DTNB Antibody

What is DTNB and how does it function in biochemical assays?

DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)), also known as Ellman's reagent, is a water-soluble compound that specifically reacts with free sulfhydryl groups at neutral pH. When DTNB reacts with a free sulfhydryl group, it produces a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB), which has a strong yellow color that can be quantified spectrophotometrically at 412 nm .

The reaction specifically targets the conjugate base (R-S-) of free sulfhydryl groups, making it highly specific for thiol detection. TNB has a high molar extinction coefficient (approximately 13,600 M-1cm-1 at 412 nm and pH 8.0), allowing for sensitive quantification of even small amounts of free thiols .

For standard laboratory assays, DTNB is typically prepared in reaction buffer (0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA). The reaction is allowed to proceed in the dark at room temperature for approximately 15 minutes before measurement .

How do I prepare and perform a basic Ellman's assay for sulfhydryl quantification?

The standard protocol for Ellman's assay involves the following steps:

• Prepare reaction buffer: 0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA .

• Prepare DTNB solution: Dissolve 4 mg of DTNB in 1 ml of reaction buffer. For long-term use, prepare a 10 mM stock solution by dissolving 40 mg DTNB in 10 ml 0.1M Tris-HCl pH 7.5 (can be stored at 4°C for up to 3 months) .

• Prepare standards: Use L-cysteine at concentrations ranging from 0.25 to 1.5 mM as a standard to confirm the functionality of the Ellman's reagent .

• Assay procedure:

  • Add 25 μl of sample or standard to 250 μl of reaction buffer

  • Add 5 μl of DTNB solution

  • Allow the reaction to proceed in the dark at room temperature for 15 minutes

  • Measure absorbance at 412 nm using a spectrophotometer

• Calculate the concentration of free sulfhydryls using the formula:
  c = A / (b × E)
  where:

  • c is the concentration

  • A is the absorbance at 412 nm

  • b is the path length

  • E is the molar extinction coefficient of TNB (13,600 M-1cm-1)

For microplate format, mix 50 μl of test sample with 950 μl of 0.1 mM DTNB working solution. The test sample should have a sulfhydryl concentration less than 0.5 mM to ensure accurate results .

How can DTNB be used to optimize antibody reduction for targeted drug conjugation?

DTNB plays a crucial role in the development of antibody-drug conjugates (ADCs) by enabling precise control and monitoring of antibody reduction processes. Studies have shown that controlled reduction of interchain disulfide bonds in antibodies is essential for creating homogeneous conjugates with optimal drug-to-antibody ratios (DAR) .

Research findings demonstrate that three key parameters influence antibody reduction:

• DTT concentration: The most significant factor affecting reduction. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. The data indicate that the number of free thiols levels off at eight, regardless of further increases in DTT concentration .

• Temperature: Significant differences in reduction patterns were observed at different temperatures. Reduction at 4°C, 25°C, 37°C, and 56°C yielded approximately 3.8, 4.6, 5.4, and 6 thiols per monoclonal antibody, respectively. Higher temperatures led to increased antibody reduction .

• Exposure time: While less impactful than the other factors, time still affects reduction. Exposure for 15, 30, 60, 90, and 120 minutes yielded approximately 4.2, 5.4, 5.7, 6.2, and 6.6 thiols per monoclonal antibody, respectively .

Based on experimental findings, to achieve specific numbers of drug molecules per antibody in ADC development, researchers should use DTT at concentrations of approximately 1.5, 3.5, 7, and 20 mM at 37°C for 30 minutes to produce ADCs with 2, 4, 6, and 8 drug molecules per monoclonal antibody, respectively .

The Ellman's test using DTNB is essential for confirming both the reduction process and subsequent alkylation of the free thiols, verified by the absence of free thiols after proper alkylation .

What methodological considerations are important when using DTNB for in-line detection of thiols in partially reduced antibodies?

For advanced research applications requiring high-throughput and precise monitoring of thiol content during antibody modification, an in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC) method coupled with DTNB detection offers significant advantages .

Key methodological considerations include:

• Separation methodology: Partially reduced monoclonal antibodies are separated on an SE-UHPLC column and monitored with a UV detector at 280 nm. The eluents are then channeled into a reaction coil and mixed with DTNB to form TNB, with thiol concentration calculated using absorption at 412 nm .

• Standard curve preparation: Prepare a standard curve using L-cysteine with concentrations between 100 and 1000 μM. This concentration range shows good linearity and provides a reliable reference for quantification .

• Sample preparation: Ensure that partially reduced monoclonal antibodies are properly separated from low-molecular weight contaminants before the DTNB reaction to avoid interference .

• Validation parameters: Evaluate the selectivity, linearity, repeatability, and robustness of the method to ensure consistent and reliable results .

This method allows for:

• Real-time monitoring of time- and temperature-dependent changes in the free-SH:protein ratio during reduction

• Evaluation of changes in drug-antibody ratio (DAR) during conjugation reactions

• Minimization of assay time and costs compared to conventional methods

How does DTNB compare with other methods for detecting and quantifying free sulfhydryls in monoclonal antibodies?

