TNFRSF1A Antibody, HRP conjugated

What is TNFRSF1A and why are antibodies against it important in research?

TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A), also known as TNF-RI, TNFR1, or CD120a, is a critical cell surface receptor that mediates signaling by tumor necrosis factor, playing essential roles in inflammation, apoptosis, and immune regulation. Antibodies targeting TNFRSF1A are valuable research tools for studying TNF signaling pathways in various physiological and pathological conditions, including inflammatory diseases, autoimmune disorders, and cancer. The receptor typically appears at approximately 55 kDa on gel electrophoresis, though variations may occur due to oligomerization, self-aggregation, or cleavage of the extracellular domain . These antibodies enable detection, quantification, and functional characterization of TNFRSF1A in diverse experimental systems, providing insights into receptor expression, localization, and signaling dynamics in different cell types and disease models.

How does the sandwich ELISA principle apply to TNFRSF1A detection using HRP-conjugated antibodies?

In sandwich ELISA for TNFRSF1A detection, the assay employs a capture antibody pre-coated onto a 96-well plate that specifically binds to TNFRSF1A in samples. After sample incubation and washing, a biotin-conjugated detection antibody is added to bind TNFRSF1A captured by the first antibody, creating a "sandwich" structure . Following another washing step, HRP-Streptavidin is introduced to bind the biotinylated detection antibody. After a final washing to remove unbound conjugates, TMB substrate is added, which is catalyzed by HRP to produce a colorimetric signal proportional to the TNFRSF1A concentration in the sample . The reaction is typically stopped with an acidic solution, changing the color from blue to yellow, with absorbance measured at 450nm. This approach offers high specificity through the use of two antibodies recognizing different epitopes of TNFRSF1A, and high sensitivity (as low as 7.81 pg/ml in some assays) through enzymatic signal amplification . The quantitative relationship between TNFRSF1A concentration and signal intensity enables precise measurement across a defined assay range, typically 7.81-500 pg/ml for human TNFRSF1A .

What are the primary applications for TNFRSF1A antibodies with HRP conjugation?

TNFRSF1A antibodies with HRP conjugation serve multiple experimental applications across immunological research. In Western Blotting (WB), these antibodies enable direct detection of TNFRSF1A from protein lysates separated by gel electrophoresis, typically revealing a band at ~55 kDa, although additional bands may appear due to receptor oligomerization or cleavage . For Immunohistochemistry (IHC), HRP-conjugated antibodies facilitate visualization of TNFRSF1A expression patterns in tissue sections through chromogenic substrates, providing insights into receptor distribution in normal and diseased states. In sandwich ELISA systems, these antibodies function as detection reagents for quantitative measurement of soluble TNFRSF1A in biological fluids, cell culture supernatants, or tissue lysates with high sensitivity . Additionally, HRP-conjugated TNFRSF1A antibodies are applicable for Immunocytochemistry (ICC) to detect cellular expression patterns, Immunoprecipitation (IP) to isolate TNFRSF1A-containing complexes, and Immunofluorescence (IF) when paired with appropriate fluorogenic HRP substrates . Each application requires specific optimization of antibody concentration, incubation conditions, and detection systems to maximize signal-to-noise ratio and specificity.

How should researchers prepare biological samples for optimal TNFRSF1A detection?

Sample preparation for TNFRSF1A detection requires careful consideration of tissue or cell type, preservation of protein integrity, and minimization of interfering substances. For tissue homogenates, researchers should: (1) Collect fresh tissue samples and rinse them with pre-cooled PBS to remove blood contaminants; (2) Cut the tissues into small pieces (approximately 1mm³) and homogenize in PBS with protease inhibitors (e.g., 1mM PMSF); (3) Perform ultrasonic disruption or freeze-thaw cycles (with ice bath cooling during sonication, repeating freeze-thaw up to twice) to achieve complete homogenization; (4) Centrifuge homogenates at 5000×g for 5 minutes and collect the supernatant; (5) Determine total protein concentration using BCA assay, aiming for 1-3mg/ml for optimal ELISA performance . For samples with high endogenous peroxidase activity (e.g., liver, kidney, pancreas), treat with 1% H₂O₂ for 15 minutes to prevent false positive results . Importantly, researchers should avoid using certain detergents (NP-40, Triton X-100) or reducing agents (DTT) that may interfere with antibody binding or HRP activity . For cell culture samples and bodily fluids (serum, plasma), centrifugation to remove cellular debris and standardization of protein concentration are essential for reproducible results.

