ALA3 Antibody

What is the ALS3 Antibody (Als3_3-A5)?

Als3_3-A5 is a mouse-derived monoclonal antibody (MIgG1, kappa light chain) specifically targeting the Agglutinin-like sequence protein 3 (Als3) of Candida albicans. This antibody was developed by Dr. L.L. Hoyer at the University of Illinois Urbana-Champaign and is available through the Developmental Studies Hybridoma Bank (DSHB). The antibody was generated using a Pichia pastoris GS115-expressed, His-tagged protein domains representing amino acids 18-329 of the Als3 protein as the immunogen. The resulting hybridoma was produced using the SP2/0 myeloma strain, creating a highly specific monoclonal antibody with confirmed reactivity against C. albicans .

What is the functional significance of the Als3 protein targeted by this antibody?

The Als3 protein is a large glycoprotein (123.7 kDa) expressed on the surface of C. albicans germ tubes and hyphae. Its biological significance stems from the peptide-binding cavity in the N-terminal domain, which mediates critical adhesive interactions with host cells, other microbes, and protein-coated abiotic surfaces. Understanding Als3 expression and function is essential for researchers investigating C. albicans pathogenicity, biofilm formation, and host-pathogen interactions . The antibody provides a valuable tool for detecting and potentially blocking these interactions in experimental settings.

How does the ALS3 antibody differ from other Candida-targeting antibodies?

The Als3_3-A5 antibody specifically targets the Als3 protein, which distinguishes it from antibodies targeting other Candida surface proteins. Unlike polyclonal antibodies, this monoclonal antibody binds to a specific epitope within amino acids 18-329 of the Als3 N-terminal domain, offering high specificity for research applications. This specificity allows researchers to precisely monitor Als3 expression without cross-reactivity to other agglutinin-like sequence family proteins that share structural similarities but have distinct functions in C. albicans virulence .

What are the validated applications for ALS3 antibody in fungal research?

The Als3_3-A5 antibody has been validated for multiple research applications, providing versatile experimental options:

Each application requires specific optimization depending on experimental conditions and sample preparation methods .

How should researchers implement immunofluorescence protocols using the ALS3 antibody?

For optimal immunofluorescence studies with the Als3_3-A5 antibody, researchers should follow this methodological approach:

• Culture C. albicans under hypha-inducing conditions (37°C, serum-containing media)

• Fix samples with 4% paraformaldehyde (10-15 minutes), avoiding methanol fixation which may disrupt epitope accessibility

• Block with 1-5% BSA in PBS (30-60 minutes) to reduce non-specific binding

• Apply Als3_3-A5 primary antibody (1:100-1:500 dilution) and incubate (1-2 hours at room temperature or overnight at 4°C)

• Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Apply appropriate fluorophore-conjugated anti-mouse IgG secondary antibody

• Include appropriate controls:

  • Isotype control (mouse IgG1)

  • Secondary antibody-only control

  • Als3-negative strain (if available)

This protocol enables visualization of Als3 distribution on hyphal surfaces, which typically shows enrichment at hyphal tips and along the lateral walls of growing hyphae .

What considerations are important for function-blocking experiments with ALS3 antibody?

When using Als3_3-A5 for function-blocking studies to investigate Als3-mediated adhesion:

• Pre-treatment approach: Incubate C. albicans cells with the antibody (1:50-1:200 dilution) for 30-60 minutes prior to adhesion assays

• Concentration determination: Perform dose-response experiments to determine optimal antibody concentration for effective blocking

• Controls must include:

  • Isotype control antibody at equivalent concentration

  • Untreated C. albicans cells

  • Known Als3-independent adhesion substrate as negative control

• Quantification methods:

  • Crystal violet staining followed by solubilization and spectrophotometric measurement

  • Fluorescence-based quantification of labeled fungi

  • CFU counting after detachment from surfaces

The peptide-binding cavity in Als3's N-terminal domain is the key mediator of adhesive interactions, making this antibody particularly valuable for investigating host-pathogen interactions and potential therapeutic blocking strategies .

How can researchers use ALS3 antibody to investigate morphology-specific protein expression?

