Mouse anti-Human TNF alpha monoclonal antibody

What is the typical affinity of Mouse anti-Human TNF alpha monoclonal antibodies?

Mouse anti-Human TNF alpha monoclonal antibodies typically demonstrate high binding affinities to their target antigen. For example, clone B-C7 exhibits an affinity of Kd = 0.5nM, indicating strong binding capacity to human TNF-alpha . The high affinity of these antibodies makes them suitable for various detection methods including ELISA, Western blotting, and functional neutralization assays. When selecting an antibody for your research, affinity considerations are crucial as they directly impact detection sensitivity and specificity in experimental applications.

What isotypes are commonly available for Mouse anti-Human TNF alpha monoclonal antibodies?

Mouse anti-Human TNF alpha monoclonal antibodies are predominantly available as IgG1 isotype with κ light chains. For instance, antibody clones like TSK114 and C2A7 are specifically characterized as IgG1 with κ light chains . The isotype information is important for secondary antibody selection, Fc receptor interactions, and potential effector functions in functional assays. When designing multi-color flow cytometry panels or immunoprecipitation experiments, isotype compatibility must be carefully considered to avoid unwanted cross-reactivity.

How do different Mouse anti-Human TNF alpha antibody clones perform in neutralization assays?

Various antibody clones exhibit different neutralization potencies against human TNF-alpha. For example, clone B-C7 demonstrates neutralizing activity against human TNF-alpha, though specific neutralization dose metrics are not provided in all cases . The MAB610 antibody shows neutralization activity with an ND50 (neutralization dose) typically ranging from 0.01-0.04 μg/mL in cytotoxicity assays using L-929 mouse fibroblast cells in the presence of actinomycin D . Another antibody, AF-410-NA, shows neutralization with an ND50 of 1.5-10 ng/mL in similar assay conditions .

When comparing neutralization potencies, researchers should:

• Use standardized cytotoxicity assays (typically with L-929 cells)

• Include appropriate controls (positive and negative)

• Calculate ND50 values under consistent experimental conditions

• Consider the presence of metabolic inhibitors like actinomycin D that enhance the sensitivity of the assay

What are the critical parameters for Western blot applications of Mouse anti-Human TNF alpha antibodies?

When utilizing Mouse anti-Human TNF alpha antibodies for Western blotting, several critical parameters should be optimized:

For optimal results, Western blot detection typically shows TNF-alpha at approximately 14-17 kDa under reducing conditions . Multiple bands may be observed, representing different glycosylation states or proteolytic fragments. When probing for TNF-alpha in cell lysates, stimulation with LPS (typically 1-10 μg/mL for 4-24 hours) significantly increases detection sensitivity in appropriate cell lines like RAW 264.7 mouse macrophages .

What is the optimal procedure for intracellular TNF-alpha staining in human PBMCs?

Intracellular TNF-alpha staining in human PBMCs requires careful methodology to preserve cellular integrity while achieving optimal staining. The following protocol provides detailed guidance:

• Isolate PBMCs using density gradient centrifugation

• Stimulate cells with LPS (1 μg/mL) and include protein transport inhibitors like monensin to prevent cytokine secretion

• After stimulation (typically 4-6 hours), fix cells using a fixation buffer

• Permeabilize cells with saponin-containing buffer to allow antibody access to intracellular compartments

• Block non-specific binding with appropriate serum

• Incubate with Mouse anti-Human TNF-alpha antibody (e.g., MAB610) at 10 μg/mL for 3 hours at room temperature

• Wash thoroughly to remove unbound primary antibody

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI if desired

• Analyze using flow cytometry or fluorescence microscopy

This approach has been validated for antibodies such as MAB610, which effectively detects TNF-alpha in LPS-stimulated human PBMCs . The inclusion of protein transport inhibitors is critical for accumulating cytokines intracellularly for optimal detection sensitivity.

How should Mouse anti-Human TNF alpha antibodies be reconstituted and stored for maximum stability?

Proper reconstitution and storage are essential for maintaining antibody activity and stability:

• Reconstitution:

  • Lyophilized antibodies should be reconstituted using double-distilled water or appropriate buffer

  • Adjust to recommended concentration (typically 1.0 mg/mL)

  • Allow complete dissolution before aliquoting

  • Filter sterilization (0.2 μm) may be appropriate for long-term storage

• Storage conditions:

  • Store reconstituted antibodies at -20°C to -70°C

  • Divide into small aliquots to minimize freeze-thaw cycles

  • Avoid repeated freezing and thawing as this may denature the antibody

  • Storage in frost-free freezers is not recommended due to temperature fluctuations

  • Working stocks can be maintained at 4°C for up to two weeks

Most Mouse anti-Human TNF alpha antibodies maintain activity for at least 12 months from the date of receipt when stored properly at -20°C to -70°C . For optimal performance in critical experiments, using freshly thawed aliquots is recommended.

How can the specificity of Mouse anti-Human TNF alpha antibodies be validated in research applications?