What controls should be included when using HRP-conjugated TNFRSF1A antibodies?

A comprehensive control strategy for experiments using HRP-conjugated TNFRSF1A antibodies should include multiple elements to ensure valid and interpretable results. Positive controls utilizing samples with confirmed TNFRSF1A expression (either recombinant protein or well-characterized cell lines) establish assay functionality and provide reference signal intensities. Negative controls, including samples known to lack TNFRSF1A expression or samples from TNFRSF1A knockout models, help establish background signal levels and confirm specificity. Isotype controls using non-specific antibodies of the same isotype and same conjugation (HRP) as the TNFRSF1A antibody help identify potential non-specific binding. Standard curves generated with purified recombinant TNFRSF1A at known concentrations (typically 7.81-500 pg/ml) are essential for quantitative applications like ELISA . Technical replicates (minimum triplicate) account for pipetting variations and other technical errors, while biological replicates from independent experimental setups address biological variability. For sandwich ELISA specifically, blank controls (all reagents except sample) determine baseline signal, while substrate-only controls (TMB and stop solution without antibodies) identify potential direct interactions between samples and detection substrates . Implementation of these controls facilitates accurate data interpretation and troubleshooting of unexpected results.

How can researchers optimize antibody dilutions for different applications?

Optimization of HRP-conjugated TNFRSF1A antibody dilutions requires a systematic approach tailored to each application. For sandwich ELISA, establish a matrix titration by testing different concentrations of both capture and detection antibodies against a standard curve of recombinant TNFRSF1A. Typical optimal dilutions for HRP-conjugated detection antibodies range from 1:99 to 1:100 in appropriate antibody dilution buffer . For Western blotting, perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) using positive control samples, evaluating signal intensity, specificity (single band at ~55 kDa), and background levels. For immunohistochemistry and immunocytochemistry, test a range of antibody dilutions on known positive tissues/cells alongside appropriate negative controls, assessing staining intensity, pattern specificity, and background. For all applications, adjust incubation parameters (temperature, time) in conjunction with antibody concentration—higher dilutions may require longer incubation periods or higher temperatures. Document all optimization experiments systematically, including images of results with different conditions, to establish reproducible protocols. Remember that optimal dilutions may vary between different lots of the same antibody and across different sample types, necessitating re-optimization when changing critical variables in experimental systems.

What factors affect the sensitivity and specificity of TNFRSF1A detection in sandwich ELISA?

Multiple factors influence the performance characteristics of sandwich ELISA for TNFRSF1A detection, with implications for assay sensitivity (minimum detectable concentration) and specificity (freedom from interference). Antibody quality represents a primary determinant—high-affinity antibodies recognizing non-overlapping epitopes maximize both sensitivity and specificity, with HRP-conjugated detection antibodies typically enabling detection limits as low as 7.81 pg/ml . Sample preparation significantly impacts results; inadequate homogenization, inappropriate buffer composition, presence of interfering substances, or protein degradation can all compromise detection. Incubation conditions affect antibody-antigen interactions; optimal temperatures (typically 37°C) and durations (90 minutes for samples, 60 minutes for detection antibody, 30 minutes for SABC) allow equilibrium binding while minimizing non-specific interactions . Washing procedures critically influence background—insufficient washing leaves residual unbound reagents, while excessive washing may reduce signal. Substrate quality and development time require careful optimization; premature reading or extended substrate incubation can lead to inaccurate quantification. Based on validation data from commercial assays, researchers can expect intra-assay precision (CV) of approximately 5.3-5.9% and inter-assay precision of 5.3-5.3% across low, medium, and high concentration ranges, with sample recovery rates of 87-105% across various biological matrices .

