IL 8 Antibody

What is IL-8 and why are antibodies against it important in research?

IL-8 (also known as CXCL8) is a crucial member of the C-X-C chemokine family that serves as a potent chemoattractant cytokine. It plays a vital role in the chemotaxis and activation of neutrophils, which are essential for immune response . IL-8 is primarily secreted by activated macrophages and epithelial cells, and its expression is significantly associated with various chronic inflammatory diseases, including inflammatory bowel disease and atherosclerosis .

IL-8 antibodies are important research tools because they enable detection, quantification, and neutralization of IL-8 in various experimental settings. This allows researchers to study IL-8's role in inflammation, cancer progression, and angiogenesis. The significance of IL-8 in disease pathogenesis makes these antibodies valuable for both basic research and therapeutic development.

What are the common forms of IL-8 antibodies available for research?

Several types of IL-8 antibodies are available for research purposes:

• Monoclonal antibodies (e.g., C-11, 8CH) - These offer high specificity and consistency between lots. For example, IL-8 Antibody (C-11) is a mouse monoclonal IgG1 kappa light chain antibody that detects IL-8 protein of human origin .

• Humanized antibodies (e.g., ABX-IL8) - Fully human anti-IL-8 antibodies like ABX-IL8 have been developed for potential therapeutic applications. ABX-IL8 is a human IgG2 monoclonal antibody directed against human IL-8 that binds with high affinity (kd = 2 × 10^10 mol/L) .

• Conjugated antibodies - Many IL-8 antibodies are available in conjugated forms, including:

  • Fluorophore conjugates (PE, FITC, Alexa Fluor®) for flow cytometry

  • Enzyme conjugates (HRP) for ELISA and Western blotting

  • Agarose conjugates for immunoprecipitation

These diverse formats allow researchers to select the most appropriate antibody for their specific experimental needs.

What forms of IL-8 protein can be detected with available antibodies?

IL-8 is synthesized as a 99 amino acid precursor protein that is further processed into one of four isoforms, with the most common being 72 or 77 amino acids in length . Available antibodies typically recognize specific epitopes within these processed forms:

• IL-8(72) is the predominant form present in adults, expressed by monocytes, macrophages, epithelial cells, and fibroblasts in response to inflammatory stimuli, environmental stress, and steroid hormones .

• IL-8(77) is secreted primarily by endothelial cells and is thought to be a less potent neutrophil activator than the other forms. It is present at high levels during fetal development, where it mediates angiogenesis rather than inflammation .

When selecting an IL-8 antibody, researchers should verify which form(s) the antibody recognizes, as this may impact experimental results depending on the biological context being studied.

What are the primary applications of IL-8 antibodies in research?

IL-8 antibodies can be utilized in multiple research applications:

• Western blotting (WB) - For detecting IL-8 protein in cell or tissue lysates

• Immunoprecipitation (IP) - For isolating IL-8 protein complexes

• Immunofluorescence (IF) - For visualizing IL-8 distribution in cells or tissues

• Enzyme-linked immunosorbent assay (ELISA) - For quantifying IL-8 levels in biological samples

• Flow cytometry - For analyzing intracellular IL-8 expression at the single-cell level

For intracellular staining followed by flow cytometric analysis, pre-titrated antibodies like the 8CH monoclonal antibody can be used at specified concentrations (e.g., 5 μL or 0.015 μg per test) .

How can IL-8 antibodies be used to study cancer progression and metastasis?

IL-8 antibodies have proven valuable in studying cancer progression, particularly in melanoma research. Studies have shown that:

• Neutralizing IL-8 with antibodies like ABX-IL8 significantly inhibits tumor growth in melanoma models. When human melanoma cells (A375SM and TXM-13) were injected subcutaneously into nude mice treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks), tumor growth was significantly inhibited compared to control IgG-treated animals .

