free thiol measurement, which is impractical and sometimes prohibitively expensive. SUMMARY OF THE INVENTION In certain embodiments, the present disclosure is directed to methods for detecting a free thiol comprising: a) contacting a thiol substrate with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically liberate TNB2−; b) contacting the liberated TNB2− with a reagent that interacts with the liberated TNB2− to produce a fluorescent signal; and c) detecting the fluorescent signal emitted by the interaction of the liberated TNB2− with the reagent to thereby detect the free thiol. In certain embodiments, the reagent employed in the context of the method is a fluorogenic probe. In certain embodiments, the reagent employed in the context of the method is a fluorescent probe. In certain embodiments, incubating the TNB2− molecules with the fluorogenic or the fluorescent probe results in 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 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-dinitrobenzene sulfonamide (DNBS) function group. In certain embodiments, the fluorogenic probe is methyl maleimido-benzochromene-carboxylate (MMBC). In certain embodiments, the fluorogenic probe is ThioFluor 623. 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 thio