Several methods exist for detecting and quantifying free sulfhydryls in monoclonal antibodies, each with distinct advantages and limitations. DTNB (Ellman's reagent) is the most widely used due to its specificity and simplicity, but other methods may be preferred in certain research contexts .

Comparison of sulfhydryl detection methods:

Advanced research applications often require peptide-level resolution to determine the exact location of free sulfhydryls. A sophisticated approach involves differential labeling of free sulfhydryls and cysteines involved in disulfide bonds with N-ethylmaleimide (NEM) and d5-N-ethylmaleimide, respectively, followed by enzymatic digestion and nanoLC-MS analysis. This method can quantify the abundance of free sulfhydryls at individual cysteine residues down to 2% with minimal non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis .

The method selection should be guided by research requirements, available equipment, and the level of detail needed in the analysis. While DTNB remains the gold standard for routine applications, more sophisticated techniques may be necessary for detailed characterization of therapeutic antibodies and ADCs .

What factors influence the accuracy of Ellman's assay in complex protein samples and how can these be mitigated?

Several factors can affect the accuracy of Ellman's assay when working with complex protein samples such as antibodies or antibody-drug conjugates:

• Sample concentration: The test sample should have a sulfhydryl concentration less than 0.5 mM. Concentrations exceeding this will result in high absorbance values and less accurate estimation based on the extinction coefficient . Mitigation: Dilute samples appropriately before analysis and record the dilution factor.

• Buffer composition: DTNB reactions are pH-dependent and optimal at slightly alkaline conditions (pH 7.5-8.0) . Mitigation: Ensure consistent pH in all samples and standards.

• Interfering substances: Compounds that absorb at or near 412 nm can interfere with readings. Mitigation: Include appropriate blanks and controls in your experimental design.

• Protein stability: Proteins with free thiols can undergo oxidation during processing. Mitigation: Work quickly, maintain samples at appropriate temperatures, and consider adding stabilizing agents.

• Reaction time: Incomplete reactions can lead to underestimation of thiol content. Mitigation: Ensure consistent reaction times (typically 15 minutes) across all samples and standards .

• Standard curve validity: Using inappropriate standards can lead to inaccurate quantification. Mitigation: Select standards that closely match the expected thiol environment in your samples. For antibodies, β-lactoglobulin A may be more appropriate than simple amino acids like cysteine in some contexts .

• Low-level detection challenges: When measuring very low levels of free sulfhydryls (e.g., 0.02 mol SH per mol protein or 2% cysteine residues with free sulfhydryls), special consideration must be given to method sensitivity. Mitigation: Consider more sensitive methods like mass spectrometry with differential labeling for very low-abundance thiols .

Research has shown that the sum of labeling on each reduced antibody chain can sometimes be slightly greater than the labeling calculated for the intact protein, particularly when working with antibodies containing very low levels of free thiols. This discrepancy highlights the importance of method validation and appropriate controls when working with complex samples .

What role does DTNB play in understanding the antiviral properties of protein disulfide isomerase inhibitors?

DTNB has been investigated not only as a tool for sulfhydryl quantification but also as a potential antiviral agent due to its ability to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry .

Research findings on DTNB's antiviral properties include:

• Fusion inhibition: DTNB showed dose-dependent inhibition of cell-cell fusion when exposed to HeLa-CD4-LTR-β-gal cells, acting as a fusion inhibitor by preventing the thiol/disulfide rearrangement in gp120 catalyzed by PDI .

• Specificity to viral strains: DTNB demonstrated antiviral activity specifically against T-tropic HIV-1 strains but not against M-tropic HIV-1 BaL. The concentration at which HIV-1 IIIB infectivity was inhibited by 50% (IC50) was 2.60 mM .

• Cell membrane and viral envelope activity: DTNB showed fusion inhibition on both CD4-expressing cells and Env-expressing cells (HL2/3), with a stronger effect on the latter, indicating possible activity on both the cell membrane and the viral envelope .

• Multiple points of action: Time-of-addition experiments revealed that DTNB not only acts on HIV entry inhibition but also at later stages of the viral cycle, suggesting multiple mechanisms of action .

• Long-lasting host cell protection: Unlike some other PDI inhibitors, DTNB provided protection against infection to cells even after removal of extracellular drug, with protection retained for up to 10 hours .

This research demonstrates DTNB's potential beyond its traditional use as a sulfhydryl detection reagent, highlighting its possible applications in antiviral research. The ability to inhibit PDI activity and provide long-lasting cell protection makes DTNB an interesting compound for further investigation in therapeutic contexts .

How can DTNB assays be optimized for quantifying free sulfhydryls in antibody-drug conjugate development?

Optimizing DTNB assays for antibody-drug conjugate (ADC) development requires special considerations due to the complex nature of these molecules and the critical importance of sulfhydryl quantification in determining drug-to-antibody ratios (DAR). Based on research findings, the following optimizations are recommended:

• Selection of appropriate buffer systems: Use 0.1 M sodium phosphate buffer at pH 8.0 containing 1 mM EDTA for optimal DTNB reactivity while maintaining antibody stability .

