DTX9 Antibody

What are the primary methods for antibody characterization in research settings?

Antibody characterization involves multiple complementary techniques aimed at understanding the binding properties, specificity, and functional effects of antibodies. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is particularly valuable for identifying conformational epitopes by measuring the exchange of hydrogen atoms in the protein backbone with deuterium from the solvent, which is affected by antibody binding . This can be complemented with Biolayer Interferometry (BLI) assays to analyze binding kinetics and affinity. For example, researchers have characterized neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying specific epitopes on diphtheria toxin responsible for antibody binding . These techniques provide critical insights into antibody-antigen interactions at the molecular level.
Immunoassays such as ELISAs are widely employed for quantitative measurements of antibodies, as demonstrated in studies measuring maternal antibody levels against diphtheria, tetanus, and pertussis . For structural characterization, techniques like X-ray crystallography and cryo-electron microscopy may provide atomic-level details of antibody-antigen complexes, although these were not specifically mentioned in the search results.

How do neutralizing antibodies against diphtheria toxin function?

Neutralizing antibodies against diphtheria toxin (DTx) operate through distinct mechanisms depending on their epitope specificity. Detailed characterization using HDX-MS and BLI assays has revealed that monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site . This mechanism prevents the initial attachment of the toxin to host cell receptors, thereby inhibiting the first step in the intoxication process.
In contrast, monoclonal antibody 2-18 employs a different neutralization strategy by binding to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis . This inhibits the enzymatic activity of the toxin rather than preventing its cellular entry. Both antibodies form conformational epitopes, highlighting the importance of tertiary structure in antibody recognition and neutralization of diphtheria toxin. Understanding these distinct mechanisms provides valuable insights for vaccine development and therapeutic antibody design.

What are the typical applications of dipeptidyl peptidase antibodies in research?

Dipeptidyl peptidase antibodies, such as the DPP9 antibody, serve multiple research applications centered around studying these enzymes' roles in cellular processes. The rabbit polyclonal DPP9 antibody (ab62025) is particularly suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and reacts with human samples . This makes it valuable for investigating DPP9 expression patterns in normal and pathological tissue sections.
DPP9 antibodies allow researchers to study the role of this enzyme as a key inhibitor of caspase-1-dependent monocyte and macrophage pyroptosis by preventing activation of NLRP1 and CARD8 . They can be employed to investigate how DPP9 sequesters the cleaved C-terminal parts of NLRP1 and CARD8 inflammasomes in a ternary complex, thereby preventing their oligomerization and activation . This application is particularly relevant for immunology research focused on inflammasome regulation and innate immune responses.
Beyond detection applications, these antibodies may also be used to modulate enzymatic activity in experimental settings, although the search results don't specifically mention inhibitory antibodies against DPP9.

What factors affect maternal antibody transfer to neonates?

Maternal antibody transfer to neonates is influenced by multiple factors, as evidenced by studies showing significant regional differences in antibody levels against diphtheria, tetanus, and pertussis. A key study in China found that antibody levels in neonates from Qianjiang (0.04 IU/ml for anti-Dtx IgG and 0.07 IU/ml for anti-Ttx IgG) were significantly lower than those from Shunyi (0.12 IU/ml for anti-Dtx IgG and 0.18 IU/ml for anti-Ttx IgG) . This disparity likely reflects socioeconomic factors and differences in maternal vaccination coverage between regions.
The prevalence of protective antibody levels also showed marked differences, with only 7.1% of neonates from Qianjiang having protective anti-Dtx levels compared to 30.5% from Shunyi . Similarly, only 7.6% of Qianjiang neonates had protective anti-Ttx levels versus 38.5% from Shunyi . These findings underscore how maternal immunization status, regional healthcare access, and socioeconomic factors significantly impact the passive immunity transferred to neonates, which is crucial for protecting infants until vaccination becomes more effective.

What is the significance of antibody concentration thresholds in protection studies?

