LCR18 Antibody

What is CD18 and what cellular functions does it regulate?

CD18 (integrin beta 2) is a 90 kDa type I transmembrane protein expressed on all leukocytes. It combines with CD11a-c integrin molecules to form heterodimers at the cell surface. These heterodimers play crucial roles in cell adhesion and cell-surface mediated signaling pathways. CD18 forms four main types of leukocyte (beta2) integrins through heterodimer formation: alphaLbeta2 (CD11a/CD18, LFA-1), alphaMbeta2 (CD11b/CD18, Mac-1, CR3), alphaXbeta2 (CD11c/CD18), and alphaDbeta2 (CD11d/CD18) . These integrins are essential for proper leukocyte migration and intercellular contacts. The absence or severe reduction of CD18 expression leads to leukocyte adhesion deficiency-1 (LAD1) or psoriasiform skin disease, respectively .

What detection methods are compatible with CD18 antibodies?

CD18 antibodies are compatible with multiple detection methods depending on the specific clone and formulation. For instance, the TS1/18 monoclonal antibody (MA1810) has been validated for flow cytometry (FACS), immunoassays (IA), and immunocytochemistry/immunofluorescence (ICC/IF) applications, with demonstrated reactivity against human samples . Other antibodies, such as anti-CD18 monoclonal antibodies used in studies of human monocytes, have been employed to investigate LPS stimulation effects and cytokine production . When selecting a CD18 antibody, researchers should consider the specific applications needed for their experimental design and verify the antibody's validation for those methods.

How do CD18 antibodies affect leukocyte function in experimental settings?

CD18 antibodies can significantly alter leukocyte function in experimental settings. Research has shown that monoclonal antibodies against CD18 (anti-CD18 MoAbs) enhance the cytokine stimulatory effect of lipopolysaccharide (LPS) on human monocytes. Overnight incubation of monocytes with anti-CD18 MoAbs increases CD14 expression as detected by certain markers (Leu-M3), indicating that CD18 participates in LPS-induced TNF-alpha production and regulates CD14 expression on monocytes . These findings suggest that anti-CD18 antibodies can be valuable tools for investigating cell signaling pathways and immune cell responses to various stimuli in experimental settings.

How can CD18 antibodies be utilized to study leukocyte adhesion deficiency (LAD)?

For studying leukocyte adhesion deficiency (LAD), CD18 antibodies serve as crucial tools for both diagnostic assessments and mechanistic investigations. Researchers can employ CD18 antibodies in flow cytometry to quantify the expression level of CD18 on patient-derived leukocytes, which is significantly reduced or absent in LAD1 cases . In experimental models, CD18 antibodies can be used to visualize the distribution and trafficking patterns of leukocytes in tissues. For mechanistic studies, researchers should design experiments that examine both the quantity (using flow cytometry with calibrated beads) and functionality (using adhesion assays) of CD18 on cell surfaces. The antibody clone selection is critical - using multiple validated clones targeting different epitopes provides more robust characterization of CD18 expression. Additionally, researchers can pair CD18 antibodies with CD11a-d antibodies to understand which specific heterodimeric complexes are affected in pathological conditions.

What controls and validation steps are essential when using CD18 antibodies in research?

When employing CD18 antibodies in research, several critical validation steps should be implemented. First, researchers must include appropriate positive controls (such as normal leukocytes known to express CD18) and negative controls (such as brain tissue samples, which are protected by the blood-brain barrier that restricts antibody movement) . Second, perform antibody titration experiments to determine optimal concentrations that provide specific staining while minimizing background. Third, validate antibody specificity through multiple detection methods including Western blotting, immunoprecipitation, and immunohistochemistry with appropriate blocking steps. Fourth, confirm antibody reactivity by testing on cell lines with known CD18 expression levels and CD18-knockout samples when available. Fifth, check for cross-reactivity with other integrin family members through competitive binding assays. Finally, ensure experimental reproducibility by maintaining consistent antibody lots and documenting complete antibody information including clone, isotype, and concentration in all publications.

How do different anti-CD18 clones compare in their epitope recognition and functional effects?

Different anti-CD18 antibody clones exhibit significant variations in their epitope recognition and subsequent functional effects, which can substantially impact experimental outcomes. For instance, the TS1/18 clone (MA1810) has been validated for multiple applications including flow cytometry and immunofluorescence, with specificity for human beta 2 integrin . Other studies have demonstrated that certain anti-CD18 monoclonal antibodies can enhance LPS-induced cytokine stimulation in human monocytes, suggesting recognition of epitopes involved in signaling regulation .

