SPCC18.20 Antibody

What is the SPCC18.20 Antibody and what epitope does it recognize?

SPCC18.20 is a monoclonal antibody that recognizes specific epitopes on the CD18 antigen, which is a 90 kDa type I transmembrane protein expressed on leukocytes. CD18 forms the β2 integrin subunit that combines with CD11a-d to form heterodimers involved in cell adhesion and cell-surface mediated signaling .

Methodologically, epitope characterization involves:

• Immunoprecipitation experiments using detergent lysates of iodinated peripheral blood leukocytes

• Competitive binding assays with other anti-CD18 antibodies

• Peptide mapping using overlapping synthetic peptides

In previously documented studies, similar CD18 antibodies have been shown to immunoprecipitate the common 95 kDa beta 2 integrin chain along with non-covalently associated alpha chains at 180 kDa (CD11a), 165 kDa (CD11b), and 150 kDa (CD11c) .

How should the specificity of SPCC18.20 Antibody be validated?

Comprehensive antibody validation requires multiple approaches:

Similar to other validated antibodies, rigorous testing should show that SPCC18.20 maintains specificity across applications and recognizes the target in its native confirmation . Avoid relying on a single validation method, as different applications may reveal different binding characteristics or potential cross-reactivity issues .

What are the optimal conditions for using SPCC18.20 in flow cytometry experiments?

Flow cytometry is a primary application for anti-CD18 antibodies. Based on documented protocols for similar antibodies:

Recommended Protocol:

• Collect cells in suspension (peripheral blood leukocytes, cultured leukocytes)

• Wash cells in PBS with 1-2% bovine serum albumin (BSA)

• Resuspend at 1 × 10^6 cells per 100 μl

• Add SPCC18.20 antibody at 1:25 to 1:200 dilution (optimize for specific lot)

• Incubate for 30 minutes at 4°C

• Wash twice with PBS/BSA

• Add appropriate fluorophore-conjugated secondary antibody

• Incubate for 30 minutes at 4°C in darkness

• Wash twice and analyze

For multicolor panels, CD18 expression can be used to differentiate between monocytes (high expression), granulocytes (intermediate-high), and lymphocytes (lower expression) . Control samples must include isotype controls at matching concentrations to determine background staining levels.

How should SPCC18.20 be used for immunohistochemistry on frozen versus paraffin sections?

The epitope recognized by anti-CD18 antibodies is typically sensitive to formaldehyde fixation and tissue processing. Therefore:

For Frozen Sections (Preferred):

• Cut 8 μm tissue sections

• Fix with acetone for 10 minutes at -20°C

• Air dry sections for 30 minutes

• Block with 2-3% serum (species of secondary antibody) for 30 minutes

• Apply SPCC18.20 antibody at optimized dilution

• Incubate overnight at 4°C or 2 hours at room temperature

• Wash 3× with PBS

• Apply detection system (secondary antibody or amplification system)

• Develop using appropriate substrate

For Paraffin Sections (If necessary):
Additional antigen retrieval is crucial:

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval with citrate buffer (pH 6.0) for 30 minutes or protease digestion

• Cool to room temperature

• Continue with blocking and immunostaining as above

When comparing results between fixation methods, acetone-fixed frozen sections typically provide stronger and more consistent staining for CD18 .

How can SPCC18.20 be used to investigate antibody-dependent cellular cytotoxicity (ADCC) mechanisms?

ADCC is a key mechanism of action for therapeutic antibodies. To investigate ADCC using SPCC18.20:

Experimental Design:

• Target cell preparation:

  • Label target cells expressing the antigen with a fluorescent dye or 51Cr

  • Opsonize with SPCC18.20 at various concentrations (0.01-10 μg/ml)

• Effector cell preparation:

  • Isolate natural killer (NK) cells or other FcR-bearing effector cells

  • Typical effector-to-target (E:T) ratios range from 5:1 to 50:1

• ADCC assay:

  • Co-culture target and effector cells for 4-16 hours

  • Measure cytotoxicity via release of label or flow cytometry

• Controls:

  • Isotype-matched control antibody

  • Target cells without antibody

  • Effector cells alone

Recent studies have shown that ADCC efficacy is typically 10-fold lower than antibody-dependent cellular phagocytosis (ADCP), with NK cells mediating ~0.04-0.1 targets/cell compared to ~0.5-3 targets/macrophage for ADCP . This should be considered when interpreting SPCC18.20 cytotoxicity data.