Antibody concentration thresholds serve as critical benchmarks for determining protective immunity in both research and clinical contexts. For antibodies against diphtheria, tetanus, and pertussis, established cut-off values include ≥0.1 IU/ml for anti-diphtheria (anti-Dtx), >0.1 IU/ml for anti-tetanus (anti-Ttx), and >40 IU/ml for anti-pertussis toxin (anti-Ptx) . These thresholds are used to assess the percentage of protected individuals and evaluate the effectiveness of vaccination strategies.
The significance of these thresholds becomes evident when comparing populations. In a study of neonatal immunity, the detectable rates of anti-Dtx and anti-Ttx IgG were significantly lower in neonates from Qianjiang (57.5% for anti-Dtx and 55.8% for anti-Ttx) compared to those from Shunyi (97.5% for anti-Dtx and 71.0% for anti-Ttx) . This demonstrates how antibody concentration thresholds can reveal disparities in protection between different populations.
When antibody levels fall below protective thresholds, individuals may become susceptible to infection despite having detectable antibodies. Therefore, these thresholds inform vaccination policies, particularly for maternal immunization strategies aimed at protecting newborns during their vulnerable early months.

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) elucidate antibody-antigen interactions?

HDX-MS provides exceptional insights into antibody-antigen interactions by monitoring the exchange rates of hydrogen atoms in the protein backbone with deuterium from the solvent. When an antibody binds to its antigen, it shields specific regions from solvent exchange, creating a "footprint" that identifies the binding epitope with high resolution. This technique was instrumental in characterizing two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, revealing that both recognize conformational epitopes with distinct mechanisms of action .
The power of HDX-MS lies in its ability to map conformational epitopes that may be challenging to identify using other methods. For example, HDX-MS demonstrated that mAb 2-25 binds selectively to the B-subunit of DTx, blocking the HBEGF binding site, while mAb 2-18 binds to the A-subunit, covering the catalytic loop region . These findings would be difficult to obtain through linear epitope mapping techniques like peptide arrays.
When combined with complementary methods such as Biolayer Interferometry (BLI), HDX-MS provides a comprehensive understanding of both the structural aspects of antibody binding and the binding kinetics. This integrated approach reveals not just where antibodies bind, but also how their binding affects antigen conformation and function, informing rational antibody design for therapeutic applications.

What mechanisms contribute to DPP9's inhibitory function on inflammasome activation?

DPP9 employs a complex mechanism to inhibit inflammasome activation, functioning as a key regulator of caspase-1-dependent pyroptosis in monocytes and macrophages. The enzyme acts by preventing activation of NLRP1 and CARD8, two important components of the inflammasome complex . Specifically, DPP9 sequesters the cleaved C-terminal parts of NLRP1 and CARD8, which constitute the active portions of these inflammasomes, in a ternary complex . This sequestration effectively prevents the oligomerization and activation of the inflammasomes, thereby inhibiting downstream pyroptotic cell death pathways.
Interestingly, while DPP9's dipeptidyl peptidase activity (cleaving N-terminal dipeptides from proteins with Pro or Ala at position 2) is required for this suppression, neither NLRP1 nor CARD8 are direct enzymatic substrates of DPP9 . This suggests the existence of other substrate(s) that are processed by DPP9 and subsequently required for NLRP1 and CARD8 inhibition . This indirect mechanism represents a sophisticated regulatory system where enzymatic activity toward one substrate affects the functional state of other proteins through complex protein-protein interactions.
The precise molecular details of these interactions and the identity of the true DPP9 substrates involved in inflammasome regulation remain active areas of investigation with significant implications for understanding innate immune responses and developing therapeutic strategies for inflammatory diseases.

How do antibody-drug conjugates overcome toxicity limitations of small molecule inhibitors?