How does sample type affect TNFRSF1A antibody performance in different experimental contexts?

Sample type significantly influences the performance of HRP-conjugated TNFRSF1A antibodies across experimental systems due to differences in matrix composition, potential interfering substances, and protein preservation. In serum samples, recovery rates for TNFRSF1A typically range from 88-104% (average 95%), demonstrating good compatibility with antibody-based detection . EDTA plasma samples show slightly higher recovery rates of 93-105% (average 98%), while heparin plasma samples exhibit 87-99% recovery (average 95%) . These variations reflect the impact of anticoagulants and plasma composition on antibody-antigen interactions. When performing serial dilutions to test linearity, serum samples maintain 87-105% recovery across dilution factors of 1:2, 1:4, and 1:8, while EDTA plasma shows 82-100% and heparin plasma 83-100% across the same dilution range . Cell culture supernatants typically offer cleaner background but may contain lower TNFRSF1A concentrations compared to biological fluids. Tissue homogenates present the greatest challenge, with potential interference from endogenous peroxidases, especially in metabolically active tissues like liver and kidney . For cross-species applications, researchers should note that many TNFRSF1A antibodies demonstrate reactivity across multiple species, including human, mouse, rat, cow, dog, monkey, and rabbit, though epitope conservation should be verified for specific applications .

What are common sources of background in TNFRSF1A detection, and how can they be mitigated?

Background issues in TNFRSF1A detection experiments stem from multiple sources that require specific mitigation strategies. Non-specific antibody binding represents a common problem, addressable through optimization of blocking procedures (using appropriate blocking buffers containing 1-5% BSA or serum) and inclusion of detergents (0.05% Tween-20) in washing buffers to disrupt weak non-specific interactions. Inadequate washing between incubation steps frequently contributes to background; researchers should perform multiple washes (3-5 times) with appropriate immersion times (1 minute per wash) as specified in protocols . Cross-reactivity with related proteins can be minimized by selecting antibodies validated for specificity, particularly those targeting unique epitopes of TNFRSF1A. Endogenous peroxidase activity in samples (especially liver, kidney, and pancreas tissues) generates false positive signals; treatment with 1% H₂O₂ for 15 minutes prior to antibody incubation effectively inactivates these enzymes . Suboptimal substrate handling (improper storage, contamination, or extended development times) may elevate background; adhere to manufacturer recommendations regarding substrate preparation and development time (typically 10-20 minutes at 37°C for TMB) . Matrix effects from complex biological samples can be addressed through appropriate dilution series testing and inclusion of matrix-matched standards when possible. Finally, reagent deterioration due to improper storage compromises performance; store antibodies according to manufacturer instructions, typically at 2-8°C for short-term (maintained at 95-100% activity for 6 months) and avoid repeated freeze-thaw cycles .

How should researchers interpret unusual banding patterns in Western blots using HRP-conjugated TNFRSF1A antibodies?

Interpretation of Western blot results with HRP-conjugated TNFRSF1A antibodies requires careful analysis of expected and observed banding patterns. The canonical band for TNFRSF1A appears at approximately 55 kDa . Multiple bands above this molecular weight often indicate receptor oligomerization or aggregation, a common occurrence with membrane-bound receptors like TNFRSF1A, particularly when samples are inadequately denatured or reduced. Bands at lower molecular weights may represent proteolytic cleavage products of the TNFRSF1A extracellular domain, which can occur both physiologically and during sample processing . To distinguish genuine signals from artifacts, researchers should: (1) Compare results with positive and negative control samples; (2) Verify specificity using peptide competition assays, where pre-incubation of the antibody with immunizing peptide should abolish specific bands; (3) Confirm findings using alternative antibodies targeting different TNFRSF1A epitopes; (4) Correlate banding patterns with sample treatment conditions—harsher denaturing conditions may reduce oligomeric forms; (5) Analyze sample integrity through general protein staining methods; (6) Consider species-specific variations in TNFRSF1A expression and processing. When validating results for publication, researchers should document all bands observed, not just those at expected molecular weights, as these may represent biologically relevant TNFRSF1A variants or processing products that could yield novel insights into receptor biology.