• Timing of measurements: Implement specific timing for measurements during the ADC development process:

  • After initial antibody reduction to confirm the desired level of free thiols

  • After purification of reduced antibody to ensure maintenance of free thiols

  • After conjugation to confirm complete reaction of free thiols with maleimide-containing drugs

• Integration with complementary analytical methods: Combine DTNB assays with:

  • SDS-PAGE under reducing and non-reducing conditions to confirm structural integrity

  • Size-exclusion chromatography to monitor potential aggregation during reduction and conjugation

• Automation for process monitoring: For large-scale or industrial applications, implement in-line SE-UHPLC-DTNB methods that allow:

  • Real-time monitoring of the reduction process

  • Immediate feedback on DAR during conjugation

  • Separation of the antibody from potential interferents prior to DTNB reaction

• Quality control parameters: Establish specific acceptance criteria for:

  • Free thiol content at each stage of the process

  • Variation in free thiol content between batches

  • Correlation between measured free thiols and final DAR

Research has shown that higher DAR ADCs not only are more toxic than lower DAR ADCs at equivalent doses of antibody but also lead to accelerated plasma clearance. Therefore, precise control of the reduction process using optimized DTNB assays is crucial for developing ADCs with the desired properties .

What are the optimal conditions for using anti-dystrobrevin beta antibodies in immunohistochemistry and western blotting?

Dystrobrevin beta (DTN-B) is a component of the dystrophin-associated protein complex (DPC) and plays important roles in cellular scaffolding and signaling. When working with anti-DTN-B antibodies, optimal conditions vary based on the experimental technique:

For Western Blotting:

• Recommended dilution range: 0.5-2 μg/mL for polyclonal antibodies or 1:200-1:2000 for others

• Sample preparation: DTN-B is expressed in multiple tissues, with notable expression in skeletal muscle tissue

• Expected molecular weight: Approximately 64 kDa as observed by Western blot

• Controls: Mouse skeletal muscle tissue serves as a positive control for validation

For Immunohistochemistry:

• Recommended dilution range: 5-20 μg/mL for polyclonal antibodies

• Sample preparation: Formalin-fixed and paraffin-embedded tissues

• Specific examples: Human glioma carcinoma tissue has been successfully labeled with anti-DTN-B antibodies at 1/200 dilution

• Visualization: Usually performed using appropriate secondary antibody conjugation systems

For Immunocytochemistry:

• Recommended dilution: 5-20 μg/mL

• Cell fixation: Standard protocols for cell fixation and permeabilization apply

Storage considerations:

• Store at 4°C for frequent use

• For long-term storage, keep at -20°C in a manual defrost freezer

• Avoid repeated freeze-thaw cycles to maintain antibody activity

Buffer formulations:

• Commercial antibodies are typically supplied in PBS, pH 7.4, containing 0.05% Proclin-300 and 50% glycerol or similar preservatives to maintain stability

What is the functional significance of dystrobrevin beta in cellular processes and how can antibodies help elucidate its role?

Dystrobrevin beta (DTN-B) serves several important biological functions that can be investigated using specific antibodies:

• Scaffolding function: DTN-B assembles dystrophin (DMD) and syntrophin alpha-1 (SNTA1) molecules to the basal membrane of kidney cells and liver sinusoids. Anti-DTN-B antibodies can be used in co-immunoprecipitation experiments to identify interaction partners and in immunohistochemistry to visualize localization patterns .

• Transcriptional regulation: DTN-B may function as a repressor of the synapsin I (SYN1) promoter through binding of repressor element-1 (RE-1), thereby regulating SYN1 expression and potentially playing a role in cell proliferation regulation during early neural differentiation. Chromatin immunoprecipitation (ChIP) assays using anti-DTN-B antibodies can help confirm these DNA-protein interactions .

• Synaptic maturation: DTN-B may be required for proper maturation and function of a subset of inhibitory synapses. Immunofluorescence studies with anti-DTN-B antibodies can reveal its localization at synaptic structures .

• Dystrophin-associated protein complex (DPC) formation: As a component of the DPC, which consists of dystrophin and several integral and peripheral membrane proteins (including dystroglycans, sarcoglycans, syntrophins, and dystrobrevin alpha and beta), DTN-B plays a critical role in maintaining membrane integrity. Disruption of the DPC is associated with various forms of muscular dystrophy. Western blotting with anti-DTN-B antibodies can help assess DPC integrity in disease models .

• Interactions with dystrophin isoforms: DTN-B is thought to interact with syntrophin and the DP71 short form of dystrophin. Co-immunoprecipitation experiments using anti-DTN-B antibodies can confirm these protein-protein interactions and help map interaction domains .

Researchers can leverage anti-DTN-B antibodies in combination with gene knockdown or knockout approaches to elucidate the precise functional consequences of DTN-B deficiency in various cellular contexts and disease states.

Human, mouse, and rat DTN-B antibodies show cross-reactivity due to conservation of the protein across species, making these antibodies valuable tools for comparative studies in different model organisms .