GL1 Antibody

What is GL1 antibody and what epitope does it recognize?

GL1 (also written as GL-1) is a rat monoclonal antibody that specifically recognizes mouse CD86, also known as B7-2, a member of the B7 family of costimulatory molecules. CD86 is an approximately 80 kDa surface receptor expressed on antigen-presenting cells. The GL1 clone was originally developed using LPS-activated CBA/Ca mouse splenic B cells as the immunogen . This antibody recognizes an epitope on the extracellular domain of CD86 that is critical for its interaction with CD28 and CTLA-4. GL1 antibody is widely used in immunological research to detect, quantify, and functionally block CD86-mediated interactions .

What is the biological significance of CD86 in immune responses?

CD86 serves as a critical costimulatory molecule during antigen presentation. Its significance in immune regulation includes:

• Functioning as a counter-receptor for the T cell surface molecules CD28 and CD152 (CTLA-4)

• Playing essential roles in T-B cell crosstalk and T cell costimulation

• Contributing to autoantibody production and Th2-mediated immunoglobulin production

• Providing the "second signal" required for effective T cell activation, with its absence potentially leading to T cell anergy or tolerance
  The kinetics of CD86 upregulation upon stimulation supports its major contribution during the primary phase of an immune response, distinguishing it from CD80 (B7-1) which plays a more prominent role in later phases .

Which cell types express CD86 and how is expression regulated?

CD86 expression follows a specific pattern across immune cell populations:

• Expressed at low baseline levels on B cells, macrophages, and dendritic cells

• Upregulated on B cells through various surface stimuli including BCR complex engagement, CD40 ligation, and certain cytokine receptor stimulation

• Also expressed by activated mouse T cells and thioglycolate-elicited peritoneal cells

• Expressed on astrocytes, providing a neuroimmune interface
  The regulation of CD86 expression is dynamic and context-dependent, with rapid upregulation occurring within hours of activation stimuli, making it a useful marker for early immune cell activation states .

What are the validated research applications for GL1 antibody?

GL1 antibody has been validated for multiple experimental applications:

• Flow cytometric analysis of CD86 expression on various cell types, particularly B cells, dendritic cells, and macrophages

• Immunoprecipitation of CD86 protein from cell lysates

• Immunohistochemical staining of acetone-fixed frozen tissue sections

• Immunofluorescence microscopy for cellular localization studies

• In vivo and in vitro blocking of T cell responses mediated by CD86-CD28/CTLA-4 interactions
  It's important to note that GL1 is not suitable for immunohistochemical staining of formalin-fixed paraffin-embedded sections, which is a significant limitation for certain experimental designs .

How should GL1 antibody be optimized for flow cytometry?

For optimal flow cytometry results with GL1 antibody, researchers should follow these methodological considerations:

• Titrate the antibody carefully, with recommended starting concentrations of ≤0.125 μg per test (where a test is defined as staining 10^5-10^8 cells in a final volume of 100 μL)

• Determine optimal cell numbers empirically, though typical ranges are 10^5 to 10^8 cells per test

• For PE-conjugated GL1, use appropriate excitation wavelengths (488-561 nm) and emission filters (578 nm)

• For APC-conjugated GL1, use red laser (633 nm) excitation

• Store the antibody solution undiluted between 2°C and 8°C, protected from prolonged light exposure, and never freeze

• Include appropriate isotype controls and single-stain controls when designing multicolor panels
  Proper titration is particularly crucial for achieving the optimal signal-to-noise ratio and ensuring reliable quantitative measurements of CD86 expression .

What functional assays can GL1 antibody be used for?

GL1 antibody is particularly valuable for functional studies of CD86-mediated immune interactions:

• Blocking mixed lymphocyte reactions in vitro

• Inhibiting the priming of cytotoxic T lymphocytes in vivo when used in combination with anti-B7-1 antibodies

• Investigating T cell costimulation requirements in various immunological contexts

• Studying the differential contributions of CD86 versus CD80 in T helper cell differentiation
  For functional blocking studies, researchers should use the Ultra-LEAF™ purified format of GL1 antibody (endotoxin <0.01 EU/μg, azide-free, 0.2 μm filtered) to avoid confounding effects from preservatives or endotoxin contamination .

How can GL1 antibody be used to investigate T cell costimulation pathways?