Antibody-drug conjugates (ADCs) represent an innovative approach to overcome toxicity limitations of small molecule inhibitors through targeted delivery and controlled release of cytotoxic payloads. This is exemplified by BCL-XL-targeting ADCs developed to address the severe mechanism-based cardiovascular toxicity observed with selective small-molecule BCL-XL inhibitors in preclinical models . These ADCs combine three critical components: altered BCL-XL-targeting warheads, unique linker technologies, and therapeutic antibodies specific for tumor-associated antigens .
The epidermal growth factor receptor-targeting ADC AM1-15 demonstrated remarkable preclinical efficacy, inhibiting tumor xenograft growth without causing the cardiovascular toxicity or dose-limiting thrombocytopenia seen with small molecule inhibitors . When an unexpected BCL-XL-mediated toxicity emerged in monkey kidneys upon repeat dosing of AM1-15, researchers further refined the drug-linker to create AM1-AAA (AM1-25), effectively mitigating this toxicity .
In combination therapy settings, these ADCs significantly enhanced the tumor growth inhibition of conventional chemotherapy. When combined with docetaxel in the PC-9 xenograft model, AM1-15 or AM1-25 improved tumor growth inhibition from 82% to 98% or 96%, respectively . Similarly, in the H1650 model, the combination therapy increased efficacy from 83% to 99% . These combinations also substantially improved response durability compared to docetaxel monotherapy .

What statistical approaches are appropriate for analyzing antibody concentration differences between populations?

Analyzing antibody concentration differences between populations requires robust statistical approaches tailored to the characteristics of immunological data. When comparing antibody levels between groups, the Wilcoxon/Krusakal Wallis test is often employed due to the typically non-normal distribution of antibody concentration data . This non-parametric approach is particularly suitable for immunological studies where data may be skewed and contain outliers.
For analyzing the prevalence of protective antibody levels (seroprevalence) between different groups, the chi-square test provides an appropriate statistical framework . When working with antibody concentration data, researchers must also address values below the assay's detection limit. A common approach involves assigning these values as half the lower limit of quantitation (e.g., 0.005 IU/ml for anti-Dtx and anti-Ttx, and 2.5 IU/ml for anti-Ptx) to enable statistical analysis .
To handle upper limits of assay detection, values exceeding the upper quantification limit are typically assigned the maximum detectable value (e.g., anti-Ttx level ≥5.0 IU/ml counted as 5.0 IU/ml) . Results are then expressed as mean concentration with 95% confidence intervals, providing both the central tendency and the precision of the estimate . Statistical significance is typically defined as P ≤ 0.05, allowing researchers to determine whether observed differences between populations are likely to represent true biological differences rather than random variation .

How can time-resolved fluorescence resonance energy transfer (TR-FRET) assays be optimized for antibody research?

Optimizing TR-FRET assays for antibody research requires careful consideration of multiple parameters to achieve maximum sensitivity and specificity. These assays exploit the distance-dependent energy transfer between donor and acceptor fluorophores to measure molecular interactions. In antibody research, TR-FRET can be used to study antibody-antigen binding or to evaluate the effects of antibodies on protein-protein interactions.
When designing TR-FRET assays, careful selection of fluorophores is critical. For example, researchers have successfully employed F-Bak [GQVGRQLAIIGDK(6-FAM)INR-amide] probe as an acceptor fluorophore in combination with a Tb-anti-GST antibody as the donor . Optimal protein concentrations must be determined empirically, with typical concentrations around 0.5 nM for tagged proteins like GST-BCL-XL, 100 nM for fluorescent probes, and 1 nM for labeled antibodies .
A significant advantage of TR-FRET assays is their compatibility with complex biological samples. They can be performed in the presence of varying concentrations of human serum to determine apparent median inhibitory concentration after accounting for serum protein binding . This feature makes TR-FRET particularly valuable for translating in vitro findings to in vivo applications. For quantitative analysis of inhibition, researchers typically determine inhibition constants (Ki) using appropriate equations like Wang's equation , allowing for precise characterization of antibody effects on molecular interactions.

What controls are essential for validating antibody specificity in research applications?