When selecting between clones, researchers should consider that:

• Epitope accessibility varies depending on the conformational state of CD18 (active vs. inactive) and its heterodimeric partners

• Some clones may preferentially recognize particular CD18-CD11 heterodimers

• Certain antibodies may act as agonists, antagonists, or have neutral effects on integrin function

• The presence of divalent cations (Mg²⁺, Ca²⁺) can alter epitope exposure and antibody binding

Researchers should perform comprehensive validation studies comparing multiple clones when initiating a new research direction involving CD18 antibodies.

How should researchers interpret contradictory results from different CD18 antibody clones?

When facing contradictory results from different CD18 antibody clones, researchers should employ a systematic troubleshooting approach. First, evaluate the specific epitopes recognized by each antibody clone, as different epitopes may be differentially accessible depending on CD18's conformational state or its association with various CD11 partners. One study demonstrated that alpha-CD14 monoclonal antibodies that inhibited LPS-induced cytokine production (3C10 and My-4) were considerably less able to detect increased CD14 expression after LPS stimulation compared to antibodies that did not inhibit cytokine production (Leu-M3 and alpha-CD14Serva) .

Second, examine the experimental conditions carefully. Factors such as buffer composition, divalent cation concentration, and sample preparation methods can significantly affect antibody binding. Third, consider the integrin activation state during your experiments, as this can dramatically alter epitope accessibility. Fourth, analyze potential heterogeneity in your cell population, as subpopulations may express different levels of CD18 or CD18-associated molecules. Evidence suggests that CD14+/CD16+ and CD14+/CD16- monocyte subpopulations respond differently to LPS stimulation regarding CD14 expression levels .

Finally, design validation experiments using complementary techniques such as functional assays, genetic knockdown/knockout approaches, or mass spectrometry to resolve discrepancies. Direct comparison experiments should include positive and negative controls, along with side-by-side testing of multiple antibody clones under identical conditions.

What statistical approaches are most appropriate for quantifying CD18 expression levels?

For quantifying CD18 expression levels, several statistical approaches should be employed depending on the experimental context and detection method. For flow cytometry data, which is commonly used to assess CD18 surface expression, researchers should report both the percentage of positive cells and the median fluorescence intensity (MFI) to provide comprehensive information about expression patterns . When comparing multiple cell populations, normalization to a standard control cell line is recommended to account for day-to-day variations in instrument settings.

For Western blot analysis of total CD18 levels, densitometric analysis with normalization to housekeeping proteins should be performed. As demonstrated in studies comparing different cancer cell lines, this approach can reveal that surface expression differences may not always correlate with total protein levels . For immunohistochemistry or immunofluorescence analyses, automated image analysis software should be employed to quantify staining intensity across multiple fields, with appropriate background subtraction.

Statistical tests should include:

• For normally distributed data: t-tests for two-group comparisons or ANOVA with post-hoc tests for multiple group comparisons

• For non-normally distributed data: Mann-Whitney U or Kruskal-Wallis tests

• Pearson's or Spearman's correlation coefficients to assess relationships between CD18 expression and functional outcomes

To enhance reproducibility, researchers should report effect sizes and confidence intervals in addition to p-values, and consider using hierarchical statistical models for complex experimental designs.

How can anti-CD18 antibodies be employed to modulate disease progression in animal models?

Anti-CD18 antibodies offer significant potential for modulating disease progression in animal models of inflammatory and immune-mediated disorders. When designing such studies, researchers should first select an antibody clone with cross-reactivity to the species being studied or use species-specific antibodies. Administration strategies should consider multiple parameters including dose optimization (typically starting with 1-10 mg/kg based on published literature), timing of intervention (preventive versus therapeutic protocols), and route of administration (intravenous for systemic effects versus local administration for tissue-specific targeting).

For inflammatory disease models, researchers should assess outcomes through multiple complementary approaches: histopathological scoring of affected tissues, quantification of inflammatory mediators, functional assays relevant to the disease model, and tracking of leukocyte infiltration using flow cytometry or immunohistochemistry. Control groups must include isotype-matched antibodies to account for non-specific effects. Moreover, researchers should evaluate potential compensatory mechanisms that may emerge following CD18 blockade, such as upregulation of alternate adhesion pathways.