For advanced investigation of T cell costimulation pathways, researchers can employ GL1 antibody in several sophisticated experimental designs:

• Combinatorial blocking studies with anti-CD80 and anti-CD86 antibodies to dissect the relative contributions of each costimulatory molecule to T cell activation

• Comparative analysis of CD86 blockade effects on different T cell subsets (Th1, Th2, Th17, Tregs) to understand subset-specific costimulation requirements

• Time-course studies exploiting the differential kinetics of CD80 vs. CD86 expression to target specific phases of immune responses

• In vivo blocking experiments to assess CD86 contributions to vaccine responses, autoimmunity models, or transplant rejection
  These approaches can yield insights into the complex interplay between CD86 and other costimulatory/inhibitory pathways that regulate T cell activation thresholds and functional differentiation .

What are key considerations for designing in vivo blocking experiments with GL1 antibody?

When designing in vivo blocking experiments with GL1 antibody, researchers should consider:

• Dosing regimen: Typically 100-200 μg per mouse, with specific optimization needed based on the experimental model

• Timing: Administration before or at the time of antigenic challenge for preventive studies, or after challenge for therapeutic assessment

• Delivery route: Intraperitoneal injection is most common, though intravenous routes may provide more rapid distribution

• Duration: Half-life considerations may necessitate repeated dosing for sustained blockade

• Combination approaches: Co-administration with anti-CD80 antibodies often provides more complete blockade of costimulation

• Controls: Isotype-matched control antibodies are essential to differentiate specific from non-specific effects

• Verification of blockade: Ex vivo assessment of CD86 accessibility on target cells to confirm effective blocking
  The Ultra-LEAF™ preparation is strongly recommended for in vivo applications to minimize endotoxin-related confounding effects .

How can multiparameter analysis incorporate GL1 antibody for comprehensive immune profiling?

For comprehensive immune profiling incorporating CD86 detection:

• Design multicolor flow cytometry panels that include GL1 antibody with markers for:

  • Cell lineage identification (e.g., CD19 for B cells, CD11c for dendritic cells)

  • Activation status markers (e.g., MHC-II, CD40, CD69)

  • Additional costimulatory molecules (CD80, CD40, ICOSL)

  • Functional markers (cytokine production, proliferation markers)

• Consider spectral compatibility when selecting GL1 conjugates:

  • PE-conjugated GL1 works well with blue (488 nm) and yellow-green (561 nm) lasers

  • APC-conjugated GL1 is optimized for red laser (633 nm) excitation

  • FITC-conjugated GL1 can be used with standard blue laser setups

• Implement dimensionality reduction techniques (tSNE, UMAP) for visualization and unsupervised clustering to identify novel CD86-expressing cell populations or correlation patterns with other markers .

What are common technical issues with GL1 antibody and how can they be resolved?

Researchers commonly encounter several technical challenges when working with GL1 antibody:

How should GL1 antibody be stored and handled to maintain optimal performance?

To maintain optimal performance of GL1 antibody preparations:

• Store undiluted between 2°C and 8°C, never freeze the conjugated antibody formulations

• Protect fluorochrome-conjugated formats from prolonged light exposure

• Maintain sterility by using aseptic technique when handling

• Avoid repeated freeze-thaw cycles with unconjugated antibody preparations

• Be aware that sodium azide (0.09%) is present in many commercial preparations as a preservative, which can inhibit certain enzymatic reactions and is toxic at high concentrations

• For functional assays, use azide-free, low-endotoxin preparations (Ultra-LEAF™)

• Centrifuge the vial briefly before opening to collect liquid at the bottom

• Observe expiration dates and recommended storage conditions specified by manufacturers
  Following these guidelines helps maintain antibody binding affinity and fluorophore activity over time .

How can researchers validate GL1 antibody specificity in their experimental systems?

To validate GL1 antibody specificity in experimental systems:

• Perform staining on known CD86-positive cells (LPS-activated B cells) and CD86-negative cells as biological controls

• Include isotype control antibodies (rat IgG2a) at equivalent concentrations to assess non-specific binding

• Conduct blocking/competition experiments with unlabeled GL1

• Compare staining patterns with alternative anti-CD86 clones

• Verify expected expression patterns on different cell populations (B cells > dendritic cells > resting T cells)

• For knockout validation, use CD86-deficient cells or tissues as the gold standard negative control

• Confirm functional blockade by assessing inhibition of T cell proliferation in mixed lymphocyte reactions
  These validation steps ensure that experimental results reflect specific detection of CD86 rather than artifacts .

How do different CD86 expression levels correlate with functional states of immune cells?