Validating antibody specificity requires a comprehensive set of controls to ensure reliable research outcomes. For antibodies like the rabbit polyclonal DPP9 antibody (ab62025), multiple control strategies should be implemented . Genetic controls are paramount – testing the antibody in samples with knockout or knockdown of the target protein should show reduced or absent signal. For the DPP9 antibody, demonstrating reduced staining in tissues from DPP9-deficient models would provide strong evidence of specificity.
Peptide competition assays represent another critical control, where pre-incubating the antibody with its immunogen (the synthetic peptide within human DPP9 conjugated to Keyhole Limpet Haemocyanin) should abolish specific staining . This confirms that the observed signal is due to antibody binding to the intended epitope rather than non-specific interactions.
Cross-reactivity controls are essential when studying proteins with homologs. For DPP9 antibody, testing against related dipeptidyl peptidases (like DPP4 or DPP8) ensures the antibody doesn't recognize these similar proteins. Additionally, testing the antibody across multiple applications and comparing results with orthogonal detection methods provides further validation. Immunohistochemistry results should correlate with Western blot or immunoprecipitation findings when using the same antibody.
Lastly, including appropriate positive and negative tissue controls in each experiment ensures consistent performance across studies. For DPP9 antibody suitable for IHC-P, tissues known to express high levels of DPP9 should show positive staining, while tissues lacking DPP9 expression should remain negative .

How should researchers approach antibody dilution optimization for immunohistochemistry?

Optimizing antibody dilutions for immunohistochemistry requires a systematic approach to balance specific signal intensity with background minimization. For antibodies like the rabbit polyclonal DPP9 antibody used in IHC-P applications, researchers should begin with a broad range of dilutions based on the manufacturer's recommendations . This typically involves testing 3-5 dilutions in a geometric series (e.g., 1:100, 1:200, 1:400, 1:800) on positive control tissues known to express the target protein.
The optimal dilution provides strong specific staining of target structures with minimal background. Signal-to-noise ratio should be quantitatively assessed across dilutions, ideally using digital image analysis to remove subjective bias. It's crucial to evaluate staining patterns at each dilution against known expression patterns of the target protein. For DPP9, which functions as a dipeptidyl peptidase in various cell types, the staining pattern should correspond to its known subcellular localization .
Environmental factors significantly impact optimal dilution. Temperature, incubation time, antigen retrieval method, detection system sensitivity, and tissue fixation all influence antibody binding. Therefore, antibody titrations should be performed under standardized conditions that will be used in the final protocol. Once the optimal dilution is determined, validation across multiple samples is essential to ensure reproducibility. If samples from different sources (e.g., different fixation protocols) will be used, optimization should account for this variability to maintain consistent staining quality across all specimens.

What methods are appropriate for measuring maternal antibody transfer to neonates?

Measuring maternal antibody transfer to neonates requires specialized methods that address the unique challenges of neonatal samples. Enzyme-Linked Immunosorbent Assays (ELISAs) represent the gold standard for quantifying antibodies against specific antigens like diphtheria toxin (DTx), tetanus toxin (Ttx), and pertussis toxin (Ptx) . Commercial ELISA kits (such as those from Euroimmun, Lübeck, Germany) provide standardized platforms with established cut-off values for protective immunity: ≥0.1 IU/ml for anti-Dtx, >0.1 IU/ml for anti-Ttx, and >40 IU/ml for anti-Ptx .
When analyzing neonatal samples, careful handling of values below detection limits is essential. A standard approach involves assigning these values as half the lower limit of quantitation (e.g., 0.005 IU/ml for anti-Dtx and anti-Ttx, and 2.5 IU/ml for anti-Ptx) to enable statistical analysis without artificially inflating or deflating results . Similarly, values exceeding the upper measurement limit should be assigned the maximum detectable value (e.g., anti-Ttx level ≥5.0 IU/ml counted as 5.0 IU/ml) .
For statistical analysis, researchers should present both antibody concentrations (as mean with 95% confidence intervals) and seroprevalence data (percentage of neonates with protective levels). Comparing these metrics between different populations using appropriate statistical tests (Wilcoxon/Krusakal Wallis test for concentrations, chi-square test for seroprevalence) provides comprehensive insights into maternal antibody transfer patterns . This approach revealed significant differences between regions in China, with neonates from Qianjiang showing much lower protection rates than those from Shunyi .

How can permeabilized cell JC-1 assays evaluate mitochondrial effects of antibody treatments?