Als3 expression is morphology-dependent in C. albicans, predominantly appearing on germ tubes and hyphae rather than yeast forms. To investigate this morphological regulation:

• Experimental design should include:

  • Time-course sampling during yeast-to-hypha transition

  • Parallel morphological assessment and Als3 quantification

  • Correlation of Als3 expression with hyphal extension rates

• Quantitative analysis approaches:

  • Flow cytometry with Als3_3-A5 to measure expression levels across populations

  • Immunofluorescence microscopy with image analysis software to quantify Als3 distribution along individual hyphae

  • Western blotting of fractionated samples to compare expression between morphological forms

• Data interpretation:

  • Als3 signal intensity typically increases with hyphal elongation

  • The peptide-binding cavity in the N-terminal domain shows specific localization patterns related to adhesive function

  • Expression patterns may vary under different hypha-inducing conditions

This approach allows researchers to correlate Als3 expression with morphological transitions, providing insights into the regulation of virulence factors during C. albicans pathogenesis .

How does ALS3 antibody complement genetic approaches in Candida research?

The Als3_3-A5 antibody provides complementary insights to genetic manipulation approaches:

• Protein-level validation: The antibody can confirm the absence of Als3 protein in knockout strains or verify overexpression in genetically modified strains

• Spatial information: Unlike transcriptional studies that show general expression levels, the antibody reveals the spatial distribution of Als3 on fungal surfaces

• Post-translational insights: Als3_3-A5 can detect mature, glycosylated Als3, providing information about post-translational processing that genetic approaches alone cannot address

• Combined methodological approach:

  • Use RT-PCR or RNA-seq to measure ALS3 gene expression

  • Confirm protein expression using Als3_3-A5 in Western blots

  • Determine protein localization through immunofluorescence

  • Assess functional impact through adhesion assays with function-blocking applications

This integrated approach provides comprehensive understanding of Als3 biology beyond what either genetic or antibody-based methods could achieve alone .

What are the considerations for incorporating ALS3 antibody into multi-parameter flow cytometry?

For advanced flow cytometry studies combining Als3 detection with other parameters:

• Panel design considerations:

  • Als3_3-A5 is a mouse IgG1 antibody, requiring appropriate secondary antibody selection

  • Avoid fluorophore spectral overlap with fungal autofluorescence (particularly in GFP range)

  • Consider viability dyes to distinguish live from dead cells

• Sample preparation optimization:

  • Gentle fixation to preserve Als3 epitope while maintaining cell integrity

  • Careful titration of antibody concentration to achieve optimal signal-to-noise ratio

  • Inclusion of blocking steps to reduce non-specific binding

• Controls for multi-parameter analysis:

  • FMO (fluorescence minus one) controls for accurate gating

  • Isotype control for each primary antibody class

  • Single-stained samples for compensation calculation

• Analysis approaches:

  • Correlation of Als3 expression with cell cycle markers

  • Relationship between Als3 levels and other virulence factors

  • Population heterogeneity assessment through clustering algorithms

This approach enables researchers to correlate Als3 expression with other cellular parameters, providing insights into the heterogeneity of C. albicans populations and the coordination of virulence factor expression .

What are common challenges when using ALS3 antibody and how can researchers address them?

Researchers may encounter several challenges when working with the Als3_3-A5 antibody:

• Variable epitope accessibility:

  • Challenge: Als3 is heavily glycosylated, potentially masking epitopes

  • Solution: Test various gentle deglycosylation methods or epitope retrieval approaches

• Background in immunofluorescence:

  • Challenge: Non-specific binding to cell wall components

  • Solution: Increase blocking agent concentration (5% BSA or 10% normal serum) and extend blocking time

• Inconsistent Western blot detection:

  • Challenge: High molecular weight (123.7 kDa) may cause transfer inefficiency

  • Solution: Extend transfer time, reduce gel percentage, or use specialized transfer methods for high molecular weight proteins

• Variable expression levels:

  • Challenge: Als3 expression depends on growth conditions

  • Solution: Standardize culture conditions and document morphological state when comparing between samples

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other Als family proteins

  • Solution: Include appropriate controls (Als3 knockout strains if available) and verify specificity through complementary methods

How should researchers interpret contradictory results between different detection methods?