CD86 expression levels provide important insights into immune cell functional states:

• Low expression on resting B cells, macrophages, and dendritic cells indicates a quiescent state

• Rapid upregulation (within hours) following activation signals such as BCR engagement, TLR stimulation, or CD40 ligation indicates early activation

• High expression on mature dendritic cells correlates with potent T cell stimulatory capacity

• Differential expression relative to CD80 can indicate the phase of an immune response, with CD86 predominating in early phases

• Aberrant expression patterns may indicate pathological states or dysregulated immune activation
  Quantitative assessment of CD86 expression using standardized measures (median fluorescence intensity, molecules of equivalent soluble fluorochrome) allows for more precise correlation with functional capabilities .

How do CD86 and CD80 cooperatively and distinctly regulate T cell responses?

CD86 and CD80 exhibit both overlapping and distinct roles in T cell regulation:

• Both interact with CD28 (costimulatory) and CTLA-4 (inhibitory), but with different binding kinetics and affinities

• CD86 is typically upregulated more rapidly than CD80 following activation stimuli

• CD86 appears to play a predominant role during the primary phase of immune responses

• CD80 shows stronger binding to CTLA-4, potentially contributing to more potent inhibitory signals

• Differential expression patterns on various antigen-presenting cells suggest specialized functions

• Combined blockade of both CD80 and CD86 typically produces more complete inhibition of T cell responses than blocking either alone

• CD86 may play a preferential role in certain Th2-mediated responses and antibody production
  Understanding these distinctions helps researchers design more targeted interventions for specific immunological scenarios .

What should researchers consider when comparing GL1 antibody data across different experimental models?

When comparing GL1 antibody data across different experimental models, researchers should consider:

• Mouse strain variations: CD86 expression patterns and regulatory mechanisms may differ between common laboratory strains

• Age-dependent effects: Immune senescence can alter costimulatory molecule expression and function

• Microbiome influences: Housing conditions and microbiota composition can affect baseline immune activation states

• Technical variables: Different flow cytometer configurations, antibody lots, and experimental protocols may introduce variability

• Biological context: Infection status, inflammatory conditions, and genetic backgrounds can significantly modify CD86 expression patterns

• Antibody format differences: Various conjugates (PE, APC, FITC) may have different sensitivities and dynamic ranges

• Quantification methods: Percentage positive versus MFI measurements provide different information about expression patterns
  Standardization of protocols, inclusion of appropriate controls, and careful reporting of methodological details facilitate meaningful cross-study comparisons .

How might GL1 antibody contribute to therapeutic research in autoimmunity and cancer?

GL1 antibody has significant potential for therapeutic research applications:

• Preclinical modeling of CD86-targeted immunotherapies for autoimmune diseases

• Investigation of CD86 blockade in combination with checkpoint inhibitors for cancer immunotherapy

• Study of selective CD86 vs. CD80 targeting to fine-tune costimulatory signals

• Development of modified GL1 derivatives with enhanced blocking or depleting capabilities

• Exploration of GL1 in establishing tolerogenic conditions for transplantation models

• Investigation of tissue-specific roles of CD86 in specialized immune microenvironments
  While primarily a research tool, insights gained from GL1 studies inform the development of humanized therapeutic antibodies targeting the CD86 pathway in clinical settings .

What novel detection technologies might enhance GL1 antibody applications?

Emerging technologies that could enhance GL1 antibody applications include:

• Single-cell RNA-seq combined with protein detection (CITE-seq) to correlate CD86 protein expression with transcriptional states

• Mass cytometry (CyTOF) incorporation of metal-labeled GL1 for high-parameter analysis without fluorescence spillover concerns

• Super-resolution microscopy to study nanoscale organization of CD86 in immune synapses

• Intravital imaging using fluorescent GL1 derivatives to track CD86 dynamics in vivo

• Bifunctional antibody constructs linking GL1 specificity to reporter enzymes or fluorescent proteins

• Engineered GL1-based chimeric antigen receptors (CARs) to target CD86-expressing cells
  These technological advances promise to extend the utility of GL1 antibody beyond conventional applications and provide deeper insights into CD86 biology .

How can computational approaches enhance data analysis from GL1 antibody experiments?

Advanced computational approaches can significantly enhance GL1 antibody experimental data analysis:

• Machine learning algorithms to identify subtle patterns in CD86 expression across cell populations

• Network analysis to map CD86 interactions with other costimulatory and inhibitory pathways

• Predictive modeling of CD86 blockade effects on immune response trajectories

• Systems biology approaches integrating CD86 expression data with transcriptomics, proteomics, and metabolomics

• Spatial analysis algorithms for quantifying CD86+ cell distributions in tissue microenvironments

• Temporal modeling of CD86 expression dynamics during immune response development

• Multi-omics data integration to contextualize CD86 expression within broader immune signatures
  These computational strategies transform descriptive GL1 antibody data into predictive models with greater biological insight and translational relevance .