What statistical approaches are appropriate for analyzing TNFRSF1A quantification data from ELISA?

Statistical analysis of TNFRSF1A ELISA data requires appropriate methods to ensure valid interpretation across different experimental contexts. Standard curve fitting should employ four-parameter logistic regression (4PL) or five-parameter logistic regression (5PL) models, which account for the sigmoidal relationship between concentration and optical density, providing more accurate interpolation than linear models, particularly at the extremes of the detection range (7.81-500 pg/ml) . For assessing technical precision, calculation of intra-assay coefficient of variation (CV) across replicates (typically <10% is considered acceptable, with published data showing ~5.3-5.9%) and inter-assay CV across independent experiments (typically <15% is acceptable, with published values around 5.3%) . When comparing TNFRSF1A levels between experimental groups, parametric tests (t-tests, ANOVA) are appropriate if data meet normality assumptions; otherwise, non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) should be employed. For correlation analyses between TNFRSF1A levels and other variables, Pearson's correlation is suitable for normally distributed data, while Spearman's rank correlation accommodates non-normal distributions. When analyzing samples below the limit of detection (typically <7.81 pg/ml), researchers should avoid simply substituting zero or arbitrary values, instead employing dedicated statistical methods for censored data or reporting results as "below detection limit." Finally, researchers should account for recovery rates (typically 87-105% across sample types) and dilution linearity (82-105% across dilution factors) when comparing absolute concentration values across different sample matrices .

How can researchers integrate TNFRSF1A detection with other TNF pathway components for comprehensive signaling analysis?

Comprehensive analysis of TNF signaling requires strategic integration of TNFRSF1A detection with assessment of other pathway components. Multiplex immunoassay panels represent an efficient approach, combining HRP-conjugated TNFRSF1A antibodies with detection systems for soluble TNF, TNFRSF1B (TNF-RII), and downstream signaling molecules (TRADD, TRAF2, RIP1) using antibodies with distinct conjugates or separated spatially in array formats. Co-immunoprecipitation experiments using HRP-conjugated TNFRSF1A antibodies can identify receptor-associated complexes, revealing active signaling modules under different stimulation conditions when analyzed by mass spectrometry or Western blotting for interacting partners. Proximity ligation assays (PLA) enable visualization of molecular interactions between TNFRSF1A and binding partners in situ, providing spatial context for signaling events at subcellular resolution. For functional studies, researchers can correlate TNFRSF1A detection with phosphorylation status of downstream kinases (IKK, JNK, p38) and transcription factors (NF-κB, AP-1) using phospho-specific antibodies in Western blots or flow cytometry. Temporal analysis of pathway activation can be achieved through time-course experiments combining TNFRSF1A detection with real-time monitoring of intracellular calcium, ROS production, or NF-κB nuclear translocation. Integration of these methods provides mechanistic insights into how TNFRSF1A levels and distribution influence downstream signaling outcomes, receptor cross-talk, and cellular responses to inflammatory stimuli in different physiological and pathological contexts.

What considerations are important when designing experiments to investigate TNFRSF1A shedding and soluble receptor dynamics?

Investigation of TNFRSF1A shedding dynamics requires specialized experimental approaches that account for both membrane-bound and soluble receptor forms. Time-course studies should examine TNFRSF1A shedding kinetics in response to stimuli (e.g., LPS, PMA, TNF itself) using sandwich ELISA with HRP-conjugated detection antibodies to quantify soluble receptor accumulation in conditioned media, paired with surface staining or Western blotting to monitor concurrent changes in membrane-bound receptor levels. When designing such experiments, researchers must carefully select antibodies that recognize epitopes preserved in the soluble form (typically in the extracellular domain) and distinguish between proteolytically shed receptors versus alternatively spliced soluble isoforms. Metalloprotease inhibitors (e.g., TAPI-0, GM6001) can be employed to discriminate between constitutive and induced shedding mechanisms, while site-directed mutagenesis of the TNFRSF1A juxtamembrane region can identify specific cleavage sites. Correlation of shedding with functional outcomes requires parallel assessment of TNF neutralizing capacity in biological fluids, typically through TNF bioactivity assays using reporter cell lines. In clinical research contexts, paired measurements of soluble TNFRSF1A and inflammatory markers in longitudinal samples enable identification of temporal relationships between receptor shedding and disease progression. Notably, sample handling significantly impacts soluble TNFRSF1A measurements; standardized collection procedures, minimal freeze-thaw cycles, and consistent processing timeframes are essential for reliable quantification and comparison across experimental conditions.