How can SPCC18.20 be humanized for potential therapeutic applications?

Humanization of antibodies reduces immunogenicity while preserving binding characteristics. Based on documented approaches:

Variable Domain Resurfacing Method:

• Sequence analysis:

  • Align SPCC18.20 variable regions with human antibody sequences

  • Identify framework residues in murine antibody that are rare in human antibodies

• Surface residue identification:

  • Calculate residue solvent accessibility using software like Swiss-PdbViewer

  • Define surface amino acids as those with relative accessibility >30%

• Guided mutation design:

  • Replace murine surface residues with human consensus residues

  • Preserve CDR regions and residues that directly contact antigen

  • Analyze potential structure changes using computational modeling

• Stepwise mutation introduction:

  • Create multiple variants with increasing numbers of mutations

  • Test each variant for retained binding properties

  • Optimize to achieve maximum humanization while maintaining specificity and affinity

• Testing humanized constructs:

  • Express in suitable systems (e.g., E. coli using pMD204 vector)

  • Purify using affinity chromatography

  • Compare binding properties to original antibody via ELISA and functional assays

Studies have shown successful humanization with 13-15 mutations at key positions while preserving functional properties . The optimal construct will retain stability, binding specificity, and affinity of the parent antibody.

What methods should be used to determine the binding affinity of SPCC18.20?

Accurate binding affinity determination requires multiple complementary techniques:

How should researchers assess cross-reactivity of SPCC18.20 with CD18 from different species?

Cross-reactivity assessment is crucial for determining potential model systems and predicting off-target effects:

Methodological Approach:

• Initial screening:

  • Flow cytometry using peripheral blood leukocytes from different species

  • Western blot analysis of leukocyte lysates from multiple species

• Quantitative assessment:

  • Compare staining intensity profiles across species

  • Calculate relative binding affinities using SPR or competition assays

• Epitope conservation analysis:

  • Sequence alignment of CD18 across species

  • Correlation of binding with sequence conservation

• Functional validation:

  • Confirm that antibody binding produces similar functional outcomes

  • Test antibody-mediated effects in different species

Based on documented patterns for similar antibodies, CD18 antibodies often show cross-reactivity with mammalian species due to high sequence conservation in certain domains. For example, some anti-CD18 clones recognize epitopes common to human, canine, bovine, and porcine CD18 .

What factors contribute to loss of SPCC18.20 functional activity during storage and how can this be prevented?

Antibody activity degradation is multifactorial:

Common Degradation Mechanisms:

• Aggregation:

  • Accelerated by freeze-thaw cycles, agitation, and high concentration

  • Monitored via size exclusion chromatography or dynamic light scattering

• Oxidation:

  • Primarily affects methionine residues in CDRs

  • Catalyzed by trace metals, light exposure, and peroxides

• Deamidation:

  • Affects asparagine residues, especially in specific sequence contexts

  • Increased rate at higher pH and temperature

• Fragmentation:

  • Common at hinge region and other susceptible sites

  • Accelerated by metal ions and extremes of pH

Recommended Storage Practices:

• Store at -80°C for long-term stability

• For working solutions, store at 4°C with preservatives (e.g., 0.09% sodium azide)

• Aliquot to avoid repeated freeze-thaw cycles

• Add carrier proteins (0.1-1% BSA) for dilute solutions

• Use amber vials to protect from light exposure

• Validate activity after extended storage using functional assays

Stability studies on similar antibodies suggest maintaining glycerol-free formulations at concentrations >0.5 mg/ml when possible, and avoiding storage in frost-free freezers where temperature cycling can occur .