Permeabilized cell JC-1 assays provide a sensitive method for evaluating mitochondrial membrane potential changes induced by antibody treatments, particularly those targeting proteins involved in mitochondrial function or apoptosis regulation. This assay utilizes JC-1, a cationic dye that forms red-fluorescent aggregates in polarized mitochondria but remains as green-fluorescent monomers when mitochondria are depolarized.
For implementation, cells are suspended in a specialized buffer such as DTEB buffer (135 mM trehalose, 50 mM KCl, 20 μM EDTA, 20 μM EGTA, 0.1% bovine serum albumin, 5 mM succinate, and 10 mM Hepes-KOH, pH 7.5) . This suspension is supplemented with 2 μM JC-1, 0.01% digitonin for controlled permeabilization, 10 mM β-mercaptoethanol, and oligomycin (20 μg/ml) to prevent ATP synthase reversal . After a brief room temperature incubation (10 minutes), the cells are mixed with additional buffer and transferred to microplate wells.
Test compounds or antibodies are then added alongside appropriate controls - DMSO as a negative control and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (10 μM) as a positive control for complete depolarization . Fluorescence at 590 nm (characteristic of JC-1 aggregates) is measured at regular intervals (every 10 minutes) at 37°C for 1-3 hours . Compounds that depolarize mitochondria decrease red fluorescence, allowing calculation of EC50 values based on the percentage of depolarization using appropriate curve-fitting software .

What analytical approaches can determine antibody-binding kinetics?

Determining antibody-binding kinetics requires sophisticated analytical approaches that can measure association and dissociation rates between antibodies and their targets. Biolayer Interferometry (BLI) represents a powerful label-free technique that has been successfully employed to characterize antibody-antigen interactions, such as those between anti-DTx monoclonal antibodies and diphtheria toxin . This method measures interference patterns of white light reflected from a biosensor surface, with changes in these patterns correlating with binding events.
BLI experiments typically involve immobilizing either the antibody or antigen on a biosensor tip, then exposing it to varying concentrations of the binding partner while monitoring real-time association. After reaching equilibrium, the sensor is transferred to buffer to observe dissociation. From these binding curves, association rate constants (kon), dissociation rate constants (koff), and equilibrium dissociation constants (KD = koff/kon) can be calculated using appropriate mathematical models.
For antibodies with complex binding mechanisms, additional analytical approaches may be necessary. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays can complement BLI by providing information about conformational changes upon binding . Surface Plasmon Resonance (SPR) offers an alternative approach with similar capabilities to BLI. For therapeutic antibodies, understanding binding kinetics is crucial for predicting in vivo efficacy, as antibodies with slow dissociation rates (small koff values) typically demonstrate prolonged target engagement and enhanced therapeutic effects.

What experimental designs best evaluate maternal antibody transfer and protection in neonates?

Evaluating maternal antibody transfer and protection in neonates requires carefully designed studies that account for multiple variables affecting passive immunity. Cross-sectional studies comparing antibody levels in neonates from different regions can reveal significant disparities in maternal antibody transfer, as demonstrated by research comparing neonates from Shunyi and Qianjiang in China . This study quantified antibodies against diphtheria toxin (DTx), tetanus toxin (Ttx), and pertussis toxin (Ptx) using standardized ELISA methods with established protective thresholds .
A comprehensive experimental design should include sufficient sample sizes from each population (e.g., 200 neonates from Shunyi and 238 from Qianjiang) . Antibody levels should be presented both as mean concentrations with 95% confidence intervals and as seroprevalence rates (percentage of neonates with protective levels) . This dual approach provides insights into both the quantity of antibodies transferred and the proportion of neonates receiving protective immunity.
Statistical analysis should employ appropriate tests for non-normally distributed data, such as the Wilcoxon/Krusakal Wallis test for comparing antibody concentrations and the chi-square test for seroprevalence comparisons . Values below detection limits should be handled consistently, typically assigned as half the lower limit of quantitation . This methodological approach revealed striking differences between regions, with protective anti-Dtx prevalence of only 7.1% in Qianjiang neonates compared to 30.5% in Shunyi neonates , highlighting the impact of socioeconomic factors on maternal antibody transfer and neonatal protection.

