US12 Antibody

What is IL-12 p70 and why is it significant in immunological research?

IL-12 p70 is a biologically active heterodimeric glycoprotein (70 kDa) composed of two subunits: p40 (40 kDa) and p35 (35 kDa), linked by disulfide bonds. It functions as a pleiotropic cytokine originally identified in activated human B lymphoblastoid cell lines, also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF). IL-12 p70 plays a critical role as a central mediator of cell-mediated immune responses through its actions on T-helper 1 (TH1) cell development, proliferation, and activity. Its significance stems from its ability to induce production of IFN-gamma and TNF by T and NK cells, enhance cytotoxic activity, induce proliferation, and act as a co-mitogen for T cells .

The p40 subunit shares extensive homology with the extracellular domain of human IL-6 receptor, while the p35 subunit shows distant but significant sequence similarity to IL-6, G-CSF, and chicken MGF, suggesting IL-12 may have evolved from a cytokine/soluble receptor complex .

How does IL-12 p70 contribute to immune system function?

IL-12 p70 is produced primarily by macrophages and B lymphocytes in response to infectious agents, serving as an initiator of cell-mediated immunity. Its key functions include:

• Inducing IFN-gamma and TNF production by resting and activated T and NK cells

• Synergizing with other IFN-gamma inducers at transcriptional and post-transcriptional levels

• Enhancing cytotoxic activity of resting NK and T cells

• Inducing and synergizing with IL-2 in lymphokine-activated killer (LAK) cell generation

• Stimulating proliferation of resting T cells as a co-mitogen

• Inducing proliferation of activated T and NK cells

Through these mechanisms, IL-12 p70 helps orchestrate immune responses against microbial pathogens, metastatic cancers, and viral infections including HIV/AIDS .

What are the standard detection methods for IL-12 p70?

IL-12 p70 can be detected through several established techniques:

• Direct ELISA: Particularly effective for quantitative measurement in biological samples

• Western blots: Detects IL-12 p70 under non-reducing conditions

• Immunohistochemistry: For visualization in tissue samples (e.g., paraffin-embedded sections)

• Cell-based assays: Including proliferation assays with PHA-activated human peripheral blood mononuclear cells (PBMC)

For optimal results in immunohistochemistry, heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic is recommended prior to primary antibody incubation .

What are the critical parameters for optimizing IL-12 p70 antibody use in immunohistochemistry?

Optimizing IL-12 p70 antibody use in immunohistochemistry requires attention to several key parameters:

When performing IHC with IL-12 p70 antibodies, specific staining should localize to plasma membrane and cytoplasm. Proper controls should always be included to confirm specificity of staining patterns .

How can researchers validate the neutralization capacity of IL-12 p70 antibodies?

Validation of IL-12 p70 antibody neutralization capacity can be performed through a cell proliferation assay:

• Stimulate PHA-activated human peripheral blood mononuclear cells (PBMC) with recombinant human IL-12 (typically 1 ng/mL)

• Add increasing concentrations of the IL-12 p70 antibody

• Measure cell proliferation using appropriate assays (e.g., MTT, BrdU incorporation)

• Calculate the Neutralization Dose (ND₅₀), which is typically 0.3-0.9 μg/mL for effective antibodies

The neutralization capacity of IL-12 p70 antibodies has been leveraged in multiple immunological studies, including research on HIV-specific CD4 T cell responses and immune checkpoint blockade .

What cross-reactivity concerns should researchers address when working with IL-12 p70 antibodies?

When selecting IL-12 p70 antibodies, researchers should consider:

• Potential cross-reactivity with IL-12 p40 subunit alone, which can exist as a monomer or homodimer

• Species specificity (human IL-12 shows minimal activity in murine systems)

• Cross-reactivity with related cytokine family members

High-quality antibodies should be tested for specificity against recombinant IL-12 p40 and other IL-12 family cytokines. For example, the MAB219 antibody shows no cross-reactivity with recombinant human IL-12 p40 in direct ELISAs and Western blots .

How are IL-12 p70 antibodies utilized in studying dendritic cell-NK cell interactions?

IL-12 p70 antibodies have been instrumental in elucidating the mechanisms of NK cell activation by dendritic cells (DCs). Research has demonstrated that:

• DC-NK cell interactions require formation of an immunological synapse

• This synapse leads to IL-12 polarization within DCs

• IL-12 p70 antibodies can be used to neutralize or detect this polarization

• The resulting IL-12 signaling activates NK cells, enhancing their cytotoxic function

A study by Borg et al. utilized IL-12 p70 antibodies in immunocytochemistry to visualize the polarization of IL-12 at the DC-NK cell interface, providing critical insights into this important immune cell interaction .