How can researchers address epitope masking when using SPCC18.20 in complex tissue samples?

Epitope masking is a common challenge in immunohistochemistry and can affect SPCC18.20 binding to CD18:

Methodological Solutions:

• Optimize fixation:

  • For CD18, acetone fixation of frozen sections typically preserves epitope accessibility

  • If using formalin fixation, limit to brief periods (≤24 hours)

• Enhanced antigen retrieval:

  • Heat-induced epitope retrieval: Test multiple buffers (citrate pH 6.0, EDTA pH 8.0, Tris pH 9.0)

  • Enzymatic retrieval: Proteinase K, trypsin, or pepsin at optimized concentrations

  • Combined approaches: Sequential enzymatic and heat-mediated retrieval

• Signal amplification strategies:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

  • Extended primary antibody incubation (overnight at 4°C)

• Blocking optimization:

  • Test various blocking agents (BSA, normal serum, commercial blockers)

  • Include blocking steps for endogenous peroxidase, biotin, and Fc receptors

• Sequential immunostaining:

  • For multilabel studies, apply SPCC18.20 first when possible

  • Use directly conjugated antibody to minimize cross-reactivity issues

For immunohistochemical applications with SPCC18.20, tissue processing steps can significantly impact epitope accessibility. In studies of prion disease samples, formic acid treatment (96% for 1 hour) after paraformaldehyde fixation has been shown to enhance epitope accessibility for certain antibodies .

How can SPCC18.20 be used to investigate leukocyte adhesion deficiency (LAD) mechanisms?

Leukocyte adhesion deficiency type I (LAD1) is caused by defects in the CD18 gene. SPCC18.20 can be instrumental in studying this condition:

Research Applications:

• Diagnostic assessment:

  • Flow cytometric quantification of CD18 expression on patient leukocytes

  • Correlation of expression levels with disease severity

  • Distinguish between complete absence and reduced expression

• Genetic variant characterization:

  • Analyze impact of specific mutations on antibody binding

  • Correlate binding patterns with structural changes in CD18

  • Compare surface expression versus intracellular retention

• Functional studies:

  • Adhesion assays under static and flow conditions

  • Migration assays in response to inflammatory stimuli

  • Formation of immune synapses and cellular interactions

• Therapeutic development:

  • Gene therapy efficacy assessment via CD18 expression restoration

  • Cell-based therapy monitoring

  • Small molecule screening for expression enhancement

Studies have demonstrated that absence of CD18 leads to LAD1, while severe reduction of CD18 expression can lead to psoriasiform skin disease . SPCC18.20 can help quantify these expression differences and correlate them with clinical outcomes.

How should SPCC18.20 be adapted for use in virus neutralization assays?

Virus neutralization assays provide important insights into antibody functionality and immune protection:

Assay Development Protocol:

• Sample preparation:

  • Dilute serum or purified antibody 1:40 (or optimize starting dilution)

  • Inactivate complement by heating at 56°C for 30 minutes

  • Prepare serial dilutions (typically 1:2 for 12 steps)

• Virus neutralization:

  • Incubate diluted samples with standardized virus dose (e.g., 1 × 10^2 PFU)

  • Maintain at 37°C for 90 minutes with gentle agitation

  • Include virus-only and antibody-only controls

• Cell infection:

  • Transfer virus-antibody mixtures to appropriate cell monolayers (e.g., Vero E6)

  • Allow virus adsorption (1 hour at 37°C)

  • Overlay with methylcellulose or similar semi-solid medium

• Endpoint detection:

  • After 48-72 hours, visualize plaques via crystal violet staining

  • Calculate neutralization titers as highest dilution reducing plaques by 50% or 80%

For analysis of immunoglobulin class-specific neutralization, samples can be pretreated with β-mercaptoethanol (0.1 M) to remove IgM before virus incubation . This allows differentiation between IgG-mediated and total neutralizing activity.

How can SPCC18.20 be used in multiplexed proteomic assays for immune complex characterization?