Validating antibody specificity is crucial for generating reliable experimental results. A comprehensive validation approach should include:

• Positive and negative control samples:

  • Use recombinant human TNF-alpha as a positive control

  • Include mouse TNF-alpha to assess cross-reactivity (unless cross-reactivity is desired)

  • Use knockout or knockdown cell lines as negative controls

• Blocking experiments:

  • Pre-incubate the antibody with recombinant human TNF-alpha

  • Observe elimination of specific staining or detection signal

• Multiple detection methods:

  • Confirm specificity across different applications (ELISA, Western blot, immunostaining)

  • Compare results with alternative antibody clones targeting different epitopes

• Stimulation experiments:

  • Compare TNF-alpha detection in unstimulated vs. LPS-stimulated cells (e.g., RAW 264.7 cells, PBMCs)

  • Expect significant upregulation after appropriate stimulation

Experimental evidence demonstrates that antibodies like MAB610 specifically detect human TNF-alpha without cross-reactivity to other TNF superfamily members such as APRIL/TNFSF13, confirming their selectivity for the intended target .

What factors affect the neutralization potency of Mouse anti-Human TNF alpha antibodies in functional assays?

Several factors can influence the neutralization potency of antibodies in TNF-alpha functional assays:

• Antibody characteristics:

  • Epitope specificity: Antibodies targeting the receptor-binding region typically show higher neutralization potency

  • Binding affinity: Higher affinity generally correlates with improved neutralization

  • Isotype: May affect stability and half-life in certain assay conditions

• Assay conditions:

  • Cell type: L-929 mouse fibroblasts are standard but sensitivity may vary

  • Presence of actinomycin D: Enhances sensitivity to TNF-alpha-induced cytotoxicity

  • TNF-alpha concentration: Must be optimized to achieve appropriate signal-to-noise ratio

  • Incubation time: Typically 18-24 hours for cytotoxicity readout

• Readout method:

  • MTT/XTT vs. crystal violet vs. LDH release assays may yield different sensitivity profiles

  • Flow cytometry-based apoptosis detection provides alternative quantification

For reliable neutralization assessment, standardized cytotoxicity assays using L-929 cells with titrated antibody concentrations against a fixed TNF-alpha concentration (typically 0.1-0.25 ng/mL) in the presence of actinomycin D are recommended .

How should researchers interpret differences in detection sensitivity between various Mouse anti-Human TNF alpha antibody clones?

When comparing detection sensitivity across different antibody clones, researchers should consider several factors that influence performance:

• Epitope specificity:

  • Different clones recognize distinct epitopes on TNF-alpha

  • Epitope accessibility may vary between applications (native vs. denatured conditions)

  • Some epitopes may be masked in protein complexes or oligomeric forms

• Affinity considerations:

  • Higher affinity antibodies (lower Kd values) generally provide better sensitivity

  • Clone B-C7 with Kd = 0.5nM represents a high-affinity antibody option

  • Affinity can affect detection limits in dilute samples

• Application-specific performance:

  • An antibody performing well in ELISA might not excel in Western blotting

  • Native conformation detection (flow cytometry, immunoprecipitation) vs. denatured detection (Western blot)

  • Fixation sensitivity in immunohistochemistry applications

• Cross-platform validation:

  • Confirm findings using alternative detection methods

  • Consider orthogonal approaches when possible

When selecting between antibody options, researchers should prioritize clones validated specifically for their intended application and experimental system rather than relying solely on general sensitivity claims.

What are the key considerations when designing TNF-alpha neutralization experiments for cytokine network studies?

TNF-alpha neutralization experiments in cytokine network studies require careful experimental design:

• System complexity assessment:

  • Simple systems: Recombinant TNF-alpha with reporter cells

  • Intermediate systems: Stimulated cell culture supernatants

  • Complex systems: Co-culture models or ex vivo tissue cultures

• Timing considerations:

  • Early neutralization: Blocks initial TNF-alpha signaling cascade

  • Late neutralization: Addresses feedback amplification loops

  • Continuous vs. pulse neutralization: Different biological outcomes

• Downstream readouts:

  • Direct TNF-alpha signaling (NF-κB activation, MAPK phosphorylation)

  • Secondary cytokine production (IL-1β, IL-6, IL-8)

  • Biological outcomes (cell death, differentiation, migration)

• Control inclusions:

  • Isotype-matched control antibodies

  • Alternative TNF-alpha neutralizing agents (soluble receptors, small molecules)

  • Genetic validation (TNF-alpha or TNFR knockout systems)

In cytotoxicity assays, both MAB610 and AF-410-NA antibodies effectively neutralize TNF-alpha-induced cell death in L-929 cells, with ND50 values of 0.01-0.04 μg/mL and 1.5-10 ng/mL respectively . These potent neutralization capabilities make these antibodies valuable tools for dissecting TNF-alpha-dependent pathways in complex biological systems.