• IL-8 blockade suppresses experimental metastasis. ABX-IL8 treatment reduced metastatic potential when melanoma cells were injected intravenously .

• IL-8 antibodies can help elucidate mechanisms of metastasis by revealing how IL-8 influences matrix metalloproteinase (MMP) expression. ABX-IL8 significantly inhibited the promoter activity and collagenase activity of MMP-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane .

• Antibody-based approaches help researchers understand how IL-8 contributes to tumor angiogenesis. In vivo vessel formation assays show that ABX-IL8 directly interferes with tubule formation by human umbilical vein endothelial cells (HUVECs) .

These findings suggest that IL-8 antibodies provide powerful tools for investigating tumor microenvironment, cancer cell invasion, and metastatic processes, potentially informing new therapeutic strategies.

What role do IL-8 antibodies play in investigating angiogenesis mechanisms?

IL-8 antibodies have been instrumental in elucidating IL-8's role in angiogenesis:

• Neutralizing antibodies like ABX-IL8 can be used in tube formation assays with HUVECs to directly assess IL-8's contribution to endothelial cell organization. Research shows that HUVECs pretreated with ABX-IL8 (100 μg/ml) for 4 days demonstrate altered tubule formation when plated on Matrigel, compared to IgG-treated controls .

• In vivo angiogenesis models utilizing IL-8 antibodies have revealed that IL-8 blockade leads to decreased vascularization of tumors. This is associated with increased apoptosis of tumor cells, highlighting the relationship between angiogenesis and tumor cell survival .

• IL-8 antibodies can be used to investigate cross-talk between inflammatory and angiogenic pathways. IL-8(77) is present at high levels during fetal development, where it mediates angiogenesis rather than inflammation—a distinction that can be explored using isoform-specific antibodies .

• Combining IL-8 antibodies with other angiogenesis inhibitors provides insights into potential synergistic therapeutic approaches. Research suggests that anti-IL-8 antibodies like ABX-IL8 may be beneficial for therapy either alone or in combination with other chemotherapeutic or anti-angiogenic agents .

These applications demonstrate how IL-8 antibodies serve as valuable tools for understanding the complex relationship between inflammation and angiogenesis in both development and disease.

How are IL-8 antibodies employed in inflammatory disease research?

IL-8 antibodies enable researchers to investigate IL-8's contribution to various inflammatory conditions:

• In bullous pemphigoid (BP) research, IL-8 antibodies have helped establish IL-8's role in neutrophil recruitment and blister formation. Studies have detected abnormally high levels of IL-8 in blisters and sera of BP patients, and in experimental mouse models, IL-8 injections facilitated blister formation in C5-or mast cell-deficient mice that were otherwise resistant to blister induction .

• IL-8 antibodies can be used to study therapeutic mechanisms of anti-inflammatory drugs. For example, researchers found that dapsone (a therapeutic agent for BP) suppresses IL-8-mediated neutrophil chemotaxis in vitro, providing insight into its mechanism of action .

• Flow cytometric analysis using PE-conjugated IL-8 monoclonal antibodies allows investigators to measure intracellular IL-8 production in specific cell populations, such as LPS-treated human blood monocytes. This helps identify which cells are responsible for IL-8 production during inflammatory responses .

• Inhibition experiments using recombinant IL-8 to block antibody staining provide a method to confirm staining specificity. This approach has been demonstrated with PE-conjugated anti-human CXCL8/IL-8 monoclonal antibody (Catalog # IC208P) in flow cytometry experiments .

These diverse applications highlight how IL-8 antibodies contribute to unraveling the complex inflammatory mechanisms underlying various pathological conditions.

What are the optimal protocols for using IL-8 antibodies in flow cytometry?

For successful flow cytometric analysis of IL-8 using antibodies, researchers should follow these methodological guidelines:

• Cell preparation: For intracellular staining, cells must be appropriately fixed and permeabilized. This can be achieved using fixation buffer followed by permeabilization/wash buffer .