Emerging multiplexed proteomic approaches offer new insights into antibody-antigen interactions:

Implementation Protocol:

• Immobilization strategy:

  • Couple SPCC18.20 to magnetic beads or other solid support

  • Alternatively, use the antibody as a bait in immunoprecipitation

• Sample processing:

  • Incubate antibody-coated beads with biological samples

  • Capture immune complexes containing target antigen

  • Wash under controlled stringency conditions

• Proteomics analysis:

  • Perform on-bead digestion with trypsin

  • Analyze peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Quantify using label-free or isobaric labeling approaches

• Data analysis:

  • Identify proteins in the immunocomplex

  • Quantify relative abundances across conditions

  • Correlate complex composition with functional outcomes

This approach can reveal not only the primary target but also associated proteins in the complex. Recent studies have demonstrated that such multiplexed, targeted-proteomic assays can characterize multiple proteins in antibody immunocomplexes, including various antibody classes, isotypes, and associated complement binding .

What considerations are important when developing SPCC18.20 as a bispecific antibody?

Bispecific antibody development requires careful engineering and characterization:

Development Considerations:

• Format selection:

  • Tandem scFv formats

  • IgG-like formats with heterodimeric Fc

  • Diabody or dual-affinity retargeting (DART) formats

• Target selection for second binding site:

  • Complementary immune effector (CD3, CD16)

  • Tumor-associated antigen for directed targeting

  • Second epitope on same antigen for enhanced avidity

• Expression system optimization:

  • Mammalian expression systems typically preferred

  • Knob-into-hole or other technologies to ensure correct pairing

  • Purification strategy to remove mispaired species

• Functional characterization:

  • Binding to each target individually and simultaneously

  • Potency in relevant cell-based assays

  • Stability assessment under physiological conditions

• Analytical characterization:

  • Size exclusion chromatography for aggregation assessment

  • Mass spectrometry for molecular integrity

  • Surface plasmon resonance for binding kinetics to each target

When developing bispecific antibodies, recent research highlights the importance of format selection to balance activity, stability, and manufacturability. The orientation and linker length between binding domains can significantly impact function, requiring empirical optimization for each target combination .

Recent studies have shown that CD18-targeting bispecifics can redirect immune effector functions with high specificity, particularly when combined with tumor-associated antigens to enhance cellular cytotoxicity against cancer cells .

What are the critical quality attributes that should be monitored for consistent SPCC18.20 performance across production lots?

Ensuring batch-to-batch consistency requires comprehensive characterization:

Regular testing against a well-characterized reference standard is essential. Implementing a stability-indicating analytical package helps detect potential degradation before it impacts experimental results. For research antibodies like SPCC18.20, endotoxin testing is particularly important as contamination can confound immunological experiments .

How should researchers validate SPCC18.20 specificity when working with samples containing variant forms of the antigen?

Validating antibody specificity for variant antigens requires systematic approaches:

Validation Strategy:

• Sequence analysis:

  • Identify variant positions relative to the epitope region

  • Predict impact on binding using structural information

  • Design experiments targeting specific variants

• Recombinant protein testing:

  • Express wildtype and variant proteins

  • Compare binding affinities using ELISA or SPR

  • Develop standard curves for each variant

• Native sample validation:

  • Source samples containing natural variants

  • Correlate antibody binding with genetic/proteomic analysis

  • Develop detection strategies for variant-specific signals

• Competitive binding assays:

  • Use known variant peptides to block antibody binding

  • Determine relative affinities for each variant

  • Identify potential cross-reactivity

• Parallel validation with alternative antibodies:

  • Compare with antibodies targeting different epitopes

  • Develop complementary detection strategies

  • Create validation panels for complex samples

This approach is particularly important when studying genetic variants or post-translational modifications. Recent studies on SARS-CoV-2 variants demonstrated that antibodies targeting the spike protein showed markedly different binding to variants like Omicron B.1.1.529, with some losing neutralizing activity completely . Similar principles apply to CD18 variants that may be present in clinical samples.