How do post-translational modifications of TNFRSF1A affect antibody recognition and functional interpretation of results?

Post-translational modifications (PTMs) of TNFRSF1A significantly influence antibody recognition, potentially affecting experimental outcomes and interpretation across different detection methods. Glycosylation, the most prominent PTM of TNFRSF1A, contributes to the heterogeneity in apparent molecular weight (beyond the expected 55 kDa) and may mask epitopes recognized by certain antibodies, particularly those targeting the extracellular domain where N-glycosylation sites predominate . Phosphorylation events, occurring primarily at intracellular domain residues following receptor activation, can alter antibody accessibility to epitopes and reflect the receptor's signaling status rather than absolute expression levels. Ubiquitination and SUMOylation, involved in receptor trafficking and turnover, may generate higher molecular weight species in Western blots that could be misinterpreted as non-specific bands rather than modified receptor forms. When designing experiments, researchers should consider these modifications by: (1) Employing enzymatic deglycosylation (PNGase F, Endo H) to normalize glycoform heterogeneity when precise molecular weight determination is critical; (2) Selecting antibodies whose epitopes are minimally affected by known PTMs, or deliberately choosing modification-sensitive antibodies when studying specific modified receptor populations; (3) Using phosphatase treatments as controls when investigating potential phosphorylation-dependent epitope masking; (4) Correlating biochemical detection with functional assays to distinguish between total receptor levels and functionally competent receptor populations. The interpretation of TNFRSF1A detection data must consider that different PTM profiles may represent distinct receptor subpopulations with unique signaling properties, trafficking patterns, or ligand responsiveness, rather than merely technical artifacts.

What performance metrics should researchers expect from HRP-conjugated TNFRSF1A antibodies in quantitative applications?

Researchers utilizing HRP-conjugated TNFRSF1A antibodies in quantitative applications should benchmark their performance against established metrics to ensure reliable results. For sandwich ELISA systems, typical sensitivity (minimum detectable concentration) ranges from 7.81 pg/ml, with a quantitative detection range of 7.81-500 pg/ml for human TNFRSF1A . Precision metrics derived from validation studies demonstrate intra-assay coefficients of variation (CV) of 5.32-5.86% across low (15.97 pg/ml), medium (64.14 pg/ml), and high (250.1 pg/ml) concentration samples tested 20 times on the same plate . Inter-assay precision shows similar consistency, with CVs of 5.28-5.3% when tested across multiple plates . Recovery performance, which assesses the accuracy of measuring known amounts of TNFRSF1A added to biological matrices, varies by sample type: serum samples show 88-104% recovery (average 95%), EDTA plasma 93-105% (average 98%), and heparin plasma 87-99% (average 95%) . Dilution linearity, measuring the consistency of detection across sample dilutions, demonstrates recovery ranges of 87-105% at 1:2 dilution, 82-104% at 1:4 dilution, and 82-104% at 1:8 dilution across different sample types . Reagent stability studies indicate that properly stored kits maintain approximately 80% activity after 1 month at 37°C and 95-100% activity when stored at 2-8°C for 6 months . These benchmarks provide reference points against which researchers can validate their own experimental systems and troubleshoot performance deviations.

How should researchers validate novel applications of TNFRSF1A antibodies beyond manufacturer-recommended uses?