• Antibody concentration: Pre-titrated antibodies should be used at manufacturer-recommended concentrations. For example, the 8CH monoclonal antibody conjugated to APC can be used at 5 μL (0.015 μg) per test, where a test is defined as the amount of antibody that will stain a cell sample in a final volume of 100 μL .

• Cell number optimization: The number of cells per test should be determined empirically but typically ranges from 10^5 to 10^8 cells/test .

• Proper controls: Include:

  • Isotype controls (e.g., Catalog # IC002G) to establish quadrant markers

  • Blocking controls using recombinant IL-8 to confirm specificity of staining

  • Unstained cells for autofluorescence assessment

• Multiparameter analysis: Combine IL-8 antibody staining with surface markers (e.g., CD14) to identify specific cell populations producing IL-8. This allows for more nuanced analysis of IL-8 expression patterns .

A well-documented example protocol involves treating human peripheral blood monocytes with LPS, then staining with Mouse Anti-Human CXCL8/IL-8 PE-conjugated Monoclonal Antibody and Anti-Human CD14 Fluorescein-conjugated Antibody. Specificity can be confirmed by inhibition of staining through addition of excess Recombinant Human IL-8 .

What controls are essential when using IL-8 antibodies in Western blot and immunoassays?

When conducting Western blots or immunoassays using IL-8 antibodies, the following controls are critical for ensuring reliable and interpretable results:

• Positive controls:

  • Recombinant human IL-8 protein at known concentrations

  • Cell lysates from IL-8-producing cells (e.g., LPS-stimulated monocytes)

  • Commercially available positive control lysates

• Negative controls:

  • Lysates from cells known not to express IL-8

  • Samples from IL-8 knockout models (if available)

  • Untreated cell lines that don't constitutively express IL-8

• Specificity controls:

  • Pre-incubation of the antibody with recombinant IL-8 to confirm binding specificity

  • Isotype-matched control antibodies to identify non-specific binding

  • Testing for cross-reactivity with related chemokines (IL-8 antibodies should fail to cross-react with closely related chemokines)

• Antibody validation controls:

  • Multiple antibodies targeting different epitopes of IL-8

  • Testing for species cross-reactivity when relevant (e.g., some antibodies show cross-reactivity with porcine CXCL8/IL-8)

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH) for Western blots

  • Equal protein loading verification using total protein stains

Implementing these controls helps ensure that observed signals are specifically due to IL-8 detection and not artifacts or non-specific interactions.

How can researchers optimize the use of IL-8 antibodies for detecting low levels of IL-8?

Detecting low IL-8 concentrations requires optimization strategies:

• Signal amplification techniques:

  • Use high-sensitivity detection systems (e.g., chemiluminescent substrates for Western blots)

  • Employ tyramide signal amplification for immunohistochemistry or immunofluorescence

  • Consider biotin-streptavidin amplification systems for ELISAs

• Sample concentration:

  • Concentrate biological fluids using ultrafiltration

  • Employ immunoprecipitation to concentrate IL-8 from dilute samples

  • Use larger sample volumes when possible

• Antibody selection and optimization:

  • Choose high-affinity antibodies (e.g., ABX-IL8 with kd = 2 × 10^10 mol/L)

  • Optimize antibody concentration through titration experiments

  • Consider using antibody cocktails targeting different IL-8 epitopes

• Reduce background and non-specific binding:

  • Optimize blocking conditions (test different blocking agents and concentrations)

  • Include detergents and carrier proteins in washing buffers

  • Extend washing steps to remove weakly bound antibodies

• Advanced detection platforms:

  • Consider Luminex-based assays for multiplex detection with high sensitivity

  • Explore single-molecule detection methods for extremely low concentrations

  • Evaluate digital ELISA platforms with improved lower limits of detection

By implementing these strategies, researchers can enhance detection sensitivity for low-abundance IL-8, enabling studies of subtle changes in IL-8 levels that may have biological significance.