What insights can CD18 antibody studies provide about cancer metastasis mechanisms?

CD18 antibody studies can provide valuable insights into cancer metastasis mechanisms, particularly regarding leukocyte-cancer cell interactions and inflammatory microenvironments that influence tumor progression. Research utilizing anti-LRP/LR specific antibody IgG1-iS18, which affects cell adhesion mechanisms similar to those involving CD18, has demonstrated significant reduction in the adhesive and invasive potential of metastatic liver cancer cells . This suggests that antibodies targeting adhesion molecules can impede critical steps in the metastatic cascade.

By applying similar methodological approaches to CD18 research, investigators can:

• Analyze how tumor-associated neutrophils and macrophages (which express high levels of CD18) influence the metastatic niche

• Examine whether CD18-mediated leukocyte infiltration differs between primary tumors and metastatic sites

• Investigate if CD18 blockade affects extracellular matrix remodeling essential for metastatic progression

• Determine whether CD18-expressing leukocytes facilitate tumor cell extravasation at distant sites

Flow cytometry analysis can be used to correlate CD18 expression levels with metastatic potential across different cancer cell lines. For instance, studies on liver cancer cells (HUH-7) have shown significantly higher cell surface levels of adhesion receptors compared to poorly-invasive breast cancer control cells (MCF-7), with direct proportionality between receptor levels and adhesive/invasive potential . These methodologies can be adapted to CD18 studies to elucidate its specific role in cancer progression.

How are antibody sequence databases enhancing therapeutic antibody development?

Antibody sequence databases are revolutionizing therapeutic antibody development through comprehensive cataloging of natural antibody repertoires. Recent advances in this field involve data mining antibody sequences for efficient database searches in proteomics data. For instance, researchers have utilized the Observed Antibody Space (OAS) database containing thirty million heavy antibody sequences from 146 SARS-CoV-2 patients to generate 18 million unique peptides through in silico digestion . These peptides were then used to create new databases for bottom-up proteomics approaches.

This methodology allows researchers to discover disease-specific antibody peptides while avoiding false positives through careful validation against negative controls, such as brain samples (which naturally restrict antibody movement due to the blood-brain barrier) . The discovered sequences provide valuable information to distinguish diseased from healthy states and can be further employed to develop therapeutic antibodies with high specificity and efficacy.

For researchers interested in applying similar approaches to CD18 antibody development, this data mining strategy offers several advantages:

• Identification of naturally occurring anti-CD18 antibody sequences from human donors

• Discovery of potential epitope hotspots that could be targeted for therapeutic development

• Comparison of antibody responses across different inflammatory conditions where CD18 plays a role

• Acceleration of lead candidate identification by starting with human-derived sequences

This approach is broadly applicable beyond infectious diseases like SARS-CoV-2 and could be adapted to develop therapeutic antibodies targeting CD18 for inflammatory disorders, autoimmune conditions, or cancer .

What role do post-translational modifications play in CD18 function and antibody recognition?

Post-translational modifications (PTMs) play critical roles in regulating CD18 function and can significantly impact antibody recognition. CD18 undergoes several PTMs including N-glycosylation, phosphorylation, and proteolytic processing that modulate its adhesive properties and signaling capabilities. When investigating CD18 antibodies, researchers must consider how these modifications affect epitope accessibility and antibody binding efficiency.

N-glycosylation of CD18 is particularly important for proper heterodimer formation with CD11 subunits and subsequent cell surface expression. Experimental approaches to study glycosylation effects include:

• Treatment with glycosidases or glycosylation inhibitors (tunicamycin, swainsonine) followed by antibody binding assessment

• Site-directed mutagenesis of glycosylation sites combined with functional assays

• Mass spectrometry analysis to characterize glycan structures under different cellular conditions

Phosphorylation of cytoplasmic domains of CD18 regulates inside-out signaling that controls integrin activation states. Researchers should employ phospho-specific antibodies in combination with standard CD18 antibodies to correlate activation state with epitope accessibility. Studies on LPS stimulation of monocytes have shown that cellular activation can dramatically alter surface marker expression and antibody binding patterns , suggesting that activation-dependent PTMs likely influence antibody recognition.

When selecting antibodies for CD18 research, investigators should critically evaluate whether the antibodies recognize conformational epitopes that might be affected by PTMs or whether they bind to linear epitopes that remain accessible regardless of modification state.