Validation of novel applications for HRP-conjugated TNFRSF1A antibodies requires systematic methodology to ensure reliability beyond manufacturer-specified uses. Initially, researchers should conduct comparative analysis using multiple antibodies targeting different TNFRSF1A epitopes to confirm consistency of detection patterns in the novel application context. Genetic validation approaches utilizing TNFRSF1A knockout or knockdown systems provide definitive specificity confirmation—signals should be absent or significantly reduced in these models compared to wild-type controls. Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide before application, should abolish specific signals while non-specific binding remains unaffected. For novel sample types or species applications, researchers should perform cross-platform validation by comparing results from the novel application with established methods (e.g., correlating flow cytometry data with ELISA quantification from the same samples). Dose-response relationships should be evaluated by testing serial dilutions of both antibody and antigen, establishing the dynamic range and detection limits specific to the novel application. When adapting sandwich ELISA antibodies to other platforms like Western blotting or immunohistochemistry, determine optimal denaturation conditions that preserve the targeted epitope. For microscopical applications, include co-localization studies with established subcellular markers to confirm expected TNFRSF1A distribution patterns. All validation experiments should be documented comprehensively, including positive and negative controls, to establish reproducibility protocols for the scientific community adopting these novel applications.

What considerations are important when comparing TNFRSF1A detection data across different studies and antibody sources?

Interpretation of TNFRSF1A data across different studies requires careful consideration of methodological variables that influence comparability. Epitope differences between antibodies significantly impact detection—antibodies recognizing the extracellular domain (such as those targeting AA 20-43) may yield different results than those recognizing intracellular domains, particularly in contexts where receptor shedding or proteolytic processing occurs. Conjugation chemistry variations between different HRP-conjugated antibodies affect signal generation efficiency; directly comparing absolute signal intensities between different antibody products without standardization is inappropriate. Assay format differences (sandwich ELISA vs. direct ELISA, Western blot conditions, immunostaining protocols) introduce systematic variations in sensitivity and specificity that complicate direct data comparison. Standard curve materials vary between commercial kits—some use recombinant receptor fragments while others use full-length proteins—creating reference standard discrepancies. Sample processing protocols significantly impact receptor preservation and epitope accessibility; variations in fixation methods, lysis buffers, or denaturation conditions contribute to systematic differences between studies. Detection system variations (chromogenic vs. chemiluminescent substrates, different microplate readers, imaging systems with varying dynamic ranges) further complicate cross-study comparisons. When conducting meta-analyses or comparative studies, researchers should harmonize data through relative quantification approaches (percent of control, fold-change) rather than absolute values, stratify analyses by antibody clone and detection method, and focus on consistent trends rather than absolute measurements. Implementation of common reference materials across laboratories would significantly enhance cross-study data comparability.

How might technological advances enhance the capability of TNFRSF1A detection beyond current HRP-conjugated antibody approaches?

Emerging technologies promise to extend TNFRSF1A detection capabilities beyond traditional HRP-conjugated antibody methods. Proximity-based enzyme complementation systems split HRP or related enzymes between TNFRSF1A-specific antibodies and TNF ligands or secondary antibodies, generating signals only when receptors and ligands interact, enabling real-time monitoring of receptor-ligand engagement in living systems. Single-molecule detection platforms, including digital ELISA technologies, may push TNFRSF1A detection sensitivity into the femtogram/ml range, enabling quantification in sample types or disease states where current methods (with 7.81 pg/ml limits) are insufficient. Mass cytometry (CyTOF) approaches using metal-tagged antibodies against TNFRSF1A and related signaling components will enable high-dimensional analysis of receptor expression in heterogeneous cell populations without fluorescence spectral overlap limitations. CRISPR-based tagging of endogenous TNFRSF1A with reporter proteins or nanobody-binding modules will facilitate live-cell imaging of receptor dynamics without potential artifacts from antibody binding. Aptamer-based detection systems may offer advantages in stability, reproducibility, and cost compared to conventional antibodies, while maintaining similar specificity and potentially higher sensitivity. Computational advances in image analysis using machine learning algorithms will enhance quantification of receptor clustering, internalization, and co-localization with signaling partners from microscopy data. Finally, microfluidic systems integrating sample processing, TNFRSF1A capture, and detection will enable point-of-care applications in clinical settings, potentially transforming research findings into diagnostic applications. These technologies collectively promise more sensitive, specific, and information-rich TNFRSF1A detection, facilitating new insights into TNF signaling biology.