What are the cross-reactivity concerns with IL-8 antibodies across species?

Cross-reactivity is an important consideration when working with IL-8 antibodies across different species:

• Documented cross-reactivity patterns:

  • Some anti-human IL-8 antibodies show cross-reactivity with porcine CXCL8/IL-8 in Western blots. For example, certain antibodies demonstrate 100% cross-reactivity with recombinant porcine CXCL8/IL-8 .

  • Cross-reactivity with mouse IL-8 homologs is generally limited due to significant sequence differences.

• Validation approaches for cross-species applications:

  • Perform parallel experiments with species-specific positive controls

  • Verify antibody specificity using recombinant IL-8 from the species of interest

  • Consider epitope mapping to identify conserved regions across species

• Alternative strategies for multi-species studies:

  • Use species-specific antibodies when available

  • Design experiments focusing on functional readouts rather than direct IL-8 detection

  • Consider genetic approaches (e.g., reporter systems) for interspecies comparisons

• Reporting standards for cross-reactivity:

  • Clearly document any observed cross-reactivity in publications

  • Include appropriate controls demonstrating specificity in the species being studied

  • Acknowledge limitations when extrapolating results across species

Understanding these cross-reactivity considerations is essential for experimental design, especially in comparative studies or when using animal models to investigate human disease mechanisms.

How can researchers troubleshoot non-specific binding in IL-8 antibody applications?

Non-specific binding is a common challenge when working with IL-8 antibodies. Here are systematic approaches to troubleshoot this issue:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blocking buffers)

  • Increase blocking time or concentration

  • Add carrier proteins (e.g., BSA, gelatin) to antibody diluents

• Validate antibody specificity:

  • Perform pre-absorption tests by incubating the antibody with recombinant IL-8 before application

  • Compare staining patterns between multiple antibodies targeting different IL-8 epitopes

  • Include negative controls (e.g., cells known not to express IL-8)

• Adjust antibody concentration:

  • Titrate primary antibody to find optimal concentration that maximizes specific signal while minimizing background

  • For flow cytometry, start with manufacturer-recommended concentrations (e.g., 5 μL or 0.015 μg per test for APC-conjugated 8CH antibody)

  • For Western blots, consider using more dilute antibody solutions with longer incubation times

• Modify washing procedures:

  • Increase number and duration of wash steps

  • Add detergents (e.g., Tween-20, Triton X-100) to wash buffers at appropriate concentrations

  • Consider using higher salt concentrations in wash buffers to disrupt weak non-specific interactions

• Sample preparation considerations:

  • Ensure complete cell fixation and permeabilization for intracellular staining

  • Pre-clear lysates before immunoprecipitation

  • Filter biological fluids to remove particulates before antibody applications

By systematically addressing these factors, researchers can significantly improve signal-to-noise ratios in their IL-8 antibody applications.

What factors affect IL-8 antibody stability and performance in experimental settings?

Several factors can impact IL-8 antibody stability and performance:

• Storage conditions:

  • Temperature considerations: Most IL-8 antibodies should be stored at 2-8°C and should not be frozen

  • Light exposure: Fluorophore-conjugated antibodies (PE, FITC, Alexa Fluor) must be protected from light to prevent photobleaching

  • Aliquoting: Divide antibodies into single-use aliquots to avoid repeated freeze-thaw cycles

• Buffer composition effects:

  • pH fluctuations can alter antibody binding characteristics

  • Certain preservatives may interfere with specific applications

  • Presence of carrier proteins may enhance long-term stability

• Application-specific considerations:

  • For flow cytometry: Optimal fixation and permeabilization buffers are critical for intracellular staining

  • For Western blotting: Denaturing conditions affect epitope accessibility

  • For immunoprecipitation: Detergent composition influences antibody-antigen complex stability

• Longevity and shelf-life:

  • Most commercial IL-8 antibodies maintain activity for approximately 12 months from date of receipt when stored properly (2-8°C)

  • Conjugated antibodies typically have shorter shelf-lives than unconjugated versions

  • Performance should be verified periodically with positive controls

• Reconstitution practices:

  • Follow manufacturer guidelines for reconstitution of lyophilized antibodies

  • Use sterile techniques to prevent microbial contamination

  • Allow antibodies to equilibrate to room temperature before opening to prevent condensation

Understanding these factors helps researchers maintain antibody integrity and optimize experimental outcomes across different applications.