How can hydrogen-deuterium exchange mass spectrometry differentiate antibody epitopes?

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers unparalleled ability to differentiate antibody epitopes by exploiting the principle that hydrogen atoms in protein backbones exchange with deuterium at rates dependent on their structural environment and solvent accessibility. When an antibody binds to its target, it shields specific regions from deuterium exchange, creating a distinctive "protection pattern" that can be detected by mass spectrometry.
This approach successfully differentiated the epitopes of two neutralizing anti-DTx monoclonal antibodies (2-25 and 2-18) . The analysis revealed that mAb 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site . In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis .
The power of HDX-MS lies in its ability to characterize conformational epitopes that might be missed by techniques focusing on linear peptide sequences. It provides spatial resolution to localize binding sites and can detect structural changes induced by antibody binding. When combined with complementary techniques like Biolayer Interferometry (BLI), HDX-MS enables researchers to connect structural binding information with kinetic parameters, providing a comprehensive understanding of how antibodies interact with their targets . This detailed epitope characterization informs vaccine design and the development of therapeutic antibodies with desired neutralization mechanisms.

What cell-based assays can evaluate functional effects of antibodies targeting enzymes?

Cell-based assays provide crucial insights into the functional effects of antibodies targeting enzymes like dipeptidyl peptidases or components of cell death pathways. For antibodies targeting DPP9, which functions as an inhibitor of inflammasome activation, pyroptosis assays represent a powerful functional readout . Since DPP9 inhibits caspase-1-dependent pyroptosis by preventing NLRP1 and CARD8 activation, antibodies that neutralize DPP9 would be expected to enhance pyroptotic cell death in relevant cell types like monocytes and macrophages .
For antibodies targeting cell death regulators like BCL-XL, mitochondrial membrane potential assays using permeabilized cells provide a direct functional readout. The JC-1 assay measures mitochondrial depolarization, an early indicator of apoptosis induction . In this assay, cells are suspended in specialized buffer supplemented with JC-1 dye and digitonin for controlled permeabilization . Fluorescence at 590 nm is measured at regular intervals to track mitochondrial membrane potential changes in response to antibody treatments .
Cell viability assays represent another critical functional evaluation method. When testing BCL-XL-targeting ADCs, researchers used cell viability assays to determine EC50 values based on concentration-response curves . For antibody-enzyme interactions, enzyme activity assays using cell lysates or purified proteins can directly measure how antibodies affect catalytic function. These complementary approaches collectively provide a comprehensive understanding of how antibodies modulate enzyme function in cellular contexts.

How do researchers analyze data from antibody seroprevalence studies?

Analyzing data from antibody seroprevalence studies requires specialized statistical approaches that account for the unique characteristics of immunological data. Researchers typically begin by establishing protective threshold values based on established standards - for instance, ≥0.1 IU/ml for anti-diphtheria (anti-Dtx), >0.1 IU/ml for anti-tetanus (anti-Ttx), and >40 IU/ml for anti-pertussis toxin (anti-Ptx) . These thresholds allow calculation of seroprevalence rates, defined as the percentage of individuals with antibody levels above the protective threshold.
For comprehensive analysis, researchers should present both antibody concentrations and seroprevalence data. Antibody concentrations are typically reported as mean values with 95% confidence intervals, while seroprevalence is presented as percentages with 95% confidence intervals . This dual approach provides insights into both the quantity of antibodies and the proportion of the population with protective immunity.
When comparing populations, the chi-square test is appropriate for seroprevalence comparisons, while the Wilcoxon/Krusakal Wallis test is suitable for antibody concentration comparisons due to the typically non-normal distribution of antibody data . This approach revealed significant differences between regions in China, with protective anti-Dtx prevalence of only 7.1% in Qianjiang neonates compared to 30.5% in Shunyi neonates (χ2 = 95.876, P < 0.0001) . These statistical techniques enable researchers to identify disparities in protection between populations and inform targeted interventions to address immunity gaps.