What role do IL-12 p70 antibodies play in investigating T helper cell differentiation?

IL-12 p70 antibodies serve as valuable tools for studying T helper cell differentiation, particularly regarding Th1/Th2 balance:

• Neutralizing IL-12 p70 antibodies can block Th1 differentiation, allowing researchers to manipulate immune responses

• Studies have shown that Toll-like receptor agonists like macrophage-activating lipopeptide-2 cooperate with IFN-gamma to reverse Th2 skewing, a process that can be blocked using IL-12 p70 neutralizing antibodies

• These antibodies help delineate the molecular mechanisms driving T helper subset commitment

In a study by Weigt et al., neutralizing IL-12 p70 antibodies were used to demonstrate the essential role of IL-12 in TLR2/6 agonist-mediated reversal of allergic Th2 responses .

How does IL-12 antibody research inform therapeutic approaches for cancer and infectious diseases?

Research utilizing IL-12 p70 antibodies has provided valuable insights for therapeutic development:

• IL-12's ability to stimulate cell-mediated immune responses makes it a candidate for immunotherapy against:

  • Microbial pathogens

  • Metastatic cancers

  • Viral infections including HIV/AIDS

• IL-12 p70 antibodies can be used to study IL-12 signaling pathways and identify potential intervention points

• The methodology developed for IL-12 p70 antibodies informs the development of other therapeutic antibodies, such as:

  • The humanized CA12-antibody (4AG4) that blocks carbonic anhydrase 12 enzymatic activity, inhibiting tumor growth in lung adenocarcinoma

  • Broadly neutralizing antibodies against SARS-CoV-2 variants

What are common challenges in IL-12 p70 detection and how can they be addressed?

Researchers frequently encounter several challenges when working with IL-12 p70:

For Western blotting applications specifically, it's critical to note that the structural integrity of the heterodimeric complex is essential for antibody recognition, which is why non-reducing conditions must be maintained .

How should researchers interpret IL-12 p70 detection in complex biological samples?

When interpreting IL-12 p70 detection results:

• Consider the presence of free p40 subunit, which is typically produced in excess of the heterodimeric p70 form

• Account for potential interference from soluble IL-12 receptors in biological samples

• Validate findings using multiple detection methods when possible

• Include appropriate positive and negative controls

• When analyzing tissue samples, compare staining patterns with established localization (membrane and cytoplasmic)

In immunohistochemical analyses, IL-12 p70 has been successfully detected in human spleen and kidney tissues, with specific staining localized to plasma membrane and cytoplasm .

What are the best practices for long-term storage and handling of IL-12 p70 antibodies?

To maintain optimal activity of IL-12 p70 antibodies:

• Store lyophilized antibodies at -20°C to -80°C

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• For short-term use (up to one month), store at 2-8°C

• Avoid exposure to light and maintain sterile conditions

• Follow manufacturer's specific recommendations for each antibody clone

• Validate antibody performance periodically, especially after extended storage

How are IL-12 p70 antibodies being utilized in COVID-19 research?

The methodologies developed for IL-12 p70 antibody research have informed broader antibody research approaches, including:

• Techniques for isolating and characterizing broadly neutralizing antibodies against SARS-CoV-2

• Methods for screening antibody effectiveness against viral variants

• Approaches for studying antibody responses in immunocompromised populations, such as patients on dialysis

Recent research has discovered SC27, a broadly neutralizing plasma antibody capable of neutralizing all known SARS-CoV-2 variants and related coronaviruses. The isolation techniques used in this research build upon methodological approaches developed in cytokine antibody research .

What new technologies are enhancing IL-12 p70 antibody research?

Several technological advances are improving IL-12 p70 antibody research:

• The Ig-Seq technology, originally developed for studying antibody responses to infections, allows closer examination of antibody responses to both infection and vaccination

• Advanced imaging techniques for visualizing cytokine-receptor interactions

• Improved methods for humanizing monoclonal antibodies, as demonstrated in the development of therapeutic antibodies against cancer targets

These technologies allow researchers to better understand IL-12 signaling pathways and develop more effective interventions targeting the IL-12/IFN-γ axis in various diseases.