How are IL-8 neutralizing antibodies being developed for therapeutic applications?

IL-8 neutralizing antibodies represent promising therapeutic strategies, particularly in inflammatory diseases and cancer:

• Development approaches:

  • Fully humanized antibodies like ABX-IL8 have been generated using XenoMouse technology, in which murine heavy and light chain loci have been inactivated and replaced with human immunoglobulin loci

  • These antibodies bind to human IL-8 with high affinity (kd = 2 × 10^10 mol/L) and fail to cross-react with closely related chemokines

  • They function by blocking IL-8 binding to its receptors (CXCR1 and CXCR2)

• Therapeutic mechanisms:

  • Inhibition of neutrophil activation, migration, and degranulation

  • Suppression of angiogenesis through interference with endothelial cell tubule formation

  • Reduction of matrix metalloproteinase-2 (MMP-2) expression and activity, decreasing cancer cell invasion

• Preclinical evidence:

  • In melanoma models, ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks) significantly inhibited tumor growth compared to control IgG-treated animals

  • ABX-IL8 treatment suppressed experimental metastasis when melanoma cells were injected intravenously

  • Decreased vascularization and increased apoptosis of tumor cells were observed in ABX-IL8-treated tumors

• Combinatorial approaches:

  • IL-8 neutralizing antibodies may be beneficial for therapy either alone or in combination with other chemotherapeutic or anti-angiogenic agents

  • This suggests potential for integration into existing treatment regimens to enhance efficacy

These developments highlight how research tools like IL-8 antibodies can evolve into potential therapeutic agents, representing an important translational aspect of IL-8 antibody research.

What role do IL-8 antibodies play in studying the relationship between inflammation and cancer?

IL-8 antibodies have been instrumental in elucidating the complex relationship between inflammation and cancer:

• Investigating inflammatory signals in tumor microenvironments:

  • IL-8 antibodies allow researchers to quantify IL-8 levels in tumor tissues and correlate expression with inflammatory cell infiltration

  • Flow cytometric analysis using IL-8 antibodies can identify which cells within the tumor microenvironment produce IL-8

• Understanding mechanistic connections:

  • Research using IL-8 antibodies has shown that IL-8 transcripts are often upregulated in tumors, and IL-8 is associated with tumor angiogenesis and metastasis

  • IL-8 antibodies have helped establish that IL-8 functions not just as an inflammatory mediator but also as an autocrine growth factor for melanoma cells and inducer of haptotatic migration

• Exploring cancer-associated inflammation:

  • Studies using IL-8 antibodies like ABX-IL8 revealed that IL-8 blockade significantly inhibits MMP-2 promoter activity and collagenase activity in human melanoma cells, decreasing invasion through reconstituted basement membrane

  • This highlights how inflammatory mediators can directly promote cancer cell invasiveness

• Therapeutic implications:

  • Experimental findings using IL-8 antibodies suggest that targeting inflammatory cytokines like IL-8 could be an effective strategy to control tumor growth and metastasis

  • The ability of IL-8 antibodies to suppress angiogenesis provides mechanistic insight into how inflammatory mediators contribute to tumor progression beyond direct effects on cancer cells

These applications demonstrate how IL-8 antibodies serve as valuable tools for understanding the inflammatory components of cancer biology, potentially informing new therapeutic approaches that target the inflammation-cancer axis.