When faced with discrepancies between different methods using the Als3_3-A5 antibody:

• Consider method-specific limitations:

  • Flow cytometry detects surface-accessible epitopes only

  • Western blotting depends on protein extraction efficiency

  • Immunofluorescence results vary with fixation and permeabilization protocols

• Systematic troubleshooting approach:

  • Verify antibody functionality with positive controls

  • Test multiple antibody concentrations and incubation conditions

  • Evaluate potential interference from sample preparation methods

• Complementary validation strategies:

  • Corroborate protein detection with gene expression analysis

  • Use alternative antibodies targeting different Als3 epitopes if available

  • Employ functional assays to validate Als3 presence

• Interpret results in context:

  • Consider the biological state of the cells (growth phase, morphology)

  • Acknowledge the limitations of each method in publications

  • Present multiple lines of evidence rather than relying on a single technique

What control experiments are essential when using ALS3 antibody?

Rigorous experimental design with Als3_3-A5 requires these essential controls:

These controls enable confident interpretation of experimental results and address potential sources of artifactual findings .

How can ALS3 antibody contribute to biofilm research?

Als3 plays a crucial role in C. albicans biofilm formation, making Als3_3-A5 antibody valuable for biofilm research:

• Methodological applications:

  • Immunofluorescence imaging of Als3 distribution within biofilm architecture

  • Flow cytometric analysis of Als3 expression in cells harvested from different biofilm regions

  • Function-blocking studies to assess Als3 contribution to biofilm initiation and maturation

• Research questions addressable with the antibody:

  • Temporal dynamics of Als3 expression during biofilm development

  • Spatial distribution of Als3 in multi-species biofilms

  • Effect of antifungal agents on Als3 expression in biofilm cells

  • Relationship between Als3 levels and biofilm matrix production

• Experimental approaches:

  • In vitro biofilm models on various substrates (polystyrene, silicone, dentin)

  • Flow cell systems for dynamic biofilm formation

  • Confocal microscopy with Als3_3-A5 for three-dimensional visualization

This antibody enables detailed investigation of Als3's role in the complex process of biofilm formation, with implications for medical device infections and oral candidiasis .

What is the potential of ALS3 antibody for in vivo infection model studies?

Als3_3-A5 can be applied to in vivo infection research with these considerations:

• Tissue section analysis:

  • Immunohistochemistry protocols adapted for infected tissue samples

  • Dual staining to correlate Als3 expression with host immune cell recruitment

  • Quantitative analysis of Als3 expression at different infection sites

• Ex vivo applications:

  • Analysis of C. albicans recovered from infection models

  • Comparison of Als3 expression between in vitro and in vivo growth

  • Assessment of Als3 expression in response to host factors

• Therapeutic investigation potential:

  • Function-blocking studies in animal models to assess virulence contribution

  • Antibody-mediated targeting of Als3-expressing C. albicans in vivo

  • Combination with antifungal agents to evaluate synergistic potential

The antibody provides a valuable tool for translating in vitro findings to more complex in vivo systems, advancing our understanding of C. albicans pathogenesis in clinically relevant models .

How might researchers use ALS3 antibody in combination with emerging technologies?

Integration of Als3_3-A5 with cutting-edge research technologies offers exciting possibilities:

• Single-cell approaches:

  • Mass cytometry (CyTOF) incorporating metal-conjugated Als3_3-A5 for high-dimensional analysis

  • Single-cell RNA-seq combined with index sorting based on Als3 expression

  • Microfluidic systems for real-time monitoring of Als3 expression during morphogenesis

• Advanced imaging techniques:

  • Super-resolution microscopy for nanoscale localization of Als3 on hyphal surfaces

  • Live-cell imaging using labeled Fab fragments of Als3_3-A5

  • Correlative light and electron microscopy to relate Als3 distribution to ultrastructural features

• Biotechnology applications:

  • Development of Als3-targeted diagnostics for invasive candidiasis

  • Exploration of Als3 as a vaccine candidate

  • Engineering of bispecific antibodies incorporating Als3_3-A5 binding domains

These emerging approaches represent the frontier of Als3 research, where the Als3_3-A5 antibody continues to serve as an essential reagent for understanding this important virulence factor .