What are the emerging research areas where TNFRSF1A antibodies play crucial investigative roles?

TNFRSF1A antibodies are becoming instrumental in several cutting-edge research domains investigating complex biological processes and disease mechanisms. In single-cell analysis, HRP-conjugated TNFRSF1A antibodies are being adapted for cellular indexing methods that correlate receptor expression with transcriptomic profiles, revealing heterogeneous TNF responsiveness within apparently homogeneous cell populations. Extracellular vesicle (EV) research is utilizing these antibodies to investigate TNFRSF1A incorporation into exosomes and microvesicles, potentially identifying novel TNF signaling mechanisms operating through intercellular communication. Within neuroinflammation research, TNFRSF1A antibodies are revealing cell type-specific receptor expression and signaling in microglia, astrocytes, and neurons, providing insights into neurodegenerative disease mechanisms. Immunometabolism studies are employing these antibodies to investigate how metabolic states influence TNFRSF1A expression, localization, and signaling efficiency in immune cells, potentially explaining context-dependent TNF responses. Cancer immunotherapy research is utilizing TNFRSF1A antibodies to assess receptor density and signaling capacity in tumor microenvironments, correlating with immunotherapy efficacy and resistance mechanisms. Tissue engineering applications are using immobilized TNFRSF1A antibodies to create biomimetic surfaces that capture and present TNF in defined orientations, creating controlled inflammatory microenvironments. Finally, drug development programs targeting TNF-TNFRSF1A interactions rely on these antibodies for screening compound libraries, validating target engagement, and monitoring receptor expression in preclinical models and clinical samples, facilitating translation of basic research insights into therapeutic approaches for inflammatory and autoimmune diseases.

What is the recommended protocol for sandwich ELISA using HRP-conjugated detection antibodies for TNFRSF1A quantification?

The following optimized protocol for TNFRSF1A quantification using sandwich ELISA incorporates evidence-based methodological details to ensure reliable results:

Day 1: Plate Coating

• Dilute capture antibody in coating buffer according to manufacturer recommendations.

• Add 100μl of diluted capture antibody to each well of a high-binding 96-well plate.

• Seal plate and incubate overnight at 4°C.

Day 2: Sample Incubation and Detection

• Wash plate twice with washing buffer (typically PBS with 0.05% Tween-20) .

• Add 100μl of standard (ranging from 7.81-500 pg/ml) or sample to appropriate wells .

• Seal plate and incubate for 90 minutes at 37°C .

• Prepare biotin-labeled detection antibody working solution by diluting concentrated antibody 1:99 in antibody dilution buffer (prepare within 30 minutes of use) .

• Wash plate twice without immersion .

• Add 100μl of diluted biotin-labeled antibody to each well.

• Seal plate and incubate for 60 minutes at 37°C .

• Prepare HRP-Streptavidin Conjugate (SABC) working solution by diluting concentrated SABC 1:99 in SABC dilution buffer (prepare within 30 minutes of use) .

• Wash plate three times with 1-minute immersion per wash .

• Add 100μl of diluted SABC working solution to each well.

• Seal plate and incubate for 30 minutes at 37°C .

• Wash plate five times with 1-minute immersion per wash .

• Add 90μl of TMB substrate solution to each well.

• Incubate for 10-20 minutes at 37°C, protected from light .

• Add 50μl of stop solution to each well when gradient appears and first few wells show deep blue.

• Read optical density at 450nm within 10 minutes of adding stop solution.

Data Analysis

• Generate standard curve using 4-parameter logistic regression.

• Calculate TNFRSF1A concentrations in samples by interpolation from the standard curve.

• Apply dilution factors if samples were diluted prior to assay.

• Evaluate technical replicates, ensuring CV <10% (expected ~5.3-5.9%) .

This protocol consistently delivers a detection range of 7.81-500 pg/ml with high reproducibility across sample types, including serum, plasma, and cell culture supernatants .